bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2084: Claudin-1 induces resistance to anoikis in Src dependent manner in colon cancer cells
Authors: Sharma Ashok , Singh Amar B. , Smith Joshua J. , Xi Chen , Beauchamp Robert D. , Dhawan Punita
Published: August 2012

Anoikis, a complete loss of cell anchorage triggers apoptosis in primary human colonic epithelial cells (CEC), is often dysfunctional in metastatic cancer cells. We along with other groups have demonstrated that expression of Claudin-1, a tight junction protein, is markedly increased and mislocalized in colon cancer and helps promote the tumor progression especially metastasis. However the underlying mechanism remains poorly understood. To explore, we examined effect of claudin-1 expression upon anoikis. Colon cancer cells genetically manipulated (stable overexpression in SW480 cells and RNA-interference based gene silencing in SW620 cells) for claudin-1 expression were cultured on polyHEMA-coated culture dishes to induce anoikis. Results demonstrated a positive correlation between claudin-1 expression and resistance to anoikis. We also observed a positive correlation between claudin-1 expression, Src (L4A1) and Akt phosphorylation (Ser 473) and Bcl-2 expression in the cells subjected to anoikis. Notably, use of PP2, a Src-kinase inhibitor, inhibited claudin-1-dependent resistance to anoikis (2.5fold) as well as the increases in Akt-phosphorylation and Bcl-2 expression, and thus suggested a causal role of Src-activation in claudin-1-dependent resistance to anoikis. Our further studies revealed that claudin-1 associates with Src in a multi-protein complex that also included ZO-1, a PDZ-domain containing tight junction protein, known to associate with claudin-1and Src. Importantly, claudin-1 contains a PDZ-binding domain in its c-terminal. Deletion of the c-terminal domain of claudin-1 resulted in the loss of the physical association between claudin-1 and Src and a significant loss in claudin-1-dependent resistance to anoikis. Thus, our findings supported a role of claudin-1 in the regulation of colon cancer progression through its association with Src and dependent cell signaling. Our further analysis using a large database of colon cancer patient samples (microarray analysis; 260 colon cancer and 10 adjacent normal colon samples) showed a significant and positive correlation between claudin-1 and Src expression. Taken together, we here report a novel partnering between claudin-1 and Src proteins in colon cancer cells which has a function implication in the regulation of anoikis in a claudin-1/Src/PI3-kinase/Bcl-2-dependent manner. Our findings have clinical significance and have the potential to open potential new therapeutic opportunity in colon cancer treatment and/or management.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2084. doi:1538-7445.AM2012-2084
Abstract 2096: Variants of uncertain clinical significance in cancer predisposition genes: Development of a family studies program for reclassification
Authors: Stuenkel Aaron J. , Lu Ira , Chao Elizabeth C.
Published: August 2012

The identification of genetic variants of uncertain significance is a fairly common, albeit unwelcomed result of gene sequence analyses. The frequency at which these variants are identified will likely continue to increase with the introduction of next-generation sequencing panels and the recent availability of clinical exome sequencing. When applied to testing for cancer predisposition syndromes, accurate variant assessment and classification helps to guide screening, treatment, family planning, and testing of at-risk family members. Multiple resources are available to assess genetic variants: population-based cohort allele frequencies, published literature, in silico prediction tools, gene-specific databases, evolutionary conservation data, and co-segregation studies. Ambry Genetics, a CAP-accredited and CLIA-certified commercial clinical laboratory providing genetic services focused on clinical diagnostics and genomic services, has developed and implemented a complementary family studies program to collect co-segregation data for variants of unknown significance identified through diagnostic testing. The Ambry family studies program is available to all specialties, but is especially active for genes associated with cancer predisposition and intellectual disability. Data collected from the cancer family studies program is combined with published literature, database information, and data compiled with Ambry Variant Analyzer (AVA©), a novel bioinformatics tool, to allow for a comprehensive interpretation of all variants of uncertain clinical significance in cancer predisposition genes. In less than one year, the program has grown to 60 active cancer family studies at the time of submission and a current rate of >5 new applications per week. Variants successfully reclassified due to family studies data include the p.Y327D missense alteration in MEN1 and in-frame deletions in both TP53 and MLH1. Variant reclassification leads to the automatic generation of amended reports for any individual previously identified by our laboratory as a carrier. Immediately distributed to health care professionals, these reports offer the most up-to-date clinical interpretation possible improving medical decision making and clarifying risk of disease. Here we provide an overview of the family studies program and discuss successful variant reclassification via the Ambry model.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2096. doi:1538-7445.AM2012-2096
Abstract 210: Cul4A is an oncogene in malignant pleural mesothelioma
Authors: Ni Jian , Hung Ming-Szu Hung , Mao Jian-Hua , Xu Zhidong , Bravo Dawn T. , Jablons David M. , You Liang
Published: August 2012

