Patent Application: US-58284104-A

Abstract:
the present invention relates to the identification a molecular signature for pten tumor suppressor . the molecular signature comprising a gene or genes that are of use for diagnosis , prognosis , drug research and development and therapeutics . specifically , the present invention relates to identification of igfbp2 gene , its mrna and / or protein products that closely associate with pten mutations . the present invention further demonstrates that igfbp2 expression is negatively regulated by pten , positively regulated by pi3k and akt activation , that igfbp2 plays a functional role in the pten signaling and is required for akt transformation . the use of igfbp2 gene , its gene product such as its rna transcript , protein and molecular probes in diagnosis , prognosis , drug discovery and validation and therapeutic target and therapeutics is also contemplated .

Description:
to identify transcriptional targets downstream of the complex signal transduction pathways of pten , we performed a gene expression profiling on prostate cancer and glioblastoma , two cancer types frequently affected by pten mutations . this global gene expression analysis identifies a molecular signature that can accurately classify tumor samples according to its pten status regardless of tumor types . we also studied igfbp2 , the most significant gene in the signature . we demonstrated that igfbp2 is biochemically regulated by pten and plays a functional role in pten function . to facilitate understanding of the invention , a number of terms are defined below : nucleotide : a monomeric unit of dna or rna consisting of a sugar moiety ( pentose ), a phosphate , and a nitrogenous heterocyclic base . the base is linked to the sugar moiety via the glycosidic carbon ( 1 ′ carbon of the pentose ) and that combination of base and sugar is a nucleoside . a nucleoside containing at least one phosphate group bonded to the 3 ′ or 5 ′ position of the pentose is a nucleotide . base pair ( bp ): a partnership of adenine ( a ) with thymine ( t ), or of cytosine ( c ) with guanine ( g ) in a double stranded dna molecule . in rna , uracil ( u ) is substituted for thymine . generally the partnership is achieved through hydrogen bonding . nucleic acid : a polymer of nucleotides , either single or double stranded . gene : a nucleic acid whose nucleotide sequence codes for an rna or a polypeptide . a gene can be either rna or dna . cdna : a single stranded dna that is homologous to an mrrna sequence and does not contain any intronic sequences . sense : a nucleic acid molecule in the same sequence order and composition as the homolog mrna . the sense conformation is indicated with a “+”, “ s ” or “ sense ” symbol . antisense : a nucleic acid molecule complementary to the respective mrna molecule . the antisense conformation is indicated as a “−” symbol or with a “ a ” or “ antisense ” in front of the dna or rna , e . g ., “ adna ” or “ arna ”. template : a nucleic acid molecule being copied by a nucleic acid polymerase . a template can be single - stranded , double - stranded or partially double - stranded , depending on the polymerase . the synthesized copy is complementary to the template , or to at least one strand of a double - stranded or partially double - stranded template . both rna and dna are synthesized in the 5 ′ to 3 ′ direction . the two strands of a nucleic acid duplex are always aligned so that the 5 ′ ends of the two strands are at opposite ends of the duplex ( and , by necessity , so then are the 3 ′ ends ). nucleic acid template : a double - stranded dna molecule , double stranded rna molecule , hybrid molecules such as dna - rna or rna - dna hybrid , or single - stranded dna or rna molecule . oligonucleotide : a molecule comprised of two or more deoxyribonucleotides or ribonucleotides , preferably more than three , and usually more than ten . the exact size will depend on many factors , which in turn depends on the ultimate function or use of the oligonucleotide . the oligonucleotide may be generated in any manner , including chemical synthesis , dna replication , reverse transcription , or a combination thereof primer : an oligonucleotide complementary to a template . the primer complexes with the template to yield a primer / template duplex for initiation of synthesis by a dna polymerase . the primer / template complex is extended during dna synthesis by the addition of covalently bonded bases linked at the 3 ′ end , which are complementary to the template . the result is a primer extension product . virtually all known dna polymerases ( including reverse transcriptases ) require