bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1056: Deregulated HDAC9 expression in breast cancer is associated with basal molecular subtype
Authors: Linares Aurélien , Lapierre Marion , Boulahtouf Abdel , Rodriguez Carmen , Orsetti Béatrice , Balaguer Patrick , Maudelonde Thierry , Cavailles Vincent , Boulle Nathalie
Published: August 2012

Estrogens play a pivotal role in the etiology of breast cancer. Estrogen receptor (ER) activity is finely regulated by various transcriptional regulatory complexes including histone acetyl transferases (HAT) and histone deacetylases (HDACs). Previous studies have underlined the potential role of some HDACs in breast tumor progression and their interactions with estrogen signalling. However, expression of the various class I, II and IV HDACs in breast tumors has not been systematically analysed. In this study, we performed a careful analysis of HDACs 1 to 11 expression in five breast cancer cell lines expressing (MCF7, ZR75, T47D) or not (MDA231, MDA436) ERα. HDAC4, HDAC11 and more strikingly HDAC9 were found to be differentially expressed in ERα+ and ERα- cells. HDAC9 mRNA levels were further measured in a panel of 14 breast tumor cell lines expressing or not ERα and classified as luminal, basal A and basal B. This showed that the difference in HDAC9 expression was more related to the luminal or basal characteristics of the breast tumor cells than to ERα expression, with basal cells harboring significantly higher HDAC9 mRNA levels than luminal ones. Comparison of mRNA levels of total HDAC9 with those of the longest HDAC9 isoforms (variants 1, 4 and 5) and the HDAC9ΔCD isoform, lacking the catalytic deacetylase domain (also known as MITR), showed a similar pattern of distribution among luminal, basal A and basal B cells. In basal cells, HDAC9 overexpression was not linked to gain in HDAC9 gene copy number but mainly to higher rates of gene transcription together with enhanced HDAC9 mRNA stability. Based on cDNA array data, the same differential expression was detected in patients’ tumors as significant higher levels of HDAC9 mRNA were measured in ERα negative and basal breast tumors as compared to ERα expressing and luminal ones respectively, suggesting the biological relevance of the results obtained using breast cancer cell lines. In contrast to luminal cells harboring very low or no detectable levels of HDAC9 protein, a 95 kDa HDAC9 protein isoform was detected in basal breast cancer cells, with a predominant and diffuse nuclear distribution as shown by immunofluorescence. Altogether, our results indicate that class IIa HDAC9 is overexpressed at the mRNA and protein levels in the most agressive breast tumors of basal phenotype. The role of this specific HDAC in breast carcinogenesis and ER signaling will be further analysed and described using mammary tumor cell models harboring HDAC9 overexpression or HDAC9 silencing.

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 1056. doi:1538-7445.AM2012-1056
Abstract 1061: EZH2 expression in different subtypes of human breast cancer
Authors: Khramtsova Galina F. , Islam Abul B.M.M.K. , Khramtsov Andrey I. , Lyman Phoebe , Chuanhong Liao , Jene-Sanz Alba , Sveen Walmy E. , Huo Dezheng , Lopez-Bigas Nuria , Benevolenskaya Elizaveta V. , Olopade Olufunmilayo I.
Published: August 2012

Introduction: Cancer is characterized by aberrant patterns of expression of multiple genes believed to be due to not only genetic but also epigenetic changes. The epigenetic changes are communicated through chemical modifications, including modifications of histone proteins. For each histone, multiple histone-modifying enzymes have been discovered in mammals. We found that the expression of genes with histone modifications was highly predictable from the level of expression of the corresponding histone-modifying enzyme. Specifically, expression of genes displaying histone H3 K27 methylation was dependent on EZH2 expression. Method: We analyzed breast cancer tissue arrays for EZH2 expression. Tissue microarrays were constructed from 95 histologically confirmed breast cancer samples. To identify breast cancer subtypes, the tissues were evaluated for the expression of ER, PR, HER2, CK5/6 and EGFR. Staining with vimentin served as a control to monitor the quality of tissue fixation in archival tumors. Breast cancer subtypes were defined as luminal A (ER+ and/or PR+, HER2-), luminal B (ER+ and/or PR+, HER2+), basal-like (ER-, PR-, HER2-, CK5/6+, and/or EGFR+), HER2+/ER- (HER2+, ER-, PR-), and unclassified (negative for all five markers). EZH2 expression was measured by staining using an anti- EZH2 antibody (Invitrogen, cat. #18-7395). Staining quantification was performed independently by two pathologists. Results: The mean age of the patients was 56 years old (SD=15.6). 63.7% of the patients had estrogen receptor positive tumors, 50.6% had progesterone receptor positive tumors, and 21.2% had HER2+ tumors. The distribution of breast cancer subtypes is luminal A (58.9%), luminal B (6.3%), HER2+/ER- (4.2%), basal-like (28.5%), unclassified (2.1%). A majority of the patients had high grade tumors (46.0% grade II and 47.1% grade III). High EZH2 expression was associated with Her2+ and basal-like tumors (P<0.0001). Conclusion: In this cohort, high expression of EZH2 was associated with the most aggressive breast cancer phenotypes and likely reflects increased expression of histone modifying proteins in the tumors. Supported by Grants: NIH R01CA138631 (E.V.B.), R01 CA142996 and P50 CA125183 (O.I.O).