Cullin 4A (Cul4A) is important in cell survival, development, growth and the cell cycle, but its role in mesothelioma has not been studied. For the first time, we identified amplification of the Cul4A gene in four of five mesothelioma cell lines. Consistent with increased Cul4A gene copy number, we found that Cul4A protein was overexpressed in mesothelioma cells as well. Cul4A protein was also overexpressed in 64% of primary malignant pleural mesothelioma (MPM) tumours. Furthermore, knockdown of Cul4A with shRNA in mesothelioma cells resulted in up-regulation of p21 and p27 tumour suppressor proteins in a p53-independent manner in H290, H28 and MS-1 mesothelioma cell lines. Knockdown of Cul4A also resulted in G0/G1 cell cycle arrest and decreased colony formation in H290, H28 and MS-1 mesothelioma cell lines. Moreover, G0/G1 cell cycle arrest was partially reversed by siRNA down-regulation of p21 and/or p27 in Cul4A knockdown H290 cell line. In the contrary, overexpression of Cul4A resulted in down-regulation of p21 and p27 proteins and increased colony formation in H28 mesothelioma cell line. Both p21 and p27 showed faster degradation rates in Cul4A overexpressed H28 cell line and slower degradation rates in Cul4A knockdown H28 cell line. Our study indicates that Cul4A amplification and overexpression play an oncogenic role in the pathogenesis of mesothelioma. Thus, Cul4A may be a potential therapeutic target for malignant pleural mesothelioma.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 210. doi:1538-7445.AM2012-210
Abstract 2105: High Galectin-1 expression correlates with poor prognosis and is involved in epithelial ovarian cancer proliferation and invasion
Authors: Lee Jeong-Won , Kim Ha-Jeong , Jeon Hye-Kyung , Cho Young Jae , Park Young Ae , Choi Jung-Joo , Lee Yoo-Young , Choi Chel Hun , Kim Tae-Joong , Bae Duk-Soo , Kim Byoung-Gie
Published: August 2012

Purpose: Galectin-1 (Gal-1) is a 14-kDa laminin-binding galectin involved in several biologic events including regulation of tumor proliferation and metastasis. In this study, we investigated the clinical significance of Gal-1 expression and its functional role in cell proliferation and invasion in epithelial ovarian cancer (EOC). Experimental Design: We evaluated the expression of Gal-1 in 52 serous, 11 endometrioid, and 3 mucinous type EOC tumor samples from 66 patients by immunohistochemistry. In vitro experiments were performed to determine the function of Gal-1 in cell survival, proliferation, and invasion in EOC cells using siRNA and anginex, a Gal-1 inhibitor, as well as recombinant Gal-1 protein. Results: Patients with strong Gal-1 peritumoral staining had poorer progression-free survival (PFS) than patients with weak peritumoral staining (P = 0.03). Inhibition of Gal-1 by siRNA or anginex resulted in the inhibition of cell growth and proliferation of HeyA8 and SKOV3ip1 cells. Moreover, the ability of cells to migrate was significantly reduced by treatment of cells with Gal-1 siRNA but was increased by treatment of cells with recombinant Gal-1. When we evaluated the interaction between fibroblasts (H HESC) and cancer cells (A2780-CP20), we found that MMP-2 expression in cancer cells was affected by Gal-1 secreted by fibroblast cells, which suggests that Gal-1 in human fibroblasts might affect the invasive abilities of tumor cells. Conclusion: Our results suggest that Gal-1 expression is a potential prognostic factor for PFS and that Gal-1 could be a novel treatment target in EOC patients.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2105. doi:1538-7445.AM2012-2105
Abstract 2118: Particulate chromate induces persistent DNA double strand breaks resulting in disruption of homologous recombination repair in human lung cells
Authors: Qin Qin , Xie Hong , Holmes Amie L. , Wise Sandy S. , Wise John P.
Published: August 2012

Hexavalent chromium (VI) is a well-documented respiratory toxicant and carcinogen. The most carcinogenic form is the particulates, which produce numerous types of DNA damage including DNA double strand breaks. The aim of this study is to determine the genotoxicity of particulate chromate and investigate cellular responses of repair proteins after chronic exposure. We used zinc chromate as a representative hexavalent chromium compound because it has been shown to be carcinogenic and induce tumors in epidemiology studies and experimental animals. We investigated the DNA double strand break formation using both comet assay and immunofluorescence staining. We exposed cells to zinc chromate for 24, 48, 72 96 and 120 hours and found it induced concentration-dependent increases in DNA double strand breaks by both measures. We further investigated the kinetics of repair protein after chronic zinc chromate exposure. We developed human lung cells expressing green fluorescent protein-tagged 53BP1 (GFP-53BP1) and generated time-lapse videos of chromate treated cells. We observed residual focal formation of 53BP1 after chronic zinc chromate exposure, which indicate some DNA double strand breaks persist. There are two repair pathways that can be utilized to repair DNA double strand breaks. Non-homologous end joining (NHEJ) is active throughout the cell cycle while homologous recombination (HR) is active in late S and G2 phases. We first investigated the sub-cellular distribution of HR proteins including MRE11, phophorylated ATM and Rad51. We found that the nuclear fraction of HR repair proteins increased in 24 hours indicating HR is active. However, it largely decreased over time. We also observed concentration- and time-dependent decrease in Rad51 foci formation, which strongly suggest that HR is inhibited. Future work will focus on elucidating NHEJ repair pathway and identifying key proteins involved in transitions between two repair pathways. This work was supported by NIEHS grant ES016893 (J.P.W.), cooperative agreement #EP-08-01 through the Maine Space Grant Consortium (J.P.W.) and the Maine Center for Toxicology and Environmental Health at the University of Southern Maine.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2118. doi:1538-7445.AM2012-2118
Abstract 213: RNF126 E3 ubiquitin ligase targets p21cip for ubiquitin-mediated degradation
Authors: Zhi Xu , Zhao Dong , Zhou Zhongmei , Chen Ceshi
Published: August 2012

