# Patent

## Title
Methods and compositions using splicing regulatory proteins involved in tumor suppression

## Abstract

Methods and compositions for diagnosis and prognosis of mammalian carcinoma or cancer derived from primary epithelial cells and tissue fibrosis are designed using newly identified epithelial cell-type specific splicing factors ESRP1 and ESRP2, which have roles in tumor suppression. Diagnostic reagents for the detection of these splicing factors in nucleotide or protein form are useful in such methods. Therapeutic compositions can provide epithelial cells with these factors to maintain FGFR2 and assist in suppressing metastasis. A high throughput splicing assay to identify compounds that change splicing events is described. RNCP1 is also identified as a splicing factor and a diagnostic for conditions characterized by inappropriate FGFR2-splicing.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This is a national stage application under 35 U.S.C. 371 of PCT/US2009/051239, filed on Jul. 21, 2009, which claims the benefit under 35 USC 119(e) of U.S. patent application Ser. No. 61/082,435, filed on Jul. 21, 2008 (now expired), all of which are incorporated by reference herein.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with government support under Grant No. CA093769 awarded by the National Institutes of Health. The government has certain rights in the invention.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made with government support under grant no. R01- CA093769 awarded by the National Institutes of Health. The government has certain rights in the invention.


BACKGROUND OF THE INVENTION
The epithelial mesenchymal transition (EMT) and the reverse process of a mesenchymal to epithelial transition (MET) are fundamental processes during embryonic development. The changes associated with cellular plasticity that characterize them have been implicated in disease processes including cancer and fibrosis. There are numerous demonstrations that the EMT is a mechanism that can contribute to the metastatic process (Thiery, 2002; Yang and Weinberg, 2008). The EMT can be a transient and reversible process by which tumor cells acquire motile properties that allow escape from the primary tumor followed by an MET at a distant site of metastasis (Chaffer et al., 2007).
Changes in the activities of trans-acting regulators of splicing and of a number of specific splice variants have been shown to contribute to tumorigenesis (Hu and Fu, 2007). For example the SR protein SF2/ASF was shown to be a proto-oncogene that can drive tumorigenesis through the induction of changes in splicing of several target transcripts (Karni et al., 2007). In additional, splicing-specific microarray platforms that facilitate global analysis of splicing have shown the potential to yield “splicing signatures” that can be used for cancer diagnosis and prognosis (Blencowe, 2006).
Fibroblast growth factor receptor 2 (FGFR2) is a protein that plays a role in cancer progression. The protein has two mutually exclusive exons IIIb and IIIc generated by alternative splicing events. A number of studies have demonstrated that disruption of the splicing pathway that leads to epithelial FGFR2-IIIb can contribute to cancer progression. Collective results from several models of tumorigenesis have led to the proposal that FGFR2-IIIb is a tumor suppressor (Gross et al, 2005). A general feature in such models posits that a switch from expression of FGFR2-IIIb to FGFR2-IIIc severs dependence of epithelia for growth and proliferation on surrounding mesenchyme and may establish autocrine growth pathways through epithelial expression of ligands for FGFR2-IIIc (McKeehan et al, 1998). In some cases, the switch in splicing of FGFR2 is followed by transcriptional downregulation of FGFR2 and activation of FGFR1-IIIc, which has similar ligand binding preferences as FGFR2-IIIc (Feng et al, 1997; Matsubara et al, 1998).
Reciprocal, compartment specific expression of FGFR2 splice variants and their ligands that participate in paracrine interactions between epithelial and mesenchymal cells regulate cell proliferation and differentiation (Acevedo et al. 2007). The epithelial FGFR2-IIIb splice variant has been suggested to function as a tumor suppressor (Savagner et al., 1994; Thiery, 2002). FGFR2-IIIb has a critical role in the maintenance of an epithelial phenotype. A switch in splicing towards the mesenchymal FGFR-IIIc isoform and/or transcriptional inactivation FGFR2 accompanies the EMT, a process involved in tumor metastasis (Hovhannisyan and Carstens, 2007). Previous studies of FGFR2 splicing regulation have identified a number of auxiliary cis-elements and non-cell type-specific regulatory RBPs that can influence exon IIIb and exon IIIc splicing combinatorially (Carstens et al., 1998; Hovhannisyan and Carstens, 2005) and references therein). One or more unidentified epithelial cell type-specific splicing regulatory proteins have been suggested to constitute a master switch that is required for FGFR2-IIIb expression (Newman et al., 2006).
There remains a need in the art for compositions and methods for the identification and use of splicing regulatory proteins and targets of such splicing implicated in cancer development for diagnosis/prognosis and treatment of certain cancers, as well as for use in screening assays enabling the identification of therapeutically desirable or undesirable properties of proposed therapeutic compounds or molecules.
SUMMARY OF THE INVENTION
The invention described herein meets the needs of the art by providing methods and compositions utilizing newly identified FGFR2 splice regulatory factors that maintain epithelial differentiation through post-transcriptional control at the level of splicing, and sequences regulated by these splicing factors.
In one aspect, a diagnostic composition involves a reagent that is capable of identifying in a biological sample from a mammalian subject

    
    
        (a) a nucleic acid sequence encoding the epithelial cell type specific splicing factor ESRP1 or ESRP2, or the protein encoded thereby;
        (b) a nucleic acid sequence splice variant that encodes a protein that is upregulated by ESRP1 or ESRP2 expression levels in a healthy mammalian control subject, or the protein encoded thereby;
        (c) a nucleic acid sequence splice variant that encodes a protein that is down-regulated by ESRP1 or ESRP2 expression levels in the healthy control or the protein encoded thereby; and
        (d) an alternative nucleic acid sequence splice variant of (b) or (c) that encodes a protein that is expressed when ESRP1 or ESRP2 expression levels are down-regulated from those of the healthy control or the protein encoded thereby.
    
    


Certain embodiments of these compositions include probes, primer sets, or other ligands that identify nucleic acid sequences, also include panels of such probes, primer sets or other biomarkers. Other embodiments are ligands capable of detecting/measuring proteins of the splicing factors or targets.
In another aspect a diagnostic kit contains one or more of the reagents identified herein.
In another aspect, a method for detecting an epithelial to mesenchymal transition (EMT) comprises contacting a biological sample obtained from a mammalian subject with a diagnostic reagent that can measure or detect the expression level of at least one of the group consisting of (a) through (d) above; and detecting an EMT when the level of expression of (a) or (b) is below the level of expression thereof in the healthy mammalian control, when the level of expression of (c) or (d) is above the level of expression thereof in the healthy mammalian control. Detection of the EMT in the sample diagnoses the presence or propensity towards metastasis of a mammalian carcinoma or cancer derived from primary epithelial cells. Detection of the EMT in the sample may also be diagnostic of tissue fibrosis.
In another aspect, a method for determining the prognosis of a mammalian carcinoma or cancer derived from primary epithelial cells, metastasis or tissue fibrosis includes obtaining a biological sample from a mammalian subject; contacting a biological sample obtained from a mammalian subject at a first point in time with a diagnostic reagent that can measure or detect the expression level of at least one of (a) through (d) above, and contacting a biological sample obtained from a mammalian subject at a later point in time after the first point in time with the same diagnostic reagent. A diagnosis of metastatic or pre-metastatic cancer or increased likelihood of progression of mammalian carcinoma or cancer derived from primary epithelial cells or tissue fibrosis is made when the level of expression of (a) or (b) in the later sample is below the level of expression thereof in the first sample, or when the level of expression of (c) or (d) in the later sample is above the level of expression thereof in the first sample.
In another aspect, a screening assay includes culturing a host cell that does not express any epithelial splicing agent selected from the group consisting of ESPR1, ESPR2 or RNCP1. The cell is transfected with a minigene comprising a nucleic acid sequence that generates a detectable signal when the minigene is contacted with the splicing agent. The host cell is cultured in the presence of a test molecule; and the generation or absence of the signal in the host cell is detected. Generation of a signal in the host cell indicates that the test molecule mimics the splicing activity of the splicing agent.
In another aspect a compound that inhibits the metastatic process of carcinomas or cancers derived from epithelial cell types, or inhibits the progression of tissue fibrosis is identified by the assay. Such compounds may upon contact with a mammalian cell permit expression of at least one variant protein encoded by a nucleic acid sequence splice variant that is regulated by ESRP1 or ESRP2 in a pharmaceutically acceptable vehicle.
In another aspect, a method for suppressing metastasis of a mammalian carcinoma or cancer derived from primary epithelial cells or blocking the progression of tissue fibrosis includes delivering to the cell at least one of ESRP1 or ESRP2 or a molecule that mimics the splicing activity of the ESRP1 or ESRP2. In another embodiment, at least one of ESRP1 or ESRP2 or a molecule that mimics the splicing activity of the ESRP1 or ESRP2 is used in the treatment of or in the preparation of a medicament for the treatment of suppressing metastasis of a mammalian carcinoma or cancer derived from primary epithelial cells or blocking the progression of tissue fibrosis.
In another aspect, a composition includes an effective amount of a protein encoded by a nucleic acid sequence splice variant that is upregulated or downregulated by the ESRP1 or ESRP2 expression levels of a healthy mammalian subject, optionally with a pharmaceutically acceptable vehicle carrier.
In another aspect, a composition includes a ligand that binds to and inhibits expression of ESRP1, ESRP2 or a protein encoded by a nucleic acid sequence splice variant that is upregulated or downregulated by the ESRP1 or ESRP2 expression levels of a mammalian subject with an epithelial cell cancer, optionally with a pharmaceutically acceptable vehicle.
In another aspect, a method for diagnosing, classifying, or determining the prognosis of, a mammalian carcinoma or cancer derived from primary epithelial cells involves measuring the level of expression of at least one epithelial cell type specific splicing factor selected from ESRP1 and ESRP2 in a biological sample from a mammalian subject. In one embodiment, the subject's level of the factor is then compared to the level of the same factor in healthy subject, or to a reference standard developed from the average level in multiple healthy subjects. In another embodiment the subject's level of the factor is then compared to the level of that factor measured in an earlier biological sample of the same subject, or to a reference standard developed from the average level in multiple subjects having various stages of the cancer.
In another aspect, methods for diagnosing, or determining the prognosis of, tissue fibrosis in a mammalian subject involve measuring the level of expression of at least one epithelial cell type specific splicing factor selected from ESRP1 and ESRP2 in a mammalian subject's biological sample. In one embodiment, the subject's level of the factor is then compared to the level of the same factor in healthy subject, or to a reference standard developed from the average level in multiple healthy subjects. In another embodiment the subject's level of the factor is then compared to the level of that factor measured in an earlier biological sample of the same subject, or to a reference standard developed from the average level in multiple subjects having various stages tissue fibrosis.
In a further aspect, a method for diagnosing the occurrence, stage or progression of a disease or condition related to inappropriate splicing of FGFR2 involves measuring the level of expression of the splicing factor RNPC1 in a biological sample from a mammalian subject. In one embodiment, the subject's level of the factor is then compared to the level of the same factor in healthy subject, or to a reference standard developed from the average level in multiple healthy subjects. In another embodiment the subject's level of the factor is then compared to the level of that factor measured in an earlier biological sample of the same subject, or to a reference standard developed from the average level in multiple subjects having various stages of disease.
In another aspect, a diagnostic reagent comprises at least one polynucleotide immobilized on a substrate, wherein the polynucleotide is a genomic probe that hybridizes to at least one splicing factor selected from the group consisting of ESRP1, ESRP2 and RNPC1. Still other diagnostic compositions contain a PCR primer-probe set that amplifies a polynucleotide sequence of at least one splicing factor selected from ESRP1, ESRP2 and RNPC1. Still other diagnostic/prognostic reagents include a composition comprising a ligand that binds to an expression product of at least one mammalian splicing factor selected from the group consisting of ESRP1, ESRP2 and RNPC1 of a mammalian subject. Diagnostic reagents for the identification of a characteristic genetic profile of disease also include panels of multiple probes or gene sequences that comprise probes, primers or sequences of at least one mammalian splicing factor selected from the group consisting of ESRP1, ESRP2 and RNPC1. These sequences may be optionally immobilized. Such reagents are useful for diagnosing the occurrence, stage or progression of a disease or condition related to inappropriate splicing of FGFR2 or to a cancer in a mammalian subject. Such reagents are useful for diagnosing or monitoring the stage or progression of a mammalian carcinoma or cancer derived from primary epithelial cells. Such reagents are useful for diagnosing or monitoring the stage or progression of tissue fibrosis.
In yet a further aspect, a method of screening a test compound or molecule includes the steps of contacting the compound with a mammalian cell expressing the gene product of at least one splicing factor selected from the group consisting of ESRP1, ESRP2 and RNPC1. In one embodiment of this method, the effect of the compound on the expression level of the gene product or of FGFR2 by the cell is measured. In another embodiment, the mutagenic effect of the compound on the nucleotide sequence or amino acid sequences of ESRP1, ESRP2, RNPC1 or FGFR2 is determined. Any effect that decreases the expression level of the factor in the cell or alters the nucleotide or amino acid sequence of the resulting ESRP1, ESRP2, RNPC1 or FGFR2 in response to the test compound is a potentially undesirable effect of use of the test compound.
Another aspect of this invention is a high throughput splicing assay used to identify compounds that change splicing events. This assay involves contacting in each individual well of a multi-well plate a different cDNA with a mammalian cell that expresses a specific splice variant associated with a specific condition, e.g., FGFR2 Mb. The cell is transfected with a minigene that expresses luciferase only when the cell expresses a specific splice variant. Cells are analyzed for luminescence after additional of luciferase reagents. A change in the expression of the specific splice variant by the cell caused by any of the cDNA is correlated with a change in the level of luminescence in each well.
Yet another aspect involves a method for maintaining normal expression of FGFR2 in a cell which involves delivering to the cell at least one of ESRP1 or ESRP2 or RNCP1. This method of delivering ESRP1 or ESRP2 is useful for treating, or suppressing metastatis of, a mammalian carcinoma or cancer derived from primary epithelial cells. This method delivering ESRP1 or ESRP2 is useful for treating, or blocking the progression of, tissue fibrosis. In another embodiment, at least one of ESRP1 or ESRP2 or a molecule that mimics the splicing activity of the ESRP1 or ESRP2 is used in the treatment of or in the preparation of a medicament for the treatment of maintaining normal expression of FGFR2 in a cell.
In still a further aspect, a composition for such delivery comprises a plasmid or viral vector comprising a polynucleotide encoding at least one of ESRP1, ESRP2 and RNPC1 under the control of a promoter operable in eukaryotic epithelial cells in a pharmaceutically acceptable vehicle or carrier.
In another aspect, a pharmaceutical composition is provided that contains an effective amount of at least one of ESRP1, ESRP2 and RNPC1 in nucleic acid or protein form in a pharmaceutically acceptable vehicle.
Other aspects of the present invention are described in the detailed description which follows.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A is a schematic representing the structural domains of the FGFR2 protein indicating Ig-like domains 1, 2 and 3, the transmembrane TM domain, and tyrosine kinase domains TK1 and TK2.
FIG. 1B is a map of the pre-mRNA with mutually exclusive splicing pathways that leads to expression of FGFR2-IIIb in epithelial cells or FGFR2-IIIc in mesenchymal cells.
FIG. 1C is a schematic representation of the bi-cistronic luciferase reporter minigene used in the over-expression screen. A fragment of FGFR2 intron 8 (IF3) known to contain all elements necessary for IIIb inclusion is cloned downstream of a synthetic 40 nt exon. Skipping of the exon generates a translation frame that stops upstream of the luciferase coding sequence. Inclusion of the exon results in translation through the luciferase open reading frame. Indicated splicing outcomes are illustrated.
FIG. 2A is a schematic of a dual fluorescent minigene stably expressed in the 293T-clone2 cells. The EGFP minigene contains no FGFR2 sequences and the mRFP minigene has IF3 cloned downstream of exon 40B, respectively. The 293T-clone2 cells were transiently transfected with the MGC clones from Table 6 or empty vector (EV) as a negative control or Fox1 as a positive control. Splicing of the minigene was analyzed by RT-PCR using the primers indicated by arrows in the schematic.
FIG. 2B is a graph indicating the percentage of exon 40B inclusion discovered as a results of the use of the minigene assay of FIG. 2A.
FIG. 2C is a schematic illustrating a reporter minigene expressed in the 293T-clone3 cells. The EGFP reporter has IF5, which lacks the Fox binding site and ISE/ISS-3 element, cloned downstream of exon 40B. Cells were transiently transfected with EV, Fox1, Esrp1, Esrp2, Rbm38, or Fusilli and exon inclusion was analyzed as described in FIG. 2A.
FIG. 2D is a Western gel of the EV, Fox1, Esrp1, Esrp2, Rbm38, and Fusilli expression showing average percentages of exon 40B inclusion with standard deviations compiled from three independent transfections are indicated below the gels.
FIG. 2E is a schematic illustrating an mRFP reporter minigene expressed in the 293T-clone3 cells, which has IF3, the Fox binding site and ISE/ISS-3 element, cloned downstream of exon 40B. Cells were transiently transfected with EV, Fox1, Esrp1, Esrp2, Rbm38, or Fusilli and exon inclusion was analyzed as described in FIG. 2A.
FIG. 2F is a Western gel of the EV, Fox1, Esrp1, Esrp2, Rbm38, and Fusilli expression from the transfectants of FIG. 2E, showing average percentages of exon 40B inclusion with standard deviations compiled from three independent transfections are indicated below the gels.
FIG. 3A are Western gels showing the results of transient transfection of 293T cells with ESRP2 or an empty vector and FGFR2 splicing was analyzed by RT-PCR protocol in which products are digested with Aval(A) or HincII(H), which specifically cuts exon IIIb- and exon IIIc-containing products, respectively. “U” indicates undigested PCR products.
FIG. 3B is a graph showing % exon inclusion in these cells.
FIG. 4A is a western gel showing that TWIST-induced EMT causes a change in splicing of FGFR2. FIGS. 4A-4C taken together show that a decrease in ESRP1 and ESRP2 expression is concurrent during an epithelial to mesenchymal transition, resulting in a IIIb to IIIc switch in FGFR2 splicing.
FIG. 4B is a gel showing expression of epithelial genes E-cadherin and Keratin 15 and mesenchymal genes N-cadherin and Foxc2 by RT-PCR of RNAs from HMLE cells expressing either the control vector pBabe-Puro or pBabe-Puro-Twist.
FIG. 4C is a graph showing the relative values of ESRP1, ESRP2, and miR-200c measured by real-time PCR in HMLE cells expressing either the control vector pBabe-Puro or pBabe-Puro-Twist.
FIG. 5A is a model for the mechanism of FGFR2 cell type-specific splicing, specifically showing that combinatorial control by ubiquitous regulatory proteins establishes a “default” splicing pathway that in mesenchymal cells silences exon IIIb and promotes inclusion of exon IIIc. Crosshatched boxes indicate known FGFR2 auxiliary cis-elements. PTB=polypyrimidine tract binding protein. A1=hnRNPA1. X indicates an unknown factor that binds an ISE downstream of exon We.
FIG. 5B is another model showing that in epithelial cells, the ESRPs collaborate with other regulatory proteins to activate exon IIIb splicing and silence exon IIIc splicing. Potential interactions between these proteins are indicated by double arrows and question marks. M represents human RNP M. TIA represents Tia1 or TiaR. Fox represents Fox family members. A base pairing interaction between two complementary sequences in the intron (ISE2 and ISAR) likely serves to position ESRP1 more closely to exon IIIb where it can act in conjunction with other factors (such as RBM38 and Tia1) to activate splicing (Muh et al., 2002). Proteins other than the ESRPs shown binding to the FGFR2 transcripts only mesenchymal oripithelial cells are expressed in both cell types and may be bound to the cognate elements in either cell type.
FIG. 6 is a graph showing validation of MGC screen hits in the 293T luciferase reporter cell line. The 293T cells stably expressing the luciferase reporter minigene were transiently transfected with an empty vector control, Fox1, or MGC cDNAs from Table 6. Luciferase activity was assayed and the mean values for three independent transfections are shown in the graph. Error bars denote standard deviation.
FIG. 7 is a graph showing that ESRP1 and ESRP2 expression is specifically observed in NCI60 cell lines classified as epithelial. Microarray data for ESRP1 and ESRP2 mRNA expression in cell lines that were classified as epithelial or mesenchymal based on the E-Cadherin/Vimentin ratio as described (Park et al., 2008) is shown. The gene profiling dataset of the NCI60 cell using the Affymetrix U133A and U133B was performed as described and was downloaded from the Cellminer website http://discover.nci.nih.gov/cellminer (Shankavaram et al., 2007). Shown are gcRMA normalized signal intensity in each cell line using probesets 219121_s_at (ESRP1/RBM35A) and 219395_at (ESRP2/RBM35B). The colon carcinoma cell line SW620 was the only “mesenchymal” cell line with significant expression of both ESRP1 and ESRP2. However, this classification displayed very low protein levels for both E-Cadherin and Vimentin and furthermore showed significant expression of epithelial-specific miR-200 family members (Park et al., 2008).
FIG. 8A is a flowchart illustrating the experimental design of an siRNA and exon based microarray approach to identify alternative splicing events regulated by ESRP1 and ESRP2.
FIG. 8B is a graph depicting quantitative RT-PCR showing a greater than 80% decrease of ESRP1 and ESRP2 mRNA in the combined knockdown samples (siESRP1/2) versus the control (siGFP). The results represent the mean of three amplifications and error bars represent the standard deviation.
FIG. 9 is a chart illustrating the numerous novel alternative splicing targets representing all known types of alternative splicing which were identified among the high confidence set of genes identified using MADS analysis of the Exon Array data. On the left there are schematics depicting each type of alternative splicing event. Splicing events that could not be definitively categorized were classified as “ambiguous or complex.” The table on the right displays the number of probesets, events, and genes that are associated with each type of splicing event.