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 1061. doi:1538-7445.AM2012-1061
Abstract 1072: The FGF18/FGFR4 amplicon: Novel therapeutic biomarkers for ovarian cancer
Authors: Wei Wei , Mohapatra Gayatry , Birrer Michael J.
Published: August 2012

Epithelial ovarian cancer is the fifth leading cause of cancer-related death among women and has the highest case-fatality rate among gynecologic cancers. High throughput genomic technologies have been instrumental in identifying new biomarkers and potential novel therapeutic targets for ovarian cancer. However, comprehensive functional validation studies on both the biological and clinical levels are needed to better understand the mechanistic basis for these biomarkers and realize their clinical significance and application. Fibroblast growth factor 18 (FGF18) has been recently identified as an aberrantly expressed gene within an expression signature predicting poor rate of survival in patients with advanced stage serous ovarian cancers. In addition, genomic amplification of both FGF18 and FGFR4 has been shown to predict for poor overall survival among women with advanced stage high grade serous ovarian cancers. siRNA mediated knock-down of FGFR4 abolishes FGF18 induced phosphorylation of FRS2 and ERK1/2 in ovarian cancer cells, indicating the FGF18 signaling is at least partially conferred by FGFR4 in ovarian cancer cells. However, the exact role of FGF18/FGFR4 in the clinicopathologic properties of ovarian cancer has not been determined. Preliminary studies by ectopic FGF18 overexpression or recombinant FGF18 treatment demonstrate that FGF18 promotes the in vitro migration and invasion of both ovarian cancer cells and endothelial cells. In SCID mice xenograft models, FGF18 expression in ovarian cancer cells results in increased tumor formation. Microarray analysis identified up-regulation of a large number of proinflammatory cytokines by FGF18 which may mediate the increases in angiogenesis and infiltration of macrophages seen in FGF18 expressing xenografts. Pathway analysis further demonstrated participation of NF-κB signaling in FGF18 mediated cytokine production. Conversely, knock-down of FGF18 in ovarian cancer cells reduces the in vitro migration; inhibits the production of cytokines and impairs the in vivo tumorigenicity in SCID mice xenograft model. Taken together, FGF18/FGFR4 axis may play an important role in ovarian cancer pathogenesis and may serve as a novel therapeutic target.

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 1072. doi:1538-7445.AM2012-1072
Abstract 1074: Keratinocyte growth factor suppresses malignant phenotype of cutaneous squamous carcinoma cells
Authors: Toriseva Mervi , Ala-aho Risto , Peltonen Juha , Peltonen Sirkku , Grenman Reidar , Kahari Veli-Matti
Published: August 2012

Keratinocyte growth factor (KGF, fibroblast growth factor-7) is a fibroblast-derived mitogen, which stimulates proliferation of epithelial cells. The expression of KGF by dermal fibroblasts is induced following injury and it promotes wound repair. However, the role of KGF in cutaneous carcinogenesis and cancer progression is not known. Here, we have examined the role of KGF in progression of squamous cell carcinoma (SCC) of the skin. The expression of KGF receptor (KGFR) mRNA was lower in cutaneous SCCs (n=6) than in normal skin samples (n=5). Expression of KGFR mRNA was detected in 6 out of 8 cutaneous SCC cell lines and the levels were downregulated by 24-h treatment with KGF. KGF did not stimulate SCC cell proliferation, but it reduced invasion of SCC cells through collagen. Gene expression profiling of three cutaneous SCC cell lines treated with KGF for 24 h revealed a specific gene expression signature characterized by upregulation of a set of genes specifically downregulated in SCC cells compared to normal epidermal keratinocytes, including genes with tumor suppressing properties (SPRY4, DUSP4, DUSP6, LRIG1, PHLDA1). KGF also induced downregulation of a set of genes specifically upregulated in SCC cells compared to normal epidermal keratinocytes, including genes associated with tumor progression (MMP13, MATN2, CXCL10, and IGFBP3). Downregulation of MMP-13 and KGFR expression in SCC cells and HaCaT cells was mediated via ERK1/2. Activation of ERK1/2 in HaCaT cells and tumorigenic Ha-ras-transformed HaCaT cells resulted in downregulation of MMP-13 and KGFR expression. These results provide evidence, that KGF does not promote progression of cutaneous SCC, but it suppresses the malignant phenotype of cutaneous SCC cells by regulating the expression of several genes differentially expressed in SCC cells, as compared to normal epidermal keratinocytes.

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 1074. doi:1538-7445.AM2012-1074
Abstract 1079: Identification of autocrine motility factor (AMF) receptor, HER2 (ErbB2) in breast cancer cells
Authors: Kho Dhonghyo
Published: August 2012

Overexpression of HER2 (ErbB2) oncoprotein is associated with approximately 25% of aggressive breast carcinoma. Although a humanized monoclonal antibody (Trastuzumab) against HER2 is currently considered one of the most effective treatments, a significant number of patients are initially or eventually become resistant to Trastuzumab based therapy. Here, we demonstrate that secreted phosphoglucose isomerase (PGI)/ autocrine motility factor (AMF), an orphan -CXXC- cytokine directly binds to HER2 and activates PI3K and MAPK through HER2 autophosphorylation, and also induces immediately HER2 cleavage in metalloprotease dependent fashion. We therefore speculate that AMF/HER2 signaling pathways play a physiological role in HER2 overexpressing breast cancer cells and contribute to Trastuzumab resistance and propose that AMF can be a novel therapeutic target in Trastuzumab resistant 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 1079. doi:1538-7445.AM2012-1079
Abstract 108: Functional characterization of WAC, a candidate tumor suppressor gene in colorectal cancer
Authors: Conboy Caitlin B. , Hatler Julia M. , Scott Pat M. , Sarver Aaron L. , Silverstein Kevin A. , Seshagiri Somasekar , Adams Matthew , Lillemoe Jenna , Dorr Casey R. , Cormier Robert T. , Starr Tim K. , Largaespada David A.
Published: August 2012