complexing of an oligonucleotide to a single - stranded template (“ priming ”) to initiate dna synthesis . a primer is selected to be “ substantially ” or “ sufficiently ” complementary to a strand of specific sequence of the template . a primer must be sufficiently complementary to hybridize with a template strand for primer elongation to occur . a primer sequence need not reflect the exact sequence of the template . for example , a non - complementary nucleotide fragment may be attached to the 5 ′ end of the primer , with the remainder of the primer sequence being substantially complementary to the strand . non - complementary bases or longer sequences can be interspersed into the primer , provided that the primer sequence has sufficient complementarity with the sequence of the template to hybridize and thereby form a template / primer complex for synthesis of the extension product of the primer . complementary or complementarity or complementation : used in reference to polynucleotides ( i . e ., a sequence of nucleotides ) related by the base - pairing rules . for example , the sequence “ a - g - t ” is complementary to the sequence “ t - c - a ,” and also to “ t - c - u .” complementation can be between two dna strands , a dna and an rna strand , or between two rna strands . complementarity may be “ partial ” or “ complete ” or “ total ”. partial complementarity or complementation occurs when only some of the nucleic acid bases are matched according to the base pairing rules . complete or total complementarity or complementation occurs when the bases are completely matched between the nucleic acid strands . the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands . this is of particular importance in amplification reactions , as well as in detection methods that depend on binding between nucleic acids . percent complementarity or complementation refers to the number of mismatch bases over the total bases in one strand of the nucleic acid . thus , a 50 % complementation means that half of the bases were mismatched and half were matched . two strands of nucleic acid can be complementary even though the two strands differ in the number of bases . in this situation , the complementation occurs between the portion of the longer strand corresponding to the bases on that strand that pair with the bases on the shorter strand . homologous or homology : refers to a polynucleotide sequence having similarities with a gene or mrna sequence . a nucleic acid sequence may be partially or completely homologous to a particular gene or mrna sequence , for example . homology may also be expressed as a percentage determined by the number of similar nucleotides over the total number of nucleotides . complementary bases : nucleotides that normally pair up when dna or rna adopts a double stranded configuration . complementary nucleotide sequence : a sequence of nucleotides in a single - stranded molecule of dna or rna that is sufficiently complementary to that on another single strand to specifically hybridize between the two strands with consequent hydrogen bonding . conserved : a nucleotide sequence is conserved with respect to a preselected ( reference ) sequence if it non - randomly hybridizes to an exact or total complement of the preselected sequence . hybridize and hybridization : the formation of complexes between nucleotide sequences which are sufficiently complementary to form complexes via complementary base pairing . where a primer ( or splice template ) “ hybridizes ” with target ( template ), such complexes ( or hybrids ) are sufficiently stable to serve the priming function required by a dna polymerase to initiate dna synthesis . there is a specific , i . e . non - random , interaction between two complementary polynucleotide that can be competitively inhibited . nucleotide analog : a purine or pyrimidine nucleotide that differs structurally from t . g . c , or u , but is sufficiently similar to substitute for the normal nucleotide in a nucleic acid molecule . dna homolog : a nucleic acid having a preselected conserved nucleotide sequence and a sequence coding for a receptor capable of binding a preselected ligand . amplification : nucleic acid replication involving template specificity . template specificity is frequently described in terms of “ target ” specificity . target sequences are “ targets ” in that they are sought to be sorted out from other nucleic acids . amplification techniques have been designed primarily for this sorting . template specificity is achieved in most amplification techniques by the choice of enzyme . enzymatic amplification : a method for increasing the concentration of a segment in a target sequence from a mixture of nucleic acids without cloning or purification . polymerase chain reaction ( pcr ): an amplification reaction is typically carried out by cycling i . e ., simultaneously performing in one admixture , the first and second primer extension reactions , each cycle comprising polynucleotide synthesis followed by denaturation of the double stranded polynucleotides formed . methods and systems for amplifying a dna homolog are described in u . s . pat . nos . 