DETAILED DESCRIPTION OF THE INVENTION
This invention involves the identification and use of splicing factors that maintain epithelial differentiation through post-transcriptional control at the level of splicing. Thus this invention provides methods for diagnosis and prognosis of diseases related to inappropriate splice events that lead to disease, as well as compositions to perform such methods, and therapeutic screens and compositions to treat and potentially reverse such splice events.
The inventors used highly advanced sensitive and specific luciferase splicing reporter minigenes to carry out a genome wide high throughput array based cDNA overexpression screen for splicing regulatory proteins that regulate splicing of fibroblast growth factor receptor 2 (FGFR2). This screen identified 28 candidates capable of conferring a splicing change in the reporter (see Table 6). The assay identified an epithelial cell type-specific RNA binding, splicing regulatory protein, Epithelial Splicing Regulatory Protein (ESRP) that was required for maintenance of FGFR2-IIIb splicing. Analysis of a panel of cell lines confirmed that expression of ESRP is limited to cells that express FGFR2-IIIb. ESRP when expressed ectopically, by itself switched splicing of endogenous FGFR2 from mesenchymal-specific exon IIIc to epithelial-specific exon IIIb in a cell line, 293T, that expresses FGFR2-IIIc. The inventors subsequently also noted that a close homolog of this gene (ESRP2) displayed the same expression pattern and function and is a functionally redundant protein. Neither of these genes nor their gene products (which were previously only identified by general gene name RNA binding motif protein 35a and 35b) had previously been shown to be regulators of alternative splicing or to be epithelial cell type-specific. At least one non-cell type-specific factor (RCNP1) was also identified.
The ESRPs co-regulate alternative splicing decisions that are epithelial-specific. In a panel of cell lines expressing either FGFR2-IIIb or FGFR2-IIIc, the inventors found exclusive expression of the mRNAs encoding these proteins in cell lines that express FGFR2-IIIb, suggesting that ESRP1 is a critical regulator of this cell type-specific splicing event. Additional regulated events comprise an epithelial cell type-specific “splicing signature” that maintains epithelial cell morphology and function.
Loss of expression of these splicing factors contributes to the epithelial-to-mesenchymal transition, a process implicated in cancer metastasis. The inventors used a model of the EMT in which the transcriptional factor Twist induces an EMT in a Human Mammary Epithelial Cell line (HMEC) (8). Upon induction of EMT with Twist, the inventors observed that ESRP mRNA that was initially detectable became undetectable by RT-PCR. This did not occur in the controls. These findings strongly support that loss of ESRP1 expression is a critical event that occurs during the EMT. Thus, the loss of the corresponding epithelial cell type-specific splicing regulatory program is also broadly compromised during cancer progression. In addition to regulating FGFR2 splicing, these factors also coordinately regulate the splicing of other alternatively spliced transcripts that are implicated in carcinogenesis. Thus, these proteins participate in the cellular processes that maintain epithelial cell differentiation. These factors themselves, as well as other targets the splicing of which is regulated by ESRP1 and ESRP2, are useful in as biomarkers for diagnosis of certain epithelial cancers, cancer metastasis and/or tissue fibrosis in biological samples, such as biopsied tissue. These factors are also useful as targets to identify other compounds and molecules useful for cancer prevention, diagnosis, and treatment, and as diagnostic and therapeutic reagents themselves.
ESRP1 and 2 thus join the list of a very limited number of mammalian cell type-specific alternative splicing regulators. The loss of these splicing factors may be universally observed during the EMT by investigating its expression in several additional EMT systems. The inventors are investigating expression of ESRP2 in a panel of human cancer specimens. In one embodiment, ESRPs are frequently lost during cancer progression, and so provide a marker of cancer progression. In another embodiment, loss of ESRP expression is more frequently seen in aggressive and rapidly progressing cancer and thus it provides a marker of cancer classification and diagnosis.
Further assays that controls ESRP1/2 expression or that may restore the factors to normal expression levels are useful for the development of therapeutic strategies. Expression levels of these ESRP1/2 factors, and of targets spliced by these factors, are useful as highly specific markers of the epithelial lineage for tracking disease development and progression.
A. Definitions
By “ESRP1” or Epithelial Splicing Regulatory Protein 1 is meant the nucleic acid sequences and encoded amino acid sequences of the factor previously identified as RBM35A. The nucleotide sequence(s) and protein sequences for the five known human isoforms of this ESRP1 are published in the NCBI database at accession numbers NM—001034915.2/NP—001030087.2 (isoform 2), NP—001122825.1/NP—001116297.1 (isoform 4); NM—001122826.1/NP—00116298.1 (isoform 3); NM—001122827.1/NP—001116299.1 (isoform 5) and NM—017697.3/NP—060167.2 (isoform 1). A murine homolog is reported at accession No. NM—194055. Other homologs are also known and, where homologous, are anticipated to be useful in the various methods and compositions of this invention. This invention involves the identification of this protein as an epithelial cell-type specific splicing factor that maintains epithelial differentiation through post-transcriptional control at the level of splicing.
By “ESRP2” or Epithelial Splicing Regulatory Protein 2 is meant the nucleic acid sequences and encoded amino acid sequences of the factor previously identified as RBM35B. The nucleotide sequence(s) and protein sequences for the known human protein are published in the NCBI database at accession numbers NM—024939.2/NP—079215.2. A murine homolog is reported at accession No. NM—176838. Other homologs are also known and where homologous are anticipated to be useful in the various methods and compositions of this invention. This invention involves the identification of this protein as an epithelial cell-type specific splicing factor that maintains epithelial differentiation through post-transcriptional control at the level of splicing.
By “RNPC1” is meant the nucleic acid sequences and encoded amino acid sequences of the factor previously identified as RBM38. The inventors are the first to demonstrate that this factor regulates splicing in mammals. The nucleotide sequence(s) and protein sequences for the two known human isoforms of this protein are published in the NCBI database at accession numbers NM—017495.4/NP—059965.2 and NM—183425.1/NP—906270.1. A murine homolog is similarly available under accession No. NM—019547. Other homologs are also known and, where homologous, are anticipated to be useful in the various methods and compositions of this invention.
By the phrase “a mammalian carcinoma or cancer derived from primary epithelial cells” is meant an epithelial cell cancer selected from the group consisting of carcinomas that derive from epithelial cell types. Among such cancers are included breast cancer, colorectal cancer, ovarian cancer, lung cancer, pancreatic cancer, kidney cancer, prostate cancer and gastric cancer, among others. Also included among these cancers are aggressive cancer subtypes, basal cancer or lobular cancer, e.g., certain aggressive breast cancers.
By the phrase “biological sample” is meant any biological fluid or tissue. In certain embodiments, a sample may be a fluid or tissue containing epithelial cells, either benign or malignant cells. Useful biological samples include, without limitation, whole blood, saliva, urine, synovial fluid, bone marrow, cerebrospinal fluid, vaginal mucus, cervical mucus, nasal secretions, sputum, semen, amniotic fluid, bronchoalveolar lavage fluid, and other cellular exudates from a mammalian subject, as well as tissue biopsies including lymph nodes. Such samples may further be diluted with saline, buffer or a physiologically acceptable diluent. Alternatively, such samples are concentrated by conventional means. Additionally, the biological sample may be a blood sample in which circulating tumor cells are found, i.e., such as a sample used in the CellSearch® Circulating Tumor Cell (CTC) Kit (Veridex).
The term “tumor,” as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
By the terms “patient” or “subject” as used herein means a mammalian animal, including a human, a veterinary or farm animal, a domestic animal or pet, and animals normally used for clinical research. More specifically, the subject of these methods and compositions is a human.
The term “prognosis” is used herein to refer to the prediction of the likelihood of cancer-attributable death or progression, including recurrence, metastatic spread, and drug resistance, of a neoplastic disease, particularly an epithelial cell-type specific cancer. The term “prediction” is used herein to refer to the likelihood that a patient will respond either favorably or unfavorably to a drug or set of drugs, and also the extent of those responses, or that a patient will survive following surgical removal of the primary tumor and/or chemotherapy for a certain period of time without cancer recurrence. These predictive methods can be used clinically to make treatment decisions by choosing the most appropriate treatment modalities for any particular patient. The predictive methods described herein are valuable tools in predicting if a patient is likely to respond favorably to a treatment regimen, such as surgical intervention, chemotherapy with a given drug or drug combination, and/or radiation therapy, or whether long-term survival of the patient, following surgery and/or termination of chemotherapy or other treatment modalities is likely. These predictive methods are also useful in identifying aggressive cancers and the occurance of metastatic cancer conditions.
The terms “splicing” and “RNA splicing” are used interchangeably and refer to RNA processing that removes introns and joins exons to produce mature mRNA with continuous coding sequence that moves into the cytoplasm of a eukaryotic cell.
The term “exon” refers to any segment of an interrupted gene that is represented in the mature RNA product. In theory the term “intron” refers to any segment of DNA that is transcribed but removed from within the transcript by splicing together the exons on either side of it. Operationally, exon sequences occur in the mRNA sequence of a gene. Operationally, intron sequences are the intervening sequences within the genomic DNA of a gene, bracketed by exon sequences and having GT and AG splice consensus sequences at their 5′ and 3′ boundaries.
By the term “functional fragment” is meant any fragment of a nucleotide or amino acid sequence that shares a common biological function of the entire sequence.
By the term “target” of ESRP1/2 is meant a nucleic acid sequence that can be spliced by ESRP1/2, or a mimic of ESRP1/2. Generally, in the presence of normal amounts of ESRP1/2 in a healthy subject, certain target proteins are expressed that contain an exon, e.g., a normal enhanced target variant or “enhanced target”. When ESRP1/2 are downregulated, e.g., in the conditions of a cancer or tissue fibrosis, the enhanced target is downregulated and an “alternative splice variant of an enhanced target” is expressed, i.e., the exon is spliced out. Additionally, in the presence of normal amounts of ESRP1/2 in a healthy subject, certain target proteins that contains an exon are silenced, e.g., a normal silenced target splice variant or “silenced target”. When ESRP1/2 are downregulated, e.g., in the conditions of a cancer or tissue fibrosis, the silenced target is downregulated and an “alternative splice variant of a silenced target” is expressed, i.e., the exon is expressed. Certain of these enhanced or silenced targets are identified herein.
Unless defined otherwise in this specification, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and by reference to published texts.
B. Diagnostic Methods and Reagents
The ESRP1/2 splicing factors and their targets are useful in diagnostic methods and as reagents for the diagnosis of cancer, metastatic cancer, tissue fibrosis or cancer fibrosis based upon the relationship of the splicing activity to the EMT.
One embodiment of such a diagnostic method detects an epithelial to mesenchymal transition (EMT) in the biological sample of a subject. In one embodiment the method includes contacting a biological sample obtained from a mammalian subject with a diagnostic reagent that can measure or detect the expression level of at least one of (a) a nucleic acid sequence encoding the epithelial cell type specific splicing factor ESRP1 or ESRP2, or the protein encoded thereby; (b) a nucleic acid sequence splice variant that encodes an “enhanced” protein that is upregulated by ESRP1 or ESRP2 expression levels in a healthy mammalian control subject, or the protein encoded thereby; (c) a nucleic acid sequence splice variant that encodes a “silenced” protein that is down-regulated by ESRP1 or ESRP2 expression levels in the healthy control or the protein encoded thereby; and (d) an alternative nucleic acid sequence splice variant of (b) or (c) that encodes a protein variant that is expressed when ESRP1 or ESRP2 expression levels are down-regulated from those of the healthy control or the protein encoded thereby. In one embodiment, the diagnostic method further involves detecting an EMT when the level of expression of (a) or (b) is below the level of expression thereof in the healthy mammalian control. In another embodiment, the diagnostic method detects an EMT when the level of expression of (c) or (d) is above the level of expression thereof in the healthy mammalian control. In another embodiment, detection of the EMT in the sample correlates with, or is indicative of, the presence or metastasis of a mammalian carcinoma or cancer derived from primary epithelial cells. In another embodiment, detection of the EMT in the sample is indicative of tissue fibrosis.
In another embodiment of a diagnostic method, the contacting step measures (a), (b), (c) or (d) as ribonucleic acid, mRNA, deoxyribonucleic acid or cDNA sequences. In another embodiment of a diagnostic method, the contacting step measures (a), (b), (c) or (d) as the target protein. In another embodiment, the contacting step of the method involves forming a physical association between the diagnostic reagent and the splicing factor or target sequence or protein in the sample. One such reagent is a nucleic acid sequence capable of hybridizing to a target-containing sequence in the sample. For example, when the reagent is a genomic probe, the physical association formed by contact of the reagent with the sample is the hybridization of the probe to the cDNA or mRNA of a sequence containing the target sequence.
Where the reagent is a PCR primer or primer pair, the physical association is the hybridization of the primer sequences to different strands or different portions of the nucleic acid (e.g., mRNA) of a marker sequence containing the target sequence. Preferably the nucleic acid probes or primers are from about 8 or more nucleotides in length, wherein the nucleotides are complementary to portions of the “non-coding” or “coding” strands of the gene sequences or non-gene sequences flanking or encompassing the selected target sequence. Such probes are, for example, oligo or polynucleotide sequences corresponding to the region surrounding (and/or comprising) any of the target sequences. Such a fragment usually has a length comprised between 8 and 50 nucleotides, preferably 12 to 35 nucleotide or 15 to 25 nucleotides. It may be a fragment of naturally occurring or synthetic DNA or RNA. In certain embodiments, each primer or probe is at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or at least 20 nucleotides in length. In other embodiments, the primers and/or probes may be longer than 20 nucleotides in length. Given the information provided herein, one of skill in the art may design any number of suitable primer/probe sequences useful for identifying the target sequences described herein. The diagnostic methods described herein can employ one or more of the diagnostic reagents or compositions described below.
Where the diagnostic reagent employed in the method is associated with a detectable label, the method further comprises transforming the detectable label's signals generated from the diagnostic reagent in association with (a), (h), (c) or (d) present in the biological sample into numerical or graphical data. In yet another embodiment, the transforming is performed by a suitably-programmed machine or instruments that can detect the detectable signals generated from the diagnostic reagents associated with the (a), (b), (c) or (d) present in the biological sample and transform same into numerical or graphical data useful in performing the diagnosis.
Another aspect of the invention is a method for determining the prognosis of a mammalian carcinoma or cancer derived from primary epithelial cells, metastasis or tissue fibrosis. In one embodiment the method comprises obtaining a biological sample from a mammalian subject; contacting a biological sample obtained from a mammalian subject at a first point in time with a diagnostic reagent that can measure or detect the expression level of at least one of the group consisting of (a) a nucleic acid sequence encoding the epithelial cell type specific splicing factor ESRP1 or ESRP2, or the protein encoded thereby; (b) a nucleic acid sequence splice variant that encodes a protein that is upregulated by ESRP1 or ESRP2 expression levels in a healthy mammalian control subject, or the protein encoded thereby; (c) a nucleic acid sequence splice variant that encodes a protein that is down-regulated by ESRP1 or ESRP2 expression levels in the healthy control or the protein encoded thereby; and (d) an alternative nucleic acid sequence splice variant of (b) or (c) that encodes a protein that is expressed when ESRP1 or ESRP2 expression levels are down-regulated from those of the healthy control or the protein encoded thereby. The method further comprises contacting a biological sample obtained from a mammalian subject at a later point in time after the first point in time with the same diagnostic reagent and diagnosing ongoing metastasis or progression of mammalian carcinoma or cancer derived from primary epithelial cells or tissue fibrosis when the level of expression of (a) or (b) in the later sample is below the level of expression thereof in the first sample, or when the level of expression of (c) or (d) in the later sample is above the level of expression thereof in the first sample.
Normally, such diagnostic methods are performed on biological samples containing mammalian cells, biological fluids containing mammalian cells, mammalian tissue, and biopsied tissue. The cancer can be characterized by inappropriate expression of splice variants of fibroblast growth factor receptor 2, or any other suitable target of the splicing factors ESRP1/2 due to inappropriate splicing. In another embodiment, the cancer is an epithelial cell cancer or carcinoma that derives from epithelial cell types consisting of breast cancer, colorectal cancer, ovarian cancer, lung cancer, kidney cancer, pancreatic cancer, prostate cancer and gastric cancer.
In another embodiment, the diagnostic method can detect an expression level of one or more of (a) through (d) that is indicative of epithelial cancer cells that have undergone a partial or complete epithelial to mesenchymal transition (EMT) and are metastatic. In another embodiment, the expression level of one or more of (a)-(d) is associated with an aggressive breast cancer subtype. In another embodiment, the cancer subtype is a basal cancer or a lobular cancer. In another embodiment, the expression level of one or more of (a)-(d) is associated with a cancer having a severe prognosis.
In another embodiment, a method for diagnosing or classifying a mammalian carcinoma or cancer derived from primary epithelial cells utilizes the splicing factors identified herein. In one embodiment, such a diagnostic method involves measuring the level of expression of at least one epithelial cell type specific splicing factor selected from ESRP1 and ESRP2 in a biological sample obtained from a mammalian subject. The expression level of the desired factor is then compared with the level of expression in a healthy mammalian subject. While such comparison can occur by direct comparison with the expression levels in one or more healthy subjects, it is more typical for a reference average expression level to be provided as a number or range. A level of expression of the splicing factor in the subject's sample that is below the level of expression in a healthy mammalian subject (or reference average) is an indication of a diagnosis or severity of a cancer. In one embodiment of such a method the measuring step includes measuring the splicing factor as ribonucleic acid, deoxyribonucleic acid, or protein using conventional assay technologies.
In a similar manner, the method for diagnosing or classifying a mammalian carcinoma or cancer derived from primary epithelial cells utilizes the impact of these splicing factors identified herein on the resulting expression of FGFR2 splice variants or another target of the splicing factors. For ease of discussion, FGFR2 will be used as a prototype of all targets of the splicing factors. Where FGFR2 is mentioned, the other targets can be referenced similarly. In one embodiment, such a diagnostic method involves measuring the level of expression of FGFR2 splice variants or a desired splice variant thereof in a biological sample obtained from a mammalian subject. The expression level of the desired FGFR2 variant is then compared with the level of expression in a healthy mammalian subject. While such comparison can occur by direct comparison with the expression level of one or more healthy subjects, it is more typical for a reference average expression level to be provided as a reference number. A level of expression of the splicing factor in the subject's sample that is below the level of expression in a healthy mammalian subject (or reference average) is an indication of a diagnosis or severity of a cancer. In one embodiment of such a method the measuring step includes measuring the splicing factor as ribonucleic acid, deoxyribonucleic acid, or protein using conventional assay technologies. In another embodiment, the cancer is characterized by inappropriate expression of fibroblast growth factor receptor 2 due to inappropriate splicing.
In another aspect, a method for determining the prognosis of a mammalian carcinoma or cancer derived from primary epithelial cells involves measuring the level of expression of at least one epithelial cell type specific splicing factor selected from ESRP1 and ESRP2 in a biological sample from a mammalian subject with the cancer. The expression level of the desired factor is then compared with the level of expression in one or more biological samples of the same subject assayed earlier in time, or before or during treatment. While such comparison can occur by direct comparison with one or more prior assessments of the same patient's status, it is also possible for a reference average expression level at specific stages or severities of the disease among other patients with the disease to be provided as a reference number or profile. A level of expression of the splicing factor in the subject's sample that is below the subject's prior level of expression (or reference average) is an indication of a bad prognosis or increasing severity of the cancer. A level of expression of the splicing factor in the subject's sample that is above the subject's prior level of expression (or reference average) is an indication of a good prognosis or decreasing severity or spread of the cancer. In one embodiment of such a method the measuring step includes measuring the splicing factor level as ribonucleic acid, deoxyribonucleic acid, or protein using conventional assay technologies. Similarly, this strategy can be employed measuring FGFR2 variants as described above.
In another aspect, a method for diagnosing tissue fibrosis in a mammalian subject involves measuring the level of expression of at least one epithelial cell type specific splicing factor selected from ESRP1 and ESRP2 or a target thereof in a mammalian subject's biological sample. The expression level of the desired factor is then compared with the level of expression in a healthy mammalian subject. While such comparison can occur by direct comparison with the expression level of one or more healthy subjects, it is more typical for a reference average expression level to be provided as a reference number. A level of expression of the splicing factor in the subject's sample that is below the level of expression in a healthy mammalian subject (or reference average) is an indication of a diagnosis or severity of the tissue fibrosis. In one embodiment of such a method the measuring step includes measuring the splicing factor as ribonucleic acid, deoxyribonucleic acid, or protein using conventional assay technologies. A level of expression of the splicing factor below the level of expression of a healthy mammalian subject is evidence of tissue fibrosis or advancing tissue fibrosis. A level of expression of the splicing factor at or above the level of expression of a healthy mammalian subject is evidence of no diagnosis of tissue fibrosis.
In another aspect, a method for determining the prognosis of tissue fibrosis in a mammalian subject includes measuring the level of expression of at least one epithelial cell type specific splicing factor selected from ESRP1 and ESRP2 in a biological sample from a subject with tissue fibrosis.
In the same manner as above for epithelial cell derived carcinoma, the expression level of the splicing factor is compared to that in an earlier biological sample of the same subject (or reference sample number derived from multiple patients at various stages of tissue fibrosis). A decrease in the factor expression over the prior or reference sample is indicative of worsening disease, while an increase in the factor expression over the prior or reference sample is indicative of a good prognosis or success of treatment.
In yet another aspect, a method for diagnosing the occurrence, stage or progression of a disease or condition related to inappropriate splicing of FGFR2 involves measuring the level of expression of the splicing factor RNPC1 in a biological sample from a mammalian subject. The expression level of the splicing factor is compared to a healthy patient or healthy patient reference average for diagnosis. An expression level below the level of expression of a healthy mammalian subject is evidence of a positive diagnosis of such disease or condition.
The specific methodologies that can be employed to perform the diagnostic methods described herein are convention and may be readily selected and adapted by one of skill in the art. Methods useful in performing the diagnostic steps described herein are known and well summarized in U.S. Pat. No. 7,081,340. Such methods include methods based on hybridization analysis of polynucleotides, methods based on sequencing of polynucleotides, proteomics-based methods or immunochemistry techniques. The most commonly used methods known in the art for the quantification of mRNA expression in a sample include northern blotting and in situ hybridization; RNAse protection assays; and PCR-based methods, such as reverse transcription polymerase chain reaction (RT-PCR) or qPCR. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Representative methods for sequencing-based gene expression analysis include Serial Analysis of Gene Expression (SAGE), and gene expression analysis by massively parallel signature sequencing (MPSS). The methods described herein are not limited by the particular techniques selected to perform them. Exemplary commercial products for generation of reagents or performance of assays include TRI-REAGENT, Qiagen RNeasy mini-columns, MASTERPURE Complete DNA and RNA Purification Kit (EPICENTRE®, Madison, Wis.), Paraffin Block RNA Isolation Kit (Ambion, Inc.) and RNA Stat-60 (Tel-Test), the MassARRAY-based method (Sequenom, Inc., San Diego, Calif.), differential display, amplified fragment length polymorphism (iAFLP), and BeadArrayTM technology (Illumina, San Diego, Calif.) using the commercially available Luminex100 LabMAP system and multiple color-coded microspheres (Luminex Corp., Austin, Tex.) and high coverage expression profiling (HiCEP) analysis.
In conjunction with the performance of the various diagnostic techniques described herein, another aspect of the invention is a variety of diagnostic reagents employing the identified splice regulators ESRP1, ESRP2 or RNCP1 or targets thereof.
In one embodiment, a diagnostic composition includes a reagent that is capable of identifying in a biological sample from a mammalian subject at least one of (a) a nucleic acid sequence encoding the epithelial cell type specific splicing factor ESRP1 or ESRP2, or the protein encoded thereby; (b) a nucleic acid sequence splice variant that encodes a protein that is upregulated by ESRP1 or ESRP2 expression levels in a healthy mammalian control subject, or the protein encoded thereby; (c) a nucleic acid sequence splice variant that encodes a protein that is down-regulated by ESRP1 or ESRP2 expression levels in the healthy control or the protein encoded thereby; and (d) an alternative nucleic acid sequence splice variant of (b) or (c) that encodes a protein that is expressed when ESRP1 or ESRP2 expression levels are down-regulated from those of the healthy control or the protein encoded thereby. For example, in one embodiment, the nucleic acid sequence splice variants are identified as described in Examples 11 and 12.
In another embodiment, the diagnostic composition comprises multiple reagents capable of identifying more than one sequence or protein of (a), (b), (c) or (d). In another embodiment, the reagent is capable of forming a physical association with one or more variant protein or nucleic acid sequence of (a), (b), (c) or (d) in a biological sample. In yet another embodiment the reagent comprises a nucleic acid sequence capable of hybridizing with a nucleotide sequence of one or more of (a) through (d). In another embodiment, the reagent is a genomic probe that hybridizes to the cDNA or mRNA of a sequence of (a), (b), (c) or (d). In another embodiment, the reagent is a PCR primer probe set that hybridizes to the mRNA of a sequence of (a), (b), (c) or (d). In another embodiment, the reagent is an antibody or functional fragment thereof that binds to at least protein of (a), (b), (c) or (d).
The diagnostic reagent nucleic acid sequence may be an mRNA sequence, a DNA sequence, or a cDNA sequence. For example, an exemplary reagent can target either the nucleic acid sequence (b) of at least one “enhanced” target gene of Table 1, wherein the nucleic acid sequence indicative of ESRP1 or 2 upregulation lacks the specified exon, or the protein encoded thereby. In another embodiment, the reagent targets either the nucleic acid sequence (c) of at least one “silenced target” gene of Table 2, wherein the sequence indicative of ESRP1 or 2 downregulation contains the specified exon, or the protein encoded thereby.
The sequences of the gene targets, exons and nucleotide positions referenced throughout the specification and in the following examples are publically known and available to one of skill in the art. All chromosomal coordinates are available publicly. See, e.g., UC Santa Cruz Genome Browser, March 2006 Assembly, among other known databases containing the sequences referenced throughout this specification.
Tables 1 through 3 identify the gene symbol identifier; the chromosomal coordinates of the identified exon; and whether ESRP1 and ESRP2 “enhances” or “silences” production of the version of the protein encoded by the nucleic acid sequence containing the exon identified by the exon coordinates, etc. By “enhances”, it is meant that expression of ESRP1 or ESRP2 increases production of the protein encoded by the nucleic acid splicing variant sequence which includes the identified exon. By “silences”, it is meant that expression of ESRP1 or ESRP2 increases production of the protein encoded by the nucleic acid splicing variant sequence which does not include the identified exon.







