bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4945: Epigenetic silencing of microRNA-34b/c in human gastric cancer
Authors: Suzuki Hiromu , Yamamoto Eiichiro , Nojima Masanori , Tokino Takashi , Imai Kohzoh , Toyota Minoru , Shinomura Yasuhisa
Published: February 2011

Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that some miRNAs are silenced in association with CpG island hypermethylation in cancer. To identify epigenetically silenced miRNAs in gastric cancer (GC), we screened for miRNAs induced by 5-aza-2′-deoxycytidine (DAC) and 4-phenylburtyrate (PBA) treatment in GC cell lines. By using miRNA microarray (Agilent technologies), we analyzed a panel of 470 miRNAs in MKN74 and AGS cells treated with or without DAC and PBA, and found that 172 and 151 of the miRNAs were significantly upregulated (>5-fold) by the drug treatment, respectively. Among them, we noted miR-34b and miR-34c, which are direct targets of p53, and are frequently silenced in colorectal cancer. Here, we show that miR-34b and -c are frequently downregulated in GC in association with DNA hypermethylation. Reporter assay revealed strong promoter activity in the upstream CpG island of miR-34b/c. Methylation of the CpG island was frequently observed in GC cell lines (13/13, 100%) and primary GC specimens (83/118, 70%), but not in normal gastric mucosa. By in situ hybridization analysis, we observed downregulated expression of miR-34b in primary GC specimens. Methylation of the CpG island was also detected in the non-tumorous gastric mucosa of the GC patients. Notably, quantitative methylation analysis by bisulfite-pyrosequencing revealed higher methylation levels in gastric mucosa with multiple GCs than those with single GCs. Transfection of precursor molecules of miR-34b or miR-34c into GC cells suppressed cellular growth. Gene expression microarray analysis revealed that miR-34b or miR-34c induced dramatic changes in the gene expression profile in GC cells. Particularly, “cell cycle” and “M phase” genes are significantly enriched among the genes downregulated by miR-34b/c. Our results suggest that miR-34b/c are novel tumor suppressors in GC, and that methylation of miR-34b/c CpG island might be a useful marker to predict GC risk.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4945.
Abstract 4949: Epigenetic silence of ASPP1 and ASPP2 genes promotes tumor growth in HBV-positive hepatocellular carcinoma
Authors: Zhao Jian , Guo Yajun
Published: February 2011

The Ankyrin-repeat-, SH3-domain- and proline-rich-region containing protein (ASPP) family of proteins regulates apoptosis through interaction with p53 and its family members. This study evaluates the epigenetic regulation of ASPP1 and ASPP2 in HBV-positive hepatocellular carcinoma (HCC) and explores the effects of down-regulation of ASPP1 and ASPP2 on the development of HCC. The expression of ASPP1 and ASPP2 was diminished in HCC cells by epigenetic silence owing to hypermethylation of ASPP1 and ASPP2 promoters as detected by methylation-specific PCR (MS-PCR) and 5-Aza-2′-Deoxycytidine treatment. Analyses of 51 paired HCC and surrounding non-tumor tissues revealed that methylation of ASPP1 and ASPP2 was associated with the decreased expression of ASPP1 and ASPP2 in tumor tissues (38.1% vs 6.7% in ASPP1, P = 0.018, and 50% vs 15% in ASPP2, P = 0.012). The correlations of the expression and the methylation of ASPP1 and ASPP2 with p53 gene status were further analyzed. HCCs harboring wild type p53 gene were more frequent to have decreased ASPP2 expression (P = 0.028). Whereas no statistic significance was found between down-regulation of ASPP1 and p53 gene status (P = 0.704). Methylation of ASPP1 and/or ASPP2 in the surrounding non-tumor tissues was closely related with the tumor size (P = 0.031) and the tumor stage (P = 0.010). This result indicates that methylation of ASPP1 and ASPP2 may participate in the early stage of neoplasia. Moreover, ASPP2 became methylated upon HBV X protein (HBx) expression. The expression of HBx enhanced the recruitment of DNMT1 and DNMT3A on ASPP2 promoter to initiate DNA methylation, and subsequent increased the binding of methyl-CpG binding proteins on its promoter to suppress ASPP2 expression. The suppressive effects on tumor growth by ASPP1 and ASPP2 were examined with RNA interference mediated gene silence. Down-regulation of ASPP1 and ASPP2 promoted the growth of HCC cells in soft agar and in nude mice, and decreased the sensitivity of HCC cells to apoptotic stimuli. In conclusion, ASPP1 and ASPP2 genes are frequently down-regulated by DNA methylation in HBV-positive HCC, which may play important roles in the development of HCC. These findings provide a new insight into the molecular mechanisms leading to hepatocarcinogenesis and may have potent therapeutic applications.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4949.
Abstract 4955: Advances in gene expression microarray profiling from small amounts of adenocarcinoma and normal total RNA samples
Authors: Lucas Anne Bergstrom , Lin Gary , Mullinax Becky , Jacobs Sharoni , Fulmer-Smentek Stephanie
Published: February 2011

