bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4098: COX-2 expression in human pancreatic cancer cells is associated with an epithelial phenotype
Authors: Freitas Gil S. , das Aditi , White Matthew , McConkey David
Published: August 2012

Epithelial to mesenchymal transition is a developmental pathway that has been shown to have a direct role in tumor progression and drug resistance. Increasing evidence suggests that EMT (and MET) leads to global changes in chromatin architecture that modifies expression of numerous genes. Interestingly, reports in the literature indicate a possible association between cyclooxygenase-2 (COX-2) expression and EMT. COX-2 is upregulated in response to inflammation, and is known to be overexpressed in multiple malignancies, including pancreatic cancer. As a critical mediator of prostaglandin production, COX-2 has proangiogenic properties associated with increased tumor growth and invasion, and is also thought to play a role in the carcinogenic process. In this study, we report that COX-2 expression is associated with an epithelial phenotype among a panel of human pancreatic cancer cells. Using chromatin immunoprecipitation (ChIP), we demonstrate increased acetylated H3 at the COX-2 promoter region in the epithelial cell lines versus mesenchymal cell lines. Treatment with a Type-I selective HDAC inhibitor SNDX-275 increased levels of acetylation at the COX-2 promoter region in mesenchymal cell lines and subsequently promoted COX-2 gene expression. Overall, these data point to an epigenetic suppression of COX-2 expression in mesenchymal pancreatic cancer cells that can be reversed by Type-I HDAC inhibitors. Our results suggest that COX2 inhibitors will preferentially target “epithelial” pancreatic cancers and that combinations of Cox2 and HDAC inhibitors may display increased anti-tumor activity.

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 4098. doi:1538-7445.AM2012-4098
Abstract 4099: Utilization of methylation levels across the H19 and IGF2 genes as a predictive marker for various types of cancer
Authors: Poulin Matthew L. , Meyer Ann , Crawford Neil , Lei Shi , Xu Jessica , Yan Liying
Published: August 2012

H19 and IGF2 are reciprocally imprinted genes found on chromosome 11. Epigenetic gene deregulation is increasingly shown to play a role in many types of cancer. Imprinted genes are especially susceptible to epigenetic regulatory changes because of silencing of one allele and the dependence on proper methylation for normal function. One of the goals in cancer research is to identify molecular markers that will predict the onset of specific cancers. The pattern of methylation across the IGF2 and H19 genes can be used to characterize the difference between normal and cancerous tissue as well as the difference between different tumor tissues. By developing methylation assays that can determine the levels of methylation for all of the CpG sites across these two genes, we can establish patterns that could be predictive in detecting the onset of specific cancer types. We have developed numerous methylation assays across the H19 and IGF2 genes and have determined the methylation levels of DNA isolated from breast, cervical, ovarian and colon cancers and their corresponding normal tissue DNAs. We show that there are unique patterns of methylation between normal and cancerous tissues, as well as distinct patterns across different types of cancers. The differences in methylation patterns between DNA isolated from tumor and normal adjacent tissue across four different tumor types in these genes indicates the critical influence of epigenetic regulation in this region on the progression of 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 4099. doi:1538-7445.AM2012-4099
Abstract 4105: Assessment of autophagosome formation in primary cells by lentiviral delivery of fluorescent protein-tagged LC3
Authors: Anderl Janet , Huryn-Selvar Karyn , Liu Haizhen , Su Kevin , Santos Mark , Hsu Matthew , Ma Jun , Kovelman Robert , Armstrong Lucas
Published: August 2012

Assessment of autophagosome formation has been greatly facilitated by monitoring redistribution of LC3-fluorescent protein fusions. The sensitivity of the technique and capability for real-time analysis of autophagosome formation are advantageous over detection of endogenous LC3 redistribution in many experimental settings. However, the necessity for transfection can be an impediment to analysis of autophagosome formation in difficult-to-transfect cells, particularly primary cultures. We have developed lentiviral vectors (employing 3rd generation technology for optimal biosafety) encoding monomeric TagGFP2 and TagRFP fused at the N-termini of wild-type LC3 and LC3G120A as tools for analysis of autophagy in a wide variety of cell types. In immortalized cell lines and in primary cells (HUVEC and human mesenchymal stem cells) transduced with the GFP- and RFP-LC3 lentiviruses, amino acid deprivation in the presence of a lysosome inhibitor induced redistribution of the fluorescence from a diffuse cytoplasmic pattern to a punctate distribution. Minimal background of punctae in fed cells was observed. 3-Methyladenine blocked the starvation-induced redistribution, and the G120A mutant did not undergo detectable redistribution upon amino acid deprivation. In addition, we demonstrate effectiveness of lentiviral GFP-LC3 in HUVECs by flow cytometry, in which the plasma membrane is selectively permeabilized such that free cytosolic fluorescent protein-tagged LC3 is released while autophagosome-bound LC3 fusion protein is retained. We anticipate that these lentiviral autophagy biosensors will facilitate analysis of autophagosome formation in physiologically relevant primary cells.