TABLE 1







Enhanced Targets of ESRP1/2













ESRP

SEQ ID NOs of 


Gene

Effect On
Percent Change
Exon Coordinates


Symbol
Exon Coordinates
Splicing
Exon Inclusion
of Col. 2














SLC37A2
chr11: 124461310-124461366
Enhance
76.5
1


FLNB
chr3: 58102625-58102696
Enhance
74
2


RALGPS2
chr1: 177127988-177128065
Enhance
65.5
3


ENAH
chr1: 223759316-223759378
Enhance
62
4


ITGA6
chr2: 173074746-173074875
Enhance
60.5
5


FNIP1
chr5: 131074170-131074253
Enhance
41
31


GOLGA2
chr9: 130069294-130069374
Enhance
39.5
6


ARFGAP2
chr11: 47150836-47150877
Enhance
35
7


TCF7L2
chr10: 114714305-114714373
Enhance
29.2
8


SLK
chr10: 105760564-105760656
Enhance
28
9


MAP3K7
chr6: 91310992-91311072
Enhance
26.5
10


MPZL1
chr1: 166011925-166012027
Enhance
26
11


HISPPD1
chr5: 102546834-102547007
Enhance
23.5
12


INTS9
chr8: 28760183-28760245
Enhance
22.2
13


ARFIP1
chr4: 154011355-154011450
Enhance
21.7
14


YAP1
chr11: 101585458-101585505
Enhance
19.9
15


CCAR1
chr10: 70186036-70186246
Enhance
19.5
16


GSK3B
chr3: 121068127-121068165
Enhance
15
17


NT5C3
chr7: 33042071-33042125
Enhance
8
18


VPS39
chr15: 40271556-40271588
Enhance
7.4
19
















TABLE 2







Silenced Targets of ESRP1/2














% Change
SEQ ID NOS of




ESRP Effect
Exon
Exon Coordinates


Gene Symbol
Exon Coordinates
On Splicing
Inclusion
of Col. 2














SCRIB
chr8: 144961710-144961772
Silence
66
20


OSBPL3
chr7: 24869344-24869436
Silence
53
21


COL16A1
chr1: 31917992-31918039
Silence
46.0
22


PLAA
chr9: 26907095-26907163
Silence
44.9
23


MAP3K7
chr6: 91284887-91285002
Silence
44
24


PQLC3
chr2: 11232545-11232586
Silence
42
25


GOLGA4
chr3: 37377738-37377800
Silence
39
26


PRC1
chr15: 89313313-89313354
Silence
36.1
27


MEST
chr7: 129927866-129927967
Silence
35
28


UBE2K
chr4: 39455697-39455825
Silence
35
29


TBC1D23
chr3: 101513367-101513411
Silence
33
30


MPRIP
ehr17: 17019332-17019451
Silence
32
32


ARHGEF11
chr1: 155174834-155174929
Silence
31.2
33


ATP13A3
chr3: 195614217-195614306
Silence
29.5
34


RIPK2
chr8: 90844194-90844347
Silence
28.5
35


WDR32
chr9: 37847238-37847348
Silence
27.5
36


MST4
chrX: 131031187-131031372
Silence
26
37


RBM34
chr1: 233390449-233390585
Silence
25
38


FAM13B1
chr5: 137320065-137320130
Silence
23
39


MRPL22
chr5: 154310574-154310691
Silence
23
40


LAS1L
chrX: 64670215-64670340
Silence
21.5
41


PARL
chr3: 185034206-185034307
Silence
21.5
42


ZNF207
chr17: 27712600-27712647
Silence
21
43


PSAT1
chr9: 80132787-80132924
Silence
20.5
44


GPR126
chr6: 142746590-142746673
Silence
20
45


STX2
chr12: 129846493-129846618
Silence
20.0
46


LAS1L
chrX: 64661169-64661219
Silence
19.5
47


SEP15
chr1: 87106324-87106373
Silence
18
48


NAE1
chr16: 65416259-65416289
Silence
16
49


CLSTN1
chr1: 9720143-9720199
Silence
15.5
50


EHBP1
chr2: 63068570-63068677
Silence
14.1
51


RBM39
chr20: 33791861-33791933
Silence
14
52


GNAS
chr20: 56907391-56907435
Silence
13.5
53


CSDA
chr12: 10753774-10753980
Silence
13.5
54


REPS1
chr6: 139289231-139289311
Silence
13.2
55


ATXN2
chr12: 110386849-110386902
Silence
9
56


FAM126A
chr7: 22953096-22953391
Silence
8.9
57


PACRGL
chr4: 20335529-20335609
Silence
8.7
58


NUMB
chr14: 72815742-72815885
Silence
8.3
59


CASK
chrX: 41301229-41301297
Silence
8
60


ANKRD36
chr2: 97153924-97154061
Silence
7.5
61


CRAMP1L
chr16: 1656075-1656179
Silence
7.3
62


ATP13A2
chr1: 17188969-17189085
Silence
6.7
63


FN1
chr2: 215953779-215954048
Silence
6.5
64


DOCK7
chr1: 62783206-62783298
Silence
5.6
65









In another embodiment, the diagnostic reagent compositions employs the nucleic acid sequences or encoded proteins of only those target genes of Table 1 or 2, wherein the percent change of exon inclusion is greater than 25%. In another embodiment, the diagnostic reagent compositions employs the nucleic acid sequences or encoded proteins of only those target genes of Table 1 or 2, wherein the percent change of exon inclusion is greater than 30%. In another embodiment, the diagnostic reagent compositions employs the nucleic acid sequences or encoded proteins of only those target genes of Table 1 or 2, wherein the percent change of exon inclusion is greater than 40%. In another embodiment, the diagnostic reagent compositions employs the nucleic acid sequences or encoded proteins of only those target genes of Table 1 or 2, wherein, for the genes of Table 1, the exons are excised or the gene variants of Table 2 wherein the exons are present.
In one embodiment, the diagnostic composition comprises multiple reagents capable of identifying more than one sequence or protein selected from the group consisting of SLC37A2 FLNB, RALGPS2, ENAH, and ITGA6. In another embodiment, the diagnostic composition comprises multiple reagents capable of identifying more than one sequence or protein selected from the group consisting of SCRIB, OSBPL3, COL16A1, PLAA, and MAP3K7. In another embodiment, the diagnostic composition comprises multiple reagents capable of identifying more than one sequence or protein selected from the group consisting of SLC37A2, FLNB, RALGPS2, ENAH, ITGA6, SCRIB, OSBPL3, COL16A1, PLAA, and MAP3K7. In another embodiment, the diagnostic composition comprises multiple reagents capable of identifying at least three, four, five, six, seven, eight, nine or ten sequences or proteins selected from the group consisting of SLC37A2, FLNB, RALGPS2, ENAH, ITGA6, SCRIB, OSBPL3, COL16A1, PLAA, and MAP3K7. In another embodiment, the diagnostic composition comprises multiple reagents capable of identifying at least three, four, five, six, seven, eight, nine, ten, fifteen or twenty sequences or proteins selected from the group consisting of SLC37A2, FLNB, RALGPS2, ENAH, ITGA6, FNIP1, GOLGA2, ARFGAP2, TCF7L2, SLK, SCRIB, OSBPL3, COL16A1, PLAA, MAP3K7, PQLC3, GOLGA4, PRC1, MEST, UBE2K.
Certain diagnostic compositions or reagents include therefor compositions, usually probes or primer sets capable of identifying multiple of these target genes as a gene signature characteristic of the epithelial cell cancer, its prognosis or its metastasis. Certain diagnostic compositions or reagents include therefor compositions, usually probes or primer sets capable of identifying multiple of these target genes as a gene signature characteristic of tissue fibrosis. One of skill in the art given this disclosure and these identified target genes can assemble various combinations of diagnostic reagents to detect or measure expression levels of the suitable target variants.
In still other embodiments, the diagnostic composition or reagent is designed to detect or measure the ESRP1/2 splicing target variants expressed by one or multiples of the genes identified in Table 3. One of skill in the art given this disclosure and these identified target genes can assemble various combinations of diagnostic reagents to detect or measure expression levels of the suitable target variants.







TABLE 3







Summary of ESRP targets tested by RT-PCR


















Reciprocal








Change in
SEQ ID NOs





Predicted
PNT2
MB231
of Genomic


Gene


ESRP
siRNA
Ectopic
Coordinates


Symbol
Event
Genomic Coordinates
Function
Validation
Expression
of Col. 3





FAM62A
Alt. 3′SS
chr12: 54814131-54814160
Promotes
Yes
No
66





upstream








3′ splice site





CHRNA5
Alt. 5′SS
chr15: 76669202-76670037
Promotes
Yes
No
67





downstream








5′ splice site





SCRIB
Cassette
chr8: 144961710-144961772
Silence
Yes
Yes
20



exon







SLK
Cassette
chr10: 105,760,564-105,760,656
Enhance
Yes
Yes
 9



exon







TRIP10
Cassette
chr19: 6697040-6697207
Enhance
Yes
No
68



exon







ADAM15
Cassette
chr1: 153301004-153301075
Enhance
Yes
Yes
69



exon







WNK1
Cassette
chr12: 859000-859458
Silence
No
No
70



exon







GADD45A
Cassette
chr1: 67924296-67924397
Enhance
No
No
71



exon







LOXL2
Cassette
chr8: 23,284,093-23,284,230
Enhance
Yes
Yes
72



exon







SCRIB
Cassette
chr8: 144961710-144961772
Silence
Yes
Yes
20



exon







SLK
Cassette
chr10: 105,760,564-105,760,656
Enhance
Yes
Yes
 9



exon







MYO1B
Dual
chr2: 191973720-191975689
Silence
Yes
Yes
73



cassette








exons







OGDH
Mutually
chr7: 44,653,552-44,653,893
Promotes
Yes
No
74



exclusive

distal exon





SF1
Alt 3′ end
chr11: 64,300,038-64,300,736
Promotes
Yes
No
75



Type I

long isoform





SF3B1
Alt 3′ end
chr2: 197,991,337-197,993,726
Promotes
Yes
No
76



Type I

long isoform





GIT2
Alt 3′ end
chr12: 108,869,527-108,873,965
Promotes
Yes
Yes
77



Type II

short isoform





EPB41L5
Alt 3′ end
chr2: 120,574,253-120,579,718
Promotes
Yes
Yes
78



Type II

short isoform





CUL4A
Alt 3′ end
chr13: 112,930,781-112,936,341
Promotes
Yes
No
79



Type II

short isoform





RBM39
Retained
chr3: 50112489-50112968
Promotes
No*
No
80



intron

splicing
















TABLE 4





ESRP targets with published roles in cell adhesion, polarity, migration, or


regulation of the actin cytoskeleton.
