Many E3 ubiquitin ligases, such as Mdm2 and Skp2, are potential therapeutic targets for cancers. To identify novel oncogenic E3 ubiquitin ligases as anti-cancer targets, we screened an E3 ubiquitin ligase siRNA library containing siRNA pools against 555 individual E3s using the SRB assay in the MDA-MB-231 breast cancer cell line and the PC3 prostate cancer cell line. RNF126 was identified and validated as a promising candidate from this screening. Depletion of RNF126 dramatically decreased cell viability in these cancer cell lines. Consistently, RNF126 depletion delayed cell cycle G1/S progression and decreased cell proliferation. By a protein array analysis, we found that RNF126 depletion increased the cell cycle dependent kinase inhibitor p21cip protein levels in both MDA-MB-231 and PC3. Knockdown of RNF126 stabilized the p21 protein rather than increased the p21 mRNA levels. We demonstrated that RNF126 interacts with p21 in cells and in vitro, and the protein-protein interaction depended on the N-terminal and middle parts of RNF126. RNF126 over-expression increased the p21 protein ubiquitination in an E3 ligase activity dependent manner. RNF126 knockdown induced loss of cell viability in MDA-MB-231 and PC-3 can be partially rescued by depletion of p21. RNF126 stable knockdown in SW527 inhibited tumor growth in SCID mice. Finally, we found that RNF126 is highly expressed in ER negative breast cancer cell lines and negatively correlated with the p21 expression levels. These findings suggest that RNF126 promotes cancer cell proliferation through targeting p21 for ubiquitin-mediated degradation. RNF126 could be a novel therapeutic target in breast and prostate cancers. This study was partially supported by a grant from the American Cancer Society (RSG-08-199-01), grants from National Nature Science Foundation of China (81072162, 81120108019, and U1132605), and a grant from Yunnan Province High-Profile Talents Program (2010CI114).

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 213. doi:1538-7445.AM2012-213
Abstract 2134: Unique and redundant roles of human RecQ homologs in cellular response to oxidative DNA damage
Authors: Sharma Sudha , Phatak Pornima , Stortchevoi Alexei , LaRocque Jeanine , Jasin Maria
Published: August 2012

RecQ helicases contribute diverse activities towards genome maintenance in response to a variety of DNA lesions. Human RecQ homologs include RECQ1, BLM, WRN, RECQL4, and RECQ5α. Although these RecQ proteins are similar in their catalytic core and share certain biochemical properties in vitro, mutations in BLM, WRN and RecQ4 lead to clinically distinct diseases of cancer predisposition. This suggests that each human RecQ protein functions in distinct molecular events, but their specialized functions are only vaguely understood. RECQ1 is the most abundant but poorly characterized RecQ homolog in humans. Biochemically, RECQ1 displayed distinct substrate specificities from WRN and BLM, indicating that these helicases likely perform non-overlapping functions. Cellular studies imply that RECQ1 is involved in cellular proliferation and maintenance of genomic stability. We have obtained key preliminary evidence suggesting a unique role of RECQ1 in repair of oxidative DNA damage. RECQ1 is quickly recruited to the chromatin when cells are exposed to hydrogen peroxide (H2O2). Remarkably, the chromatin localization of RECQ1 is more robust and rapid than that of WRN helicase which has been shown to function in oxidative DNA damage repair. Deficiency in WRN or BLM helicase result in reduced homologous recombination and over-activation of PARP under basal condition; however, RECQ1-deficiency did not lead to PARP activation in undamaged cells and resulted in a comparatively modest, but significant, reduction in relative homologous recombination repair. In stark contrast to what is seen in WRN-deficiency, RECQ1-depleted cells over-activate PARP in a specific response to H2O2 treatment. Nonetheless, similar to WRN, RECQ1 associates with PARP-1 in human cells. Our results suggest a novel and non-overlapping role of RecQ1 in exogenously induced-oxidative DNA damage repair via modulation of PARP-1. Given that several RecQ proteins exhibit interaction with PARP-1, this study should provide a paradigm to delineate specialized and redundant functions of human RecQ homologs in DNA repair mechanisms.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2134. doi:1538-7445.AM2012-2134
Abstract 2135: Proline, glutamic acid, leucine rich protein 1 (PELP1) is essential for optimal p53-mediated DNA damage response
Authors: Nair Binoj C. , Krishnan Samaya R. , Sareddy Gangadhara R. , Mann Monica , Xu Bo , Natarajan Mohan , Tekmal Rajeshwar Rao , Vadlamudi Ratna K.
Published: August 2012

