bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3000: MIIP regulates histone H3 phosphorylation at serine 10 and securin degradation during mitosis in colon cancer cells
Authors: Ji Ping , Hu Limei , Zhang Wei
Published: June 2012

Chromosome condensation and segregation are highly orchestrated during mitosis. In early mitosis, dramatic structural changes occur to produce metaphase chromosomes, each consisting of a pair of compacted sister chromatids (condensation), At anaphase onset, a signal is produced to disrupt the linkage between sister chromatids (segregation), allowing them to be pulled apart to two daughter cells. Recently, we discovered that the MIIP protein, encoded by the Migration and invasion inhibitory protein gene located on chromosome 1p36, plays a critical role in mitotic transition through negatively regulating APC/CCdc20 activity and stabilizing cyclin B1 in glioma cells. Here we report that MIIP inhibits chromosome condensation and segregation resulting in G2/M arrest and polyploidy cell formation in colon cancer cells. Gain-of-function studies showed that the MIIP expression inhibited colony formation of isogenic HCT116 cells independent of the TP53 and PUMA status. To understand the potential role of MIIP in colon cancer cells, a panel of six colon cancer cell lines (DLD1, SW620, SW480, HCT116, NCI-747 and SW837) was used to elevate MIIP expression by adenovirus expression vector (Ad-MIIP). The G2/M arrest and polyploidy cell formation were observed in all the six cell lines we tested irrespective of the status of microsatellite stability and TP53 mutation. By using double thymidine block strategy, we synchronized HCT116 cells at G1/S boundary and released the cells in fresh medium to enrich the cell population in different stage during the cell cycle. We found that the expression of MIIP is cell cycle dependent and peaks at mitosis. Two major mitotic regulators, cyclin B1 and securin, were induced by MIIP. The cycloheximide (CHX) chase experiments showed that MIIP blocked cyclin B1 and securin degradation, which is consistent with our previous finding that MIIP directly interacted with Cdc20 and inhibited APC/CCdc20 activity. We further found that MIIP inhibited histone H3 phosphorylation at serine 10. Taken together, our studies provide evidence that MIIP plays a critical role in chromosome condensation and segregation in mitosis. Because chromosome 1p36 region is commonly deleted in colon cancer cells, the MIIP gene may represent a novel negative cell cycle regulator that is abnormally altered thus contributing to genomic instability in colon cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3000. doi:10.1158/1538-7445.AM2011-3000
Abstract 3022: Disruption of the nuclear matrix attachment domain of the DNA replication factor Ciz1 in common solid tumors and its potential as a biomarker
Authors: Sercombe Heather , Higgins Gillian , Munkley Jennifer , Copeland Nikki , Roper Katherine , Rahman Faisal Abdel , Ainscough Justin , Coverley Dawn
Published: June 2012

The DNA replication protein Ciz1 promotes initiation of mammalian DNA replication in cooperation with cyclin A-dependent kinase, most likely by delivery cyclin A to sites where cyclin E-dependent pre-replication complex assembly has taken place. Normally, Ciz1 is anchored within nuclear matrix-associated foci that co-localize with sites of DNA replication, but in the absence of anchor domain Ciz1 retains the ability to promote initiation of DNA replication in isolated nuclei so attachment to the nuclear matrix is not essential for function. Here, we show that expression of DNA replication and nuclear matrix anchor domains of Ciz1 are uncoupled and uneven at the transcript level in a panel of common cancer cell lines, giving rise to excess DNA replication domain protein that is not attached to the nuclear matrix. Moreover Ciz1 domain expression is also uncoupled in a wide range of common solid tumours, including breast and lung, so that anchor domain transcript is elevated over control tissues, and exceeds replication domain in all primary stage I, II and III tumours tested. Notably, more than half of all stage IV tumours tested resemble established cell lines, having more replication domain than anchor domain. In cell based assays, recombinant anchor domain protein interferes with attachment of endogenous Ciz1 to the nuclear matrix, revealing a dominant negative effect that also impacts on nuclear matrix-recruitment of a key component of the pre-replication complex, Cdc6. This shows that Ciz1 normally plays a role in localizing Cdc6 to the nuclear matrix and suggests that cancer-associated uncoupled expression influences recruitment of mammalian pre-replication complexes to the nuclear matrix. These findings implicate spatially unconstrained DNA replication as a source of nuclear disorder in cancer cells.