OSBPL3 (ORP3)
oxysterol binding protein-like 3


EPB41L5
erythrocyte membrane protein band 4.1 like 5


MYO1B
myosin IB


LAMC2
laminin, gamma 2


SCRIB
scribbled homolog (Drosophila)


PXN
paxillin


CD99
CD99 molecule


KLHL5
kelch-like 5 (Drosophila)


DOCK7
dedicator of cytokinesis 7


SLK
STE20-like kinase (yeast)


ENAH
enabled homolog (Drosophila)


TGFB2
transforming growth factor, beta 2


ADAM15
ADAM metallopeptidase domain 15


CASK
calcium/calmodulin-dependent serine protein kinase



(MAGUK family)


SPTAN1
spectrin, alpha, non-erythrocytic 1 (alpha-fodrin)


ITGA6
integrin, alpha 6


ITGB1
integrin, beta 1(fibronectin receptor)


ITGB3BP
integrin beta 3 binding protein


ITGB1BP1
integrin beta 1 binding protein 1


MACF1/ACF7
microtubule-actin crosslinking factor 1


GIT2
G protein-coupled receptor kinase interactor 2


VEGFA
vascular endothelial growth factor


CTNND1
p120-catenin; catenin (cadherin-associated protein),



delta 1


LOXL2
Lysyl oxidase-like 2


CDH3
cadherin 3, type 1, P-cadherin (placental)


FLNB
filamin B, beta (actin binding protein 278)


JAG2
jagged 2


FAT
FAT tumor suppressor (Drosophila)


PTPRM
protein tyrosine phosphatase, receptor type, M









In another embodiment of the invention, the diagnostic reagent is immobilized on a substrate. In a further embodiment, the reagent enables detection of changes in expression in at least one of (a), (b), (c) or (d) from that of a reference expression profile, the changes correlated with the diagnosis, stage or progression of a disease selected from the group consisting of an epithelial cell cancer or carcinoma, metastasis thereof, tissue fibrosis or a disease or condition related to inappropriate splicing of a target such as FGFR2. In another embodiment, the composition is presented in a microfluidics card, chip or chamber. In another embodiment, the reagent is associated with a directly-detectable, or indirectly-detectable, label. In another embodiment, the diagnostic composition may comprise a microarray or panel of two or more the reagents capable of identifying the presence of two or more sequences of (b), (c) or (d) in a biological sample. In a further embodiment, two or more of (b), (c) or (d) form a signature diagnostic of an epithelial cell cancer or carcinoma, metastasis thereof, or tissue fibrosis.
In a further embodiment, the any of the above compositions or reagents is contained within a kit. In another embodiment, the kit contains a positive or negative control.
One embodiment of a diagnostic reagent comprises at least one polynucleotide immobilized on a substrate. The polynucleotide is a genomic probe that hybridizes to at least one splicing factor selected from ESRP1, ESRP2 or RNPC1 or suitable target variant thereof. The reagent can contain additional splicing factors or target variants useful as a genetic signature of a disease involving inappropriate FGFR2 splicing, such as tissue fibrosis or an epithelial cell cancer. In one embodiment the reagent enables detection of changes in expression in at least one splicing factor selected from ESRP1, ESRP2 and RNPC1 or target variant from that of a reference expression profile. Differences between the expression of these factors or targets in a subject that of the signature profile can indicate a diagnosis of cancer, tissue fibrosis or another related disorder. The changes correlate with the diagnosis, stage or progression of a disease or condition related to inappropriate splicing of FGFR2.
Still another diagnostic reagent or composition or kit for diagnosing the occurrence, stage or progression of a disease or condition related to inappropriate splicing of a target such as FGFR2 in a mammalian subject includes one or more PCR primer-probe sets that amplifies a polynucleotide sequence of at least one splicing factor selected from ESRP1, ESRP2 and RNPC1 or one target thereof.
The diagnostic compositions of the invention can be presented in the format of a microfluidics card, a microarray, a chip or chamber employs the PCR, RT-PCR or Q PCR techniques described above. In one aspect, such a format is a diagnostic assay using TAQMAN® Quantitative PCR low density arrays. When a biological sample from a selected subject is contacted with the primers and probes in the diagnostic composition, PCR amplification of genes in the gene expression profile from the subject permits detection of changes in expression in the splicing factor genes or targets thereof in the gene expression profile from that of a reference gene expression profile. Significant changes in the gene expression indicating a decrease in the expression level of these splicing factors or targets from that of the reference gene expression profile can correlate with a diagnosis of disease, e.g., tissue fibrosis, cancer or a particular stage of a cancer.
The selection of the particular polynucleotide sequences useful as primers or probes for the splicing factors or their targets (a)-(d) above, the length and labels used in the composition are routine determinations made by one of skill in the art in view of the teachings of which genes can form the gene expression profiles suitable for the diagnosis and prognosis of epithelial cell cancers.
Still another diagnostic reagent includes a composition or kit comprising at least one ligand that binds to an expression product of at least one mammalian splicing factor selected from ESRP1, ESRP2 and RNPC1 or one target thereof. In one embodiment the ligand is associated with a detectable marker. In another embodiment, the splicing factor is a protein and the ligand is an antibody or functional fragment thereof, such as a Fab fragment, a complementarity determining region “CDR”, an scFv, among other known sequences. Such reagents are useful in immunohistochemistry diagnostic methods. Antibodies or antisera, preferably polyclonal antisera, and most preferably monoclonal antibodies, or other protein-binding ligands specific for each factor or target are used to detect expression. The antibodies can be detected by direct labeling of the antibodies themselves, for example, with radioactive labels, fluorescent labels, hapten labels such as, biotin, or an enzyme such as horse radish peroxidase or alkaline phosphatase. Alternatively, unlabeled primary antibody is used in conjunction with a labeled secondary antibody, comprising antisera, polyclonal antisera or a monoclonal antibody specific for the primary antibody. Protocols and kits for immunohistochemical analyses are well known in the art and are commercially available. In still another embodiment, the splicing factor or target is a nucleic acid sequence and the ligand is an antisense sequence or polynucleotide as discussed above.
Such diagnostic reagents and kits containing them are useful for the measurement and detection of ESRP1, ESRP2 or RCNP1 or targets thereof in the methods described herein for diagnosis/prognosis of tissue fibrosis, epithelial celltype specific cancers, and other FGFR2-related conditions. In such composition, the antibodies or peptides or nucleic acid sequences may be immobilized on suitable substrates, e.g., bound to an avidin-coated solid support, plates, sticks, or beads. Of course, other binding agents known to those of skill in the diagnostic assay art may also be employed for the same purposes. Other reagents include conventional diagnostic labels or label systems for direct or indirect labeling of the antibodies, peptides or nucleic acid sequences, with e.g., radioactive compounds, radioisotopes, such as 32P, 125I, tecnhicium; fluorescent or chemiluminescent compounds, such as FITC, rhodamine or luciferin; and proteins such as biotin or enzymes and enzyme co-factors, such as alkaline phosphatase, beta-glactosidase or horseradish peroxidase; and/or molecular labels such as FLAG, etc. Other elements of the label systems include substrates useful for generating the signals upon interaction with the other components of the label system, e.g., a streptavidin and horseradish peroxidase system. Any method known in the art for separately conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al, 1962 Nature 133:945; Pain et al 1981 J. Immunol., Meth. 40:219 and other conventional texts.
Alternatively, a diagnostic kit thus also contains miscellaneous reagents and apparatus for reading labels, e.g., certain substrates that interact with an enzymatic label to produce a color signal, etc., apparatus for taking blood samples, as well as appropriate vials and other diagnostic assay components.
C. Screening Assays For The Identification Of Useful Compounds
The splicing factors identified by the inventors may also be used in the screening and development of chemical compounds, small molecules, nucleic acid sequences, such as cDNAs, or proteins which affect the normal splicing of FGFR2 or of another enhanced target or silenced target of these splicing factors. Such compounds, which preferably mimic the activity of ESPR1/2 have utility as therapeutic drugs for the treatment of diseases related to FGFR2 or the enhanced/silenced target disruption, such as tissue fibrosis and epithelial cell cancers.
As one aspect of the invention, there is provided a method for screening a test compound or molecule to determine its impact, if any, upon splicing activity, in the production of specific FGFR2 splice variants. In one embodiment, a test compound, including small molecules, peptides or polypeptides, nucleotide sequences, such as cDNAs, are selected for testing. In one embodiment, the selected test compound is contacted or exposed to a mammalian cell expressing the splicing factor. In another embodiment, the selected test compound is contacted or exposed to a mammalian cell expressing the gene product of the target of a splicing factor, e.g., FGFR2. In a specific embodiment, the splicing factor is ESRP1, ESRP2 or RNPC1. After suitable culture conditions, e.g., 37° C. for about 48-60 hours, the effect of the compound on the expression level of the gene product is assessed by any suitable means. The effect of the compound to alter the normal expression or variant of the target, such as FGFR2, is inversely related to its impact on the expression level of the splicing factor by the cell. Where the effect is that the test compound allows maintenance of normal expression levels or increases levels of the target of the splicing factor, the test compound mimics the activity of the splicing factor. Where the effect is that the test compound increases the expression of the target, that test compound is indicated to be useful as a potential therapeutic compound in the treatment of disorders requiring maintenance of normal expression of FGFR2. Thus, that test compound may be used to treat, prevent, or reduce metastasis of carcinomas or cancers derived from epithelial cell types, and to treat, block or reduce tissue fibrosis.
Where the effect is that the compound decreases the expression level of normal target (e.g., decreasing expression level of specific FGFR2 splice variants) and/or increases expression of the alternate splice variant and/or decreases expression of the splicing factor itself, the compound is indicated to have a potential adverse effect on conditions requiring maintenance of normal expression of the target, e.g., FGFR2. Thus, that test compound may be carcinogenic or not indicated for the treatment of certain carcinomas or cancers derived from epithelial cell types, and not desired to reduce tissue fibrosis.
In another related embodiment, a method of screening a test compound involves measuring the mutagenic effect of the compound on the expression of the target gene product or of the splicing factor itself. For example, the determination of the impact of the test compound is observed where it has mutagenized the splice regulatory factor or resulted in a mutant target or alternate splice variant of a target, e.g., FGFR2. Thus, a therapeutically desirable effect of the test compound is inversely related to its mutagenic effect on the expression of the gene product by the cell. If a compound causes a mutation in the splicing factor or normal target, e.g., FGFR2, after being contacted with a cell expressing the normal target, e.g., FGFR2, it is indicated to be possibly carcinogenic or to have a potential adverse effect on conditions requiring maintenance of normal expression of the target, e.g., FGFR2.
The high throughput splicing assay used to identify compounds that change splicing events, such as the assay described in Example 1 below, may also be used to screen compounds for the ability to alter normal splicing activity of ESRP1/2. In one embodiment such an assay involves contacting in each individual well of a multi-well plate a different selected test compound (e.g., nucleotide sequence, amino acid sequence, small molecules, etc) with a mammalian cell that expresses a specific target splice variant associated with a specific condition, e.g., cancer or tissue fibrosis. In one embodiment that cell is transfected with a minigene that expresses luciferase (or another marker gene) only when the cell expresses a specific splice variant. As one example, the minigene can have the structure outlined in the examples below. After the compound has been exposed to the expressing cell under appropriate culture conditions, the level of the marker gene (or luminescence) is conventionally measured. A change in the expression of the specific target splice variant normally expressed by the cell caused by any of the test compounds is correlated with the expression or lack of expression of the marker in each well. For example, where the assay of Example 1 is utilized to screen test compounds, the maintenance or an increase in expression level of ESRP1 or ESRP2 or a normal target thereof, e.g., FGFR2, is indicative that the compound has an anti-carcinogenic effect on the epithelial cancer or tissue fibrosis. A result in which there is a decrease in expression level of the splice variant is indicative that the compound has a potential carcinogenic effect or adverse effect on tissue fibrosis.
The use of the high throughput assay of Example 1 in identifying splicing regulators also reveals the potential for this screening method to uncover a larger set of splicing regulators of diverse mammalian transcripts. While the relative merits and liabilities of an RNAi screen versus a cDNA overexpression screen have been described elsewhere, functionally redundant protein homologs or paralogs that affect the screening assay equally, as shown here, will elude detection in an arrayed RNAi screen (Rines et al., 2006). A particular advantage of the array based approach over pooled expression screening is the ability to identify multiple genes that function in a pathway or process. In addition to the ESRPs, the inventors discovered several additional splicing regulators, which while individually unable to affect endogenous FGFR2 splicing, are likely to be a part of the combinatorial code regulating this transcript. This high throughput, cell-based methodology, used to screen cDNAs or other forms of compound is highly desirable in this context.
Another aspect of the invention comprises a screening assay to determine compounds which mimic the splicing activity of ESPR1, ESPR2 or RNCP1. In one embodiment, the method comprises culturing a host cell that does not express any epithelial splicing agent (e.g., any of ESPR1, ESPR2 or RNCP1), the cell transfected with a minigene comprising a nucleic acid sequence that generates a detectable signal when the minigene is contacted with the splicing agent; culturing the host cell in the presence of a test molecule; and detecting the generation or absence of the signal in the host cell, wherein generation of a signal in the host cell indicates that the test molecule mimics the splicing activity of the splicing agent. In one embodiment, the test molecule is a chemical reagent, a protein or a nucleic acid sequence that blocks the epithelial mesenchymal transition and inhibits the metastatic progress of carcinomas or cancers derived from epithelial cell types, or inhibits the progression of tissue fibrosis. In another embodiment, the assay is a high throughput assay. In another embodiment, the detectable signal is generated by luciferase. In another embodiment of the assay, the mammalian cell expresses a nucleic acid sequence splice variant encoding a protein associated with a specific disease condition and the minigene expresses luciferase only when the cell expresses the specific splice variant. Compounds discovered to mimic the activity of the splicing factors are discussed below related to therapeutic methods and reagents.
Other conventional assays and techniques also exist for the identification and development of compounds and drugs which impact the expression or activity of a splicing factor described herein, such as methods described in publications referenced herein. Such other assay formats may be used and the assay formats are not a limitation.
D. Therapeutic Methods And Reagents
The splicing factors identified herein, desired target variants described herein or compounds determined to mimic the biological splicing activity of these factors, may also be employed as therapeutic compositions or in therapeutic methods.
1. Methods
In one embodiment, a method for suppressing metastatis of a mammalian carcinoma or cancer derived from primary epithelial cells or blocking the progression of tissue fibrosis involves delivering to the cell at least one of ESRP1 or ESRP2. In one embodiment, the delivery comprises administering a plasmid or viral vector comprising a polynucleotide encoding ESRP1 or ESRP2 or a functional fragment thereof under the control of a promoter operable in eukaryotic epithelial cells. In another embodiment, the delivery comprises administering at least one of ESRP1 or ESRP2 as a protein ectopically to the cell. In still a further embodiment, delivery occurs under conditions permitting the uptake of the ESRP1 or ESRP2 by the cell. In another embodiment, at least one of ESRP1 or ESRP2 in an amount sufficient to provide maintain epithelial identity or prevent epithelial mesenchymal transition.
In another embodiment, a method for suppressing metastasis of a mammalian carcinoma or cancer derived from primary epithelial cells or blocking the progression of tissue fibrosis comprises delivering to the cell at least one of ESRP1 or ESRP2 or a molecule that mimics the splicing activity of the ESRP1 or ESRP2. In one embodiment, the delivery comprises administering a plasmid or viral vector comprising a polynucleotide encoding ESRP1 or ESRP2 or a functional fragment thereof under the control of a promoter operable in eukaryotic epithelial cells. In another embodiment, the delivery comprises administering at least one of ESRP1 or ESRP2 of a molecule that mimics the activity of ESRP1 or ESRP2 ectopically to the cell. In another embodiment, the delivery occurs under conditions permitting the uptake of the ESRP1 or ESRP2 or the molecule by the cell. In yet another embodiment, the delivering supplies the at least one of ESRP1 or ESRP2 or the molecule in an amount sufficient to provide maintain epithelial identity or prevent epithelial mesenchymal transition.
Another aspect of the invention comprises a method for diagnosing the occurrence, stage or progression of a disease or condition related to inappropriate splicing of FGFR2 comprising measuring the level of expression of the splicing factor RNPC1 in a biological sample from a mammalian subject, wherein the expression level of the splicing factor below the level of expression of a healthy mammalian subject is evidence of the disease or condition.
2. Compositions
Thus, in one embodiment a therapeutic composition for administration to a human subject for treatment of a disease related to inappropriate splicing of a splicing factor target such as FGFR2 or for tissue fibrosis, or a epithelial cell type specific carcinoma includes a plasmid or viral vector comprising a polynucleotide encoding at least one of ESRP1, ESRP2 and RNPC1 or a desired target variant, under the control of a promoter operable in eukaryotic epithelial cells in a pharmaceutically acceptable vehicle or carrier. The selection of a suitable plasmid backbone or viral vector, of which many are well known in the art and commercially available may be done by a person of skill in the art. Examples of such vectors include recombinant adenoviral vectors, herpes simplex virus (HSV)-based vectors, adeno-associated viral (AAV) vectors, hybrid adenoviral/AAV vectors, recombinant retroviruses or lentiviruses which are constructed to carry or express a selected nucleic acid composition of interest, modified vaccinia virus Ankara (MVA), Vaccinia, Adeno-associated virus (AAV), Alphavirus etc. Retrovirus vectors that can be employed include those described in EP 0 415 731; International Patent Publication Nos. WO 90/07936; WO 94/03622; WO 93125698; and WO 93/25234; U.S. Pat. No. 5,219,740; International Patent Publication Nos. WO 93/11230 and WO 93/10218; GB Patent No. 2,200,651; and EP 0 345 242, among others. Alphavirus-based vectors may also be used as the nucleic acid molecule encoding the transgene. Such vectors can be constructed from a wide variety of alphaviruses, including, for example, Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532). Representative examples of such vector systems include those described in U.S. Pat. Nos. 5,091,309; 5,217,879; and 5,185,440; and International Patent Publication Nos. WO 92/10578; WO 94/21792; WO 95/27069; WO 95/27044; and WO 95/07994.
Examples of adenoviral vectors include those described in International Patent Publication Nos. WO 94/12649; WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655. Other particularly desirable adenoviral vectors include those derived from chimpanzee adenoviruses, such as those described in U.S. Pat. No. 6,083,716. Another viral vector is based on a parvovirus such as an adeno-associated virus (AAV). Representative examples include the AAV vectors disclosed by Srivastava in International Patent Publication No. WO 93/09239, Samulski et al., 1989 J. Viral. 63:3822-3828; Mendelson et al., 1988 Viral. 166:154-165; and Flotte et al., 1993 PNAS 90:10613-10617. Other particularly desirable AAV vectors include those based upon AAV1; see, International Patent Publication No. WO 00/28061, published May 18, 2000. Other desirable AAV vectors include those which are pseudotyped, i.e., contain a minigene composed of AAV 5′ ITRS, a transgene, and AAV 3′ ITRs packaged in a capsid of an AAV serotype heterologous to the AAV ITRs. Methods of producing such pseudotyped AAV vectors are described in detail in International Patent Publication No. WO 01/83692. Such selection of vector and expression system is not believed to limit this invention.
In another embodiment, in one embodiment a therapeutic composition for administration to a human subject for treatment of a disease related to inappropriate splicing of a splicing factor target such as FGFR2 or for tissue fibrosis, or a epithelial cell type specific carcinoma includes a naked DNA containing at least one of ESRP1, ESRP2 and RNPC1 or a desired target variant, under the control of a promoter operable in eukaryotic epithelial cells in a pharmaceutically acceptable vehicle or carrier.
In still another embodiment, a pharmaceutical composition for administration to a human subject for treatment of a disease related to inappropriate splicing of a target such as FGFR2 or for tissue fibrosis, or a epithelial cell type specific carcinoma includes contains an effective amount of at least one of ESRP1, ESRP2 and RNPC1 or the desired target variant in protein form in a pharmaceutically acceptable vehicle.
Another compound or compounds useful in these methods are those that inhibit the metastatic process of carcinomas or cancers derived from epithelial cell types, or inhibit the progression of tissue fibrosis. In one example, such a compound upon contact with a mammalian cell permits expression of at least one variant protein encoded by a nucleic acid sequence splice variant that is regulated by ESRP1 or ESRP2 expression levels and may be used in a composition with a pharmaceutically acceptable vehicle. In one embodiment the compound is one or more compounds selected from the group consisting of phorbol 12-myristate 13-acetate (PMA), phenylazo-3-phyridinol, podophyllotoxin, R(−)-isoproterenol (+)-bitartrate, beclomethasone ethylcarboxamido adenosine. See, e.g., Example 22 and Table 5 below. Table 5 lists compounds that activated splicing in the luciferase reporter screen using the LOPACTM library of compounds, a group of high purity, small organic ligands. In one embodiment desirable compounds exhibit a greater than 1.75 fold-increase over the Average Median norm for each plate exclusive of the wells in which Esrp1 cDNAs were spotted as positive controls (col 1) or the Average control (DMSO) norm (col 2) of Table 5. In another embodiment, desirable compounds exhibit a greater than 1.9 fold-increase over the Average Median norm for each plate exclusive of the wells in which Esrp1 cDNAs were spotted as positive controls (col 1) or the Average control (DMSO) norm (col 2) of Table 5. In another embodiment, desirable compounds exhibit a greater than 2.0 or 2.5 fold-increase over the Average Median norm for each plate exclusive of the wells in which Esrp1 cDNAs were spotted as positive controls (col 1) or the Average control (DMSO) norm (col 2) of Table 5. In another embodiment, desirable compounds exhibit a greater than 3.5 fold-increase over the Average Median norm for each plate exclusive of the wells in which Esrp1 cDNAs were spotted as positive controls (col 1) or the Average control (DMSO) norm (col 2) of Table 5. In another embodiment, the compound is determined using the above described screening assay.