DNA Damage Response (DDR) is a vital ameliorative action undertaken by cells against various intrinsic and extrinsic genotoxic agents. p53 is an important transcription factor that plays a critical role in mediating cell cycle arrest or apoptosis upon DNA damage. PELP1 is a proto-oncogene and scaffolding protein that functions as a coregulator for a number of nuclear receptors such ERα and transcription factors such as E2F1. In this study, we discovered that PELP1 plays a vital role in p53-mediated DNA damage response. Using p53 wild-type breast cancer cells and PELP1-specific shRNAs, we show that cells lacking PELP1 are less sensitive to various genotoxic agents including etoposide, cisplatin, camptothecin or α-irradiation. PELP1 interacts with p53 upon DNA damage and ChIP analysis revealed that PELP1 is recruited to p53 target gene promoters. Reporter gene assays demonstrated that PELP1 functions as a coactivator of p53 and is required for optimal activation of p53 target genes under genomic stress. Mechanistic studies revealed that resistance to cell death in PELP1-depleted cells upon genotoxic stress was due to significant decrease in expression of p53 target genes. Furthermore, we demonstrated that PELP1 is phosphorylated at Serine 1033 upon DNA damage. Using a novel phospho-specific PELP1 antibody that specifically recognizes phosphorylated Serine 1033 on PELP1, we show that various genomic insults can induce PELP1 phosphorylation in vivo. Using cell lines that are either proficient or deficient in DDR kinases like ATM, ATR or DNAPKc, we have also established that PELP1 is phosphorylated by all three kinases depending on the type of DNA damage. Interestingly, we also found that the phosphorylation of PELP1 at Ser-1033 is important for optimal p53's transcriptional functions and that the phosphorylated form of PELP1 is recruited to the site of DNA damage when analyzed by laser micro-irradiation and confocal imaging, suggesting a potential role of PELP1 in the DNA repair process. Overall, these studies established a previously uncharacterized role of PELP1 in DNA damage response. Since PELP1 is as proto-oncogene with deregulated expression in many cancers, these findings will have implications in our understanding of cancer progression and therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2135. doi:1538-7445.AM2012-2135
Abstract 2137: Using Rac1 isogenic cells to model tumor metastasis
Authors: Wiggins Ceri , Astley Holly , Mahoney Claire , Sorrell David , Torrance Chris , Foster Rebecca
Published: August 2012

The Rho family of GTPases regulate a wide range of normal cellular functions including cell growth and cytoskeleton dynamics, but it is becoming increasingly apparent that when deregulated, proteins such as Rac1 contribute to tumorigensis, tumour invasion and metastasis. Overexpression of Cdc42 and Rac1 have been reported in human breast cancer, and coincide with breast cancer progression, metastasis and resistance to chemotherapy. The aim of this study was to generate a model system to investigate how Rac1 activation affects the migratory propensity of human breast epithelial cells. We utilised X-MAN isogenic cells derived from MCF10A mammary epithelial cells which harbour a single Q61L Rac1 mutation introduced by AAV-mediated homologous recombination, paired with a parental cell line expressing the wildtype allele. This system offers several advantages over conventional methods; protein expression is driven from endogenous promoters, thereby eliminating the caveats associated with transfection-based approaches. Using the X-MAN isogenic cells we showed that introduction of the Rac1Q61L mutation results in a profound effect on cell morphology. The most striking phenotype was the appearance of large intracellular vacuoles, but immunofluorescence studies also revealed cytoskeletal abnormalities with Rac1Q61L cells showing evidence of microtubule disorganisation and accumulation of newly polymerised actin fibers. These observations, in conjunction with previous reports that activated Rac is associated with cell migration, prompted us to address whether these cells may also have a migratory phenotype. Using Transwell and scratch assays, we confirmed that Rac1Q61L cells are more motile than their parental counterparts, and this correlated with increased adhesive properties. Pathway analysis revealed Rac1Q61L cells express higher basal levels of activated MEK and ERK, both of which have been implicated in the migration of numerous cell types. In conclusion, we have generated a pair of isogenic cell lines to demonstrate how constitutive Rac1 activation results in cytoskeletal changes and increased motility. Our data highlights the advantages of being able to study clinically-relevant pathways in a phenotypically-relevant assay, and how this platform could be exploited to profile molecularly targeted agents aimed at reducing tumour invasion and metastasis.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2137. doi:1538-7445.AM2012-2137
Abstract 2140: Regulator of G-protein signaling 2 blunts cellular viability mediated by a stabilized lysophosphatidic acid analogue (2S-OMPT) in the presence of chemotherapy in an ovarian cancer cell model of chemoresistance
Authors: Altman Molly K. , Ali Mourad W. , Callihan Phillip C. , Hooks Shelley B. , Murph Mandi M.
Published: August 2012