Gene expression profiling by microarray analysis provides an important avenue for understanding biological mechanisms, classifying tissue and tumor types, and identifying signs for diagnosis and prognosis. To address the need for high sensitivity gene expression profiling of low quantities of total RNA, we have developed a modified linear amplification procedure that generates quantities of Cy-labeled cRNA suitable for oligonucleotide microarray experiments from total RNA input amounts as low as 10 nanograms. This new procedure, available in the new Low Input Quick Amp Labeling Kit, employs the AffinityScript Reverse Transcriptase, a mutant MMLV-RT that binds primer-template complexes with 10-fold higher efficiency than wild type MMLV-RT, resulting in increased cDNA yields and improved sensitivity from smaller sample inputs. The protocol uses a single round of IVT amplification without purification of the cDNA product resulting in labeled cRNA in less than one day, enabling gene expression profile comparisons in less than two days. Comparisons of probe signals from technical replicate samples demonstrate high reproducibility with wide dynamic ranges, and generally comparable signals across a broad range of input amounts. This new labeling approach was used to detect differences in gene expression between cancerous and normal cells using new gene expression arrays with updated content resulting in gene expression profiling results consistent with the current literature.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4955.
Abstract 4962: ERG expression levels in prostate tumors reflect functional status of the androgen receptor as a consequence of fusion of ERG with AR regulated gene promoters
Authors: Mohamed Ahmed A. , Dobi Albert , Sun Chen , Shaheduzzaman Syed , Tan Shyh-Han , Petrovics Gyorgy , Hu Ying , Chen Yongmei , Furusato Bungo , Sesterhenn Isabell , Sreenath Taduru , McLeod David G. , Srivastava Shiv
Published: February 2011

Background: Androgen dependent overexpression of the TMPRSS2:ERG fusion is the most prevalent chromosomal rearrangement in prostate cancer. During our recent evaluations of the ERG downstream transcriptional targets, we identified 15-Hydroxyprostaglandin Dehydrogenase gene (HPGD) in response to ERG knock-down. HPGD is an enzyme that catalyzes the oxidation of prostaglandins and lipoxins by converting them into inactive keto-metabolites, and physiologically antagonizes COX-2, a prostaglandin-synthesizing enzyme. HPGD also behaves as a tumor suppressor in many cancers as overexpression of HPGD resulted in marked inhibition of cell growth. The objective of this study was to evaluate potential connection between ERG overexpression and prostaglandin signaling.

Experimental procedures: Small interference RNA (siRNA) oligo duplex was used to knock-down endogenous ERG and HPGD expressions. Western blot assay and Immunofluorescence was used to detect protein expressions. QT-PCR (normalized to GAPDH) was used for transcriptome analysis. GeneChip, Chromatin immunoprecipitation assays and cell proliferation assay were utilized for functional analysis.

Results: ERG knock-down in VCaP prostate tumor cells results in overexpression of HPGD, down regulation of the prostaglandin E2 receptor 4 (EP4), and inhibition of prostaglandin E2 induced Urokinase Plasminogen Activator (uPA) expression and cell growth. Comparison of HPGD between TMPRSS2:ERG positive and negative prostate tumors revealed a trend towards decreased HPGD expression in fusion positive tumors.

Conclusions: Taken together, these data suggest that ERG over-expression in CaP cells may positively influence prostaglandin mediated signaling. Funding sources: NIH Grants RO1 DK065977

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4962.
Abstract 4964: Transcriptional repression of MYCN is mediated by TAp63 in human neuroblastomas
Authors: Suenaga Yusuke , Kaneko Yoshiki , Yamamoto Mami , Matsumoto Daisuke , Ichikawa Tomoe , Moritan Toshiyuki , Kamijo Takehiko , Nakamura Yohko , Ohira Miki , Ozaki Toshinori , Nakagawara Akira
Published: February 2011

p53 is one of the major tumor suppressors and promotes apoptotic cell death by transcriptional activation of pro-apoptotic target genes. In malignant human tumors, however, its tumor suppressive activity is frequently inactivated by somatic mutation and/or chromosomal deletion. To enhance the efficacy of chemotherapeutic agents in p53-deficient tumors, it is necessary to elucidate the p53-independent mechanisms of apoptotic cell death.

Recently, we have shown that TATA-binding protein-like protein (TLP) transactivates one of p53 family members, TAp63, and induces apoptosis in a p53-independent manner. In response to DNA damage by etoposide, TLP is induced to translocate from cytoplasm to nucleus and enhance promoter activity of TAp63 through direct recruitment onto the promoter. TAp63 stimulated by TLP promotes apoptotic cell death in Hep3B cells null for the p53 alleles. The expression levels of TLP mRNA in human primary non-small lung cancers is low compared with corresponding normal tissues and the downregulation is significantly correlated with low levels of TAp63 and poor outcome of patients’ survival. These results suggest the potential for TLP-TAp63 pro-apoptotic pathway as a therapeutic target for malignant lung cancers.