TABLE 5







LOPAC Compounds Screened for ESPR1 Activity

















Avg
Avg

mol









Med
DMSO

weight

Sec







norm
Norm
CATNUM
Structure
Name
Name
Class
Enzyme
Action
Selectivity
Description




















7.032680952
6.87358611
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1


6.591126212
6.406361632
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1


5.65991068
6.104789004
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1


5.272125178
5.684993578
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1


5.026824151
4.935146648
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1


4.915565088
5.29410053
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1


4.879419641
5.212778295
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1


4.683539048
4.509093136
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1


4.304648879
4.636619909
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1


3.896729388
3.792033799
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1


3.825421503
4.121797359
P 8139
616.84292
Phorbol
PMA
Phospho-
Enzyme
Activator
PKC
Activates






12-

rylation



protein






myristate





kinase C






13-acetate





in vivo












and in












vitro;












strong












NO












promoter;












promotes












expression












of












iNOS in












cultured












hepatocytes; T












lymphocyte












activator


3.588748546
3.549019763
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1


3.39328163
3.61912973
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1


3.31180299
3.52749457
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1


3.227318922
3.164027962
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1


2.760668436
2.688986369
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1


2.598412207
2.802816598
S 9186
213.24097
SIB 1757
6-
Glutamate

Antagonist
mGluR5
Highly







Methyl-




selective







2-




mGlu5







(phenylazo)-




metabotropic







3-




glutamate







pyridinol




receptor












antagonist


2.584686163
2.743086853
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
Espr1
















2.035659347
1.984651029
P 4405
414.41584
Podo-

Cytoskeleton and
Inhibitor

Antineo






phyllotoxin

ECM


plastic











glucoside;











inhibitor











of











microtubule











assembly


1.995538081
1.94333614
I 2760
361.35186
R(−)-Iso-

Adrenoceptor
Agonist
beta
Sympatho-






proterenol(+)-




mimetic amine






bitartrate




acting











almost











exclusively











on











beta











adrenoceptors;











broncho











dilator;











active











enantiomer











of











Isoproterenol


1.941646182
2.094814676
P-108
343.34499
N6-

Adenosine
Agonist
A1
Potent






Phenyl-




A1






adenosine




adenosine











receptor











agonist


1.860506666
1.995858795
B 0385
408.92643
Beclo-
9alpha-
Hormone

Gluco-
Anti-






methasone
Chloro-


corticoid
inflammatory







16beta-



glucocorticoid







methyl-











1,4-











pregnadiene-











11beta,











17alpha,-











21triol-











3,20-











dione






1.821987263
1.953058981
B-152
398.42499
N6-
N6-
Adenosine
Agonist
A3
Selective






Benzyl-5′-
Benzyl-



A3






N-ethyl-
NECA



adenosine






carbox-




receptor






amido-




agonist






adenosine







1.782972743
1.923013117
P-215
267.28691
PD 98,059
2-(2-
Phosphorylation
Enzyme
MEK2
Specific







Amino-

Inhibitor

inhibitor







3-



of the







methoxy



activation







phenyl)-



of







4H-1-



mitogen-







benzopyran-



activated







4-



protein







one



kinase











kinase











(MAPK











K)


1.714439944
1.674176693
E 4642
219.66983
(±)-
(±)-
Adrenoceptor
Agonist

Adrenoceptor






Epinephrine
Adrenalin



agonist






hydrochloride
hydrochloride






1.712519854
1.662983152
I 5627
247.72401
(±)-

Adrenoceptor
Agonist
beta
Sympatho-






Isoproterenol




mimetic amine






hydrochloride




acting











almost











exclusively











on











beta











adrenoceptors;











broncho











dilator


1.696018387
1.654418765
E 4375
333.29768
(−)-
Adrenaline
Adrenoceptor
Agonist

Endogenous






Epinephrine
bitartrate



hormone






bitartrate




and











neuro-











transmitter


1.670563788
1.792037261
C 6019
344.84719
Clotrimazole
1-(o-Chlorotrityl)-
K+ Channel
Inhibitor
Ca2+-
Specific







imidazole


activated
inhibitor










K+
of Ca2+-










channel
activated











K+











channels


1.664787743
1.795025781
S 0441
287.74848
SB-
Vanilloid
Vanilloid
Antagonist
VR1
Vanilloid






366791
receptor-1



receptor-







antagonist



1 (VR1)











antagonist


1.655508435
1.786285871
S 8502
398.30028
(−)-
Hyoscine
Cholinergic
Antagonist
Mus-
Competitive






Scopolamine
methyl


carinic
muscarinic






methyl
bromide



acetylcholine






bromide




receptor











antagonist















1.643127846
1.762160022
A 9561
311.77656
5-(N,N-
Ion Pump
Inhibitor
Na+/H+
Na+/H+






hexamethylene)amiloride


Antiporter
antiport










inhibitor









In another embodiment, these therapeutic compositions are useful to block the epithelial to mesenchymal transition (EMT) and inhibit metastasis. In another embodiment, the composition blocks tissue fibrosis.
Still other useful compositions comprise an effective amount of a protein encoded by a nucleic acid sequence splice variant that is upregulated or downregulated by the ESRP1 or ESRP2 expression levels of a healthy mammalian subject, optionally with a pharmaceutically acceptable vehicle carrier.
Another aspect of the invention comprises composition comprising a plasmid or viral vector comprising a polynucleotide encoding at least one of ESRP1, ESRP2 and RNPC1 or encoding at least one variant protein encoded by a nucleic acid sequence splice variant that is regulated by ESRP1 or ESRP2 expression levels under the control of a promoter operable in eukaryotic epithelial cells in a pharmaceutically acceptable vehicle or carrier.
Another aspect of the invention comprises a composition comprising an effective amount of at least one of ESRP1, ESRP2 and RNPC1 in a pharmaceutically acceptable vehicle.
Another aspect of the invention comprises a composition comprising a ligand that binds to and inhibits expression of ESRP1, ESRP2 or a protein encoded by a nucleic acid sequence splice variant that is upregulated or downregulated by the ESRP1 or ESRP2 expression levels of a mammalian subject with an epithelial cell cancer, optionally with a pharmaceutically acceptable vehicle. In one embodiment, the ligand is an antisense nucleic acid sequence that binds to ESRP1, ESRP2, the regulated protein encoding sequence, or a nucleic acid sequence that expresses a protein variant that is changed by the downregulation of EPRP1 or ESRP2. In another embodiment, the ligand is a nucleic acid sequence that silences expression of a protein variant that is upregulated by the downregulation of ESRP1 or ESRP2. In another embodiment, the composition is linked to a carrier to targeted delivery agent for use in gene therapy.
As defined herein, pharmaceutically acceptable carriers suitable for use in these compositions are well known to those of skill in the art and may be readily selected by same. In one embodiment, a preferred pharmaceutical carrier contains water for injection with mannitol added for tonicity at a concentration of about 45 mg/mL. Other possible carriers include, without limitation, and depending upon pH adjustments, buffered water, buffered saline, such as 0.8% saline, phosphate buffer, 0.3% glycine, hyaluronic acid, alcoholic/aqueous solutions, emulsions or suspensions. Other conventionally employed diluents, adjuvants and excipients, may be added in accordance with conventional techniques. Such carriers can include ethanol, polyols, and suitable mixtures thereof, vegetable oils, and injectable organic esters. Buffers and pH adjusting agents may also be employed. Buffers include, without limitation, salts prepared from an organic acid or base. Representative buffers include, without limitation, organic acid salts, such as salts of citric acid, e.g., citrates, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid, Tris, trimethanmine hydrochloride, or phosphate buffers. Parenteral carriers can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous carriers can include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose and the like. Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like may also be provided in the pharmaceutical carriers. These immunogenic compositions are not limited by the selection of the carrier. The preparation of these pharmaceutically acceptable compositions, from the above-described components, having appropriate pH isotonicity, stability and other conventional characteristics is within the skill of the art. See, e.g., texts such as Remington: The Science and Practice of Pharmacy, 20th ed, Lippincott Williams & Wilkins, publ., 2000; and The Handbook of Pharmaceutical Excipients, 4th edit., eds. R. C. Rothe inventors et al, APhA Publications, 2003.
As used herein, the term “effective amount” or “pharmaceutically effective amount” refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes one or more of the following: (1) preventing the disease; e.g., preventing a disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease; (2) inhibiting the disease; e.g., inhibiting a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting or slowing further development of the pathology and/or symptomatology); and (3) ameliorating the disease; e.g., ameliorating a disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology). For example, an effective amount,” when administered to a subject to treat cancer, is sufficient to inhibit, slow, reduce, or eliminate tumor growth in a subject having cancer.
The amounts of splicing factors/desired target variants in the pharmaceutical formulations can vary widely, i.e., from less than about 0.1 mg/mL, usually at or at least about 2 mg/mL to as much as 20 mg/mL, or alternatively up to 50 mg/mL or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. Dosages will also be adjusted for the severity of disease, type of disease, and physical condition of the subject as determined by a physician.
A human unit dose form of the immunogenic composition is typically included in a pharmaceutical composition that comprises a human unit dose of an acceptable carrier, preferably an aqueous carrier, and is administered in a volume of fluid that is known by those of skill in the art to be used for administration of such compositions to humans (see, e.g., Remington's Pharmaceutical Sciences, 17th Edition, A. Gennaro, Editor, Mack Publishing Co., Easton, Pa., 1985).
These compositions may be sterilized by conventional, well known sterilization techniques, such as sterile filtration for biological substances. Resulting aqueous solutions may be packaged for use as is. In certain embodiments, the aqueous solutions are lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
Similarly as therapeutic compositions, the mode of administration for in vivo administration may be selected by one of skill in the art, and can include any suitable route. Such routes may be selected from, e.g., oral, intravenous (i.v.), respiratory (e.g., nasal or intrabronchial), infusion, parenteral (aside from i.v., such as intralesional, intraperitoneal and subcutaneous injections), intraperitoneal, transdermal (including all administration across the surface of the body and the inner linings of bodily passages including epithelial and mucosal tissues), and vaginal (including intrauterine administration). Other routes of administration are also feasible and include, without limitation, liposome-mediated delivery, topical, nasal, sublingual, uretheral, intrathecal, ocular or otic delivery, implant, rectal, or intranasal.
E. EXAMPLES OF THE INVENTION
The following examples illustrate of the compositions, and method described herein as well as experiments performed to generate the information giving rising to the embodiments of this invention. It will be readily understood that alterations or modifications, e.g., in the formulation of the components, the routes of delivery, and the dosing, can be made for reasons known to those of skill in the art.
Example 1
Identification of Novel FGFR2 Splicing Regulatory Proteins Using Luciferase-Based Minigene Reporters in a High Throughput cDNA Expression Screen
To comprehensively identify the splicing factors required for FGFR2-IIIb expression, a near genome-wide, high-throughput cDNA overexpression screen was performed. Among the previously uncharacterized splicing regulators identified using this novel approach were the essential epithelial-specific FGFR2 splicing regulators.
To carry out a high throughput cDNA expression screen for FGFR2 splicing regulators, a previously described fluorescent reporter minigene was modified by substitution of a coding sequence for firefly luciferase for that of EGFP (Hovhannisyan et al., 2006; Newman et al, 2006). The following description refers to FIGS. 1A-1C.
As shown in FIGS. 1A and 1B, the PKC-neg minigene PKC-neg-40B-IF3-Luc contains an open reading frame derived from rat protein kinase C (PKC) in a 5′ terminal exon. The ORF contains FGFR2 cDNA sequences from the natural start codon through the end of exon 7 fused with a cassette containing intron 7, exon IIIB, intron 8, exon IIIc, intron 9 and exon 9. This heterologous minigene also contains a 40 nt artificial cassette exon (40B) whose inclusion in spliced transcripts is required to translate the luciferase coding sequence in frame. Exon 40B is included in only 5-10% of spliced transcripts in stably transfected cells, but insertion of downstream ISEs enhances exon inclusion with a corresponding increase in luciferase activity. Exon 40B together with flanking introns separates the PKC ORF from a 3′ terminal exon containing a firefly coding sequence for luciferase from the pGL4 vector, an IRES, and a puromycin resistance marker gene (PuroR).
Splicing of the 40B exon maintains an ORF that yields the enzymatic marker, wherein skipping generates a frame terminating in a stop codon upstream of the luc (see FIG. 1C). When PKC-neg minigenes are stably expressed in a cell type, e.g., 293T, that expresses FGFR2-IIIc and where the FGFR2 auxiliary cis-elements from intron 8 do not promote exon inclusion in the absence of exogenous factors, an increase in luminescence results in only in the presence of a factor that promotes exon inclusion.
Insertion of an FGFR2 intron 8 fragment (Intron Fragment 3: IF3) containing ISEs that promotes splicing of FGFR2 exon IIIb similarly enhances splicing of the heterologous 40B exon in cells that express FGFR2-IIIb in an epithelial cell type-specific manner (Newman et al., 2006). However, transient transfection of cDNAs for Tia-1 or Fox-1 enhanced inclusion of the heterologous exon in 293T cells in the presence of these FGFR2 ISEs (Gerhard et al., 2004).
Using a mesenchymal 293T cell clone (which expresses predominantly FGFR2-IIIc) which and stably expresses the PKC-neg-40B-Luc minigene, a reverse transfection approach was used in 384 well format to screen the entire mammalian genome collection (MGC) collection of full length cDNAs (>14,000) available in CMV-driven pCMV-Sport6 expression vectors (Newman et al., 2006) (data not shown). Empty pCMV-Sport vector was used as a negative control and pCMV-Fox-1 vector as a positive control. Each plate is transfected in duplicate.
An automated Wellmate liquid handler (Matrix) was used to mix 0.12 μL of Mirus transfection reagent with 20 μL of serum free media and applied to each well of the plates containing 40 ng of individual cDNAs. Subsequently 293T cells stably expressing the splicing reporters are mixed with serum containing media and dispensed to each well using the WellMate apparatus. The well plates were incubated at 37° C. for about 48-60 hours. Cells were analyzed for luminescence after additional of luciferase reagents.
Because the entire population of cells in a given well is transfected with the same cDNA, even small induced changes in the readout (e.g., luminescence) can be identified. Thereafter the data was analyzed, clones were picked, and cDNA identity was confirmed and validated.
Using a six-fold increase in luciferase activity across two duplicate wells as the cutoff, a total of 28 cDNAs was identified in the primary screen, corresponding to 22 unique genes. See Table 6 below and FIG. 6. Plasmid preps for these cDNAs were prepared and confirmed by DNA sequencing. Subsequent validation steps determined that four of the hits were false positives. Nine of these hits were RNA binding proteins previously shown to regulate alternative splicing. The remaining 18 proteins were predominantly RNA binding proteins that have not previously been implicated in splicing regulation.
These remaining 18 cDNAs were independently validated to enhance luciferase activity and increase exon 40b splicing by RT-PCR confirming that the screen was specific for identification of splicing regulatory proteins. 15 of the 18 validated hits encode RNA binding proteins (RBPs) or contain consensus RNA binding domains or motifs. Eight of these RBPs have previously been shown to function in the regulation of mammalian alternative splicing. The remaining 7 RBPs and the other three proteins therefore represent novel mammalian splicing regulators. Three of these regulators were identified as RNPC1 (previously Rbm38), ESRP1 (previously Rbm35A) and ESRP2 (previously Rbm35B).
This experiment indicates the potential of this cell-based genetic screening strategy to directly uncover additional heretofore unrecognized alternative splicing regulators in mammalian cells.







TABLE 6







cDNAs that promoted a = 6-fold change in luciferase


activity in the screen.












Fold


Validated



Change
MGC Clone
RNA Binding
Change in


Gene Symbola
in Screen
Number
Domain
Splicing














Zgpat (1)b
30.4
BC021513.1
CCCH Zinc
Yes





finger, G-Patch



Rbm38
26.6
BC006687.1
RRM
Yes


Esrp1
25.5
BC031468.1
RRM
Yes


(Rbm35a)






Zgpat (2)b
14.1
BC027218.1
CCCH Zinc
Yes





finger, G-Patch



SFRS3b,c
13.5
BC000914.1
RRM
Yes


Cugbp2c
12.5
BC026856.1
RRM
Yes


SEPT9
12.0
BC054004.1

Yes


Vgll4
11.4
BC048841.1
RRM
No


BOLL
11.0
BC033674.1
RRM
Yes


Sfrs3b,c
10.6
BC071196.1
RRM
Yesd


Esrp2
10.6
BC031444.1
RRM
Yes


(Rbm35b)






Khdrbs1c
10.5
BC002051.1
KH Domain
Yes


RBM4b,c
9.5
BC032735.1
RRM
Yes


Rcc1
9.2
BC019807.1

Yes


Prkacab
8.6
BC054834.1

Yes


Brunol4c
8.4
BC048405.1
RRM
Yes


Ssb
8.4
BC003820.1
RRM
Yes


Rbm4bb,c
8.1
BC019488.1
RRM
Yes


BRUNOL5c
7.8
BC028101.1
RRM
Yes


RNPS1
7.7
BC108316.1
RRM
Yes


Zcchc10
7.4
BC025078.1
CCHC
Yes





Zinc finger



SNRP70c
6.8
BC001315.1
RRM
Yes


PRKACAb
6.8
BC039846.1

Yes


ZGPATb
6.4
BC032612.1
CCCH Zinc
Yes





finger, G-Patch



WNT1
6.3
BC005449.1

No


Pspc1
6.1
BC026772.1
RRM
No


Zbtb7c
6.1
BC018187.1
C2H2 Zinc
No





finger



Zgpat (2)b
6.0
BC027218.1
CCCH Zinc
Yes





Finger, G-Patch





aAll caps denotes a human orthologue, the others are mouse orthologues.


bGenes represented by multiple, independent cDNAs or transfections.


cKnown regulators of mammalian splicing.


dNot tested directly, but inferred from the results obtained with human SFRS3.