4 , 683 , 195 and 4 , 683 , 202 , both to mullis et al . amplifiable nucleic acid and amplified products : nucleic acids that may be amplified by any amplification method . dna - dependent dna polymerase : an enzyme that synthesizes a complementary dna copy from a dna template . examples are dna polymerase 1 from e . coli and bacteriophage t7 dna polymerase . under suitable conditions a dna - dependent dna polymerase may synthesize a complementary dna copy from an rna template . dna - dependent rna polymerase or transcriptase : enzymes that synthesize multiple rna copies from a double stranded or partially double stranded dna molecule having a promoter sequence . examples of transcriptases include , but are not limited to , dna - dependent rna polymerase from e . coli and bacteriophage t7 , t3 , and sp6 . rna - dependent dna polymerase or reverse transcriptase : enzymes that synthesize a complementary dna copy from an rna template . all known reverse transcriptases also have the ability to make a complementary dna copy from a dna template . thus , reverse transcriptases are both rna - dependent and dna - dependent dna polymerases . rnase h : an enzyme that degrades the rna portion of an rna / dna duplex . rnase h may be an endonuclease or an exonuclease . most reverse transcriptase enzymes normally contain an rnase h activity . however , other sources of rnase h are available , without an associated polymerase activity . the degradation may result in separation of the rna from a rna / dna complex . alternatively , the rnase h may simply cut the rna at various locations such that pieces of the rna melt off or are susceptible to enzymes that unwind portions of the rna . reverse transcription : the synthesis of a dna molecule from an rna molecule using an enzymatic reaction in vitro . for example , the rna molecule may be primed with a primer that is complementary to the rna molecule and the dna molecule is synthesized by extension using a reverse transcriptase such as the dna polymerase with reverse transcription activity , mmlv reverse transcriptase , amv reverse transcriptase , and any other enzyme that has the ability to synthesize a dna molecule from an rna molecule template . in vitro transcription : the synthesis of an rna molecule from a dna molecule using an enzymatic reaction in vitro . for example , the dna molecule may be double stranded and comprises an rna polymerase promoter such as t7 , sp6 , t3 , or any other enzyme . promoter for synthesis of rna from dna . vector : a recombinant nucleic acid molecule such as recombinant dna ( rdna ) capable of movement and residence in different genetic environments . generally , another nucleic acid is operatively linked therein . the vector can be capable of autonomous replication in a cell in which case the vector and the attached segment is replicated . one type of preferred vector is an episome , i . e ., a nucleic acid molecule capable of extrachromosomal replication . preferred vectors are those capable of autonomous replication and / or expression of nucleic acids to which they are linked . vectors capable of directing the expression of genes encoding for one or more polypeptides are referred to herein as “ expression vectors ”. particularly important vectors allow cloning of cdna from mrnas produced using a reverse transcriptase . functional parts : a portion of an intact molecule that retains one or more desired properties of the intact molecules . thus , for example , an antibody binds an antigen . in that context of the property of binding that antigen , a functional part of an antibody can be any portion of an antibody that binds the cognate antigen . similarly , a functional part of a nucleic acid that encodes an antibody that binds that antigen is any portion of that nucleic acid that encodes a polypeptide that binds to that antigen . antibody : in various grammatical forms as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules , i . e ., molecules that contain a combining site for antigen or paratope . exemplary antibody molecules are intact immunoglobulin molecules , substantially intact immunoglobulin molecules and portions of