Broadening our understanding of the genetic basis of cancer will facilitate construction of relevant pre-clinical models and provide novel targets for drug development. To identify genes that contribute to the initiation or progression of colorectal cancer (CRC) when mutated or dysregulated, we recently conducted a set of forward genetic screens using transposon insertional mutagenesis to drive intestinal tumor formation in mice. These studies were designed to identify novel cancer genes that function in a wildtype genetic context or cooperate with known predisposing alleles (ApcMin/+ or Trp53R270H/+). Over 150 genes were identified, including several known to be important in human CRC (eg. APC, PTEN, and FBXW7) and many novel candidate cancer genes. WW domain containing adaptor with coiled-coil (WAC), is a gene not previously associated with cancer that was identified in all three screens. While the function of WAC is incompletely understood, previous reports indicate that the WAC protein participates in multiple cellular processes, including golgi biogenesis, modulation of gene expression, and induction of p21CIP1 expression and cell cycle arrest in response to DNA damage. In this study, we evaluated the role of WAC in cellular transformation by depleting WAC in mouse colonic epithelial cells conditionally immortalized by expression of a temperature sensitive SV40 large T antigen. In this model, loss of WAC expression increased anchorage-independent growth of colonic epithelial cells in cooperation with mutant APC. In human CRC samples, resequencing the WAC gene identified non-silent mutations in 2 of 74 samples. Further, CRC-associated WAC mutants were found to be functionally deficient in induction of p21CIP1 in a zebrafish embryo model. These data are consistent with the model that WAC functions as a tumor suppressor in the colonic epithelium.

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 108. doi:1538-7445.AM2012-108
Abstract 1193: A single phosphorylation site mutant of the Retinoblastoma protein blocks apoptosis induced by UV
Authors: Lentine Brandon , Hunce Raymond A. , Antonucci Lisa , Krucher Nancy A.
Published: August 2012

The Retinoblastoma protein (Rb) is important in the control of cell proliferation and apoptosis. Its activity is controlled by reversible phosphorylation on several serine and threonine residues. When Rb is hypophosphorylated, it inhibits proliferation by preventing passage through the G1- S phase transition. Hyperphosphorylated Rb promotes cell cycle progression. The role of Rb phosphorylation in the control of apoptosis is largely unknown, although several apoptotic stimuli result in dephosphorylation of Rb. It may be the case that dephosphorylation of specific amino acids may signal apoptosis versus cell cycle arrest. Using glutamic acid mutagenesis, we have generated 15 single phosphorylation site mutants from serine/threonine to glutamic acid, to mimic the phosphorylated state. By calcium phosphate transfection mutant plasmids were introduced into C33A Rb-null cells and apoptosis was induced using UV. Apoptosis was measured by ELISA detection of degraded DNA and by immunoblotting to assess proteolytic cleavage of PARP. We found that only threonine-821 when mutated to glutamic acid blocked apoptosis by 50% whereas other sites tested had little effect. In addition, endogenous Rb is dephosphorylated on threonine-821 when cells are undergoing apoptosis. Experiments to determine the effect of this mutation in response to additional apoptotic stimuli are underway.

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 1193. doi:1538-7445.AM2012-1193
Abstract 1201: Hypoxia promotes not CXCR7 but CXCR4 expression and its biological activity in gastric cancer cell through activation of hypoxia-inducible factor-1β
Authors: Lee Hyo Jin , Oh Yun Sook , Kim Ha Yon , Yoon Hwan Jung , Jo Deog Yeon , Kim Samyong
Published: August 2012

Background: The chemokine receptor, CXCR4 and CXCR7, which bind with high affinity same chemokine CXCL12/SDF-1, are associated with the biological behavior in several kinds of cancer, but few studies have addressed the expression and regulation of CXCR4 and CXCR7 in gastric cancer. Methods: Gastric cancer cell line KATO III was subjected to hypoxia or normoxia. The expression of CXCR4 and CXCR7 were investigated using RT-PCR, Westerning blotting, and flow cytometry before and after hypoxic conditioning. The regulation of CXCR4 and CXCR7, and their biological effects by hypoxia were evaluated using gastric cancer cells transfected with hypoxia-inducible factor (HIF)-1α shRNA. Result: mRNA and proteins of both CXCR4 and CXCR7 were detectable by RT-PCR and Western blot analysis, respectively, in KATO III cells. A small population of KATO III cells expressed membrane CXCR4 and CXCR7 as determined by flow cytometry. Hypoxia up-regulated CXCR4 proteins and enhanced membrane expression of CXCR4 in human gastric cancer KATO III cells, which constitutively expressed membrane CXCR7 and CXCR4 in a steady state, as revealed by Western blotting and flow cytometry, respectively. In contrast, CXCR7 proteins remained unchanged under hypoxia, and promotion of CXCR7 surface expression on KATO III cells could not be demonstrated by flow cytometry. Hypoxia-induced expression of CXCR4 was mediated through activation of HIF-1α with phosphorylation of AKT and ERK, as revealed by HIF-1α knockdown using shRNA. The chemical inducer of HIF-1α, cobalt chloride, induced upregulation of CXCR4 in KATO III cells under normoxic conditions. In addition, KATO III cells exposed to hypoxic condition showed enhanced wound healing, transwell migration, and invasion in response to stromal cell-derived factor (SDF)-1α, a specific ligand for CXCR4. Conclusions: These findings provide evidences for differential regulation and different role of chemokine receptor, CXCR7 and CXCR4, in gastric cancer cell in vitro.