Example 2
Seven of the 18 Splicing Regulators Identified in the Screen are Dependent on FGFR2 Intronic Cis-Elements to Enhance Exon Splicing
The inventors next determined whether enhanced inclusion of exon 40B was dependent on the FGFR2 cis-elements present in IF3 using a stable 293T clone co-expressing dual color EGFP and mRFP-based minigenes (Ladd et al., 2001); see FIG. 2A. The control EGFP minigene does not contain any FGFR2 sequences, whereas the experimental mRFP minigene with FGFR2 IF3 is similar to the minigene used for the screen. Transient transfection of all 18 cDNAs into this cell line revealed that a requirement for FGFR2 IF3 to significantly enhance exon 40B inclusion was limited to seven (Rbm38, Rbm35a, Rbm35b, Cugbp2, BOLL, Brunol4 and BRUNOL5) of the 18 confirmed hits. See FIG. 2B. Three (CUGBP2, Brunol4, and BRUNOL5) of these seven genes corresponded to the Brunol/CELF family of RBPs that have previously been shown to regulate diverse alternative splicing events (Kuroyanagi et al., 2007).
The gene that displayed the greatest enhancement of splicing was RBM38 (RNPC1), which has not previously been shown to regulate splicing in mammals.
Example 3
Two Gene Paralogues, Esrp1 and Esrp2, Require ISE/ISS-3 to Enhance Exon Splicing
The recent studies have focused on the role of an auxiliary cis-element called ISE/ISS-3 (Intronic Splicing Enhancer/Intronic Splicing Silencer-3), that functions specifically in epithelial cell types to coordinately enhance splicing of the upstream exon IIIb and silence the downstream exon IIIc (Hovhannisyan and Carstens, 2007). Among numerous RBPs shown to bind ISE/ISS-3 using biochemical approaches only one protein, hnRNP M, was identified that was shown to influence an FGFR2 splicing reporter (Hovhannisyan et al., 2006 and data not shown). However, hnRNP M was highly abundant in cells expressing either FGFR2 isoform, and its function was limited to the silencing of exon IIIc. Thus, identification of a critical epithelial protein that coordinately switches splicing from exon IIIc to exon IIIb through binding to ISE/ISS-3 eluded detection using standard biochemical approaches.
To determine whether any of the seven genes identified in Example 2 that were functionally dependent upon FGFR2 intronic sequences furthermore displayed a specific requirement for ISE/ISS-3 to enhance splicing, the inventors transfected the respective cDNAs in another 293T cell clone (clone 3) that also contained the same experimental mRFP-based minigene. However, the stably co-expressed control EGFP minigene in this cell line contains all of the same FGFR2 cis-elements except for the UGCAUG Fox binding motif and ISE/ISS-3 (Intron Fragment 5; IF5). Unlike IF3, IF5 cannot confer cell type-specific enhancement of a heterologous exon in cells that express FGFR2 (Hovhannisyan et al., 2006). See, FIGS. 2C, 2D, 2E and 2F.
As predicted, enhanced exon inclusion upon transient transfection of cDNAs for Fox proteins was observed only in the minigene that contained its binding site. Five of the seven cDNAs (Rbm38, Cugbp2, BOLL, Brunol4, and BRUNOL5) enhanced splicing of the heterologous exon from both minigenes, indicating that they did not require ISE/ISS-3 or the UGCAUG Fox binding motif and thus interact with sequences further upstream in the intron.
However, two cDNAs for two previously uncharacterized mammalian RBP paralogs, Rbm35a and Rbm 35b, required ISE/ISS-3 and/or the UGCAUG motif to enhance exon 40B splicing. Furthermore, a cDNA encoding the single D. Melanogaster ortholog for these factors, Fusilli (Fus), demonstrated the same phenotype.
To investigate whether these novel proteins bound specifically to ISE/ISS-3 the inventors performed ultraviolet crosslinking experiments with radiolabelled RNAs corresponding to the wild-type ISE/ISS-3 and a previously defined functional mutant (Ponthier et al., 2006). As a control for specificity the inventors also used an RNA previously shown to bind Fox-2 (Park et al., 2008). The RNAs were incubated with nuclear extracts from 293T cells transiently transfected with cDNAs encoding FLAG-tagged Rbm35a, Rbm35b, Fox-2, or empty Flag vector. Crosslinked bands corresponding to the predicted size of FLAG tagged Rbm35a and Rbm35b proteins were observed using the wild-type ISE/ISS-3, but not with the mutant or the unrelated Fox-1 RNA target, upon incubation with nuclear extracts transfected with these cDNAs). The same crosslinks were not observed with extracts from empty vector or Fox-2 transfected cells.
In contrast, a clearly evident crosslinked band corresponding to transfected Fox-2 was specifically observed upon incubation of the Fox-2 target RNA with extracts from Fox-2 transfected cells. The identity of the crosslinked FLAG-tagged bands was validated by immunoprecipitation of the crosslinked extracts with anti-FLAG resin. These results therefore indicate that ESRP1 and ESRP2 bind directly to ISE/ISS-3.
Example 4
Ectopic Expression of cDNAs Encoding Either ESRP1 (Rbm35a) or ESRP2 (Rbm35B) in Cells that Express FGFR2-IIIc is Sufficient to Switch Splicing of Endogenous FGFR2 Transcripts from Exon IIIc to Exon IIIb
The inventors also analyzed splicing of endogenous FGFR2 in 293T cells transiently transfected with cDNAs for the subset of genes with FGFR2 intron 8 dependent function. In the initial experiments, only the cDNA for Rbm35b affected splicing of endogenous FGFR2 with a switch from nearly 100% exon IIIc splicing to between 20-50% inclusion of the epithelial IIIb exon. The inventors noted, however, that the MGC cDNA clone for Rbm35a was truncated at the 5′ end and obtained a full length Rbm35a cDNA clone that was likewise sufficient to induce a similar switch in endogenous FGFR2 splicing. These results indicate that these paralogous FGFR2 splicing regulators are functionally redundant.
Establishment of cell lines stably expressing either Rbm35a or Rbm35b was complicated by apparent selection against their continued expression. Therefore, to investigate the effects of more prolonged expression of these cDNAs in 293T and other cells that express the mesenchymal FGFR2-IIIc isoforms, the inventors used a retroviral expression system. Analysis of endogenous FGFR2 splicing one week after transduction of 293T cells with vectors encoding these cDNAs demonstrated a more substantial switch in splicing from less than 5% exon IIIb inclusion with empty expression vector to 50% with the cDNA encoding ESRP1/Rbm35a and 66% with the cDNA encoding ESRP2/Rbm35b. The ability of either factor to switch splicing from exon IIIc to IIIb was also shown in three other FGFR2-IIIc expressing cell lines, including the rat AT3 prostate cancer cell line. At the same time the inventors also observed that transduction with virus encoding a cDNA for D. melanogaster caused a similar, or even higher degree of switching of endogenous FGFR2 splicing, further indicating the conserved function of this gene family. See, e.g., FIGS. 3A and 3B.
Example 5
ESRP1 (Rbm35a) and ESRP2 (Rbm35b) are Epithelial Cell Type-Specific Splicing Regulatory Proteins
In view of the discovery of the induction of a robust switch in FGFR2 splicing from the mesenchymal to epithelial pathway by Rbm35a and Rbm35b, the inventors examined whether these two proteins were epithelial cell type-specific factors whose expression is limited to cells and cell lines that express FGFR2-IIIb. The inventors analyzed Rbm35a and Rbm35b mRNA expression in a panel of cell lines commonly used in the lab by RT-PCR. The inventors discovered that both proteins were expressed in FGFR2-IIIb expressing cell lines, but not in those expressing FGFR2-IIIc (PNT2, AT3, DT3, 293T, HMLE).
The inventors further confirmed that these splicing factors are epithelial specific using a microarray database that examined mRNA expression across the NCI60 panel of cell lines used in a study showing that endogenous miRNA-200 family members are powerful epithelial cell markers whose expression closely correlate with E-Cadherin/Vimentin ratios (Park et al., 2008). All cell lines classified as “epithelial” based on E-cadherin/vimentin ratio that also expressed the miR-200 expressed substantially higher levels of RBM35A or RBM35B (usually both), than cells classified as “mesenchymal,” most of which displayed nominal expression (Shankavaram et al., 2007). See FIG. 7.
To provide further verification that expression of these proteins correlates directly with expression of FGFR2-IIIb, the inventors performed qRT-PCR with RNAs from an additional subset of cell lines, including several from the NCI60 collection, to independently validate that expression of RBM35A and/or RBM35B correlated with FGFR2-IIIb expression. FGFR2 splice variant analysis has previously been shown for several of these lines and included DU-145, which demonstrates predominant FGFR2-IIIc, but some FGFR2-IIIb, expression (Carstens et al., 1997; Cha et al., 2008; Luqmani et al., 1996). Consistent with this observation, DU-145 was classified as being indeterminate for epithelial or mesenchymal type based on co-expression of E-cadherin and vimentin (Park et al., 2008).
This analysis confirmed that significant expression of RBM35A or RBM35B was limited to those cells that express exclusively FGFR2-IIIb, with modest expression of RBM35B in DU-145.
To further test whether expression of RBM35A or RBM35B is predictive of the FGFR2 splicing pattern, the inventors carried out splice variant analysis using RNA from two ovarian cancer cell lines, Ovcar3 and Ovcar5, for which the FGFR2 splicing pattern was not known. As predicted, Ovcar 3, which expressed relatively high amounts of both proteins expressed FGFR2-IIIb, whereas Ovcar5, which did not, expressed FGFR2-IIIc. Thus, while expression of these FGFR2 splice variants are not known for most of the NC160 cell lines, a direct correlation between Rbm35a and/or Rbm35b and expression of FGFR2-IIIb is supported by all examples in which the inventors or others have determined FGFR2 isoform expression.
The inventors anticipate that testing of additional cell lines with high levels of these factors (e.g. Colo205, HCC-2998) will reveal exclusive FGFR2-IIIb expression.
The epithelial specificity of these proteins is further supported by data in which their mRNAs have been identified in vivo. For example, distinct epithelial-specific expression of Rbm35a in epithelia overlying the facial skeleton was shown using in situ analysis and the same study supported an epithelial-specific expression pattern for Rbm35b as well (McKee et al., 2005). Furthermore, Rbm35a was shown to be a member of a group of “endodermal signature genes” during early mouse embryogenesis (Sherwood et al., 2007). By contrast, expression of both genes is turned off during induced differentiation of human embryonic stem cells into mesenchymal stem cells (Barberi et al., 2005).
Collectively, these data support that these factors constitute evolutionarily conserved epithelial cell type-specific splicing proteins.
Example 6
RNA Interference Demonstrates that the ESRPs are Required for Expression of the FGFR2-IIIb Splice Variant in Epithelial Cell Types
To provide proof that expression of at least one of these factors is required for expression of FGFR2-IIIb, the inventors performed RNA interference in cells that express this isoform. Given functional redundancy in the ectopic expression experiments the inventors first determined the effect of knockdown of either factor alone as well as combined knockdown using siRNAs in the human prostate cancer PNT2 cell line (Cussenot et al., 1991). Although knockdown with 2 effective siRNAs against ESRP1 alone caused a partial switch from exon IIIb to exon IIIc splicing, 2 separate combinations of effective siRNAs against both ESRP1 and ESRP2 caused a nearly complete switch from exon IIIb to exon IIIc splicing in the endogenous FGFR2 transcript.
The inventors also performed the same siRNA mediated knockdown in a human mammary epithelial cell line, HMLE, and also observed a switch from exon IIIb to exon IIIc splicing although the switch was less complete, most likely due to decreased efficiency of RNAi in these cells. HMLE cells were transfected with siRNAs against GFP as a control or against ESRP1 and ESRP2 in tandem. FGFR2 splicing was analyzed by RT-PCR. Average exon IIIb inclusion with standard deviations was compiled from three independent transfections. For the siGFP control the % IIIb inclusion was 97+/−0.06; for the tandem experiment, the % IIIb inclusion was 56+/−0.38 (gel not shown). Although knockdown by independent siRNAs against ESRP1 and ESRP2 constitutes strong evidence that the switch in splicing is due to knockdown of these factors rather than off target effects, the inventors further carried out a “rescue” with mouse cDNAs for ESRP1 and ESRP2 that were immune to knockdown by these human siRNAs. PNT2 cell were transduced with empty retroviral vector or vector containing the cDNAs for mouse Esrp1 or Esrp2. After 9 days, stable cells were transfected with siRNAs against human ESRP1 and ESRP2 and RNA was harvested for FGFR2 isoform analysis. In cells transduced with empty vector, the inventors again observed a nearly complete switch in FGFR2 splicing. In contrast, cells expressing RNAi resistant cDNAs for either Esrp1 or Esrp2 maintained predominant FGFR2-IIIb expression.
These data provide definitive evidence that these proteins are essential for FGFR2-IIIb expression. These data therefore support that these paralogous gene products are important regulators of this developmentally essential alternative splicing choice.
Example 7
The ESRP Family of Splicing Regulators is Evolutionarily Conserved and Developmentally Essential RRM Domain Proteins
The mouse ESRPs and orthologs in humans, D. melanogaster, and C. elegans contain three RNA Recognition Motifs (RRMs) that constitute the only readily identifiable protein domain common to all orthologs; most phylogenetic sequence conservation is present in these domains. The sequences of the three human RRMs can be found in SEQ ID NOs: 1-3. Clustal W (DNASTAR) alignment of the three ESRP RRM domains was performed using the accession numbers of the sequences as follows: NP—918944 (Mm Esrp1), NP—789808 (mM Esrp2), NP—060167 (Hs ESRP1), NP—524691 (Dm Fusilli), NP—495960 (Ce Sym-2) XP—418338 (Gg Esrp1), NP—001025737 (Gg Esrp2) (data not shown). The boundaries of the RRM domains were defined by the SMART computer system (sequence motif analysis and retrieval tool) (http://smart.embl-heidelberg.de/). The highest sequence identity is observed within RRM1. Drosophila fusilli was originally identified in a screen for dominant maternal enhancers of a dorsalizing mutation in the cactus gene and a potential splicing regulatory function was speculated based on homology with the mammalian splicing regulators hnRNP F and H (Wakabayashi-Ito et al., 2001). Fusilli expression was required in ovarian follicle cells for dorsal-ventral patterning and subsequently was noted to be specifically expressed in the epithelium of the stomodeum and proctodeum in stage 9 embryos. These data, together with the observation that Fusilli can functionally substitute for mammalian Esrps, make it evident that the functions of these orthologs as splicing regulators as well as their epithelial-specific expression are highly conserved evolutionarily. This conserved function as a splicing regulator extends to C. Elegans sym-2 (Synthetic Lethal with Mec-8), which was previously suspected based on developmental redundancy with the splicing regulator Mec-8, was recently confirmed to regulate splicing of a number of transcripts during several different stages of worm development (Barberan-Soler and Zahler, 2008).
Example 8
Downregulation of ESRP Expression Coincides with a Switch from FGFR2-IIIb to FGFR2-IIIc in the EMT
A loss of FGFR2-IIIb is known to occur during the EMT. The inventors investigated whether loss of ESRP is associated with the EMT. The inventors initially analyzed FGFR2 isoform expression and ESRP expression in an immortalized human mammary epithelial cell line, HMLE, induced to undergo EMT in response to the transcription factor Twist1 (Yang et al., 2004). Twist is one of several EMT inducing transcription factors that have been shown to correlate with tumor invasion and metastasis and whose expression has been associated with a poorer prognosis for several types of carcinoma. Analysis of FGFR2 splicing following transduction with a retrovirus containing the Twist cDNA showed that a partial switch from predominant FGFR2-IIIb to IIIc occurred that coincided with a morphological EMT, decrease in established epithelial markers, and increase in mesenchymal markers. Consistent with their previously observed epithelial-specific expression, the inventors detected both ESRP1 and ESRP2 in control HMLE cells, but loss of mRNA for both factors following Twist induced EMT as determined by qRT-PCR. See FIGS. 4A-4C. Interestingly, knockdown of E-Cadherin by RNAi was recently shown to induce an EMT in the same cell line and microarray analysis by these authors likewise revealed a significant downregulation of both ESRP1 and ESRP2 (Onder et al., 2008). Significantly, a reduction in total FGFR2 expression was also observed which was also apparent in the analysis of FGFR2 isoform expression following Twist-induced EMT.
These findings are consistent with previous studies which have shown that changes consistent with an EMT are accompanied by a switch from FGFR2-IIIb to FGFR2-IIIc as well as an overall transcriptional downregulation of FGFR2 (Feng et al., 1997). The epithelial specificity of the ESRPs and these results implied that they constitute an important component of an epithelial cell type-specific signature. Also, the expression level of the ESRPs has an inverse correlation with mesenchymal markers in certain tumors, including those associated with a poorer outcome.
To examine this further the inventors generated a list of a number of previously published epithelial and mesenchymal markers and performed gene cluster analysis on the microarray data set derived from cells that underwent an EMT upon E-cadherin knockdown. The inventors also performed a similar analysis on microarray data from a breast cancer model in which cells selected for invasive properties in vitro produced invasive, poorly differentiated tumors in SCID mice (Huang et al. 2007). These invasive cells and the tumors they produced were likewise shown to display features consistent with an EMT. In both analyses ESRP1 and ESRP2 clustered with epithelial markers and showed similar down regulation during the EMT that coincided with upregulation of mesenchymal markers, including Twist.
Thus, a decrease in ESRP expression is predicted to be a general feature associated with the EMT associated with metastasis and tissue fibrosis. Furthermore, the inventors anticipate that post-transcriptional targets of these proteins contribute to epithelial cell differentiation. Therefore the patterns of alternative splicing they control are themselves a component of such an epithelial expression program and signature.
Example 9
Viral Transduction of cDNAs Encoding ESRP1 or ESRP2 can Prevent and Reverse Twist Induced EMT in Human Mammary Epithelial Cells
To determine whether downregulation of ESRP expression is a requirement for cells to acquire a mesenchymal phenotype, the inventors transduced viral vectors expressing cDNAs encoding ESRP1 and ESRP2 in Twist-transformed HMLE cells that had undergone a clear morphologic EMT. In these preliminary experiments, the inventors found that ESRP1 caused these cells to revert to a more epithelial morphology with a cobblestone appearance, whereas the control EGFP transduced cells maintained a more fibroblastic appearance. However, attempts to transduce HMLE-Twist cells with a cDNA encoding ESRP subsequently resulted in complete cell death. These experiments were carried out using dual selection to maintain expression of both ESRP1 and Twist, suggesting that the reversion to an epithelial phenotype was due at least in part to downstream events controlled by these factors and not merely a loss of Twist expression.
Furthermore, the inventors also performed the same experiment in reverse and tested whether HMLE cells transduced with ESRP1 or ESRP2 could prevent Twist induced EMT. The control transduction Twist was able to induce a morphological EMT, but maintenance of an epithelial phenotype as well as epithelial markers and lack of mesenchymal markers was seen in cells already expressing ESRP1 or ESRP2. These results therefore provide further evidence that expression of at least one of these factors controls a number of cellular processes that prevent an EMT. Thus, expression of these proteins is essential in the prevention of metastasis and tissue fibrosis.
As demonstrated by the examples above, the inventors have identified two novel epithelial cell type-specific splicing regulatory protein paralogs that are unequivocally required for the expression of the epithelial FGFR2-IIIb splice variant. In addition to a complete switch in splicing from exon IIIb to exon IIIc by knockdown of ESRP1 and ESRP2 in an epithelial-type cell line, the inventors also demonstrated a robust switch from exon IIIc to exon Mb when ectopically introduced into cells that express FGFR2-IIIc. Among the FGFR2 splicing factors described to date, these proteins constitute the most essential master switch that regulates the splicing of this developmentally essential alternative splicing event.
The inventors are not aware of a previous example in which ectopic introduction of a single factor into a cell line where the factor is not expressed is sufficient to switch splicing of an endogenous target to the degree seen here.
These results with the ESRPs demonstrate that a single protein is sufficient to shift the balance of these factors towards an epithelial cell type-specific pattern of splicing. The switch from FGFR2-IIIb to the mesenchymal FGFR2-IIIc isoform in through depletion of the ESRPs alone indicates suggests that exon IIIc splicing can indeed be considered a “default” splicing pathway that does not require the expression of mesenchymal-specific factors nor a specific ratio of combinatorial regulators. However, such a default pathway does not merely arise from the relative strength of the splice sites flanking the competing exons, but rather reflects the additional contribution of an underlying combinatorial layer of auxiliary cis-elements and the factors that bind them (Carstens et al., 1998; Carstens et al., 2000). The function of the ESRP proteins, however, can override this layer and, through interactions with some of these ubiquitous factors, shift splicing towards the epithelial pathway. It is noted, however, that the ESRPs do not induce a complete splicing switch in mesenchymal cell types; an observation that supports a combinatorial effect of splicing regulators in mesenchymal cells that favors exon IIIc inclusion and Hub silencing.
The previous association of a switch from FGFR2-IIIb to FGFR2-IIIc during the EMT led the inventors to consider whether a loss of ESRP1 and/or ESRP2 might accompany this process (Savagner et al., 1994). Such an association would be consistent with a proposed role of FGFR2-IIIb as a tumor suppressor and further implicate these epithelial splicing proteins themselves as tumor suppressors via regulation of FGFR2 splicing as well as in the regulation of other post-transcriptional targets (Ricol et al., 1999). The previous studies have largely constituted use of cell lines from the Dunning prostate cancer model in which a switch from FGFR2-IIIb to Mc accompanies the progression from androgen dependent to independent tumors (Yan et al., 1993). Such findings also reflect a reversal of normal development process in which signaling via FGFR2 is required for the establishment of homeostatic control of prostatic epithelial cells (Lin et al., 2007). Similar observations were made in a bladder cancer model associated with an EMT and recently a switch from FGFR2-IIIb to FGFR2-IIIc was also shown to correlate with invasiveness and an associated EMT in breast cancer cell lines (Cha et al., 2008). However, the ability of FGFR2-IIIb to function as a tumor suppressor is context dependent as in some settings dysregulated signaling through this receptor can promote cancer progression (Itoh et al., 1994; Moffa and Ethier, 2007).
By virtue of promoting an epithelial-specific expression program that extends beyond FGFR2, the inventors hypothesize a more general function of the ESRPs as potential tumor suppressors. The identification of bi-allelic mutations in ESRP1 (RBM35A) in several colon cancer cell lines with microsatellite instability suggest that mutation of these factors may indeed be one mechanism by which their loss can be implicated in cancer progression (Ivanov et al. 2007) However, as a part of an epithelial cell type-specific expression program that is lost during the EMT, a loss of expression may more commonly occur in the absence of mutation, but rather as reflecting changes in cellular plasticity that represent aberrant reversal of normal developmental changes in gene expression programs. A number of studies suggest that upregulation of mesenchymal transcription factors such as Twist or Snail are associated with tumor invasion and metastasis in experimental cancer models and associated with a worse prognosis when present in patient tumor specimens is associated with an cancer progression and metastasis (Blanco et al., 2002; Cano et al., 2000; Moody et al. 2005; Yang et al., 2004).
The current data support the role of ESRPs as markers of primary tumors with less metastatic potential and a more favorable prognosis. In addition to serving as a novel epithelial cell marker, these factors are further capable of preventing or reversing the EMT induced by Twist, thereby suggesting that they represent excellent targets for future therapies directed towards maintaining or restoring their expression. However, the ability of the ESRPs to induce an EMT also suggests that they are likely to be mediators of this process in established metastases in which case their expression may no longer be favorable.
Experiments to determine whether ectopic introduction of the ESRPs into invasive tumor cell types can prevent tumor metastases in animal cancer models are being conducted. Finally there are several interesting parallels between the identification of ESRP and the recent identification of the miR-200 microRNAs that similarly enforce an epithelial phenotype and an ability to induce the EMT (Gregory et al., 2008; Park et al., 2008). These collective findings providing clear evidence of the important role of coordinated post-transcriptional regulation as a critical contributor to cellular differentiation, in this case at two separate steps in the RNA life cycle by epithelial specific regulators.
The findings also represent the first example of a splicing regulatory protein that is a likely tumor suppressor and/or whose expression is lost during cancer progression and metastasis via the EMT. These data therefore add an important addition to the evidence that alterations in alternative splicing can play an important role in the development and progression of cancer. Identification of additional splicing events regulated by ESRP may thus characterize a splicing signature that will have clinical utility as well as identify additional transcript variants that play coordinated roles in either cancer progression or prevention.
Example 10
The ESRPs Regulate Splicing of CD44, CTNND1, and ENAH
The profound switch in endogenous FGFR2 splicing from the epithelial to mesenchymal isoform upon depletion of ESRP1 and ESRP2 suggested that these cell-type-specific factors might regulate additional epithelial-specific transcript variants. Inclusion of several “variable” exons of CD44 transcripts, including exons 8-10 (V8-V10), has been shown to be epithelial specific (Ponta et al., 2003). Depletion of ESRP1 and ESRP2 in PNT2 cells resulted in a significant decrease in the inclusion of CD44 exons 8-10 and increase in the standard isoform (CD44s) in which all of the variable exons are skipped (data not shown). Delta catenin (CTNND1), also known as p120-Catenin, expresses mesenchymal specific splice variants that contain alternative exons 2 and 3 (Keirsebilck et al., 1998). Skipping of these exons in epithelial cells results in a shorter protein isoform that initiates translation in exon 5. Expression of the mesenchymal p120-Catenin isoform is induced during the epithelial-to-mesenchymal transition (EMT) (Ohkubo and Ozawa, 2004). Knockdown of ESRP1 and ESRP2 in PNT2 cells also induced expression of the mesenchymal isoform of p120-Catenin (data not shown). ENAH contains an alternative exon 11a that is predominantly included in epithelial cell lines and skipped in mesenchymal cells (Pino et al., 2008). Knockdown of ESRP1 and ESRP2 led to a significant decrease in ENAH exon 11a inclusion (data not shown).
These three examples of additional targets of the ESRPs suggest that they regulate a larger number of epithelial versus mesenchymal splice variants. Furthermore, they illustrate examples of regulated targets which can promote epithelial-specific exons (CD44 and ENAH), silence mesenchymal exons (CTNND1), or both (FGFR2). Because the proteins encoded by these gene transcripts have well documented rolls in the EMT, the inventors sought to further expand the list of alternative splicing events under the regulation of the ESRPs. Additional gene transcripts that are regulated by these proteins similarly play important roles in epithelial to mesenchymal transitions during development as well as pathophysiologic conditions such as cancer metastasis and tissue fibrosis.
Example 11
An Approach Using siRNA-Mediated Depletion and Exon Microarrays to Identify Splicing Events Regulated by ESRP1 and ESRP2