In this study, we analyzed expression of p53 family genes in 106 primary neuroblastomas using quantitative real time RT-PCR, and found that the levels of only TAp63 expression was significantly high in favorable neuroblastomas as compared with unfavorable tumors (p=0.00696) and that its expression was inversely correlated with MYCN expression (p=0.0350). MYCN oncogene is frequently amplified and overexpressed in unfavorable neuroblastoma, and in vitro studies have demonstrated that downregulation of MYCN is the critical steps for neuronal cell death induced by retinoic acids (RA) regardless of p53 status in tumors. However, the precise molecular mechanisms of the down-regulation of MYCN by RA have not been elusive. Intriguingly, TLP and TAp63 are induced in response to RA treatment and TAp63 directly represses MYCN transcription. In addition, RA treatment also inhibits positive auto-regulation of MYCN, leading to further down-regulation of MYCN mRNA levels.

Collectively, our results suggest that TAp63 is highly expressed in favorable neuroblastomas and contributes to repression of MYCN by RA treatment.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4964.
Abstract 4976: Crif1, a new negative regulator of Nrf2
Authors: Bae Insoo , Kang Hyo Jin , Kim Hee Jeong , Hong Yong Bin , Chang Young Soo
Published: February 2011

Abstract

Free radicals generated by oxidative stress cause damage that can contribute to numerous chronic diseases. Mammalian cells respond to this damage by increased transcription of cytoprotective phase II genes, which are regulated by Nrf2. Previously, it has been shown that Nrf2 protein levels increase after oxidative stress because its negative regulator, Keap1, loses its ability to bind Nrf2 and cause its proteasomal-mediated degradation during oxidative stress. Here, we show that Crif1 is also able to negatively regulate Nrf2 protein stability. However, and in contrast to Keap1, which regulates Nrf2 stability only under normal reducing conditions, Crif1 regulates Nrf2 stability, accumulation and target gene expression under both reducing and oxidative stress conditions. Thus, Crif1-Nrf2 interactions and their consequences are redox-independent. In addition, Crif1, but not Keap1 (which only interacts with N-terminal region of Nrf2), physically interacts with both N- and C-terminal regions of Nrf2 and promote Nrf2 ubiquitination and proteasomal-mediated degradation. Finally, Crif1 can modulate cells’ sensitivity to oxidative stress-induced cytotoxicity.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4976.
Abstract 4980: Myocardin functions as an effective inducer of growth arrest and differentiation in human leiomyosarcoma cells
Authors: Kimura Yasunori , Morita Tsuyoshi , Hayashi Ken'ichiro , Miki Tsuneharu , Sobue Kenji
Published: February 2011

Myocardin is an important transcriptional regulator in smooth and cardiac muscle development. We noticed that the expression of myocardin was markedly downregulated in human uterine leiomyosarcoma cells. Restoration of myocardin expression induced the re-expression of smooth muscle marker proteins and the formation of well-developed actin fibers. A concomitant increase in the expression of a CDK inhibitor, p21, led to a significant slowing of cell proliferation, via p21's inhibition of the G1/S transition. A p21 promoter-reporter assay showed that myocardin markedly increased p21's promoter activity. Furthermore, a serum response factor (SRF)-binding cis-element CArG-box in the p21 promoter region was required for this myocardin effect. Chromatin immunoprecipitation and DNA-protein binding assays showed that myocardin indirectly bound to the CArG-box in the p21 promoter through an interaction with SRF. Furthermore, immunohistochemistry revealed that the levels of myocardin and p21 were both lower in leiomyosarcoma samples than in normal smooth muscle tissue. Taken together, our results indicate that the downregulation of myocardin expression facilitates cell-cycle progression via the reduction of p21 expression in human leimyosarcomas, and suggest that myocardin could be a useful therapeutic target for this disease.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4980.
Abstract 4994: Association of protein inhibitor of activated STAT protein 4 with the development of pancreatic ductal adenocarcinoma identified by chromosomal analysis from single nucleotide polymorphism arrays
Authors: Chien Wenwen , Kawamata Norihiko , Okemoto Kazu , Ogawa Seishi , Koeffler H. Phillip
Published: February 2011