Lysophosphatidic acid (LPA) enhances growth, survival, viability and proliferation in numerous models. Thus, the receptors for LPA are also implicated in oncogenic signaling and represent a group of “druggable” therapeutic targets in cancer. Regulator of G protein Signaling 2 (RGS2) is a GTPase activating protein (GAP) that deactivates the G alpha subunits of heterotrimeric G proteins, turning off G protein-coupled receptor (GPCR) signaling. In our previous studies examining computational bioinformatics, we demonstrated that reduced RGS gene expression develops with the occurrence of drug resistance in ovarian cancer cell lines. The purpose of our study was to investigate whether the LPA receptors are also involved in mediating viability as a result of enhanced GPCR signaling due to reduced RGS proteins in this chemoresistance model. This is important since RGS proteins can deactivate G alpha subunits of many different GPCRs, and are not specific to merely the LPA receptors. If a “druggable” GPCR is mediating viability in chemoresistance, it is critical to identify which ones are responsible. To test this idea we used the metabolically stabilized LPA analogue L-sn-1-O-oleoyl-2-O-methylglyceryl-3-phosphothionate (2S-OMPT) as a potent non-hydrolyzable agonist for LPA receptors at a concentration (10 µM) sufficient to activate multiple LPA receptors. We hypothesized that if LPA signaling was not involved, then OMPT would override any contribution of RGS2 on modulating the viability response in the presence of chemotherapy. Using a tet-off promoter system, we generated an inducible stable model of RGS2-expressing HeyA8-MDR cells, which we have previously confirmed are resistant to taxanes. After 48 h OMPT pre-treatment, HeyA8-MDR-RGS2-inducible cells were exposed to cisplatin for 24 h and evaluated for viability. IC50 values for cisplatin were lower in the RGS2-induced HeyA8-MDR cells that were not pretreated with OMPT, a demonstration of enhanced sensitivity to cisplatin in the presence of RGS2. All conditions pre-treated with OMPT demonstrated enhanced viability even in the presence of chemotherapy; however, ovarian cancer cells that were pretreated with OMPT and cultured in tet-free medium to induce RGS2 expression showed a significant blunting of viability. This supports the interaction between LPA receptors and RGS2 in the control of drug sensitivity in ovarian cancer chemoresistance and provides a mechanism for our current and previous observations.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2140. doi:1538-7445.AM2012-2140
Abstract 2141: cAMP/PKA signaling enhances activity of Deleted in Liver Cancer 1 (DLC1) tumor suppressor in suppressing liver cancer tumorigenesis
Authors: Ko Chi Fat , Chan Lo Kong , Tse Edith Yuk-Ting , Yeung Yin-Shan , Sze Karen Man-Fong , Ng Irene Oi-Lin , Yam Judy Wai Ping
Published: August 2012

Deleted in Liver Cancer 1 (DLC1) which encodes a RhoGTPase-activating protein (RhoGAP) is a tumor suppressor frequently inactivated in a wide spectrum of human cancers. The RhoGAP activity has been shown to play a predominant role in the biological activities of DLC1. It has been shown that cells with silenced DLC1 exhibit increased active RhoA level. This finding provides evidence about the activation of RhoA as the consequence of deregulated DLC1 and points to the importance of RhoGAP activity in the biological activity of DLC1. In this regard, it is important to comprehend how RhoGAP activity of DLC1 is related. Here, we show that DLC1 was robustly phosphorylated by cyclic AMP (cAMP)/Protein kinase A (PKA) signaling in cells. Phosphorylation of DLC1 was enhanced by forskolin, a known activator of PKA while suppressed when H-89, an inhibitor of PKA was added. Direct phosphorylation of DLC1 by PKA was further confirmed by the in vitro kinase assay. Using specific phospho-DLC1 antibodies, PKA was shown to phosphorylate DLC1 at S431 and S549. Functional assays demonstrate that phosphorylation at S549 plays a critical role in provoking the inhibitory activity of DLC1 in suppressing growth and motility of Ras-transduced p53 null mouse hepatoblasts. When compared with the stable clone of wild-type DLC1, stable clone of DLC1 phosphomimetic mutant, S549D, displayed a largely reduced growth of subcutaneous and orthotopic liver implanted tumors and an enhanced apoptosis. The migration and invasion rates of S549D cells were also significantly inhibited. Furthermore, S549D expression abolished stress fiber formation but failed to alter filopodia protrusions. These functional effects exerted by S549D were ascribed to the enhanced RhoGAP activity against RhoA. To further investigate the mechanism through which the RhoGAP activity is enhanced, we found that DLC1 dimerized upon S549 phosphorylation. Our findings have revealed for the first time about the regulation of RhoGAP activity of DLC1 via dimerization. Our study suggests a molecular link between PKA and DLC1/Rho pathways and underscores the importance of S549 phosphorylation in the regulation of RhoGAP activity of DLC1. (This study was funded by the Small Project Funding Program, The University of Hong Kong 200907176125)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2141. doi:1538-7445.AM2012-2141
Abstract 2146: The Rho GTPase RhoE is a p53-regulated candidate tumor suppressor
Authors: Bi Feng , Zhu Yajie , Tang Qiulin
Published: August 2012