The inventors previously demonstrated that depletion of ESRP1 and ESRP2 from the human prostatic epithelial cell line PNT2 is sufficient to robustly alter the epithelial splicing pattern of four gene transcripts towards the mesenchymal splicing pattern. The inventors therefore made use of this experimental system to carry out a near genome-wide based approach to identify a broader set of splicing events regulated by the ESRPs. Combined depletion of ESRP1 and ESRP2 in PNT2 cells was carried out with siRNAs against each transcript or with siRNAs against green fluorescent protein (GFP) as a control. Each condition was performed in four biological replicates and total RNA, each processed and prepared separately, was converted into labeled cDNA for hybridization to the Affymetrix Human Exon Array 1.0 ST Microarray (FIG. 8A). The efficacy of siRNA-mediated knockdown of ESRP1 and ESRP2 was demonstrated by real-time RT-PCR (FIG. 8B). Because there are presently no suitable antibodies to specifically detect endogenous ESRP protein, the inventors were unable to verify protein depletion by immunoblotting. However, the inventors were able to demonstrate that ESRP1 and ESRP2 were functionally depleted by showing a nearly complete splicing switch from use of the mutually exclusive epithelial IIIb exon, to the mesenchymal IIIc exon in the FGFR2 transcript in all four replicates of ESRP knockdown compared to the control knockdowns (data not shown). Furthermore, the inventors previously validated the specificity of this approach as being due to protein depletion by showing that the epithelial splicing pattern could be rescued by expression of a mouse Esrp1 protein from an RNAi resistant cDNA.
Example 12
Exon Array Analysis Identifies Many Novel Splicing Targets of ESRP1 and ESRP2
In order to identify novel ESRP regulated exons and alternative splicing events the MADS tool was used to identify probe sets that were differentially expressed between the two conditions, subtracting out probe set differences that were the result of differences in overall transcript abundance. The resulting probe sets were ranked according to their MADS p-value and the top 500 differentially expressed probe sets were selected for further analysis and validations. The inventors first manually examined all probe sets using the UCSC genome browser and discarded any probe sets that did not correspond to annotated alternative splicing events as supported by mRNA or EST evidence. Examples of alternative promoters, including cases in which the exon array data could not distinguish between alternative promoters and true alternative splicing events, were discarded. For the remaining probe sets, graphical output from the MADS algorithm as well as the Gene View of all filtered probe sets in Partek Genomics Suite were analyzed. Cases in which neither analysis supported an interpretation of a true splicing change were also discarded. This assessment included a somewhat subjective interpretation of differential probe set signal, determination whether probe set differences were more consistent with overall transcript differences, and a general analysis of the overall pattern of probe set signals. Such an assessment thus also included, in some cases, an assessment as to whether reciprocal changes in several transcripts probe sets also supported differential splicing. For example, several cases of mutually exclusive exons or alternative 3′ ends in which opposite changes in probe set signal were supportive of alternative splicing events were included in the final set of high confidence alternative splicing events. While some of this assessment necessarily was partly subjective, only examples in which the inventors had high confidence that the alternative splicing events were indeed regulated by the ESRPs in this experiment were included. Some of the discarded probe sets may correspond to novel, or poorly annotated examples of alternatively spliced exons. However, most of the discarded probes were present in intronic regions, the majority of which did not represent bona fide alternative splicing events and which would complicate analysis. The validation rate from the limited number of validated events strongly suggests that most of these values are true positives.
After manual annotation of the top 500 MADS probe sets and deletion of poorly supported alternative events, 148 alternative splicing events were identified from 171 probe sets, in a total of 134 different genes (data not shown). This list included detection of ESRP-regulated alternative splicing events that the inventors previously demonstrated in the CD44, CTNND1, and ENAH transcripts described in Example 10. In the case of CD44, probe sets corresponding to tandem variable exons 2, 4, 9, 5, and 7 ranked 21st, 26th, 100th, 101st, and 122nd, respectively. For CTNND1 (p120-catenin), a probe set for alternative exon 2 ranked 139th. The ENAH alternative cassette exon 11a ranked 206th (data not shown). Probe sets corresponding to exons IIIb and IIIc of FGFR2 were filtered from the analysis because the overall transcript level of FGFR2 did not pass the expression threshold, and hence, did not appear in the list of top 500 probe sets. Nonetheless, the p-values associated with both of these mutually exclusive exons likewise strongly supported differential splicing of these exons as shown by RT-PCR.
The inventors categorized the different types of alternative splicing events and found that there is at least one example of every known type of alternative splicing event in the resulting list of high-confidence ESRP regulated splicing events (FIG. 9). Notably, the computational Alternative Conserved Exon (ACEScan) tool predicts 25 of the 143 events were ACEScan positive (including 21 out of the 68 cassette exons), indicating that they were highly likely (or known) to be evolutionarily conserved. The ACEScan data set was derived based on intronic sequence conservation between human and mouse genes that is indicative of conserved splicing events in both species and likely also reflects the presence of intronic binding sites for splicing regulatory factors. The enrichment of such exons indicates that alternative splicing of these exons is likely to be physiologically relevant. The largest group of splicing events consists of alternative cassette exons. However, the inventors also noted a large class of alternative splicing or polyadenylation events that result in the use of alternative 3′ ends. These events can be sub-divided into alternative polyadenylation (polyA) sites or alternative 3′ terminal exons. The inventors further categorized the alternative 3′ terminal exons into two types. The inventors defined Type I events as those where an alternative 5′ splice site is in competition with a polyA signal in the immediate downstream intron. Thus, in these events, when the 5′ splice site is not used, the associated exon becomes the 3′ terminal exon, whereas its use leads to use of an exon further downstream as the 3′ terminal exon, often also including numerous additional downstream cassette exons. Type II events are defined as those in which competing 3′ splice sites in different exons lead either to an upstream 3′ terminal exons, or selection of a downstream exon as either the 3′ terminal exon or continued use of additional 3′ cassette exons (see schematic in FIG. 9).
Example 13
The ESRPs Regulate Inclusion and Skipping of Internal Cassette Exons
The ESRPs were predicted by the Exon Array data to both enhance and repress splicing of alternative cassette exons. A single probe set in the SLK transcript corresponding to exon 13 showed decreased expression in the ESRP knockdown (data not shown). This suggests that inclusion of SLK exon 13 is enhanced by the ESRPs. RT-PCR confirmed this prediction, showing that exon 13 inclusion decreases from 30% in the control to 2% when ESRP1 and 2 are down regulated (data not shown). The inventors further validated examples of ESRP enhanced cassette exons in the LOXL2 and TRIP10 transcripts. Inclusion of the LOXL2 and TRIP10 regulated exons decreased from 4.9% to 1.3% and 2.6% to 0.2%, respectively, upon ESRP knockdown (data not shown). An example of a cassette exon predicted to be repressed by the ESRPs is exon 16 in the SCRIB transcript. The single probe set associated with this exon showed increased levels of inclusion in the ESRP knockdown compared to the control (data not shown). RT-PCR shows the inclusion level of the exon increasing from 11% to 77% upon ESRP knockdown, validating this exon as being a target of ESRP repression (data not shown).
Example 14
The ESRPs, Regulate the Utilization of Alternative 3′ and 5′ Splice Sites within an Exon
In addition to exon inclusion or skipping, the analysis also revealed that the ESRPs can regulate alternative 5′ or alternative 3′ splice site selection. CHRNA5 exon five has three alternative 5′ splice sites and the use of these splice sites results in exons of 49 nt, 350 nt, and 836 nt. Four probe sets corresponding to the portion of the exon created by utilization of the most downstream 5′ splice site, three of which were in the top 500 MADS probe sets, show increased expression upon ESRP knockdown (data not shown). This data indicates that the ESRPs repress splicing at the most downstream 5′ splice site in CHRNA5 exon five. RT-PCR confirmed this prediction as the splice variant of CHRNA5 with the longest form of exon five increases from 5% to 18% upon ESRP knockdown. Utilization of both upstream 5′ splice sites decreased when the ESRPs were down regulated. The amount of the shortest form and intermediate form of exon five decreased from 19% to 10% and 76% to 72%, respectively (data not shown).
As an example of an alternative 3′ splice site choice regulated by the ESRPs, the inventors validated the predicted change in 3′ splice site utilization in FAM62A exon 14. A single probe set that corresponds to the portion of exon 14 formed by splicing at the upstream 3′ splice site showed decreased expression upon ESRP knockdown relative to the control suggesting that ESRP promotes splicing of this exon at the upstream 3′ splice site. RT-PCR analysis clearly confirmed this prediction, with the level of transcripts including the long form of exon 14 decreasing from 23% to 5% (data not shown). The inventors also observed that in four of the five examples of alternative 5′ splice site selection, the ESRPs are predicted to favor splicing at the upstream 5′ splice site. Similarly, in four of the five examples of alternative 3′ splice site selection, the ESRPs are predicted to favor splicing at the upstream 3′ splice site. Such a preferential function provides clues as to how the ESRPs regulate splicing at the molecular level.
Example 15
The ESRPs Regulate Splicing of Mutually Exclusive Cassette Exons
Within the high-confidence list of ESRP regulated splicing events (See, e.g., Tables 1-5) was one example of a mutually exclusive splicing event. A single probe set from the MADS data predicted increased expression of an exon directly upstream of exon 4 in the OGDH transcript. OGDH mRNA and EST alignments strongly suggest that the MADS exon, which the inventors termed exon 4a, and exon 4 are spliced in a mutually exclusive manner. Despite the probe set for exon 4 being absent from the MADS top 500 list the inventors performed an RT-PCR assay to validate the predicted increase in exon 4a inclusion and the reciprocal decrease in exon 4 inclusion. To distinguish between the two amplicons of similar size, the inventors digested the PCR products with Pvu II and BstE II restriction enzymes that uniquely cut exon 4 and exon 4a, respectively (data not shown). Splicing of exon 4a is increased in the ESRP knockdown, from 19% to 48% inclusion, as predicted by MADS. Conversely, exon 4 is included in 81% of the OGDH transcripts in the control but decreases to 52% inclusion when the ESRPs are down regulated. Furthermore, there was no indication of double-inclusion or double-skipping of the two exons. This result validates the MADS prediction as well as the interpretation that the alternative splicing event detected by the MADS is of the mutually exclusive type. Interestingly, this is not analogous to the role the ESRPs play in the regulation of FGFR2 mutually exclusive splicing where the ESRPs favor splicing of the upstream mutually exclusive exon and silencing of the downstream exon. In the case of OGDH, the ESRPs appear to promote splicing of the downstream exon over that of the upstream exon (but see also below).
Example 16
The ESRPs Co-Regulate Splicing of Tandem Sets of Exons
The inventors previously demonstrated that the ESRPs regulated multiple, tandem exons within the CD44 and CTNND1 gene transcripts. The MADS analysis revealed additional tandem alternative exons regulated by the ESRPs and two examples are illustrated here. Two probe sets in MYO1B corresponding to exons 24 and 25, showed increased inclusion in the ESRP knockdown, indicating that these exons are normally silenced by the ESRPs (data not shown). RT-PCR confirmed that both of these exons are targets of ESRP repression. In the control, products containing both exons 24 and 25 make up 5% of the MYO1B transcripts compared to 26% in the ESRP knockdown sample (data not shown). A PCR product representing single exon inclusion is also seen at a low level and the inventors verified that this single inclusion product is a mix of products including either the upstream or the downstream exon alone (data not shown).
The second example of a pair of tandem exons regulated by the ESRPs is in the ADAM15 transcript where the probe sets for exons 20 and 21 were shown to decrease in the knockdown sample compared to the control as detected in our MADS analysis (data not shown). Unlike the case of the MYO1B exons, the two exons in ADAM15 were predicted to be enhanced by the ESRPs. RT-PCR analysis revealed three major splice isoforms corresponding to skipping of both exons, single inclusion of one or the other exon, and inclusion of both exons (data not shown). As predicted by the array data, the splice variants containing one or both of these exons decrease in the ESRP knockdown, while those that skip both increase in abundance. This result confirms that splicing of exons 20 and 21 in ADAM15 are enhanced by the ESRPs.
Example 17
The ESRPs Regulate Splicing of Alternative Terminal Exons
An unexpectedly prevalent regulatory function of the ESRPs unveiled by the MADS analysis was alternative splicing of 3′ terminal ends or alternative polyadenylation (see description above). A recent study showed that these events are widespread, occurring in up to 20% of human genes. Many of these types of events dramatically change the size and sequence of the resulting proteins more than is typically achieved through alternative splicing of cassette exons. Thus, it is perhaps more likely that some of these events may more significantly affect the physiological functions of the alternative isoforms. A total of 36 of these events were in our list of high-confidence list of alternative splicing targets (data not shown). Identification of these events was made possible not only by the exon probesets identified by MADS, but by using the complete set of probe sets for each transcript to detect reciprocal changes in expression between the upstream and downstream terminal exons as analyzed in the Geneview feature of the Partek software program. The inventors selected several alternative 3′ terminal exon events, including both Type I and Type II events, for independent RT-PCR validation and present two examples here. The inventors used a competitive PCR approach that employed a common forward primer and reverse primers specific to each alternative 3′ end. The disadvantage of this method is that it cannot be used as accurately to determine the true ratio of the alternative splice variants as in the case of cassette exons using common PCR primers. However, this approach was sufficient to detect robust changes in splicing mediated by the ESRPs in the validation assays.
Exon 2 of the SP1 gene transcript has a 5′ splice site in competition with a polyA signal. When the 5′ splice site in exon two is used, it is spliced to exon three and the remaining downstream exons. Conversely, when this 5′ splice site is not used, the SF1 transcript is polyadenylated and terminates in the intron immediately downstream of exon two, thereby encoding a significantly truncated isoform of SF1 (data not shown). Our analysis of the MADS data predicted the Type I alternative 3′ end splicing of the SF1 gene to be a target of ESRP regulation. Three of the five probe sets corresponding to the portion of exon two created by skipping of its 5′ splice site were present in the MADS top 500 and show increased expression in the knockdown while the probe sets corresponding to the downstream exons display decreased expression (data not shown). Together, these results suggest that the ESRPs enhance splicing of exon two to the downstream exons and when they are depleted there is increased polyadenylation in the intron downstream of exon two. This prediction was confirmed by RT-PCR. In the control, the predominant splicing product is the full length SF1 transcript. When the ESRPs are down regulated, the inventors observe a decrease in the full-length transcript and an increase in the truncated splice variant (data not shown). The inventors validated an additional Type I alternative 3′ end splicing target of the ESRPs in the SF3B1 gene transcript (data not shown).
The EPB41L5 gene transcript undergoes Type II alternative 3′ end splicing. In the EPB41L5 transcript, exon 16 can be spliced to the upstream terminal exon 17, with polyadenylation and termination occurring in the intron downstream of exon 17. Alternatively, exon 17 can be skipped and exon 16 is spliced to exon 18 and the numerous remaining exons in the transcript. Similar to SF1, splicing to exon 17 in the EPB41L5 transcript would result in a significantly truncated protein. Four probe sets corresponding to the downstream exons in EPB41L5 were in the MADS top 500 list. The MADS probe sets as well as the rest of the probe sets corresponding to the EPB41L5 exons downstream of exon 17 showed increased expression. The two probe sets corresponding to the alternative terminal exon 17 showed a converse decrease in expression. These data indicate that the ESRPs enhance splicing of exon 17, therefore promoting the shorter EPB41L5 isoform. RT-PCR confirmed this to be true. In the control, almost all EPB41L5 terminates at exon 17. However, when the ESRPs are down regulated, the inventors observe a mix of both the short and long splice variants of EPB41L5. The inventors also validated two additional Type H alternative 3′ end splicing targets of the ESRPs (CUL4A, and GIT2) and established that the ESRPs promote splicing of the upstream terminal exon in these two cases as well (data not shown).
Example 18
Additional Validation of Predicted ESRP Regulated Splicing Events
In total, the inventors tested 18 alternative splicing events from the list of high-confidence ESRP targets ourselves by RT-PCR. Of these 18, the inventors were able to validate 15 novel alternative splicing events regulated by the ESRPs (Table 3). We note, however, that one of the events that was not validated was a predicted retained intron in RBM39. While this retained intron was not validated, an increase in the inclusion of a known alternative cassette exon in RBM39 flanked by this intron was observed in response to ESRP knockdown. To further validate changes in splicing associated with down-regulation of the ESRPs, the inventors submitted RNA samples for high-throughput (HT) RT-PCR using high-resolution capillary electrophoresis (data not shown). This analysis was used to investigate an additional 35 targets with two RT-PCR reactions per target. To facilitate direct analysis of the products and simplify the design of PCR primers for this approach, the inventors necessarily focused our use of this method mostly towards analysis of simple, single alternative cassette exons. These examples demonstrate clear changes in splicing of the ESRP enhanced exon in the FNIP1 gene transcript and the ESRP silenced exons in OSBPL3, and GOLGA4 gene transcripts. In total, 25 of the 35 events the inventors tested were confirmed to be valid changes in splicing as predicted by the array data. Four of the ten targets that the inventors were unable to validate (NASP, TGFBR2, PRMT1, GPR126 [probe set 2928530]) turned out to suffer from errors or ambiguities in primer design and were thus removed from the overall analysis as no conclusions could be drawn. Exon two of the NT5C3 gene transcript (probeset 3045024) was predicted to be enhanced by the ESRPs, but RT-PCR results showed that exon inclusion increased upon ESRP depletion. This contradiction might be explained by the presence of alternative first exons, only one of which was targeted by the PCR primers. Four events demonstrated (OSBPL3, DZIP1, ASPH, and TIA1 [probe set 2558539 small changes in splicing that did not pass our threshold of an 1.0% change in splicing or a change was only detected by one of the two primer pairs, but the changes did match the corresponding prediction. Thus, several of these exons may in fact be regulated by the ESRPs, but without additional replicates, the inventors cannot confidently conclude that these are validated splicing targets.
Combining this data with the validations summarized in Table 3, the inventors validated a change in splicing of a total of 38 out of 49 tested splicing events that were identified in the top 500 of the MADS analysis. Thus, at a minimum 78% of the splicing events that the inventors subjected to further validation could be confidently concluded to represent true ESRP regulated splicing events. Specifically, the splicing events indicated in Tables 1-2 above, particularly those over 30% or more inclusion represent true ESRP regulated splicing events.
Example 19
Depletion of ESRP1, but not ESRP2, is Sufficient to Induce Switching Towards Mesenchymal Splicing Patterns
To further investigate functional redundancy between ESRP1 and ESRP2 as well as the relative requirements for each factor in maintaining epithelial splicing pathways, mRNAs encoding each protein were individually depleted as well in combination, as was previously done for the exon array. The inventors then assayed splicing of two gene transcripts with simple cassette exons that displayed robust changes in splicing in response to ESRP1 and ESRP2 depletion (data not shown). Depletion of ESRP1 alone was sufficient to induce a partial increase in splicing of the SCRIB exon and combined depletion of both ESRP1 and ESRP2 caused a further increase in exon inclusion. In contrast, depletion of ESRP2 alone was not sufficient to induce a change in splicing of this exon. In the case of SLK, depletion of ESRP1 alone partially induced exon skipping which was similarly augmented by combined depletion of ESRP1 and ESRP2 (data not shown). However, depletion of ESRP2 alone was unable to induce exon skipping and if anything a slight increase was observed. The specificity and efficacy of knocking down each factor was validated by quantitative RT-PCR (data not shown). These results thus suggest that while ESRP1 and ESRP2 generally have functionally redundant activities, expression of ESRP1 would appear to have more essential functions in splicing regulation (see also below).
Example 20
Ectopic Expression of mEsrp1 in a Mesenchymal Cell Line Induces Reciprocal Changes in Splicing of ESRP Target Gene Transcripts
To further investigate the role of the ESRPs in regulation of these novel splicing targets, the inventors tested whether ectopic expression of the ESRPs was also sufficient to promote the opposite changes in splicing in a mesenchymal cell line as those seen in epithelial cells upon ESRP depletion. The inventors previously showed (Example 10) that ectopic expression of a mouse Esrp1 cDNA in the mesenchymal human breast cancer cell line MDA-MB-231 was in fact sufficient to induce the opposite changes in splicing of FGFR2, CD44, ENAH, and CT7VND1 from those seen with ESRP depletion in the PNT2 cell line. Using this experimental system, the inventors tested the effect of mEsrp1 expression on splicing of the 18 splicing events that we could test ourselves using the primers sets we had for RT-PCR (Table 3). The ESRP regulated exons in the FLNB and SLK transcripts are included at low levels in the control 231 cells (EV). Expression of mEsrp1 greatly enhances splicing of these exons (data not shown). Two examples of exons that are suppressed by the ESRPs are also shown (data not shown). The SCRIB alternative exon is included in 58% of transcripts in the control cells where cells expressing mEsrp1 only splice this exon in 9% of transcripts. In the case of MYO1B, inclusion of the tandem exons is strongly silenced as the percentage of transcripts containing one or both exons decreases from 31% to 8% upon mEsrp1 expression. Finally, an example of mEsrp1 being sufficient to alter alternative 3′ end splicing is shown for the EPB41L5 transcript (data not shown). In the control cells, there is a nearly equal mix of the short and long splice variant of EPB41L5. However, when mEsrp1 is expressed in these cells, the long splice variant is almost completely down-regulated in favor of the truncated splice variant. In total, out of the 15 targets that were confirmed in the PNT2 ESRP knockdown experiment, 8 showed reciprocal changes in splicing upon ectopic expression of mEsrp1 in the MDA-MB-231 cells (data not shown). Interestingly, while not a true “validated” splicing change, the cassette exon in RBM39 also showed a reciprocal change from that observed upon ESRP knockdown (data not shown). Unexpectedly, the direction of change in splicing of the mutually exclusive exons in the OGDH transcript was the same in the mEsrp1 expression system as it was in the ESRP knockdown experiment (data not shown). One possible explanation for this unexpected finding could be that the ESRPs have differential effects on splicing of one or both exons in different cellular milieus, or that they may indirectly regulate these exons. Given that most exons are under regulation by complex combinatorial control by multiple regulatory factors, it is possible that antagonistic functions of the ESRPS may be due to interactions with different regulatory proteins in different cell types. However, at present we cannot provide a simple explanation to account for this seemingly contradictory observation.
Six of the validated splicing changes observed in response to ESRP knockdown showed no change in response to mEsrp1 expression in mesenchymal cells. One possible explanation was that some of these events might instead be specifically regulated by ESRP2. To address this possibility, the inventors analyzed the splicing of these targets in MDA-MB-231 cells when mEsrp1, mEsrp2, or both proteins together were ectopically expressed. In five of these six cases (FAM62A, CUL4A, TRIP10, SF3B1, and SF1) neither mEsrp1, mEsrp2, nor a combination of both induced splicing changes. In the case of the CHRNA5 alternative 5′ splice site there did appear to be evidence that mEsrp2 could induce a change in splicing that was not achieved by mEsrp1, but the effect was small (data not shown). As controls, the inventors also tested the effects of ectopic expression of both paralogs on splicing of SLK and SCRIB. In both cases the inventors noted that mEsrp1 induced more profound changes in splicing than mEsrp2 and co-expression of both proteins showed no evidence of additive functions. (Data not shown). Thus, together with the results from independent ESRP1 and ESRP2 knockdown, these results suggest that these proteins have similar, redundant functions, but that ESRP2 may be a less robust splicing factor than ESRP1. It is possible, particularly for the alternative 3′ terminal exon splicing events that are often difficult to validate, that these assays may have been unable to detect some mEsrp1 induced splicing changes. However, it may be more likely that for some of these types of splicing events, the ESRPs are necessary for expression of epithelial isoforms, but ectopic expression of mEsrp1 alone may not be sufficient to promote a switch in splicing from the mesenchymal to the epithelial pathway. Here too it is possible that some of the discrepancies might be due to indirect effects on splicing in one or both experimental systems. Nonetheless, taken together the results from the ectopic expression experiments support the conclusion that the ESRPs are in fact bona fide regulators of most of the splicing changes identified in the MADS analysis of the microarray data.
Example 21
ESRP Target Genes Share Common Functions and Biological Processes, Including Numerous Examples of Genes that Function in Cell-Cell Adhesion and Cell Motility
The inventors were interested in investigating whether the set of ESRP splicing targets the inventors identified could be shown to function in any common pathways are have related biological roles. As a first step in such analysis, the 134 genes from the list of high-confidence splicing events within the MADS top 500 probe sets were analyzed for enriched Gene Ontology (GO) terms using the DAVID Bioinformatics Resource. DAVID was unable to map the following four genes (MNAT, AGFG1, PMS2L14, ARFGAP2) and thus, these are absent in the analysis. Compared to the background set of all genes represented by probesets on the Exon Arrays, the inventors noted several GO terms that were enriched among the genes in the high confidence list. Though not all of the predicted splicing events in the 134 genes analyzed have been validated, the validations performed suggest that most of the genes in this list are true targets. Furthermore, any false positives would not be predicted to cluster in a way that would further support the observed enrichment.
This analysis, summarized in Table 7 below and Table 4 above suggested that many of the ESRP target genes encode proteins involved in cytoskeleton structure, cell adhesion, RNA splicing, and the other categories presented. Tables 4 and 7 depict a subset of ESRP target genes which regulate cytoskeleton structure and cell adhesion. Gene Ontology (GO) analysis was performed on the ESRP targets. The number of ESRP targets in each GO category, the enrichment for each category, and the corresponding p-values are listed.