We identified a variant Ciz1 isoform with alterations in nuclear matrix attachment domain and tumour-restricted expression. Variant Ciz1 is elevated at the transcript and protein level with high frequency in small cell lung tumours, and with lower frequency in a range of other tumour types. Selective suppression of variant Ciz1 expression using inducible shRNA restricts proliferation of lung cancer cells that express it in vitro and in vivo, identifying a novel, exploitable therapeutic target with potential application in the treatment of lung cancer. Recent evidence will also be presented on the application of variant Ciz1 as a serum biomarker.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3022. doi:10.1158/1538-7445.AM2011-3022
Abstract 3028: A multiplexed, bioluminescent HDAC assay for determining target-specific, anti-cancer potency
Authors: Evans Nathan , Niles Andrew L. , Kirkland Thomas A.
Published: June 2012

Histone deacetylase (HDAC) class I and II enzymes are zinc dependent protein deacetylases. They play many roles in normal gene regulation events of development and homeostasis, but their dysregulation has been linked to a variety of solid tumors and hematological malignancies. We have recently developed a one-step, homogeneous, luminescent assay for activity detection from multiple HDAC Class I and II isoforms, thereby providing a useful tool for cell culture, enriched or recombinant sources of lysine deacetylases. This luciferase-containing, “glow-type” assay reagent is cell-permeable and can directly measure HDAC activity in living attachment-dependent and suspension cells. Furthermore, this luminescent HDAC assay can be paired in same well multiplex formats with fluorescent assays that measure biomarkers associated with changes in viability and cytotoxicity. Here we correlate HDAC inhibition with cellular fate using representative inhibitors from the hydroxamate, short-chain fatty acid, benzamide and cyclic peptide classes. We will also demonstrate relative on-target anti-cancer efficacies and off-target safety using transformed, primary or iPS-derived cell lineages.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3028. doi:10.1158/1538-7445.AM2011-3028
Abstract 3033: NKX2-5, a potential tumor suppressor gene in prostate cancer
Authors: Daremipouran Mohammad , Wang Song Ping , Ellis Alexander , Bamji Zebalda , Kwabi-Addo Bernard
Published: June 2012

Background: Prostate cancer (PCa) is a common malignancy and a leading cause of cancer deaths among men in the United States. Abundant evidence has accumulated to suggest that epigenetic DNA methylation changes may appear earlier during PCa development than genetic changes, as well as more commonly and consistently suggesting that DNA methylated genes can be explored as DNA-based biomarker for PCa disease detection. Recently, we have identified NKX2-5 as a novel that is hypermethylated in prostate cancer. However, there is little information about the biological significance of this gene in prostate carcinogenesis. We hypothesize that NKX2-5 is a potential tumor suppressor gene that is frequently inactivated in prostate cancer. Methods: We carried out gain-and-loss functional studies of NKX2-5 in prostate cancer cell lines and validated expression at the RNA transcript level using quantitative RT-PCR. Protein expression was analyzed by western blotting and cell cycle analysis investigated by flow cytometer. Results: Over-expression of NKX2-5 was detrimental to prostate cancer cell proliferation as evidenced by significant inhibition of prostate cancer cell proliferation in comparison to control (vector only transfection) and this was due to cell arrest in Go/G1 phase and increase apoptosis. In contract, successful knockdown of NKX2-5 by shRNA transfection increased prostate cancer cell proliferation. Western blot analysis demonstrated that NKX2-5 plays a key regulatory role in the expression of several genes including p53, PTEN, Histone H1 and the androgen receptor. Conclusion: Our observation suggests that NKX2-5, a member of the homeobox gene family of plays an important tumor suppressor activity in prostate carcinogenesis. Because this gene plays important role in several signal transduction pathways, this gene can be exploited as potential biomarker for the early detection of prostate cancer and could be an attractive target to explore for drug investigation or gene therapies of prostate cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3033. doi:10.1158/1538-7445.AM2011-3033
Abstract 3035: Functional roles of HS6ST3 in human breast cancer in vitro
Authors: Iravani Omid , Yip George W. , Bay Boon-Huat
Published: June 2012

Introduction: Heparan sulfate is a sulfated polysaccharide that is found on the cell surface and extracellular matrix of all human cells. It interacts with a variety of proteins and is thus involved in numerous biological processes such as growth and development. Heparan sulfate's function critically depends on the number and the position of sulfate groups, which modulate the binding sites for proteins such as growth factors, cytokines, receptors, enzymes, and inhibitors. However, little is known about their roles both in vitro and in vivo. Alterations in heparan sulfation may be associated with pathologic conditions such as cancer. Heparan sulfate 6-O-sulfotransferases (HS6STs) catalyze the transfer of sulfate groups to the carbon 6 position in heparan sulfate. Three isoforms of these enzymes have been discovered in humans. Recently, several studies have proposed that dysregulation of 6-O-sulfation patterns may be involved in carcinogenesis. This study aimed to analyze the functional role of HS6ST3 in T47D breast cancer cell line. Procedures: HS6ST3 was silenced using siRNA by more than 85%. Functional studies such as proliferation, cell cycle, apoptosis, adhesion and migration assays were then performed. Results: The study demonstrated that silencing HS6ST3 in T47D breast cancer cell line enhanced apoptosis and cellular adhesion; and diminished proliferation with cell cycle arrest at G0/G1. Taken together the results demonstrated that silencing HS6ST3 decreased the tumoral progression in breast cancer. Conclusions: This could be happen through alteration of heparan sulfate interactions with ligands such as growth factors. HS6ST3 could be used not only as a valuable biomarker for diagnostic and prognostic purposes but in cancer gene therapy in near future.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3035. doi:10.1158/1538-7445.AM2011-3035
Abstract 3063: Development of a sensitive COLD-PCR method for the detection of EGFR mutations in DNA
Authors: Richardson Katherine Richardson A. , Candau Reyes , Legendre Benjamin , Wu Grant , Shandilya Harini , Ekenkamp Karissa , Eastlake Philip , Gerard Gary
Published: June 2012

COLD-PCR is a method to exponentially amplify mutations in DNA while linearly amplifying wild type sequences. The method can be adapted to multiple mutation detection platforms including Sanger Sequencing and is amenable to development as a diagnostic kit.