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 4105. doi:1538-7445.AM2012-4105
Abstract 4115: Depletion of unsaturated fatty acids by stearoyl-coA desaturase inhibition results in endoplasmic reticulum stress-mediated tumor cell death
Authors: Roongta Urvashi V. , Pabalan Jonathan G. , Wang Cindy , Ryseck Rolf-Peter , Fargnoli Joseph , Henley Benjamin , Yang Wen-Pin , Zhu Jun , Madireddi Malavi , Lawrence Michael , Wong Tai W. , Rupnow Brent A.
Published: August 2012

Over the past few years, a more detailed picture of the importance of metabolic pathways to the maintenance and progression of human cancers has begun to emerge. In particular, recent studies have implicated lipid biosynthesis and desaturation as a requirement for tumor cell survival. The endoplasmic reticulum-resident Stearoyl CoA Desaturase (SCD) is the rate limiting enzyme that catalyzes conversion of saturated fatty acids to monounsaturated fatty acids, a key step in the fatty acid metabolic network. In the studies reported here, we show that inhibition of SCD1 by siRNA or a small molecule antagonist results in strong induction of apoptosis and growth inhibition when tumor cells are cultured in reduced (2%) serum conditions but has little impact on cells cultured under normal (10%) serum conditions. Consistent with the observed dependence on SCD1, levels of SCD1 protein increased in response to decreasing serum levels. Both induction of SCD1 protein and sensitivity to growth inhibition by SCD1 inhibition could be reversed by supplementing growth media with unsaturated fatty acids. Transcription profiling of cells treated with an SCD inhibitor revealed strong induction of markers of endoplasmic reticulum stress. This finding was confirmed using siRNA specifically targeting SCD1. Underscoring its importance in cancer, SCD1 protein was found to be highly expressed in a large percentage of human cancer specimens. SCD inhibition resulted in tumor growth delay in a human gastric cancer xenograft model. Altogether, these results suggest that unsaturated fatty acids are required for tumor cell survival and that SCD may represent a viable target for the development of novel agents for cancer therapy.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4115. doi:1538-7445.AM2012-4115
Abstract 4128: miR203 induces mesenchymal to epithelial transition via repressing SNAI2 and activating P-cadherin in prostate cells
Authors: Qu Yi , Zhang Jigang , Li Wen-Cheng , Hellem Margrete R. , Rostad Kari , Liu Run-hui , Zhang Wei-dong , Oyan Anne Margrete , Kalland Karl H. , Ke Xi-Song
Published: August 2012

MicroRNAs play critical roles in tumorigenesis and metastasis. Here we report the functions of miR203 in prostate cancer based upon an experimental cell culture model. While highly expressed in prostate epithelial cells, miR203 became completely repressed following their epithelial to mesenchymal transition (EMT). Forced re-expression of miR203 in the mesenchymal cells induced many epithelial features, including morphological change in 2D and 3D culture, decreased abilities of invasion and migration, as well as increased epithelial markers and decreased mesenchymal markers. By computer prediction combined with microarray profiling and luciferase reporter target assays, we identified SNAI2 as target of miR203. Knock-down of SNAI2 can mimic the epithelial phenotypes induced by miR203. Most of the affected genes by miR203 were also found to be changed with the same pattern by knock-down of SNAI2. Furthermore, P-cadherin (CDH3), an important epithelial adhesion component, was identified as the downstream target of SNAI2. CDH3-promoter-Luc reporter assay demonstrated that 3 E-boxes in the CDH3 promoter mediated direct targeting by SNAI2. Decreased and increased expression of CDH3 were found in culture cells with overexpression and knock-down of SNAI2, respectively. We concluded that miR203 represses SNAI2, thus activating P-cadherin in induction of epithelial features.