TABLE 7






% of ESRP
% of Non-





targets within
ESRP targets




Number of
GO
within GO




ESRP targets
category
category
GO term
p-value



















Molecular Function






 9
7.1
2.3
Cytoskeletal protein binding
8.7E−3


12
9.4
3.8
RNA binding
9.0E−3


 4
3.1
0.5
Structural constituent of
2.2E−2





cytoskeleton



 4
3.1
0.5
Protein tyrosine phosphatase
3.1E−2





activity



Cytoplasmic






Component






68
53.5
35.7
Cytoplasm
2.0E−5


16
12.6
6.3
Cytoskeleton
2.1E−2


Biological Process






12
9.4
4.1
Cell adhesion
1.6E−2


 6
4.7
1.2
Protein targeting
1.9E−2


 5
3.9
0.8
Dephosphorylation
1.9E−2


 6
4.7
1.3
RNA splicing
2.3E−2









The inventors wished to further investigate whether relevant biological functions or themes could be identified among the list of ESRP target genes and thus the inventors also carried out an extensive literature review of these gene products. A subset of these genes could be shown to encode proteins with important roles in the regulation of the actin cytoskeleton, cell-cell adhesion, and cell motility and migration (data not shown). Among these gene products were a number that have well documented and essential roles in epithelial to mesenchymal transitions and maintenance of cell polarity in addition to the previously reported FGFR2, p120-catenin, CD44, and ENAH. Such genes include, for example, EPB41L5, SCR1B, MACF1 (ACF7), GIT2, LOXL2, FAT, and PTPRM. See also Table 4. The inventors also noted a number of cases in which these gene products have been shown to physically associate with one another in the regulation of these cell behaviors. These observations thus suggest both that many of these targets function in common pathways and imply that the different splice variants may well prove to have different, or even opposing functions in epithelial and mesenchymal cells as has already been demonstrated for FGFR2 and p120-catenin.
Example 22
Validated Target Genes
Subsequent to validation of the experimental method described in the preceding examples, the inventors used siRNA-mediated depletion and exon microarrays to identify additional splicing events regulated by ESRP1 and ESRP2. The data described above represent the use of the Affymetric Exon Array ST1.0 splicing sensitive array to detect changes in splicing upon siRNA mediated depletion of ESRP1 and ESRP2 in the epithelial PNT2 cell line. We have since extended this analysis using another splicing sensitive microarray platform. This platform uses both exon and exon junction probesets to profile splicing changes and is referred to as the Exon Junction array. In total we used both the Exon Array and Exon Junction array to identify changes in splicing in PNT2 cells with ESRP1 and ESRP2 depletion. We also used both arrays to profile splicing changes induced by ectopic expression of a cDNA for mouse Esrp1 in the mesenchymal MDA-MB-231 cell line. Numeorus validations of these changes in splicing were carried out using RT-PCR or high throughput capillary RT-PCR. The most significant changes that were validated from primary data achieved through any of these approaches or platforms are summarized in Tables 1, 2 and 3.
Example 23
ESRP-Like Compounds
Another aspect of the invention is to identify compounds that affect the epithelial mesenchymal transition in a way similar to that of ESRP1 and ESRP2. To accomplish this, the inventors identified compounds that activated splicing in the luciferase reporter screen using the LOPAC 1280TM library of compounds, a group of high purity, small organic ligands.
Briefly, compounds from the LOPACTM library were dissolved in DMSO. 293T cells (which express predominantly FGFR2-IIIc) which stably express the PKC-neg-40B-Luc minigene, as described in Example 1, were incubated with the compounds or with a DMSO control for 24 hours. After the incubation period, BriteLite luciferase readout reagent was added to each well and the plates were read on an Analyst HT multimode plate reader (Molecular Devices). As positive controls, we also added a cDNA encoding the mouse Esrp1 protein to individual wells on each plate.
The results were then ranked in order from largest fold change over background down to the DMSO blank. Any average reading over the DMSO control (approximately 1.75 fold over background) was considered to be a possible ESPR mimicking compound. After analysis, the top ranking wells were, not surprisingly, the controls expressing the Esrp1 protein. Additionally, phorbol 12-myristate 13-acetate (PMA), phenylazo-3-phyridinol, podophyllotoxin, R(−)-isoproterenol (+)-bitartrate, and beclomethasone ethylcarboxamido adenosine were found to have luciferase readings significantly over the background reading (See, Table 5).
All publications cited in this specification, including the references identified below and the provisional application U.S. No. 61/082,435, are incorporated herein by reference. In addition, Warzecha, et al, Molecular Cell, 33:591-601 (March 2009) including all of the supplemental materials published online as of the publication date, is specifically incorporated in its entirety by reference. While the invention has been described with reference to a particularly preferred embodiment, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims
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## Claims

1. A screening method for identifying a compound that causes an epithelial to mesenchymal transition (EMT) in a normal mammalian cell comprising:
(a) adding a test compound to an in vitro biological sample obtained from a mammalian subject, which consists of normal healthy mammalian cells;
(b) contacting said biological sample containing the test compound with reagents that can measure or detect the expression level of alternative splice variants of each of genes SLC37A2, FLNB, RALGPS2, ENAH, FNIP1, ARFGAP2, SLK, SCRIB and ARHGEF11 or the proteins encoded thereby;
(c) performing an assay to measure the levels of expression of the splice variants; and
(d) identifying a test compound as causing an EMT in the sample when
i. the levels of expression of the splice variants of SLC37A2, FLNB, RALGPS2, ENAH, FNIP1, ARFGAP2, and SLK in the sample containing the test compound are below the level of expression of those splice variants in the sample in the absence of the test compound, and
ii. when the level of expression of the splice variants of SCRIB and ARHGEF11 in the sample containing the test compound are above the level of expression of those splice variants in the sample in the absence of the test compound,

wherein the splice variant of:
SLC37A2 includes the exon sequence SEQ ID NO: 1,
FLNB includes the exon sequence SEQ ID NO: 2,
RALGPS2 includes the exon sequence SEQ ID NO: 3,
ENAH includes the exon sequence SEQ ID NO: 4,
FNIP1 includes the exon sequence SEQ ID NO: 31,
ARFGAP2 includes the exon sequence SEQ ID NO: 7,
SLK includes the exon sequence SEQ ID NO: 9;
SCRIB excludes the exon sequence SEQ ID NO: 20, and
ARHGEF11 excludes the sequence SEQ ID NO: 33.

2. The method according to claim 1, wherein the contacting step comprises measuring the splice variants as ribonucleic acid, deoxyribonucleic acid, cDNA sequence or protein.

3. The method according to claim 2, wherein the reagents that can measure or detect the expression of the splice variants are PCR primers or probes that identify the nucleic acid sequences of the specified splice variants, or antibodies or ligands that identify the variant proteins encoded by the splice variant nucleic acid sequences.

4. The method according to claim 3, wherein the contacting comprises forming a physical association between the reagent and the nucleic acid sequence or protein in the sample.

5. The method according to claim 3 further comprising transforming a signal generated from the reagent in association with the splice variants present in the biological sample into numerical or graphical data.

6. The method according to claim 3, further comprising extracting RNA from the sample and contacting the extracted RNA sample with the reagents.

7. The method according to claim 1, wherein the biological sample is selected from the group consisting of mammalian cells, biological fluids containing mammalian cells, and mammalian tissue.

8. The method according to claim 7, wherein the sample is blood.

9. The method according to claim 1, wherein the assay is performed using polymerase chain reaction (PCR).

10. The method according to claim 9, wherein the assay is performed using quantitative real-time polymerase chain reaction.

11. The method according to claim 9, wherein said assay is performed using a reverse transcriptase PCR exon splicing assay.