The purpose of this study is to identify novel genomic alterations in pancreatic ductal adenocarcinoma (PDAC) by single nucleotide polymorphism arrays (SNP-chip). Genomic DNA from 12 tissues and 15 cell lines of PDAC was examined using 250K SNP-chip. Regions with gains and losses of DNA copy number and loss of heterozygosity (LOH) were identified. Additional 10 normal and 6 tumor samples were used in quantitative real-time PCR to validate a subset of genes in the region with copy number alterations. Our results were compared with previously reported data. Frequent gains of chromosomes: 1q, 2, 3q, 5, 7, 8q, 11, 12p, 14, 17q and 20q (>50% of cases) and losses of 8p, 9p and 18q (>33% of cases) were detected. LOH regions were identified in chromosome arms: 6p, 6q, 8p, 9p, 9q, 13q, 17p, 18q, 19p, 19q and 21q (>25% of cases). On 19p LOH region, a common deletion occurred at 19p13 which included the gene known as protein inhibitor of activated STAT protein 4 (PIAS4). The deletion of the gene and expression levels of PIAS4 were correlated in the PDAC samples. The effects of exogenous over-expression of PIAS4 and short hairpin RNA (shRNA)-mediated knockdown of endogenous PIAS4 on cell proliferation and apoptosis were examined, as well as identification of cell signaling pathway linking PIAS4 and pancreatic tumorigenesis. Over-expression of PIAS4 decreased pancreatic cancer cell growth as well as decreased expression of genes involved in cell proliferation such as Bcl-2, Bcl-xL, cyclin D1 and XIAP. Activated PIAS4 decreased glycogen synthase kinase 3beta (GSK3beta) activity and inhibited transcriptional activity of nuclear factor kappa B (NFkB). Our data indicated that deletion of PIAS4 is associated with development of PDAC. Mechanistic studies showed that PIAS4 acted as a tumor suppressor gene and was a novel modulator of GSK3beta signaling in pancreatic tumorigenesis.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4994.
Abstract 5008: Loss of DEC1 (Deleted in esophageal cancer 1) during esophageal cancer progression and functional characterization of its downstream target
Authors: Wong Victor CL , Qi Robert Z. , Chan Kwok Wah , Li Ping Jun , Wang Li Dong , Li Ji Lin , Law Simon , Tsao Sai Wah , Lung Maria Li
Published: February 2011

Background and aims: Deleted in esophageal cancer1 (DEC1) is a frequently down-regulated candidate tumor suppressor gene in esophageal cancer (EC). We previously showed that this gene can suppress tumor growth and up-regulate Dual specificity phosphatase 6 (DUSP6), as a downstream target of DEC1, in esophageal squamous cell carcinoma (ESCC). In this study, we attempt to detect the expression of DEC1 during cancer progression and to identify subcellular localization of DEC1. We also evaluated the tumor suppressive role of DUSP6 in tumor development. Methods: As yet, there are no other published studies of DEC1 protein. Thus, DEC1-specific polyclonal antibody was generated and utilized for Western blotting, tissue microarray study, and immunostaining. We utilized cell functional assays such as colony formation, cell proliferation, and wound healing assays, as tools to evaluate tumor suppressive role of DUSP6. Results: Loss of DEC1 expression is observed in ESCC tumors, as compared to their matched normal counterparts in Western blot analysis. Significant loss of DEC1 during cancer progression was found. The expression of DEC1 is significantly related to cancer family history and age of patients. By immunostaining, DEC1 was localized to the cytoplasm, nucleus, and perinuclear region. In addition, we observed an important role of DUSP6 in suppressing cell growth and migration. Conclusions: We further validated the crucial role of DEC1 expression in EC tumorigenesis by use of tissue microarray. We identified subcellular localization of DEC1, providing a basis for further study of the tumor suppressive mechanism of DEC1. In addition, a tumor suppressive role of DUSP6 was confirmed.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5008.
Abstract 5012: REST is lost in aggressive breast cancer through an aberrant splicing loop
Authors: Wagoner Matthew P. , Gunsalus Kearney TW , Schoenike Barry , Dang Christina , Alexander Caroline M. , Richardson Andrea L. , Friedl Andreas , Roopra Avtar
Published: February 2011

Transcriptional profiling of breast tumors has lead to new diagnostic tools allowing better prediction of disease recurrence and response to treatment, but does little to elucidate mechanisms responsible for the differential disease course. Using transcriptional profiling we show for the first time that a subset of breast tumors lack the tumor suppressor RE1 silencing transcription factor (REST), a master repressor of neuronal genes. Loss of REST is sufficient to induce anchorage independent cell growth in human mammary epithelial cells, and we find that these REST-less tumors show a highly aggressive disease course independent of tumors grade, size or subtype. Importantly, these aggressive tumors overexpress the tumor promoter and REST target gene Lin28, which may be responsible for the aggressive phenotype of these tumors (Gunsalus et al. poster, AACR 2010 CB4). We find that loss of REST function occurs in breast cancer via alternative splicing of the tumor suppressor to a truncated form normally observed in the brain following seizures. Here, we show that REST regulates its own aberrant splicing in an intriguing positive-feedback loop that results in a loss of REST function. These results identify a previously unknown subset of aggressive cancers, the transcription factor responsible for unique molecular profile, and the novel positive feedback pathway responsible for the aberrant splicing.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5012.
Abstract 5022: Roles of endoplasmic reticulum factor ERLIN2 in malignancy maintenance and therapy resistance in breast cancer
Authors: Wang Guohui , Liu Gang , Ethier Stephen P. , Zhang Kezhong , Yang Zeng-Quan
Published: February 2011