Rho family proteins are prominent members of the well-known Ras superfamily and are deeply involved in almost all the malignant behaviors of cancer. As Small GTPases, Rho proteins can cycle between active GTP-bound state and GDP-bound state by regulating of GEFs, GAP and GDI. RhoE is a unique member of Rho family proteins that can bind but do not hydrolyze GTP, thus remain constitutively active upon expression without the need of regulation by the GTP/GDP conformational changes of other Rho GTPases. Previous studies have shown that RhoE may play a role opposite that of RhoA in regulating cell proliferation. Here, for the first time, we systematically investigate the expression patterns of RhoE by systemic immunocytochemistry analysis of 74, 32, 62, and 34 gastric, colorectal, lung, and breast carcinomas, respectively, and found that RhoE protein level was significantly decreased in most cancer cases compared with that of adjacent normal tissues. RhoE expression also appeared to inversely correlate with the clinical grade of gastric, colorectal, lung cancer tissues. Increasing RhoE expression in MKN45, SW480, HepG2 and SPCA1 cancer cell lines that have relatively low level of endogenous RhoE could markedly inhibit proliferation, migration and invasion activities and induced apoptosis of the cancer cells. Interestingly, p53 transcription activity was reduced in these RhoE under-expressed cancer cells that contain p53 mutations, and increasing p53 activity significantly increased RhoE expression in the cells. Significantly, p53 transcription activity, measured by the p53-responsive p21Cip1, MDM2, and GADD45 expressions, paralleled RhoE expression in gastric cancer tissues. Thus, RhoE may participate in human cancer progression as a candidate tumor suppressor under the regulation by p53. This work also enriches our knowledge in the role of small G protein network in cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2146. doi:1538-7445.AM2012-2146
Abstract 2148: Exploitation of androgen receptor splice variant signaling by guanine nucleotide exchange factor Vav3 in castration resistant prostate cancer
Authors: Peacock Stephanie , Fahrenholtz Cale , Burnstein Kerry L.
Published: August 2012

The treatment for advanced or metastatic prostate cancer is androgen deprivation therapy. Patients inevitably relapse and the cancer is now termed androgen-independent or castration resistant (CRPC). CRPC remains dependent on androgen receptor (AR) signaling. One of the proteins implicated in the reactivation of AR transcriptional activity in CRPC is Vav3, a Rho GTPase guanine nucleotide exchange factor. Vav3 is up-regulated in human prostate cancer tumors compared to benign tissue and in preclinical models of prostate cancer progression to CRPC. Vav3 enhances AR activity at both physiological and as well as at subnanomolar androgen concentrations, similar to prostatic microenvironment levels demonstrated in CRPC. Vav3 confers castration resistant growth to a classically androgen dependent cell line in a mouse xenograft model. Thus, elevated levels of Vav3 seem to be pivotal in promoting continued AR signaling in CRPC. We examined the contributions of Vav3 to AR activity and cell proliferation in the CRPC cell lines CWR-22Rv1 and CWR-R1. Knockdown of Vav3 attenuated cell growth and reduced AR activity as compared to the shGFP control. However knockdown of full length AR had little effect, perhaps due to impaired ligand-mediated transcriptional activity of the mutated AR expressed in these cell lines. Because CWR-22Rv1 also express AR splice variants that lack the ligand binding domain and are proposed to underlie CRPC growth, we examined the possibility that Vav3 enhances AR splice variant signaling. The AR splice variant, ARV7 (also known as AR3), exhibits constitutive ligand independent activity and is linked to poor prognosis and survival. Depletion of ARV7 in 22Rv1 and CWR-R1 cell lines resulted in slowed growth and lowered AR activity. Soft agar assays revealed that knockdown of either Vav3 or ARV7 resulted in greatly decreased anchorage independent growth. Vav3 potently enhanced the activity of ARV7 in reporter gene assays. Further, knockdown of Vav3 resulted in lowered levels of nuclear AR splice variants in CWR-22Rv1. Co-immunoprecipitation revealed that ARV7 and Vav3 directly interact. These results suggest that Vav3 may enhance ARV7 activity through increased nuclear localization and that the interaction of Vav3 and ARV7 may be required for robust growth of CRPC cells. These novel data demonstrating interaction between a versatile AR coactivator and an AR splice variant provide insights into the possible mechanisms by which Vav3 exploits and enhances AR splice variant signaling in the progression of prostate cancer malignancy and castration resistance.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2148. doi:1538-7445.AM2012-2148
Abstract 2154: CDCP1 links RAS and Src signaling pathways as a common effector for the promotion of tumor metastasis
Authors: Sakai Ryuichi , Uekita Takamasa
Published: August 2012