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 1201. doi:1538-7445.AM2012-1201
Abstract 121: Novel epigenetic mechanisms of resveratrol regulation of PTEN/Akt pathway in prostate cancer
Authors: Dhar Swati , Li Kun , Dias Steven J. , Levenson Anait S.
Published: August 2012

Resveratrol (Res) (3,5,4′-trihydroxy-trans-stilbene), a natural compound found in grapes and berries, is one of the most promising diet-derived chemopreventive agent with potential chemotherapeutic capabilities. Chemopreventive and anticancer effects of Res may be exerted in part by modulating various components of the epigenetic machinery. We recently demonstrated that Res represses nucleosome remodeling and deacetylation (NuRD) co-repressor complex by inhibiting metastasis associated protein 1/ histone deacetylase 1 (MTA1/HDAC1) subunit, which plays an essential role in governing deacetylation of histone and non-histone proteins. Since Res-induced upregulation of tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) is concomitant with MTA1-downregulation, we hypothesized that in the absence of Res MTA1 works in cooperation with PI3K/Akt survival pathways. We further hypothesized that Res-induced restoration of PTEN expression and function may occur by dual mechanism through MTA1/HDAC inhibition and regulation of miRNA network. Our data indicate that shRNA-mediated MTA1 knockdown in Du145 cells (wt PTEN) enhances acetylation of PTEN, and that HDAC inhibitor trichostatin (TSA) further potentiates PTEN acetylation. We also showed that Res treatment simultaneously decreases MTA1 and pAkt levels in LNCaP cells, the effect which is enhanced in combination with PI3K inhibitors, LY294002 and BEZ235. To further understand Res-induced MTA1-mediated epigenetic alterations, we analyzed MTA1-associated and Res-induced microRNA (miRNA, miR) changes in LNCaP cells expressing and silenced for MTA1 (shMTA1). Interestingly, Res downregulated miRs (oncomiRs) included members of miR17-92 cluster (miRs-17, 20a, 20b) and miRs 106a and 106b which target PTEN. These findings were further confirmed by real time PCR analysis which showed that Res specifically down-regulated these miRNAs in PCa cells (LNCaP and Du145) but not in normal prostate cell line RWPE-1. Direct interaction of miR-17, miR-20a and miR-106b with PTEN 3′UTR were analyzed in the presence and absence of Res using pmiRGLO luciferase reporter. In summary, we propose novel epigenetic mechanisms of Res regulation of PTEN/ Akt signaling through inhibition of MTA1/NuRD co-repressor complex and MTA1-associated miRNAs.

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 121. doi:1538-7445.AM2012-121
Abstract 1215: Orexin and their 7-membrane spanning receptors OX1R: a new colon cancer therapeutic target
Authors: Voisin Thierry , Firar Aadil El , couvineau alain , laburthe marc
Published: August 2012

Orexins are hypothalamic peptides involved in sleep/wake control. Unexpectedly, we have shown that orexins promote robust apoptosis in various colorectal cancer cells1. The cell death is mediated by the orexin 1 receptor (OX1R) through an original mechanism. OX1R, a G protein-coupled receptor class I (GPCR), is aberrantly expressed in primary colorectal tumors and liver metastases2. We next investigated the molecular mechanism of OX1Rmediated cell death. OX1R makes use of “tyrosine-based motifs” ITIM (Immunoreceptor Tyrosine-based Inhibitory Motif)3 and ITSM (Immunoreceptor Tyrosine-based Switch Motif)4 to drive apoptosis. These “tyrosine-based motifs” are mandatory for recruitment and activation of the tyrosine phosphatase SHP-2, leading to apoptosis. Orexins promote cell death through Mitogen-/Stress-activated Protein Kinase (MAPK/SAPK) p38 activation in a SHP-2 dependent manner. The present work is original for two reasons: 1) OX1R is the first example of the involvement of both ITSM and ITIM in GPCR signal transduction. ITIM and ITSM sequences are more frequent than initially thought in GPCRs. This emphasizes the need to explore this mechanism of signal transduction in various GPCRs for regulating cellular processes including at least cell apoptosis and proliferation; 2) we provide evidence that OX1R is aberrantly expressed in all primary colorectal tumors and liver metastases and that its activation by exogenous orexins result in robust apoptosis of colon cancer cells in culture and strong decrease of tumor development in mice xenografted with colon cancer cells in vivo. OX1R represent a new promising target in colorectal cancer therapy and/or for metastases imaging. 1 J.Biol.Chem, 279: 45875 (2004) 2 Can. Res. 71: 3341 (2011) 3 FASEB J. 22: 1993 (2008) 4 FASEB J. 23: 4069 (2009)