EGFR EXONS 20 & 21: Conditions for non-biased, selective amplification of DNA with mutations in the tyrosine kinase activation regions of the EGFR gene (exon 19 deletions and the T790M and L858R point mutations) have been developed. Ice COLD-PCR methodology was used for both exon 19 deletions and the exon 21 L858R mutation. Fast COLD-PCR methodology was used for the T790M mutation in exon 20. Using control plasmids containing the T790M and the L858R mutations, COLD-PCR amplification with SURVEYOR® Nuclease/WAVE® HS confirmation, demonstrated a limit of detection (LOD) of 0.05% mutant DNA in wild type DNA. COLD-PCR amplification followed by confirmation of these mutations using Sanger sequencing had a limit of detection of 0.2%. Only 5-10 copies of amplifiable DNA containing the mutation are needed for amplification using COLD-PCR followed by downstream mutation determination. Similar results were obtained using DNA isolated from the cell lines, H1650 and A549, and serum spiked with DNA containing a T790M mutation.

EGFR EXON 19: For detection of deletions in EGFR exon 19, another series of plasmids containing 3, 6, 9, 15, and 21 base-pair deletions were constructed. The two 15 base-pair deletions were those which result in the most commonly observed mutation in non-small cell lung cancer, p.E746_A750del15 (c.2235-2249del15 or c.2236-2259del15). For these exon 19 deletions, the SURVEYOR Nuclease/WAVE HS was able to detect the presence of a mutation at 0.05% and Sanger sequencing was able to confirm these deletions at 0.2%.

CONCLUSION: These data demonstrate that COLD-PCR enriches low number and low abundance EGFR mutations including point mutations and deletions, and suggests applicability to serum/plasma. COLD-PCR coupled with mutation detection methods such as Sanger sequencing is a new model for EGFR mutation testing with potential applicability for detection of mutations in other genes.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3063. doi:10.1158/1538-7445.AM2011-3063
Abstract 3067: Improved detection of BRAF mutation in clinical samples using PNA-mediated real-time PCR clamping techniques
Authors: Oh Miae , Cho Minhye , Choi Jae-Jin , Park Heekyung
Published: June 2012

The BRAF mutation is a somatic mutation with the highest pathogenic cancer-associated prevalence and a biomarker used for detecting cancer and monitoring prognosis of cancer patients. Usually, mutation in the Exon15 codons V600E mutation (1799 T>A) represent more than 90% of BRAF mutations.

However, techniques using PCR and direct sequencing have limitations and are unable to detect low-level mutations in cancers to give false negative diagnoses.

We have developed a highly sensitive and simple method for detecting BRAF mutations using PNA-mediated real-time PCR clamping, PNAClampTM.

PNAClampTM method is a simple, reliable and sensitive method with a detection limit of approximately 0.1% mutant alleles. The turnaround time for this method was only 2 hours. The method was utilized to detect BRAF mutations in fine needle aspiration biopsy (FNAB) specimens from papillary thyroid cancer (PTC) patients. V600E mutation was detected in 62% (52/84) of the FNAB specimens in the PTC samples, 10% higher rate compare with PCR and direct sequencing methods. In this report, we will discuss the results of clinical studies in detail including the comparison data. In summary, PNAClampTM method can serve a rapid, reliable, and economical alternative for BRAF mutation detection in clinical settings.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3067. doi:10.1158/1538-7445.AM2011-3067
Abstract 3069: One-step sensitive PNA mediated real-time PCR clamping assay to detect PIK3CA mutations
Authors: Kim Hyunsun , Choi Jae-Jin , Park Heekyung
Published: June 2012

Phosphatidylinositol 3-kinases (PI3K) are important regulators of cellular growth, transformation, adhesion, apoptosis, survival and motility. Mutations in the PIK3CA oncogene encoding PI3K have been frequently found in various human cancers. The presence of PIK3CA mutations in a tumor has been suggested as a prognostic factor and may predict responses to select treatments. Therefore, detection of PIK3CA mutations may help predict drug responses and prognosis of cancer patients.

We have developed a highly sensitive and simple method to detect 11 mutations in exons 9 and 20 of PIK3CA using one-step PNA-mediated real-time PCR clamping, PNAClampTM. PNAClampTM relies on unique properties of PNA probes that PNA oligomers are not recognized by DNA serving as sequence-selective clamps during PCR amplification. This method can also detect 11 mutations through only three tube reactions each with 10 ng of the target DNA. PNAClampTM is simple and reliable with a detection limit of approximately 0.1% mutant alleles using 10 to 50 ng of normal DNA as a template. The turnaround time for performing this assay was only 2 hours.