The oncogenic RAS is one of the most intensively studied proteins which are well recognized as a master regulator of cancer initiation and progression, however rather limited information is available as for their contribution to the cell properties associated with tumor metastasis such as anoikis resistance, cell migration and invasion. We have revealed that CUB domain-containing protein 1 (CDCP1) protein, a substrate of Src family kinase (SFK), in solid cancers is involved in the regulation of anoikis resistance, cell migration and invasion. CDCP1 contributes significantly to the metastatic potential of various cancers including lung, stomach, and pancreatic cancers in a tyrosine phosphorylation dependent manner. It was recently observed that human lung cancer cell lines with K-RAS mutations show significantly higher expression of CDCP1 than those without, while the other common mutations such as EGFR, LKB1 and p53 did not show such relationship. The introduction of the activating mutation of K-RAS clearly induced the expression of CDCP1 and that this enhancement is also affected by the expression levels of Myc protein. Treatment of PD98059, an inhibitor of MEK downstream of Ras, as well as expression of dominant-negative Ras, reduced the expression of CDCP1, and at the same time suppressed cell survival in suspension culture, cell migration and invasion. However, these properties were rescued only when both CDCP1 and Fyn, a member of SFKs were expressed. On the contrary, metastatic potentials caused by expression of activated K-Ras in lung cancer cells was blocked by knockdown of in CDCP1 expression induced by Ras. Using three dimensional organotypic culture assay of human cervical keratinocytes (HCKs) which mimics in vivo tissue invasion of cervical cancers, marked expression of CDCP1 was detected at the site of cell invasion into the collagen raft. The treatment of CDCP1 siRNA or SFKs inhibitor PP2 repressed cell invasion into the collagen raft induced by Ras and Myc. As a summary, it was revealed that expression of CDCP1 is induced by oncogenic Ras, and upregulated CDCP1 triggers cancer invasion and metastasis when phosphorylated by SFKs. Metastatic potential induced by Ras is dependent on the CDCP1 induction, while CDCP1 is not responsive for proliferative change also induced by Ras. These findings demonstrated the central role of CDCP1 as the functional link between RAS and SFKs signaling during the progression of human malignant tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2154. doi:1538-7445.AM2012-2154
Abstract 2157: A novel role for Giα proteins in differentially regulating activation of RTK-mediated signaling
Authors: Chu Wen-Ming , Wang Zhanwei , cruz Rica Dela , Chu Catherine , Zhang Jianhua , Jiang Meisheng
Published: August 2012

The activation of the extracellular-signal-regulated protein kinase (ERK) pathway and the PI3K/Akt/mTORC pathway by growth factors is mediated by the receptor tyrosine kinases (RTKs), and plays a crucial role in physiological and pathological processes including cell proliferation, growth, survival, migration, tumorigenesis and metastasis. We previously reported that the activation of the PI3K/Akt/mTORC1 in mouse embryonic fibroblasts (MEFs) by epidermal growth factor (EGF) and its family growth factors is mediated by the heterotrimeric G protein alpha inhibitory (Giα). Herein we demonstrated that Giα proteins are able to differentially regulate RTKs-mediated activation of the ERK and the Akt/mTORC1 pathways. Loss of Giα proteins in MEFs impaired activation of the ERK pathway by EGF, insulin-like growth factor (IGF) and Insulin, but not basic fibroblast growth factor (bFGF). Intriguingly, while activation of the Akt/mTORC1 pathway by EGF and bFGF was severely defective in Giα protein deficient cells, this activation was only slightly reduced in response to insulin and IGF. Also, Giα proteins were able to alter activation of the Akt/mTORC1 and the ERK pathways by serum and platelet-derived growth factor (PDGF) in a dose dependent manner. To further understand that how Giα proteins differentially regulate RTK-mediated activation of the Akt/mTORC1 and ERK pathways, we performed an immuno-precipitation study. Our results showed that growth factors were able to induce the association of Giα proteins with growth factor receptors and their downstream adaptor proteins. Loss of Giα proteins in MEFs impaired the association of growth factor receptors with their adaptor proteins indicating that Giα proteins act downstream of RTKs but upstream of the adaptor proteins. Finally, we examined a role of Giα proteins in cancer biology. Similar to results obtained from Giα deficient MEFs, knockdown of Giα proteins in several human cancer cell lines diminished activation of the Akt/mTORC1 and the ERK pathways by growth factors. As a result, Giα protein deficiency inhibited cancer cell growth and migration. Given the fact that growth factors and their receptor pathways play pivotal roles in tumorigenesis and metastasis of cancer, our findings may promote further understanding the molecular mechanism underline RTK signaling regulation in cancer biology and may have implications for cancer therapy. Supported by Department of Defense, Leukemia and Lymphoma Society and NIH.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2157. doi:1538-7445.AM2012-2157
Abstract 2163: RSK signaling in HER2-positive breast cancer cells
Authors: Fink Marc Y. , Singh Shawn , Cho Yohan , Mestry Daksha
Published: August 2012