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 1215. doi:1538-7445.AM2012-1215
Abstract 1221: Microscopy based high throughput screen for the mechanistic analyses of ErbB2 degradation in response to Hsp90-targeted therapeutics
Authors: Bailey Tameka A. , Luan Haitao , Wells Amy L. , Kelly David L. , Naramura Mayumi , Raja Srikumar , Band Vimla , Band Hamid
Published: August 2012

ErbB2 expression is observed in 25 - 30 % of invasive breast cancers and is associated with reduced response to chemotherapy, and shorter time to relapse and reduced overall survival of breast cancer patients. Malignant mammary epithelial cells that overexpress ErbB2 exhibit enhanced proliferation, invasion of mammary tissue and metastasis. The stability of overexpressed mature ErbB2 at the plasma membrane of mammary epithelial cells is critically dependent on its interaction with the chaperone protein, heat shock protein 90 (Hsp90). Thus, Hsp90 promotes the proliferation and survival of malignant mammary epithelial cells and interruption of this interaction is expected to inhibit the growth of ErbB2-overexpressing tumors. 17-AminoAllyl-Geldanamycin (17-AAG), an Hsp90 inhibitor, is a semi-synthetic ansamycin antibiotic currently in phase II clinical trials for breast cancer treatment (www.clinicaltrials.gov). Because it preferentially binds to Hsp90 of the chaperone complex in malignant mammary epithelial cells, treatment of ErbB2-overexpressing breast cancer with 17-AAG is expected to destabilize ErbB2 and inhibit tumor growth. While a number of Hsp90 client proteins have been identified, ErbB2 is amongst the most sensitive to Hsp90 inhibition. These facts provide strong rationale for using 17-AAG as a part of the targeted therapy against ErbB2-overexpressing breast cancer. However the cellular machinery responsible for 17-AAG induced degradation of ErbB2 is not completely understood. At present it is unclear whether internalization of ErbB2 occurs via Clathrin-dependent or -independent endocytosis. Furthermore it is unresolved if the degradation of ErbB2 is dependent upon proteasomal or lysosomal proteolysis. And although C-terminus of Hsp70 Interacting Protein (CHIP) and Cullin 5 were identified as E3 ligases responsible for the ubiquitination of ErbB2, it is unknown if these ligases are the sole E3s responsible for the ubiquitnation of ErbB2. Also vague is if the rerouting of ErbB2 from the recycling pathway to the degradative pathway in response to 17-AAG is exclusively dependent upon ubiquitnation. In this current work we attempt to bridge these critical gaps in our knowledge of the mechanisms of 17-AAG induced degradation of ErbB2 using an automated microscopy-based analysis to identify genes whose siRNA-dependent suppression reduces 17AAG-induced ErbB2 degradation. We expect that this work will identify additional targets for therapeutic intervention and potential biomarkers of response to breast cancer drug therapy with Hsp90 inhibitors.

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 1221. doi:1538-7445.AM2012-1221
Abstract 1229: Fibroblast growth factor signaling in ovarian cancer
Authors: Brantner Manuel , Wagner Renate , Grusch Michael , Berger Walter , Holzmann Klaus , Marian Brigitte , Grunt Thomas W.
Published: August 2012

Epithelial ovarian cancer (OC) is a rapidly progressive, highly lethal disease. It is the fifth leading cause of cancer-related death among women. Over the last decades, there has been only modest improvement in the treatment of OC. Nevertheless, recent clinical trials with inhibitors of transmembrane receptor tyrosine kinases such as fibroblast growth factor (FGF) receptors (FGFRs) yielded some promising results in OC. FGFRs are involved in malignant transformation, angiogenesis and chemoresistance. Inhibition of FGFR-dependent signaling can overcome resistance to standard therapies. Thus, FGFRs represent potential targets for cancer therapy. Unfortunately, however, the biological and therapeutic relevance of the FGF-FGFR system has not been elucidated yet in OC. The aim of this study was to characterize the FGF-FGFR system, its influence on malignancy-related cell properties and its potential role as a druggable signaling pathway in OC cells. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that expression of FGFs and FGFRs, leading to autocrine signaling loops with a strong mitogenic potential, is a common event in OC. Immunoblotting proved that the FGF signaling system is functionally active in these cells. Conditioned growth medium was able to induce phosphorylation of ERK1/2. Moreover, recombinant FGF-2 significantly elevated the growth rate in approximately 50% of the OC cell lines as demonstrated by formazan dye assay, whereas FGF-1 markedly induced migration in 66% of the cell lines. The dependency of OC cells on FGFR signaling was further evaluated by growth inhibition assays using the two small molecular FGFR inhibitors PD173074 and dovitinib (TKI-258, CHIR-258). A-2780 cells were found to be extremely sensitive to FGFR inhibition, while HEY and SKOV-3 cells showed moderate sensitivity. In contrast, OVCAR-3 cells were not inhibited by these compounds. Together, our data suggest that FGFR inhibitors reveal anticancer activity in a significant proportion of OC cells even as single drugs. Thus, combination of conventional cytotoxic intervention with blockade of the FGF-FGFR system might represent a promising future strategy for the treatment of OC.