an immunoglobulin molecules , including those portions known in the art as f ab , f ab &# 39 ; s ( f ab ′ ) 2 , f v and scf v . immunoreact : in various forms means specific binding between an antigenic determinant - containing molecule and a molecule containing an antibody combining site such as a whole antibody molecule or a portion thereof . cistron : a sequence of nucleotides in a dna molecule coding for an amino acid residue sequence and including upstream and downstream dna expression control elements . promoter : a nucleic acid to which a polymerase molecule recognizes , perhaps binds to , and initiates synthesis . for the purposes of the instant invention , a promoter can be a known polymerase binding site , an enhancer and the like , any sequence that can initiate synthesis by a desired polymerase . knockdown : a method to which a rna made from a dna sequence ( shrna ) introduced into a cell or a rna sequence ( sirna ) introduced into a cell to initiate degradation of the mrna of a protein of interest . the present invention provides a novel method for identifying and selecting genes that associate with pten gene abnormalities and / or pten - related cellular process , and / or pi3k - and / or akt - related signal transduction pathway . the present invention combines the following elements as discussed in details thereafter : 1 . isolation of nucleic acids ( genomic dnas or mrnas ) from tumor cells , tumor specimens ; 5 . identifying genes with predictive power for association with pten status tumor and cancer cells . the invention now will be exemplified further in the following non - limiting examples . we have used microarray technology to identify overexpression of androgen receptor as the general mechanism for hormone refractory prostate cancer . the data indicate that overexpression of androgen receptor is a diagnostic and therapeutic target for hormone refractory prostate cancer and can be used as a screening method for hormone refractory prostate cancer drug development ( fig7 ). this is consistent with microarray technology being a useful tool for a variety of purposes . however , it has been difficult in identifying molecular signatures for signal transduction pathways . one of the reasons is that microarray experiments are usually performed on a relatively few samples , therefore , data analysis on these experiments requires specific statistical tools . in this report , we described a novel unsupervised learning algorithm , called random forest , to identify a molecular signature for the signaling pathway of pten . this statistic tool can deal effectively with small data sets involving relatively a few observations ( samples ) and a large volume of variables ( gene expression values ). it can calculate a predictive power for each gene . when a set of genes is used to predict the pten status , it can also generate an error rate by a three - fold cross - validation , in which one - third of the samples are left out as test set therefore , identification and verification of signatures , and identification of significant genes can be achieved using this algorithm . to identify transcriptional targets associate with the pten tumor suppressor function , we compared the gene expression profiles of 11 tissue samples that have the wild - type pten gene to those of 14 samples that have mutated pten gene ( table 1 ). these 25 samples include 12 advanced prostate cancer xenografts and 13 glioblastoma tissue samples . the pten status of the prostate cancer xenografts were characterized previously ( 21 ) and those of the glioblastoma were determined by western blot and genomic dna sequence analysis . six of the glioblastoma samples do not express the pten protein and , therefore , were defined as pten mutant samples . the other seven samples have the wild - type pten because they express the pten protein and they do not carry point mutation , which was determined by genomic dna sequence analysis . we used both prostate cancer and glioblastoma tissue samples in order to increase a tissue - independent signature associated with the pten status . we adopted a statistic technique called random forest ( rf ) because this method has been designed to analyze data that contain many covariates and relatively few observations ( breiman l , 1999 ). this technique is ideal to analyze microarray data , in which expression of a large number of genes is observed in a relatively few samples . this approach identified 490 genes that have statistic power in predicting the pten status , and ranked each gene according to the significance of its predictive power , which is represented by gini index ( fig1 a ). we