In clinical studies with breast cancer patients in Korea, PIK3CA mutations were detected utilizing PNAClampTM in 12 (35.3%) of clinical samples, and the results were compared with direct sequencing method. Among 34 patients, 1 (2.94%) had mutations in codon 542, 6 (17.65%) had mutations in codon 545 and 5 (14.71%) had mutations in codon 1047. The results will be discussed in detail in this report including the method development and optimization.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3069. doi:10.1158/1538-7445.AM2011-3069
Abstract 3084: The role of RNA-binding protein HuR in lung metastasis of triple-negative breast cancer
Authors: Techasintana Patsharaporn , Gubin Matthew Michael , Magee Joseph David , Dahm Garrett MacKenzie , Atasoy Ulus
Published: June 2012

Breast cancer is the second most common cancer among women worldwide. It is estimated that approximately one million women are diagnosed with breast cancer annually. Furthermore, more than 410,000 women will die each year from the disease primarily due to tumor metastasis. Currently, even the most effective treatments often result in recurrence and metastasis, in part due to genetic factors. Therefore, understanding the mechanism of breast cancer related gene regulation is crucial for the development of novel treatments and preventive strategies to those who are predisposed to developing breast cancer. Previously, we have shown that the RNA-binding protein HuR posttranscriptionally regulates various proto-oncogenes by stabilizing their mRNAs and facilitating their translation into proteins. Additionally, HuR upregulation and cytoplasmic localization has been associated with invasive cancer progression and poor prognosis. HuR has been described to control genes in multiple areas of the acquired capabilities model of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established. HuR regulates genes involved in angiogenesis, cell growth and cell cycle regulation including VEGFα, TSP1, HIF1α, CDKN1A(p21) and β-catenin. In this study, we investigated the role of HuR in an aggressive triple-negative (estrogen receptor ER-, human epidermal growth factor receptor 2 (HER2)- and progesterone receptor (PR)-) breast cancer cell subline LM-2. The LM-2 cells were derived from triple-negative breast cancer cells MDA-MB-231 which were isolated from two rounds of in vivo selection of the cancer cells which metastasized to the lungs. The LM-2 cells were retrovirally transduced with triple-fusion protein reporter construct encoding thymidine kinase1, green fluorescent protein (GFP) and firefly luciferase in order to obtain nuclear imaging, fluorescent and bioluminescent properties for in vivo and in vitro tracking. LM2 cells were further transfected with a plasmid containing HA-HuR or empty vector control to investigate the function of HuR in LM2 cells. Two clones of HuR overexpressing LM2 cells were shown to grow faster in vitro compared to the empty vector control. Also, HuR overexpression significantly facilitates tumor invasion in vitro by matrigel invasion assay. Further analysis of HuR overexpressing LM2 cells using in vivo imaging system (IVIS) will reveal the role of HuR in breast cancer growth and metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3084. doi:10.1158/1538-7445.AM2011-3084
Abstract 31: Isoform level gene expression signature is a better discriminator of cancer from normal cells than gene level expression signature
Authors: Zhang ZhongFa , Davuluri Ramana V.
Published: June 2012

The majority of mammalian genes generate multiple transcript variants and protein isoforms, which usually are differentially expressed in different cellular and disease conditions. We hypothesize that the isoform level gene expression profiles can better discriminate normal and cancer cells with different sub-types than gene-level expression profiles. We analyzed Affymetrix exon array data sets, collected from public domain (ENCODE and TCGA datasets from NCBI GEO database), comprising 160 cell lines of either normal or tumor tissues of origin. The unsupervised clustering of genome-wide gene expression estimates at isoform-level effectively grouped the normal and cancer cell lines with 97.5% homogeneity, regardless of the tissue origins of the cell lines, with only 4 exceptions out of 160 samples. However, at gene level, this homogeneity grouping rate was much lower, and was about 75% at best. The supervised method revealed genes and isoforms that are differentially expressed between pairs of tumor and normal samples, which can serve as signatures to effectively differentiate tumor and normal cell lines. Finally, the pathway analyses both at gene and isoforms-level indicate that there are subtle differences of derived pathways. The study reveals that there exists a common isoform-level signature that can be used not only for effective discrimination of oncogenic cell lines from non-oncogenic cell lines, but may also have important biological implications for a better understanding of common defining characteristics of tumors of varied types. Finally, we present the identified isoform-level gene signatures and associated pathways for melanoma, breast cancer and glioblastoma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 31. doi:10.1158/1538-7445.AM2011-31
Abstract 3104: Stromal fibroblasts promote progression of esophageal squamous cell carcinoma cells both in vitro and in vivo
Authors: Saito Shin , Morishima Kazue , Matsubara Daisuke , Hosoya Yoshinori , Sata Naohiro , Yasuda Yoshikazu , Ishikawa Shumpei , Niki Toshiro
Published: June 2012

Objectives: The purpose of the study was to clarify the role of fibroblasts in the proliferation and growth pattern of esophageal squamous cell carcinoma and to experimentally study interactions between cancer cells and fibroblasts.