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 1229. doi:1538-7445.AM2012-1229
Abstract 1230: Syntaxin 6- and microtubule-mediated intracellular trafficking contributes to Golgi and nuclear translocation of EGFR
Authors: Du Yi , Shen Jia , Hsu Jennifer L. , Han Zhenbo , Wang Ying-Nai , Yamaguchi Hirohito , Miller Stephanie A. , Hung Mien-Chie
Published: August 2012

Receptor mediated endocytosis is well known for its degradation and recycling trafficking. Recent evidence shows that these cell surface receptors translocate from cell surface to different cellular compartments, including the Golgi, mitochondria, endoplasmic reticulum (ER) and the nucleus to regulate physiological and pathological functions. Although some trafficking mechanisms have been resolved, the completed mechanism of intracellular trafficking from cell surface to the Golgi is still unknown. Herein, we report a mechanism of the Golgi translocation of EGFR in which EGF-induced EGFR travels to the Golgi via microtubule (MT)-dependent movement by interacting with dynein and fuses with the Golgi through syntaxin 6 (Syn6) mediated membrane fusion. We also demonstrate that the Golgi translocation of EGFR is necessary for its consequent nuclear translocation and transcriptional activity. Interestingly, foreign protein such as bacterial cholera toxin, which is known to activate its pathological function through the Golgi/ER retrograde pathway, also utilizes the MTs/Syn6 pathway. Thus, the MTs/Syn6/Golgi/ER pathway defines a comprehensive retrograde trafficking route for both cellular and foreign molecules to travel from cell surface to the Golgi and the nucleus.

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 1230. doi:1538-7445.AM2012-1230
Abstract 1246: ELK-1 regulates interferon-alpha8 expression via a polymorphic region in the interferon-alpha8 promoter associated with outcome of glioma patients
Authors: Kohanbash Gary , Ishikawa Eiichi , Fujita Mitsugu , Mckaveney Kayla , Sakaki Masashi , Scheurer Michael , Bondy Melissa , Sarkar Saumendra Narayan , Okada Hideho
Published: August 2012

Several studies have demonstrated a significant immunological impact of single nucleotide polymorphisms in Interferon (IFN)-related genes, such as Toll-Like Receptor (TLR)3 and TLR4. We recently reported that patients with the AA-genotype of a SNP in the IFN-alpha8 (IFNA8) promoter region was associated with better overall survival of patients with WHO grade 2 and 3 gliomas compared to patients with the AC-genotype. Based on these observations we hypothesized that the A-allele in the promoter of IFNA8 allows for enhanced transcription factor binding and expression levels of IFNA8 compared with the C-allele. Analyses of THP-1 cells transfected with a luciferase gene downstream of a short sequence containing the SNP in the IFNA8 promoter demonstrated that the A-allele results in enhanced promoter activity. In silico analysis suggested c-Krox and ELK-1 as likely transcription factors that bind to the IFNA8 polymorphic region. Co-transfection of plasmids encoding the c-Krox or ELK-1 and the luciferase constructs revealed that ELK-1 negatively regulated promoter activity with the A but not C-allele, while c-Krox could not affect activity regardless of allele. Consistently, transfection of ELK-1 siRNA into THP-1 cells resulted in increased promoter activity. To further identify factors that contribute to the enhanced A-allele promoter activity we assessed the effect drugs that inhibit intracellular signaling pathways: PD98059 for inhibiting MEK/ERK, SB203580 a p38 inhibitor, and PPase-2B for de-phosphorylating small molecules. Use of any of the inhibitor resulted in decreased activity of the A-allele promoter activity indicating that multiple proteins or a protein common to multiple pathways seems to be involved. Taken together our data demonstrates that the A-allele in the IFNA8 promoter, which is associated with better glioma patient survival, allows for enhances promoter activation which is negatively regulated by ELK-1 and further regulate by multiple signaling pathways.

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 1246. doi:1538-7445.AM2012-1246
Abstract 1250: Crosstalk between Notch and MAPK signaling regulates proliferation in papillary thyroid cancer
Authors: Yamashita Alex S. , Geraldo Murilo V. , Kimura Edna T.
Published: August 2012