next ask how many genes must be included in order to correctly predict the pten status , since most of the 490 genes have weak predictive power ( their gini indexes are near zero ). we generated an error rate for each gene set by a three - fold cross - validation , in which one - third of the samples were left out as test sets . the first gene in the gene set was the one with the most predictive power ( the highest gini index ), and a following gene was added each time according to its ranking of the gini index . a gene list cannot predict the pten status accurately until 12 genes with the highest gini indexes were included ( fig1 b ). the accurate prediction was maintained in gene lists composed of the “ top ” 18 genes and was lost when more genes were included , consistent with the fact that each gene has different gini index and , therefore , carries different weights in predicting the pten status . the accuracy of the 12 genes to predict the pten status in the test set was confirmed by multidimensional scaling analysis ( fig1 c ) and by hierarchical clustering ( fig1 d ). glioblastoma and prostate cancer were clustered into pten mutant or wild type tumor regardless of cancer types . further studies are required to determine whether this signature can be exploited more broadly as a tool to define pten status of tumors using larger , independent datasets with characterized pten status tumors . having identified several genes whose expression patterns correlate with the pten status , we wish to investigate biochemical regulation and biological role of one of these genes in pten function . because both probe sets in the microarray were identified and both have the highest power in predicting the pten status , igfbp2 was chosen for further study . as the first step , we confirmed the relationship between pten mutations and igfbp2 expression by western blot analysis using whole tissue lysates from prostate cancer xenografts . igfbp2 protein was detected in pten mutated xenografts lapc9 , lucap 35 , and lncap but not in pten wild - type tumors lapc4 and lucap23 ( fig2 a , table 1 ), consistent with the microarray analysis . the relationship was extended to other tumors that were not included in the microarray analysis . igfbp2 was highly expressed in pten mutated tumors lapc3 , lapc12 , and lucap41 , but was not detected in pten wild - type tumor lapc14 ( fig2 a , table 1 ). this association also holds true for 23 of the 24 glioblastoma samples examined , of which 13 samples were included in the microarray analysis and 10 of them were independent samples ( fig2 b , table 1 ). igfbp2 protein was detected in samples whose pten expression was low or lost , but was not detected in samples whose pten expression was high . genomic sequence analysis indicate that the pten protein detected in the western blot analysis was wild - type . the correlation was confirmed by immunohistochemical analysis ( unpublished data , paul mischel ). there is one exception for the association between the pten mutations and igfbp2 expression in glioblastoma samples ( fig2 b , table 1 ). this sample (# 429 ) has pten protein expression while igfbp2 is also highly expressed . genomic sequence analysis indicates that this sample has the wild - type pten gene , suggesting that mechanisms other than pten mutations are responsible . indeed , this sample has high levels of akt and akt activation , as indicated by western blot analysis on total akt and phosphorylation of ser 473 of akt ( fig2 c ). the mechanism of elevated akt level in this specific patient is unknown . to examine if high level of igfbp2 is secreted by cells with pten mutations , glioblastoma ( 9l and u251 ) and prostate cancer cells ( lapc4 and lncap ) were grown in tissue culture and the igfbp2 levels were measured by radioimmunoassay . in contrast to less than 20 ng / ml of secreted igfbp2 by cells with wild - type pten genes , the levels of secreted protein in cells with mutated pten gene are more than 70 ng / ml ( fig2 d ). the high levels of secreted igfbp2 were also detected in pten mutated breast cancer cell ( mda - mb - 468 ), but not in pten wild - type breast cancer cell ( skbr3 ). to examine if serum igfbp2 levels correlate with pten status in tumors , serum levels of human igfbp2 were measured from mice carrying human prostate and breast cancer xenografts ( fig2 e ). while serum levels of human igfbp2 levels were low in mice carrying human tumors with the wild - type pten genes , mice with pten mutated tumors contained high levels of human igfbp2 in serum . these data indicate that human tumors with pten mutations secreted high levels of igfbp2 , and