Methods: We used 22 esophageal squamous cell carcinoma cell lines (mainly, TE series and KYSE series obtained from the RIKEN Cell Bank and the Japanese Collection of Research Bioresources, respectively) and human esophageal fibroblasts that had been established in our laboratory from surgical specimens. Cancer cells were cultured on Matrigel in the presence or absence of fibroblasts in the Matrigels, and their growth patterns were examined. Cell count assays were performed to examine the effects of the fibroblast culture supernatant, growth factors, cytokines, and various inhibitors on cancer cell growth. We also examined the influence of co-injection of fibroblasts on tumor growth in immunodeficient mice.

Results: When plated on Matrigel, nearly all cell lines spread and grew in an invasive manner in the presence of fibroblasts, while in the absence of fibroblasts, most cell lines formed round or spheroidal colonies. Cell-count assay showed that in most cancer cells, cell proliferation was stimulated by addition of fibroblast supernatant. In particular, cell proliferation increased by more than 50% in 11 cell lines. Addition of hepatocyte growth factor (HGF) or interleukin-6 elicited a definitive proliferative response in only 2 and 3 cell lines, respectively. Both KYSE-220 and KYSE-50 proliferated in the presence of HGF, but the addition of MET inhibitor (PHA665752) suppressed the proliferation of only the former. In vivo tumor growth was stimulated by co-injection of fibroblasts in 9 of 22 cancer cell lines.

Conclusions: Fibroblast had a profound influence on cell growth pattern on Matrigel. In many esophageal cancer cell lines, cell proliferation was promoted by the presence of fibroblasts both in vivo and in vitro. The stimulatory effect of fibroblast could be accounted for by HGF and/or interleukin-6 in some, but not in all, esophageal cancer cells.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3104. doi:10.1158/1538-7445.AM2011-3104
Abstract 3105: Extracellular matrix density and the development of breast acini and ducts in 3D cultures
Authors: Swamydas Muthulekha , Ricci Krista , Burg Karen J.L. , Dreau Didier
Published: June 2012

Breast epithelial cells develop into polarized and highly organized acinar and ductal structures in response to stromal cues, including extracellular matrix composition and density, which can in part be reproduced in 3D culture conditions. Here we present the effects of increasing density of stroma composition on the ability of heterotypic cultures of epithelial and mesenchymal stem cells to organize into acinar and tubular structures in in vitro 3D cultures. Normal murine mammary gland (NMuMG) cells were cultured in combination (30:70) with mouse mesenchymal stem cells (D1), in 3D matrices composed of increasing concentrations (0.8 to 8 mg/ml) of collagen I and growth-factor reduced Matrigel® (1:1). The organization of the breast-like structures was assessed by immunohistochemistry. After 3-5 days in culture, the organization and the presence of acinus-like and tubule-like structures were determined. By immunohistochemical analysis, NMuMG cells co-cultured with D1 cells formed acinar and tubular structures with the NMuMG epithelial cells surrounding a lumen composed of dead cells while the D1 cells were mostly peripheral and separated by laminin-positive basement membrane. Collagen I /Matrigel® matrices with density of 1.6 and 2.4 mg/ml were associated with significantly higher concentrations of acinus-like structures compared to other densities (p<0.05). In contrast, the high concentration (8mg/ml) matrix prevented the development of acinus-like structures (p<0.05). The number of tube-like structures was drastically reduced as the density of the collagen I / Matrigel matrix increased from 0.8 to 3.2 mg/ml (p<0.05). These results indicate that the density of the matrix influences the organization and the generation of acinus-like and duct-like structures in the mammary tissues. Whether the biomechanical and density properties of the breast extracellular matrix can be harnessed to build improved tissue culture systems to benefit breast cancer patients remains to be determined.

This work was supported by grants from the Department of Defense Era of Hope program (BC044778) and the National Science Foundation EFRI program (CBE0736007).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3105. doi:10.1158/1538-7445.AM2011-3105
Abstract 3110: Microglia-stimulation of glioma cell invasion is dependent on EGFR and CSF1R signaling
Authors: Coniglio Salvatore J. , Dobrenis Kostantin , Stanley Richard , Symons Marc H. , Segall Jeffrey E.
Published: June 2012