Background: RET/PTC, RAS and BRAF are present in about two thirds of papillary thyroid carcinomas (PTC), activating constitutively the mitogen activated protein kinase (MAPK) signaling pathway. There is practically no overlap between RET/PTC, RAS and BRAF mutations in PTC, the lack of concordance for these mutations provides compelling genetic evidence for the requirement of this signaling system for transformation to PTC. However, the effect of these genetic alterations in other signaling pathways remains unclear, which could, at least in part, cooperates in transformation. Recently, Notch signaling has been implicated in tumorigenesis in other cancer types. In this study, we test the hypothesis that RET/PTC and BRAFT1799A oncogenes crosstalks and regulates Notch pathway. Methods: TPC-1 and BCPAP (human PTC cells harboring RET/PTC1 rearrangement and BRAFT1799A mutation, respectively) were treated with gamma secretase inhibitor (GSI) and proliferation was analyzed by MTT assay and growth curve; cell cycle and apoptosis were analyzed by flow cytometry. PCCL3 cells harboring Doxycycline-inducible RET/PTC3 and BRAFT1799A (PC-BRAF) were used to analyze protein and gene expression by Western blotting and real-time PCR, respectively. Notch1 protein expression was analyzed in transgenic mice (Tg-BRAF) which harbor targeted expression of BRAFT1799A restricted to thyroid gland. Results: PTC cells treated with GSI decreased the percentage of viable cell in a dose-dependent manner and reduced the growth rate. Cell cycle analysis in PTC cells showed enhancement of DNA fragmentation and increased apoptotic cells. Furthermore, the GSI treatment in PTC cells modulates cell cycle related genes, increasing CDKN1A and CDKN1B gene expression and reducing CCND1, MCM6, CKS2, and MADL2 expression. Conditional activation of RET/PTC3 and BRAFT1799A in PTC3-5 and PC-BRAF cells, respectively, showed a sustained enhancement in pERK protein expression, increased Notch1 protein and gene expression and Hes1 gene expression, the target gene of Notch signaling. Furthermore, transgenic mice Tg-BRAF showed a stronger stain for Notch1 in follicular and stromal cells. Treating PTC cells with MEK inhibitors, U0126 or PD96059, reduces NOTCH1 and HES1 gene expression. The combination of GSI with MEK inhibitor enhances the growth suppression promote by MAPK signaling inhibition in PTC cell proliferation. CONCLUSION: Taken together, targeting Notch signaling resulted in anti-proliferative effect, suggesting an important role of Notch signaling in tumorigenesis of MAPK-induced PTC and our results indentify Notch signaling as a potential therapeutic target in thyroid 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 1250. doi:1538-7445.AM2012-1250
Abstract 1253: Fer is required for mammary tumorigenesis
Authors: Greer Peter A. , Chi Changnian , Gao Yan , LeBrun David , Golbourn Brian , Sangrar Waheed
Published: August 2012

Fer is a ubiquitously expressed cytoplasmic protein-tyrosine kinase (PTK) which is inducibly activated upon engagement of the epidermal and platelet-derived growth factor receptors (EGFR/PDGFR). Fer - and its homologous family member Fps/Fes - are structurally distinguished from other PTKs by a membrane-binding F-BAR domain implicated in regulating membrane-cytoskeletal dynamics during receptor endocytosis. We are investigating the role of Fer in EGFR internalization using both non-transformed mammary epithelial cells (MCF10A) and mammary tumor epithelial cells (MTEC) isolated from an ErbB2 mouse model of breast cancer. MCF10A Fer-knockdown cells (MCF10A-F2) and MTECs harboring a kinase-inactivating knock-in mutation at the fer locus (ferDR/DR) displayed elevated rates of EGF-induced endocytosis suggesting that Fer inhibits EGFR internalization. Elevated endocytic rates correlated with enhanced short-term MAPK signaling in both cell culture models and elevated steady-state Erk activity in ferDR/DR ErbB2 mammary tumors. Systems-level analyses using a physiochemical model of EGFR-MAPK signaling suggested that up-regulation of receptor endocytic rates could potentiate MAPK signal strength and sensitize this pathway to pharmacological inhibition at the receptor level. Inhibition of MAPK signaling with the EGFR/ErbB2 inhibitor, Lapatinib, confirmed this prediction, showing increased Erk signal-sensitivity to this drug in endocytosis-enhanced ferDR/DR MTECs. This correlated with a 10-fold reduction in the EC50 for Lapatinib cytotoxicity on ferDR/DR MTECs. Both the signaling and cytotoxicity phenoytypes were reversible by rescued expression of wild type Fer in ferDR/DR MTECs. These data reveal a novel mechanism by which potentiation of EGFR endocytosis enhances the sensitivity of downstream signaling to inhibition by ErbB inhibitors. In this respect, Fer is a unique kinase target because it provides a direct means of pharmacologically intervening with the mechanism of EGFR endocytosis. The therapeutic significance of potentiating receptor endocytosis by targeting Fer is highlighted by observations of: (1) delayed onset of ErbB2-mediated breast tumorigenesis in ferDR/DR genetic backgrounds in vivo; and; (2) impaired tumor growth of Fer-deficient MDA-MB-231 cells in mammary-fat pad transplantation models. We envisage that anti-Fer based treatments may sensitize breast - and potentially other carcinoma types - to anti-ErbB therapies. These observations provide biological proof-in-principle for developing small molecule inhibitor-based therapies against Fer in 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 1253. doi:1538-7445.AM2012-1253
Abstract 1275: Quantitative proteomic analysis of tumor growth associated proteins in cutaneous malignant melanoma
Authors: Serada Satoshi , Kotobuki Yorihisa , Tanemura Atsushi , Katayama Ichiro , Naka Tetsuji
Published: August 2012

BACKGROUND: The identification of proteins responsible for tumor development and regulation is of great value. In this study, we aimed to identify novel functional proteins associated with tumor progression in malignant melanoma. MATERIAL AND METHODS: The proteins extracted from malignant melanoma and normal skin tissue were labeled with iTRAQ reagent and performed a quantitative mass spectrometric analysis using nano LC-MS/MS system. The detected protein expression and localization in cutaneous malignant melanoma tissue was confirmed by immunohistochemistry. Human melanoma cell line (Mewo) was subcutaneously injected into Rag2-/- mice and POSTN-/-Rag2-/-mice, thereafter implanted tumor growth was compared. RESULTS: Among 1314 identified proteins, 118 proteins were increased more than 5.0-fold and 37 proteins were decreased <0.5-fold in malignant melanoma compared to normal tissue. In these proteins, we found significant upregulation of an extracellular matrix protein, periostin (POSTN). POSTN expression was robustly induced in fibroblast cultured with Mewo and evident on the stroma of advanced melanoma tissue, suggesting the importance of interaction of these cells. As an assessment of functional implication in POSTN in malignant melanoma, we observed significant increase in melanoma cell number in the treatment with recombinant POSTN. Moreover, tumor growth in POSTN-/-Rag2-/- mice was significantly suppressed compared with that in Rag2-/- mice.CONCLUSIONS: We newly identified upregulation of POSTN expression in malignant melanoma tissue. POSTN was induced in the co-culture of fibroblast with melanoma cell and showed the growth promoting effect. These results indicate that periostin might be a potential therapeutic target in malignant melanoma.