raise the possibility that serum igfbp - 2 levels could serve as a biomarker for pten status . to establish a causal role of pten loss in igfbp - 2 upregulation , we extended our analysis to an isogenic model . western blot analysis was performed using the lysates from the pten wild - type and deleted isogenic mouse embryonic fibroblasts ( mef ) ( 22 ). while igfbp2 protein was barely detectable in pten wild - type mef , pten mutant cells produced a high level of igfbp2 ( fig3 a ). to determine if upregulation of the igfbp2 expression by the pten mutations is dependent upon the pi3k / akt pathway , pharmacological and genetic approaches were employed . when pten mutated cells were treated with a pharmacological drug ( ly294002 ) that inhibits the pi3k kinase activity , the production of igfbp2 was reduced to the basal level ( fig3 b ). furthermore , igfbp2 was induced in cells with the wild - type pten gene when a constitutively active akt allele was expressed ( fig3 c ). these results indicate that igfbp2 expression is induced by the pi3k / akt pathway , which is antagonized by the pten tumor suppressor . to determine if igfbp2 plays a functional role in the pten signal transduction pathway , we introduced either pten or igfbp - 2 into pten null cell lines by lentiviral infection , which gives highly efficient infection rates in prostate cancer epithelial cells (& gt ; 90 %). as reported , re - introduction of the wild - type pten decreased growth of pc3 cells by 36 % ( fig4 a ), with a concomitant decrease of the endogenous igfbp2 expression ( fig4 c ). forced expression of exogenous igfbp2 rescued the growth inhibition of pten by 47 % ( fig4 a ). a similar effect was observed with cell cycle analysis ( fig4 b ). re - introduction of the wild - type pten into pc3 cell reduced the percentage of cells in s phase , and the effect is partially rescued by forced expression of exogenous igfbp2 . these data suggest that down - regulation of igfbp2 may partially contribute to the pten tumor suppressor function . to examine if igfbp2 is involved in the pi3k signaling , a pharmacological approach was employed . consistent with the result in fig3 b , lncap cells have a high basal level of akt activation . treatment of ly294002 , a specific pi3k inhibitor , resulted in reduced akt phosphorylation and igfbp2 expression ( fig5 a , bottom panel ). this treatment caused a decrease of the percentage of cells in s phase by 40 %, and the reduction was partially ( 28 %) rescued by the forced expression of exogenous igfbp2 ( fig5 a , top panel ). to examine if igfbp2 plays a biological role for akt function , clonogenic assay was performed using igfbp2 knockout mef ( 23 ). while akt promoted colony formation in igfbp2 wild type mef ( fig5 b , top and left panel ), deletion of igfbp2 abrogated this promoting activity . the requirement of igfbp2 is specific to akt because c - myc promoted colony formation in both cells ( fig5 c ). the inability of akt to promote colony formation in igfbp2 knockout mef can be rescued by re - introduction of igfbp2 ( fig5 b ). taken together , these results indicate that one of the effects of the pten tumor suppressor is to suppress the expression of igfbp2 , which is involved in the function of the pi3k / akt signal transduction pathway . to determine how igfbp2 is involved in akt function , we made use of gene knockdown technology by shrna . the shrna efficiently knockdown igfbp2 expression , as shown by western blot analysis ( fig6 ). this knockdown reduced the growth of pc3 and the activation of akt , effects identical to re - introduction of the wild - type pten ( fig6 ). these data suggest that igfbp2 may regulate akt activation through an autoloop mechanism . through random forest and other statistical analysis , we identified upregulation of igfbp2 expression as the most consistent change associated with pten mutations . among 12559 probe sets in the microarray , both probe sets representing igfbp2 were identified as the most and the second most significant gene to predict the pten status . we demonstrated that igfbp2 is biochemically regulated by pten and pi3k - akt pathway . consistent with our finding , it was reported that overexpression of igfbp2 was only observed in glioblastoma , but not in low - or intermediate - grade gliomas ( 24 ). in addition , igfbp2 overexpression was observed in 50 % of glioblastoma . the stage in which igfbp2 is overexpressed and the percentage of tumors with this gene overexpression coincide with the frequency of pten mutations in advanced gliomas ( 25 ). overexpression of igfbp2 was also identified as the most distinct progression - related