Glioblastoma multiforme is a deadly cancer whose current treatment options are limited. The ability of glioblastoma tumor cells to infiltrate the surrounding brain parenchyma makes complete removal of these tumors nearly impossible. We investigated how microglia, the resident macrophages of the brain, stimulate glioblastoma cell invasion. We examined the ability of normal microglia from C57Bl/6J mice to stimulate GL261 glioblastoma cell invasion in vitro. The two cell populations were differentially labeled with cell tracker dyes and invasion was quantified by confocal imaging. The effect of inhibiting EGFR and CSF1R (c-fms) on microglia-stimulated glioblastoma invasion using pharmacological agents was tested. We also determined the level of EGF and CSF1 ligand expression by glioblastoma and microglial cells using ELISA, western blotting, and immunofluorescence. We found that microglia stimulate the invasion of GL261 glioblastoma cells by approximately 8 fold in an in vitro invasion assay. Pharmacological inhibition of EGFR strongly inhibited microglia-stimulated invasion. Furthermore, using a pharmacological CSF-1R kinase domain inhibitor, we showed that CSF-1R signaling is also required for microglial enhancement of glioblastoma invasion. GL261 cells were found to constitutively secrete CSF-1, the levels of which are unaffected by EGF stimulation, EGFR inhibition or coculture with microglia. We also demonstrated that microglia express EGF which is largely associated with the plasma membrane. These data indicate that during invasion, glioblastoma and microglia cells signal to each other using EGF and CSF-1. The results of this study indicate the possibility of inhibiting glioblastoma invasion by targeting glioblastoma-associated microglia.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3110. doi:10.1158/1538-7445.AM2011-3110
Abstract 3115: Role of nuclear coactivator AIB1 in endothelial cell function and angiogenesis
Authors: Al-Otaiby Maram A. , Zhangzhi Hu. , Riegel Anna , Wellstein Anton
Published: June 2012

The nuclear receptor coactivator oncogene AIB1 (Amplified In Breast Cancer 1) has a clearly defined role in steroid and growth factor signaling in epithelial cells of cancers. Recently, we have demonstrated that AIB1 is functionally required for HER2-mediated mammary tumorigenesis in MMTV/Neu mouse model for breast cancer through regulation of HER2 phosphorylation and signaling. Generation of neu/HER2 transgenic mice with either loss of one or both copies of the AIB1 gene resulted in reduced or complete abolition of mammary tumor development, respectively. One very interesting aspect of this study was that the tumors that arose in HER2 AIB1 HET (+/-) mice were phenotypically different from those in HER2 AIB1 WT (+/+) mice in that they were smaller, more necrotic and significantly less vascularized. This data indicated to us the possibility that the role of AIB1 was not confined to the mammary epithelium proliferation but also had major effects on the surrounding stromal cells. In addition, recent expression array data indicated that AIB1 is also overexpressed in human breast cancer stroma which includes the endothelium. In here, we examined the role of AIB1 in endothelial cell function. We demonstrate that reduction of AIB1 levels in endothelial cells cultured in vitro prevents their ability to form endothelial tubes, in conjuction with reduced proliferation and motility. Furthermore, in ECIS (Electric Cell-substrate Impedance Sensing) assay, HUVEC cells with low AIB1 expression loss the ability to form a monolayer on the surface of gold-film electrodes. Consistent with the reduced proliferation that we observed, cDNA array analysis that compared gene expression patterns of HUVEC cells +/- AIB1 shRNA indicates that a number of key cell cycle regulatory genes and cytokines were regulated by reduction in AIB1 levels. Overall these data describe a new role for nuclear receptor coactivator AIB1 in the endothelium.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3115. doi:10.1158/1538-7445.AM2011-3115
Abstract 3132: C6orf69 is down-modulated by protein kinase C epsilon and exhibits increased expression in breast cancers
Authors: Garczarczyk Dorota , Hinger Doris , Gruber Peter , Hofmann Johann
Published: June 2012

Protein kinase C (PKC) comprises a serine/threonine kinase family of at least ten related isozymes. Protein kinase C epsilon (PKCε) has been shown previously to act as an oncogene, contributing to malignancy by tumor cell invasion, metastasis and inhibiting cell death. However, the precise function is not known at present. We investigated whether PKCε expression leads to alterations in the expression of other genes.

We found by gene array after overexpression of constitutively active PKCε that EST AA167550, of which 348 bases were known, was down-modulated. We obtained the full-length sequence of AA167550. It was identified as chromosome 6 open reading frame 69 gene (C6orf69). At least two distinct mRNAs were found to be transcribed from this gene. We generated an antibody and as shown by Western blotting, two proteins (47 and 40 kDa) were detected. The full length sequence of C6orf69 contains a BTB-BACK-Kelch domain usually involved in protein-protein interactions and implicated in the stability of actin filaments. C6orf69 co-immunoprecipitated with PKCε and bound to actin. PKCε/C6orf69 interaction was additionally confirmed by a yeast two-hybrid screen using C6orf69 full length as bait.

C6orf69 is expressed in many tissues except in heart, ovary, small intestine and leukocytes. In nine out of nine breast cancers, the expression levels were higher than in matched normal mammary tissue. C6orf69 contains four putative PKC phosphorylation sites. The C6orf69 protein is phosphorylated but is not a direct substrate of PKC isozymes, as demonstrated by in vitro PKC assays. Recently, BTB-domain containing proteins have been reported to regulate protein stability by recruiting proteins to the Cullin3 (Cul3) ubiquitin ligase leading to their degradation via ubiquitin/proteasome. By a yeast two-hybrid screen and co-immunoprecipitation we could demonstrate that C6orf69 is interacting specifically with Cul3. In addition, an in vivo ubiquitylation assay showed that overexpressed C6orf69 protein is efficiently polyubiquitylated, indicating to be a substrate of E3 ligase itself.