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 1275. doi:1538-7445.AM2012-1275
Abstract 1277: Antigen-specific markers for paraneoplastic neurological syndromes obtained by proteomic analysis of patient immunoglobulins
Authors: vanDuijn Martijn M. , Maat Peter , Dekker Lennard J. , Luider Theo M. , Smitt Peter A. Sillevis
Published: August 2012

In the antibody response, as occurs in antitumor immunity, germline sequences are rearranged and mutated to generate a sequence with specificity for an antigen. In earlier work, it was shown that the antigen specificity of immune sera from rats can be distinguished by the proteomic analysis of immunoglobulins. It is hypothesized that the sequence of the antigen binding sites converges to a similar subset in individuals exposed to a given antigen. We therefore tested the applicability of our approach in patients with Paraneoplastic Neurological Syndrome (PNS). In these patients, a tumor triggers a well-characterized auto-immune response against onconeural antigens expressed in tumor and the affected part of the nervous system. These antibodies serve as markers for both the underlying tumor and specific neurological syndromes. Sera of 60 patients with PNS associated with paraneoplastic antibodies were analysed: 20 anti-HuD (18 lung cancer), 20 anti-Yo (6 breast, 9 gynecological and one other cancer), 10 anti-amphiphysin (4 lung, one breast and one other cancer), and anti-CV2 (6 lung cancer) antibodies. Antigen-specific immunoglobulins were collected by affinity enrichment on beads coated with recombinant antigen. The antigen-specific immunoglobulins were digested with trypsin and analyzed by nano-LC and mass spectrometry. From the resulting dataset, peptides were selected that uniquely identified one of the patient groups, and the peptide sequence was deduced from fragmentation spectra. 28 specific and unique peptides were found; 0 specific for amphiphysin, 2 specific for CV2, 11 for HuD and 15 for Yo. Several marker peptide sequences showed homology to known immunoglobulin sequences, and no relations were found to other known proteins from the NCBInr protein database. The data show that immunoglobulin-derived biomarkers can indeed be found in samples from patients and can serve as early markers of cancer and auto-immunity.

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 1277. doi:1538-7445.AM2012-1277
Abstract 1278: Determining the proteomic changes that define progressor and non-progressor clear cell renal cell carcinoma
Authors: Kurban Ghada , Tsavachidou Dimitra , Gallie Brenda , Evans Andrew , Finelli Antonio , Jonasch Eric , Jewett Michael
Published: August 2012

Renal cell carcinoma (RCC) is the most common type of kidney cancer, is increasing in incidence and is the most lethal genitourinary cancer. Due to the increased use of abdominal imaging, small renal masses (SRMs), mostly RCC, are now commonly detected in asymptomatic individuals and are the most common presentation of kidney cancer. Many of these SRMs grow slowly or not at all, while about 30% progress to advanced stages. However, in the absence of factors distinguishing masses that need treatment from those that can be managed by surveillance alone, most SRMs are treated by surgery. Clear cell RCC (ccRCC) is the most common form of RCC, has the worst prognosis and is characterized by inactivation of von Hippel-Lindau tumor suppressor gene (VHL). Systemic therapies developed to treat patients at advanced stages have targeted pVHL regulated genes particularly hypoxia inducible factor (HIF) downstream targets. However, these drugs rarely achieve cures suggesting that other pathways are implicated in ccRCC initiation and progression. Our national clinical trial, “Active Surveillance of Small Renal Masses” provides a unique opportunity to study the genetics and biology of the early stage ccRCC and characterize the genomic and proteomic signatures distinguishing non-progressor from progressor ccRCC-SRMs. Patients with ccRCC-SRMs (<4cm in size) are followed with time and if their mass progresses, they undergo surgical or other forms of intervention as appropriate. In this study, we determine the proteomic differences between a) non-progressor and progressor ccRCC-SRMs by characterizing biopsies taken at the time of diagnosis and recruitment into the surveillance protocol from both groups, and b) progressor ccRCC-SRM (at time of recruitment) and advanced ccRCC by comparing the initial, diagnostic biopsy of ccRCC-SRMs that subsequently progress to the surgical specimen at the time of treatment for progression (advanced stage). Using reverse phase protein array (RPPA), we identify proteomic signatures that distinguish progressor and non-progressor groups. We observed changes mostly in the PI3 kinase and ERK pathways as well as alterations in apoptotic pathways. Our results provide important insight for management of ccRCC-SRMs and define pathways involved in their initiation and progression. This could furthermore identify potential therapeutic targets that would lead to better management of ccRCC 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 1278. doi:1538-7445.AM2012-1278