expression change in high - grade gliomas in another similar study through cdna microarrays and tissue arrays ( 26 ). this study uncovered that igfbp2 is a poor prognostic marker for patients with gliomas . while patients with igfbp2 negative tumors had a mean survival of 75 months , patients with tumors of strong igfbp2 expression had a mean survival of 23 months . this also coincides with the aggressiveness of pten mutated tumors . these data suggest that upregulation of igfbp2 in pten mutated tumors may play an important role in tumor formation and progression . indeed , forced expression of igfbp2 partially rescued the inhibitory effect of pten and a pi3k inhibitor as well ( fig4 ). serum igfbp2 can be developed as a surrogate marker for pten mutations and akt activation we and other have recently demonstrated that tumors with pten mutations are more sensitive to drugs such as cci - 779 that targets mtor , a downstream effector of the pi3k / akt pathway ( 22 , 27 ). this effect is later observed in several other studies . these studies suggest that drugs targeting the pi3k / alkt pathway may only benefit patients who have aberrant pten / akt activities . since pten mutations are carried in less than 50 % of tumors even for the most frequently mutated cancer type , the pharmaceutical benefit can be masked by an unselected population . this may explain why cci - 779 and some other drugs targeting the pi3k - akt pathway fail in clinical trials , even though this drug effectively inhibits pten mutated cancer cells . because igfbp2 is a serum protein , we envision that the serum level of igfbp2 can be used to predict pten mutations and akt activation . in support of this notion , we detected high concentrations of human igfbp2 in condition medium of pten mutated cells and also in sera of mice carrying pten mutated human tumors . in addition , serum concentration of igfbp2 was shown to be elevated in 50 % of patients with advanced prostate cancer ( personal communication , pinchas cohen ). the stage in which igfbp2 is overexpressed and the percentage of tumors with this gene overexpression coincide with the frequency of pten mutations in advanced prostate cancer in patients . furthermore , it was reported that patients treated with igf - l , a stimulus for akt activation , caused an elevated level of igfbp2 in serum ( 28 ). serum level of igfbp2 can also be used to predict if drugs hit targets because overexpression of igfbp2 can be inhibited by a pi3k inhibitor . the smallest gene expression signature associated with the pten status contained eight down - regulated and four up - regulated genes in pten mutated tumors ( fig1 d ). several of the identified genes were involved in different pathways implicated in tumor formation and progression . human neuralized belongs to a family of the neurogenic genes and is an e3 ligase for the notch signal transduction pathway that is associated with tumorigenesis ( 29 , 30 ). this protein mediates proteosome - dependent degradation of the notch ligand delta ( 31 ). loss - of - function mutations of the neurogenic genes produce hyperplasia of the embryonic nervous system ( 32 ), which is reminiscent of phenotype of the brain - specific pten knockout mice ( 33 ). furthermore , expression of human neuralized is high in normal human brain tissue , but low or absent in advanced gliomas ( 34 ), consistent with our finding . these data suggest that the notch pathway may play an important role in pten tumor suppressor function . dual specificity phosphatase 10 , also called mkp - 5 , selectively dephosphorylates jnk and reduces its activity ( 35 ). the level of this phosphatase is reduced in tumors with pten loss , suggesting that upregulation of the jnk signal transduction pathway is a key element for cancer development and progression in pten - null tumors . this hypothesis is supported by our unpublished data . curiously , two proteins identified in the signature specifically bind pip3 ( 36 , 37 ), the established substrate for the pten tumor suppressor . cytohesin - l belongs to a family of guanine nucleotide - exchange proteins for the 20 - kda adp ribosylation factor ( arf ) ( 38 ). it also associates with integrin beta2 and regulate cell adhesion that is important for tumorigenesis and cancer metastasis ( 39 ). regulator of g - protein signaling 1 belongs to a family of gtpase - activating protein and is inhibited by pip3 ( 37 ). these data suggest that a feedback control may be invoked to maintain the pi3k signaling , consistent with a published report that expression of pten causes feedback upregulation of irs - 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