In summary, C6orf6 is down-modulated by PKCε, shows elevated expression in breast cancers compared to corresponding normal breast tissue, contains a BTB domain, interacts with PKCε, actin and Cul3. C6orf69 is phosporylated but not substrate of PKC isozymes and it is ubiquitylated.

This work was supported by grants P15039 and P19878 from the Austrian Science Fund (FWF).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3132. doi:10.1158/1538-7445.AM2011-3132
Abstract 3140: Analysis of candidate phosphatidylinositol 3’ kinase inhibitor biomarkers in matched synchronous primary and metastatic breast cancer tumors
Authors: Boyd Zachary S. , O'Brien Carol , Spoerke Jill , Mohan Sankar , Pandita Ajay , Fridlyand Jane , Punnoose Elizabeth , Koeppen Hartmut , Friedman Lori , Amler Lukas , Hampton Garret , Lackner Mark
Published: June 2012

The class I phosphatidylinositol 3’kinase (PI3K) is activated in a wide variety of human malignancies and inhibitors targeting the PI3K pathway hold great promise in the treatment and management of cancer. Successful development of such inhibitors will be enhanced by identification of responsive patients through the use of predictive biomarkers. Using cell lines and xenograft tumor models to evaluate a collection of putative biomarkers predictive of response to the selective inhibitor GDC-0941 in breast cancer, we have found excellent activity of the PI3K inhibitor GDC-0941 in luminal and HER2 amplified models, and that PIK3CA mutations and HER2 amplification are highly specific biomarkers of response to this agent. PTEN was predictive in some but not all cases, suggesting a need for further biomarkers of response in PTEN-driven disease. Here we report expanded analysis of human breast tumor samples, including matched primary tumor and lymph node metastases from 59 patients, suggesting primary and metastatic sites were generally concordant in terms of status of key biomarkers such as PTEN and PIK3CA, though several notable exceptions were discovered. We also compared PTEN status determined by immunohistochemistry (IHC) with a chromosomal fluorescence in situ hybridization (FISH) assay in a subset of tumors and found evidence for chromosomal deletion of the PTEN locus in several ER+ breast cancers, suggesting that some but not all of the PTEN loss observed in breast occurs at the chromosomal level and that both FISH and IHC may have utility as diagnostic assays. While we report that PIK3CA mutations and PTEN loss have reasonable prevalence to be considered as biomarkers for GDC-0941 in ER+ breast cancer, we have found a number of breast cancer models with no apparent pathway alterations but sensitivity to GDC-0941, indicating the need for additional predictive biomarkers. We identified a collection of genes for which expression levels indicate PI3K pathway activation and correlate with in vitro sensitivity to GDC-0941 as well as apparent prognostic significance in retrospective analysis of breast tumors. We report here expression analysis of these genes across matched primary breast tumors and lymph node metastases. In summary, our data provide strong rationale for developing a selective PI3K inhibitor in the ER+ breast cancer setting, and a framework for clinical implementation of predictive biomarker assays with potential utility in patient selection.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3140. doi:10.1158/1538-7445.AM2011-3140
Abstract 3148: Immunohistochemical analysis of HB-EGF expression in breast, colon, gastric, hepatocellular, lung, ovarian, pancreatic and prostate cancer
Authors: Halim Abdel-baset , Dauffenbach Lisa M. , Kerfoot Christopher A. , Stull William R. , Zwick-Wallasch Esther , Scheyer Richard , Senaldi Giorgio
Published: June 2012

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the epidermal growth factor (EGF) family of proteins. It is synthesized as a transmembrane protein, which can be cleaved at the cell surface to release the soluble ectodomain sHB-EGF. HB-EGF has been implicated in several physiological and pathological processes, including tumorigenesis and metastasis. Anti-HB-EGF therapeutic monoclonal antibodies are currently under investigation and hold promise for treating multiple types of cancer. In order to investigate the relationship between HB-EGF expression in cancer and response to therapy, an immunohistochemical assay to HB-EGF was developed and validated. Antigen retrieval and antibody titration studies were performed for six antibodies in order to select an antibody with proper performance characteristics. The antibodies were tested in characterized xenografts, characterized cell lines, placenta (HB-EGF-positive) and/or ovarian cancer. Of the antibodies tested, the AF-259-NA goat polyclonal antibody demonstrated the appropriate staining pattern in xenografts, cell lines and placenta. The optimized immunohistochemistry assay was used to stain cancer samples, and the frequency of moderate (2+) or strong (3+) staining in cancer cells was as follows: gastric = 50%, hepatocellular = 50%, lung = 40%; ovarian = 38%, colon = 37%, breast-ER+ = 20%, bladder = 10%; breast-HER2+ = 10%, pancreatic = 10%, breast-triple negative = 0%, prostate = 0%. When compared by average H-score, the cancer ranked in the following order (H-score in parenthesis): Hepatocellular (69), ovarian (37), gastric (23), breast-HER2+ (14), breast-ER+ (12), pancreatic (10), lung (5), breast-triple negative (2), colon (2), bladder (1), prostate (1). For comparison, H-scores for normal tissues were as follows: Normal bladder epithelium (74), stomach (24), colon (8), prostate (3), ovary (1), liver (1), breast (0), lung (0) and epidermis (0). In summary, immunohistochemistry using the AF-259-NA antibody produces the expected staining pattern in characterized samples and can be applied to human cancer samples to evaluate the relationship between clinical response and HB-EGF expression.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3148. doi:10.1158/1538-7445.AM2011-3148
Abstract 3149: OncoPredictor: A systematic approach for predicting responsive cancer populations from large scale cell line screening
Authors: Tomilo Mark , Eddy Sean F. , Banka Wendy Lockwood , Sadis Seth E. , Williams Paul D. , Wyngaard Peter J. , O'Day Christine , Ovechkino Yulia , Warrior Usha , Rhodes Daniel R.
Published: June 2012