Endoplasmic reticulum (ER)-associated degradation (ERAD) components are highly induced in various tumors and are associated with cancer cell survival and resistance to anti-cancer treatments. The ERAD pathway is crucial for the cell to adapt to ER stress by degrading misfolded, unassembled and even some normal proteins in the ER lumen. Recent work in our group has led us to identify the ER lipid raft-associated 2 (ERLIN2, also known as SPFH2, C8ORF2) gene as one of the candidate oncogenes within the 8p11-12 amplicon in breast cancer based on statistical analysis of copy number increase and over expression. ERLIN2 protein, which localized specifically to the ER, is the key component of ERAD pathway. To elucidate the oncogenic properties of the ERAD factor ERLIN2 in human breast cancer, we established the MCF10A-ERLIN2 cell line, which stably over expresses ERLIN2 in human nontransformed mammary epithelial cells (MCF10A) using the pLenti6/V5-ERLIN2 construct. ERLIN2 over expressing cells and their respective parental cell lines were assayed for alterations in growth rates, growth factor-independent proliferation and other phenotypes expressed by cancer cells. From these experiments, we found that over expression of ERLIN2 in MCF10A cells results in growth factor-independent proliferation in insulin-like growth factor deficient media and formed large, highly-proliferative, and abnormally-structured colonies in three-dimensional morphogenesis assays. However, over expression of ERLIN2 in MCF10A cells did not result in cells with anchorage independent growth potential. To further elucidate the molecular basis that ERLIN2 is linked to the ER-stress response pathways in breast cancer cells, we knocked down the ER stress sensor IRE1α activity in the breast cancer cell lines in which ERLIN2 is amplified and over expressed. The knockdown of the IRE1α RNase activity, but not the kinase activity, significantly reduced protein levels of ERLIN2 in ERLIN2 amplified breast cancer cells. In addition, forced expression of wild-type IRE1α or spliced XBP1, the IRE1α target, led to increased expression of ERLIN2 in MCF10A cells. We also found that ERLIN2 protein was induced in response to the ER stress-inducing reagents Tunicamycin, Thapsigargin or growth factor depletion. Together, our findings indicate that ERLIN2 plays an unconventional oncogenic role through modulating ER stress response and ERAD pathways in a subset of breast cancer. Targeted inhibition of ERAD factor ERLIN2 in cancer could provide potential new avenues for therapeutic development.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5022.
Abstract 5029: RasGRP3- a new therapeutic target in human melanoma
Authors: Yang Dazhi , Juan Tao , Kedei Noemi , Li Luowei , Yuspa Stuart , Blumberg Peter
Published: February 2011

RasGRP3, a RasGEF (Ras guanine nucleotide exchange factor), is an activator of H-Ras, R-ras and Rap1, Here, we report that its expression level was increased in multiple human melanoma cell lines, reaching a level of protein expression approaching that of Ramos cells in the case of the SK-MEL-5 melanoma line. Likewise, RasGRP3 expression was variably elevated in human melanoma tissue samples. The importance of RasGRP3 expression for the melanoma cell lines was demonstrated by down regulating its expression, which inhibited cell proliferation and blocked both colony formation in soft agar and xenograft tumor formation in immunodeficient mice in the case of both the M14 and SK-Mel-5 cell lines. Finally, RasGRP3 was involved in downstream signaling in these cells. Suppression of RasGRP3 expression reduced both basal and HGF induced AKT phosphorylation in the M14 and SK-Mel-5 cells. Consistent with these results, we found that overexpression of RasGRP3 in human primary melanocytes changed their morphology, enhanced cell proliferation, and caused xenograft tumor formation. Suppression of the RasGRP3 overexpression in these cells inhibited downstream RasGRP3 responses and suppressed cell growth, confirming the functional role of RasGRP3 in the altered behavior of these cells. We conclude that RasGRP3 represents a potential therapeutic target in melanoma. Even more generally, the high level of expression and functional role of RasGRP3 in melanoma emphasizes that this RasGEF may play a more general role in regulation of Ras family members than had been initially appreciated.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5029.
Abstract 5032: MK8745, an Aurora A kinase-specific inhibitor induces apoptosis in a p53-dependent manner
Authors: Nair Jayasree S. , Schwartz Gary K.
Published: February 2011