Heregulin (HRG) is a soluble growth factor involved in the control of proliferation, survival, invasion, and differentiation of normal and malignant mammary epithelial cells. HRG binds its receptor, HER3, leading to activation of the HER2-HER3 heterodimer, which acts as an oncogenic factor in breast cancers. Pharmacological inhibitors to HER2 are currently being used in the clinic to treat HER2-positive breast cancer, however, they are associated with resistance after one year of therapy. A more comprehensive understanding of the signaling network downstream of HRG, in HER2-positive breast cancer cells, will be crucial for further development of inhibitors that do not elicit resistance. This study assessed HRG signaling in the HER2-positive breast cancer cell lines BT474 and SKBR3. The RSK (p90 ribosomal S6 Kinase) pathway was activated in an ERK-dependent manner. HRG stimulation increased phosphorylation of RSK at S380, T359, and S363. HRG stimulated the phosphorylation of CREB (cAMP Response-Element Binding Protein), an established substrate of RSK, at S133 and this was blocked by pharmacological inhibition of RSK. HRG induced the expression of c-FOS in an RSK-dependent manner. These data demonstrate that RSK has an important role in transducing HRG signaling to the gene expression program and suggest that inhibitors of RSK may be useful in the treatment of HER2-positive breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2163. doi:1538-7445.AM2012-2163
Abstract 2186: Function and regulation of Polo-like Kinase 3 in human pancreatic cancer
Authors: Li Zhongkui , Chang Zhe , Chiao Paul J.
Published: August 2012

Pancreatic cancer is currently the fourth leading cause of cancer death in the United States. Despite the recent advances in pancreatic cancer research, patients with this devastating disease still have a poor prognosis. Therefore, deciphering pancreatic cancer-related gene expression and defining the individual gene's specific functions will make a significant impact on the diagnosis and treatment of pancreatic cancer. Recently, our lab has demonstrated the tumorigenic transformation in an immortalized pancreatic cell line, HPNE/hTERT by sequential introduction of multiple genetic alterations. We found that Polo-like kinase 3 (Plk3) is significantly down-regulated in transformed tumor cell lines. Plk3 belongs to the Polo-like kinase family and is a multi-functional kinase. We previously reported that Plk3 played a pro-apoptotic role in pancreatic cancer lines when it was overexpressed. Plk3 down-regulation in transformed HPNE cells suggests that Plk3 may have tumor suppression function during pancreatic tumor development. In addition, we found Plk3 expression is regulated by Pten. In Pten-knockout MEF cells, Plk3 expression was significantly down-regulated. So Plk3 may be downstream of Pten in the tumor suppression pathway. These data suggest that Plk3 play an important role in pancreatic cancer development.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2186. doi:1538-7445.AM2012-2186
Abstract 2189: Novel transcriptional regulation of Notch1 by EZH2 in mammary stem cells
Authors: Gonzalez Maria E. , Li Xin , Kruger Heather , Toy Kathy , Kleer Celina G.
Published: August 2012

Background: EZH2 has oncogenic functions in the breast and other organs, and has been recently shown to regulate the differentiation of tissue specific stem cells. However, the underlying mechanisms are largely unknown. We hypothesized that EZH2 may regulate mammary stem cells by transcriptional mechanisms of key signaling pathways. Methods: We developed EZH2 KD in SUM149 breast cancer cells, and an inducible and transient cell culture model of EZH2 overexpression, (EZH2-OV), and its deletion mutants in MCF10A and in primary breast cells. Mammary stem cells were identified using ALDH1+ and CD44+/CD24- by FACS. ChIP assays were used to determine EZH2 recruitment to specific areas in NOTCH1 promoter. Ectopic expression of EZH2 and deletion mutants leads to identify the EZH2 domain responsible for the transcription up regulation of NOTCH1. Results: We identified that EZH2 KD in SUM149 significantly decreased NOTCH1 signaling in the stem cell population. We observed preferential expression of EZH2 in the stem cell population of normal primary breast cells compared to non-stem cells. In MCF10A cells, EZ-OV was predominantly increased in the ALDH-1+ population. We found that EZH2-OV increased the stem cell population of MCF10A cells. Consistently, EZH2-OV in MCF10A and primary breast cells increased Notch1 mRNA specifically in the ALDH1+ population. EZH2-OV increased Notch1 protein levels and promoter activity in MCF10A and primary cells. Mechanistically, Notch1 inhibition by GSI reversed the effect of EZH2-OV in the stem cell population of MCF10A cells. ChIP assays revealed that EZH2 is recruited to specific regions of the NOTCH1 promoter depending on EZH2 specific context. Moreover, ChIP assays with EZH2 deletion mutants identify that the Nter- EZH2 HII domain is responsible for the NOTCH1 promoter transcriptional up regulation and that this function is independent of the SET domain (methytransferase activity) and as shown by comparing the EZH2 tri-methylation mark (3H3K27) in the NOTCH1 promoter. Conclusion: Collectively, these results indicate that EZH2 regulates mammary stem cells and requires, at least in part the activation of Notch1 signaling by recruitment of EZH2 to the NOTCH1 promoter through the Nter domain.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2189. doi:1538-7445.AM2012-2189