Investigational new drugs for cancer must demonstrate convincing preclinical efficacy and a compelling strategy to translate preclinical observations to the clinical setting. Personalized medicine approaches are gaining wider acceptance, and large scale cell line databases have demonstrated utility in identifying biomarkers of drug response that can inform clinical development strategies. However, the lack of an integrated platform to translate preclinical biomarker profiles to clinical populations limits the power of this approach. To solve this problem we developed, first, a cell line screening and genomic analysis pipeline that associates drug response across 200+ cell lines with mutation, DNA copy number, and gene expression biomarkers; and second, a parallel database of biomarker frequencies in clinical tumor samples, compiled from all available published genomic data. In the present study, we tested 8 targeted anti-cancer agents and identified cell line biomarkers representing each of the genomic data types – mutation, DNA amplification, and gene over-expression – and then assessed the distribution of these biomarkers across tumor samples. In each case the tumor populations predicted to be responsive by this unsupervised approach were validated by results from clinical trials. We also present an example of biomarker results leading to potential new indications for an approved drug.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3149. doi:10.1158/1538-7445.AM2011-3149
Abstract 3150: Epithelial mesenchymal transition (EMT) and IGF-2 expression as determinants of sensitivity to the dual IGF-1R/IR inhibitor OSI-906 in hepatocellular carcinoma
Authors: Zhao Hui , Desai Vidhi , Wang Jian , Epstein David , Miglarese Mark , Rizzo Elizabeth
Published: June 2012

Hepatocellular carcinoma (HCC) affects over 600,000 patients worldwide and is the third leading cause of cancer-related death. Understanding signaling pathways that may be targeted for the treatment of HCC has been a major goal. A growing body of data indicates that inhibiting the type 1 insulin-like growth factor receptor (IGF-1R) might be an especially effective treatment strategy for HCC. Increased expression of IGF-2 as well as phospho-IGF-1R is observed in a subset of HCC tumors, and blockade of IGF-1R results in inhibition of proliferation for preclinical HCC cell models.

OSI-906 is a small molecule dual IGF-1R/IR inhibitor currently in advanced clinical development. In this report we sought to determine the effect of OSI-906 on proliferation for a panel of 21 HCC tumor cell lines as well as to investigate molecular determinants of responsiveness to OSI-906. A subset of HCC tumor cell lines was sensitive to OSI-906 in proliferation assays. Sensitivity to OSI-906 correlated with epithelial-mesenchymal-transition (EMT) status where the majority of epithelial HCC cell lines, but none of mesenchymal cell lines, were sensitive to OSI-906. We also found differential expression of IGF axis components, including IGF-2, in epithelial and mesenchymal tumor cell lines, which correlated with sensitivity to OSI-906. Induction of EMT upon treatment with TGFβ reduced sensitivity to OSI-906. Within a human HCC tumor database, epithelial tumors were identified to be enriched in one subgroup with higher IGF2 expression.

We have previously shown that both IGF-1R and IR can maintain tumor growth in a mouse mammary tumor model, and co-inhibition of both receptors is required for maximal inhibition of tumor growth. Herein, we find that OSI-906 sensitive HCC tumor cell lines expressed both phosphorylated IGF-1R and IR, and dual targeting of both receptors appears to be important for maximal inhibition of proliferation. Treatment of HuH-7 tumor cells with an IGF-1R neutralizing antibody was associated with increased IR signaling. In contrast OSI-906 fully inhibited both phosphorylated IR and IGF-1R and achieved greater inhibition of the IRS-AKT pathway compared to the IGF-1R antibody. Collectively, these data support the concept for dual IGF-1R/IR targeting HCC, where EMT status and expression of IGF-2 could be utilized to identify those patients most likely to benefit from treatment. A phase 2 clinical study evaluating OSI-906 in HCC is currently ongoing.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3150. doi:10.1158/1538-7445.AM2011-3150