Aurora kinases are mitotic serine/threonine protein kinases and are attractive novel targets for anticancer therapy. Many small molecule inhibitors are being developed and few are currently undergoing clinical trials. Aurora A kinase is essential for the successful mitotic transition. MK8745 is a novel and selective small molecule inhibitor of Aurora A kinase. Treatment of tumor cells with MK8745 (1 to 5 µM) resulted in accumulation of cells in mitosis with an increase in Aurora A expression. Despite this increase in Aurora A in the cells, dose dependent inhibition of Aurora A kinase activity was confirmed by an in vitro kinase assay using a PLK1, a specific substrate of Aurora A kinase. Also, treatment of HCT116 (p53−/−) cells ectopically overexpressing wild type p53 with MK8745 showed a decrease in phospho p53 (ser315), another known substrate of Aurora A. We then explored the mechanism by which MK8745 induces proliferation inhibition in cell lines of multiple lineages such as colon, melanoma, sarcoma and pancreatic according to p53 status. In general, the cell lines expressing wild type p53 showed a short delay in mitosis followed by cytokinesis with significant apoptosis (15%) upon exposure to MK8745. However, cell lines with null or mutant p53 underwent endoreduplication resulting in 60% polyploidy after a prolonged delay in mitosis with no apoptosis. This was further confirmed in the isogenic HCT-116 cell lines such that HCT p53−/− cells underwent polyploidy, whereas HCT p53 +/+ and the HCT p21 −/− cells underwent apoptosis. Under all of these conditions phospho histone H3 (Ser10) was not inhibited, indicating these phenotypic effects are a result of Aurora A inhibition and not Aurora B. P53 dependence for apoptosis was also confirmed in p53 null cells overexpressing wild type p53. Transient knock down of Aurora A kinase by specific SiRNA resulted in high levels of apoptosis and again showed p53 dependence. These results suggest that determining the p53 status may be important when using Aurora A kinase targeted therapies in the clinic. Further studies are underway to determine the role of p53 in the induction of apoptosis that results from inhibition of Aurora A kinase.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5032.
Abstract 504: First in vitro radiobiological characterizations of cells from chordoma origin
Authors: Kato Takamitsu , Tsuda Akihisa , Fujimori Akira , Kamada Tadashi , Tsujii Hirohiko , Okayasu Ryuichi
Published: February 2011

Although chordoma, a rare bone cancer, has been treated with surgery and/or radiation including heavy ions, few in vitro characterizations of chordoma cells are available. The main reason for this is the extremely long doubling time associated with the only recognized cell line, U-CH1. Here, we derived a cell line (designated as U-CH1-N) from U-CH1 and made the in vitro characterization possible, and results with U-CH1-N were compared with those with HeLa (cervical cancer) and U87-MG (glioblastoma) cells. The cell doubling times for HeLa, U87-MG and U-CH1-N were 18 h, 24 h and 3 days respectively. U-CH1-N had the most DNA content among the three, was near tetraploid, and showed an abnormal karyotype. U-CH1-N and U87-MG were more sensitive to x-irradiation than HeLa cells. Heavy ion irradiation more efficiently killed all three cell lines than x-rays did. Relative biological effectiveness (RBE) at 10% survival for U87-MG and HeLa cells was about 2.5 for 70keV/μm carbon ions and 3 for 200keV/μm iron ions, while for U-CH1-N it was 2.5 for carbon and 4 for iron ions. Four different chemical agents producing various DNA lesions were used for further characterization of these cells. Although camptothecin, mitomycin C or cisplatin did not reveal strong cytotoxity to U-CH1-N compared with other cells, bleomycin, which produces DNA strand breaks, showed marked cytotoxic effect on U-CH1-N. Our data provide the first chronological cell survival information using cells of chordoma origin and also help explain the successful chordoma treatment by heavy ions.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 504.
Abstract 5041: Aspirin acetylates and activates p53 in HT-29 and HCT-116 colon cancer cells
Authors: Alfonso Lloyd F. , Chivukula Raghavender , Marimuthu Srinivasan , Gunaje Jayarama B.
Published: February 2011

Increasing evidences from human epidemiological studies, animal models and in vitro experiments suggest that regular use of aspirin may reduce the risk of cancer of the colon; however, the mechanisms are not well understood. Since aspirin's protective effect is mainly observed in epithelial cell types which are more resistant to chemotherapy, an urgent need exists to identify the primary targets and cancer preventive pathways affected by aspirin. We hypothesized that aspirin's anti-cancer effect may involve acetylation of the tumor suppressor protein p53 and modulation of target gene expression. In the present study, we demonstrate that exposure of HT-29 and HCT-116 colon cancer cells to aspirin caused acetylation of p53 at lysine 382 (K382). HT-29 cells harbor a mutant form of p53 (R273H), whereas HCT-116 contain wild type p53. Acetylated p53 was mainly localized to the nucleus. In both cell types aspirin induced acetylation of p53 in a concentration dependent manner, and this was associated with increased p53 DNA binding activity. Consistent with this, we also observed induction of p21, a protein involved in cell cycle arrest as well as Bax, a mitochondrial proapoptotic protein. Aspirin also caused acetylation of bacterially expressed recombinant p53 in vitro, suggesting that it is a non-enzymatic reaction. Our observation that aspirin acetylates p53 leading to induction of target gene expression suggest that its anti-cancer effect may involve activation of the p53 pathway. Since p53 is mutated in nearly 50% of all tumors which leads to loss of its function, our observation also suggests that aspirin may find use in chemotherapy to reactivate mutant p53.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5041.
Abstract 5047: Synergistic anti-cancer activity of the CDK4/6 inhibitor PD-0332991 in combination with 5-fluorouracil-based chemotherapy in human colon cancer cells
Authors: Pishvaian Michael J. , Yang Shaoxian , Zouhairi Majed El , Wu Christina S. , Mishra Lopa , Avantaggiati Maria-Laura
Published: February 2011

Background: The cell cycle activator cyclin-dependent kinase 4 (CDK4) is overexpressed in many cancers and is a promising target for novel anti-cancer therapies. PD-0332991 (Pfizer, Inc) is a highly specific CDK4/6 inhibitor that has proven anti-cancer efficacy in multiple pre-clinical models, and is currently undergoing Phase II clinical testing in humans. PD-0332991 inhibits tumor cell growth by inducing cell cycle arrest. Sequential therapy with PD-0332991 followed by DNA damaging cytotoxic chemotherapy could produce additive or synergistic anti-cancer effects. Our goal was to assess the anti-tumor efficacy of a combination of PD-0332991 and cytotoxic chemotherapy in vitro, and in vivo. We also sought to determine the mechanisms of anti-cancer activity seen with combination therapy.

Methods: First, using human colorectal cancer (CRC) cell lines, we performed in vitro cell growth assays to determine the effects of a combination of PD-0332991 and 5-fluorouracil (5-FU) or oxaliplatin. Next, we tested the activity of PD-0332991, oral capecitabine (an oral 5-FU prodrug), or both in mice bearing human CRC xenografts. Finally, we assessed the mechanisms of cell death in vitro and in resected xenograft tumor samples.

Results: The combination of PD-0332991 and 5-FU or oxaliplatin exhibited additive/synergistic anti-cancer activity, in vitro. Furthermore, synergistic anti-cancer activity was observed in mice bearing xenografts of HCT 116 human CRC cells upon treatment with a combination of PD-0332991 and capecitabine. Virtually no anti-tumor activity was observed in mice treated with PD-0332991 or capecitabine alone. In assessing the mechanism of anti-tumor activity, in vitro and in resected xenograft samples, no increase in apoptosis was observed, as determined by annexin V and TUNEL staining. Even when cells were synchronized in G1 with PD-0332991 and then released and treated with 5-FU or oxaliplatin, there was no increase in apoptosis. Thus, assessment of alternative mechanisms of cell death, including cell senescence and autophagy are ongoing.

Conclusions: Our results demonstrate an additive/synergistic anti-tumor activity with the combination of PD-0332991 and traditional cytotoxic chemotherapy. The mechanisms of anti-tumor activity have yet to be determined, but are not dependent upon apoptosis. Given the synergistic anti-tumor activity, these results suggest that a clinical trial of PD-0332991 and 5-FU-based chemotherapy is warranted.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5047.
Abstract 5057: The insulin-like growth factor receptor I promotes motility and invasion of bladder cancer cells through Akt- and MAPK-dependent activation of paxillin
Authors: Metalli David , Lovat Francesca , Tripodi Farida , Xu Shi-Qiong , Alberghina Lilia , Vanoni Marco , Baffa Raffaele , Iozzo Renato V. , Gomella Leonard G. , Morrione Andrea
Published: February 2011

The insulin-like growth factor receptor I (IGF-IR) plays an essential role in transformation by promoting cell growth and protecting cancer cells from apoptosis. Aberrant IGF-IR signaling is implicated in several types of tumors, including carcinomas of the lung, breast, prostate, pancreas, liver and colon. However, whether the IGF-IR contributes to the transforming phenotype of urothelial cells has not been clearly established but recent data suggest that the IGF-IR is over-expressed in bladder cancer.

In this study we demonstrated by immunohistochemical analysis that the IGF-IR is over-expressed in invasive bladder cancer (T3/T4) tissues compared to non-malignant controls. We have also characterized the mechanism of action of the IGF-IR in cancer urothelial cells using urothelial carcinoma-derived 5637 and T24 cells. Although activation of the IGF-IR did not appreciably affect their growth, it did promote migration and stimulate in vitro wound closure and invasion through extracellular matrix. These effects required the activation of the Akt and MAPK pathways and IGF-I induced Akt- and MAPK-dependent phosphorylation of paxillin, which relocated at dynamic focal adhesions at the protruding edge of migrating urothelial cancer cells. Using siRNA strategies we also demonstrated that paxillin was necessary for promoting IGF-I-mediated motility and invasion in bladder cancer cells.

In conclusion, our results provide the first evidence for a role of the IGF-IR in motility and invasion of bladder cancer cells, and support the hypothesis that the IGF-IR may play a critical role in the establishment of the invasive phenotype in urothelial neoplasia. The IGF-IR could represent a novel molecular target in bladder cancer and could also serve as a novel tumor biomarker for diagnosis and possibly prognosis of bladder tumors.

This work has been supported by the Benjamin Perkins Bladder Cancer Fund, the Martin Greitzer Fund and National Institutes of Health Grants RO1 DK068419 (A.M.) and RO1 CA39481 and RO1 CA047282 (R.V.I.).

§Present address: Medimmune, One Medimmune Way, Gaithersburg, MD 20878

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5057.
Abstract 5060: Downregulation of THAP11 by Bcr-Abl promotes CML cell proliferation through c-Myc expression
Authors: Yokota Daisuke , Nakamura Satoki , Takemura Tomonari , Hirano Isao , Ono Takaaki , Shigeno Kazuyuki , Fujisawa Shinya , Takeshita Kaori , Misawa Kiyoshi , Ohnishi Kazunori
Published: February 2011

[Background and Purpose] THAP11 (thanatos-associated protein 11) is one of the zinc-dependent, sequence-specific DNA-binding factors, which regulate cell proliferation, apoptosis and cell cycle. It has been reported that THAP11 is ubiquitously expressed in normal tissues and frequently downregulated in several tumors. In CML cells, we found that THAP11 expression was inhibited. We investigated the mechanisms of the suppression of THAP11 and its function in CML cell proliferation. [Methods] The cells used in this study were human CML cell lines, K562 and Meg01 cells. Primary CML cells were obtained from the bone marrow of CML (CP) patients. Human normal mononuclear cells (MNCs) were isolated from bone marrow of healthy volunteers after obtaining informed consents. For analysis of THAP11 mRNA expression, quantitative RT-PCR was performed in all cell lines treated with Abl kinase inhibitors (STI571, AMN107, and BMS354825). For proliferation analysis and the expression of c-Myc in CML cells, MTT assays, western blot and cell cycle analysis were performed in all cell lines transfected with Bcr-Abl siRNA, THAP11 siRNA, or THAP11 cDNA respectively. Moreover, THAP11 expression, c-Myc expression, and the colony counts of CFU-GEMM, CFU-GM, and BFU-E were analyzed in CML stem/progenitor cells transfected with Bcr-Abl siRNA or treated with Abl kinase inhibitors. [Results] In CML cell lines treated with Abl kinase inhibitors or transfected with Bcr-Abl siRNA, the expressions of THAP11 and c-Myc mRNA and protein were significantly increased compared to untreated cells. On the other hand, in CML cells transfected with the THAP11 cDNA, the c-Myc expression was suppressed. The overexpression of THAP11 induced G1 cell cycle arrest through p27 and p21 accumulation, and inhibited the CML cell proliferation. Moreover, in CML stem/progenitor cells obtained from patients with CML, the transfection with Bcr-Abl siRNA or treatment with Abl kinase inhibitors increased the expression of THAP11 mRNA and protein, and decreased the c-Myc expression and the counts of CFU-GEMM, CFU-GM and BFU-E. [Conclusion] Our results demonstrated that the Bcr-Abl suppressed the expression of THAP11, the depletion of THAP11 induced c-Myc expression, and induced the proliferation of CML cells through cell cycle progression. Moreover, induction of THAP11 expression inhibited the proliferation of CML stem/progenitor cells.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5060.
Abstract 5064: The non-receptor tyrosine kinase Fer is required for activated ErbB2-mediated tumorigenesis
Authors: Sangrar Waheed , Shi Changnian , Gao Yan , Greer Peter A.
Published: February 2011

The fer proto-onogene encodes a non-receptor protein-tyrosine kinase (PTK) which together with the parologous Fps/Fes kinase makes up a distinct F-BAR domain containing subclass of PTKs. F-BARs have recently been shown to mediate membrane binding and bending properties that confer roles in receptor endocytosis and vesicle trafficking. Among known substrates of the Fer PTK are cortactin and F-BAR/SH3 containing adaptor proteins that regulate cytoskeletal remodeling and membrane dynamics important to cellular functions such as polarity determination, migration and mitogenesis. Mice deficient in Fer, Fps/Fes or both kinases are overtly healthy; although they do display enhanced responses to endotoxin challenges. Biochemical studies have implicated Fer in growth factor signaling, cadherin-dependent cell-cell adhesion, integrin-mediated adhesion and redox signaling. Cell biological studies have suggested a role for Fer in potentiating proliferation and migration. Recent results in our laboratory have provided compelling biological proof-in-principle that Fer is required for mammary tumorigenesis. RNAi-based knockdown of Fer reduced the tumorigenesis of the metastatic MDA-MB-231 breast cancer cells in mouse xenografting studies. In addition, tumor development in a transgenic mouse model of Her2/Neu/ErbB2-mediated breast tumorigenesis was significantly delayed in Fer-deficient mice by virtue of a targeted knock-in kinase-inactivating mutation in the mouse fer locus. Biochemical internalization assays suggested that EGFR endocytosis was enhanced in Fer-deficient tumor-derived epithelial cell cultures. These data point to a tumor promoting function for Fer in mammary tumorigenesis through a mechanism involving prolongation of ErbB2 signaling. Taken together, these studies suggest that pharmacological intervention of Fer may have therapeutic benefit in human ErbB2 positive breast cancer and potentially, in other epithelial cancers.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5064.