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 4128. doi:1538-7445.AM2012-4128
Abstract 4140: MicroRNA expression in Osteosarcoma: potential role of miR-1 and miR-133b
Authors: Pazzaglia Laura , Novello Chiara , Cingolani Chiara , Conti Amalia , Quattrini Irene , Magagnoli Giovanna , Manara Maria Cristina , Benassi Maria Serena
Published: August 2012

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and young adults. Conventional therapy for OS has reached a plateau of 60-70%, a 5-year survival rate that has remained almost unchanged in two decades, highlighting the need for new therapeutic approaches by finding new targets. This study presents a comparative analysis of miRNA expression profile in clinical OS samples and paired normal tissues with microarray approach. The Unsupervised Hierarchical Clustering, performed on log2(Hy3/Hy5) ratios, revealed 100 miRNAs with the highest standard deviation across all samples. The Supervised Hierarchical Clustering showed that 40 miRNAs were differentially expressed with a p-value <3,95E-5, and 80% of these were underexpressed in OS samples when compared with normal tissues. In particular, the 5-fold underexpression of miR-1 and miR-133b (p<3.95E-5) was confirmed by Real Time PCR. When U2OS OS cells were transfected with miRNA precursor molecules, we demonstrated that the ectopic expression of miR-1 and miR-133b respectively decreased to 75% and 68% the cell proliferation as compared with negative control, concomitantly with strong slowing down of cell migration and invasion and reduced expression of Met protein that it is considered a potential target of both miRNAs. It is well known that miR-1 and miR-133b are expressed in skeletal muscle and are transcriptionally regulated by myogenic differentiation factors. Our data demonstrate a deregulation of miRNA profiling in OS clinical specimens and highlight the function of miR-1 and miR-133 in the control of OS cell growth and invasiveness. Further studies should be addressed to investigate the role of these miRNAs and their targets as potential prognostic and therapeutic markers.

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 4140. doi:1538-7445.AM2012-4140
Abstract 4146: The expression of exosomal microRNA in serum of patients with esophageal squamous cell carcinoma
Authors: Tanaka Youhei , Watanabe Masayuki , Kamohara Hidenobu , Kurashige Junji , Mima Kousuke , Okabe Hirohisa , Baba Hideo
Published: August 2012

Background: Exosomes are 40 -100 nm membrane vesicles secreted by various cells such as T cells, platelets, and epithelial cells by exocytosis. Exosomes are found in blood, urine, amniotic fluid, and malignant ascites, and contain distinct subsets of microRNAs (miRs) depending upon the tumor from which they are secreted. Exosomes play a major role in cell-to-cell communication, targeting cells to transfer exosomal molecules including proteins, mRNAs, and microRNAs by an endocytosis-like pathway. Recent reports imply that miRNAs can be stably detected in circulating plasma and serum since miRNAs are packaged by exosomes to be protected from RNA degradation. We have previously reported that overexpression of miR-21 was observed in patients with esophageal squamous cell carcinoma. miR-26a is also known to be overexpressed in esophageal cancer. Objective: Aim of this study is to clarify the expression levels of miR-21 and miR-26a in exosomes from the serum of esophageal cancer patients. Patients and method: We extracted exosomes from serum of 20 esophageal cancer patients (ESCC group) and of 20 benign disease cases without inflammation (control group) by September, 2010 from October, 2009. We separated exosomes from serum, and then extracted total RNA including microRNA from exosome. Furthermore, using quantitative quantitative RT-PCR, we analyzed expression of miR-21, miR-26a where overexpression was confirmed in esophageal cancer in a past report. Results: The expression levels of miR-21 in exosomes were significantly higher in ESCC than those in control (P <0.001), while miR-26a expression levels in ESCC were tended to be higher than those in controls. Furthermore, we analyzed the supernatant which extracted exosomes from serum. There was almost no expression of both miRs in the supernatant after extraction of exosomes. When we compared exosomal and serum levels of miR-21, no correlation was observed between them. Conclusion: We have confirmed the elevated levels of exosomal miR-21 in the serum of patients with ESCC compared with the control. Although further analysis is needed to clarify the biological meaning of the elevated exosomal miR in the serum, the exosomal miR may become new biomarkers for ESCC.

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 4146. doi:1538-7445.AM2012-4146
Abstract 4151: Real-time imaging of inter-cellular communication via exosomes
Authors: Homma Kimi , Suetsugu Atsushi , Ochiya Takahiro , Hoffman Robert M.
Published: August 2012

Growing evidence suggests that extracellular microRNAs (miRNAs) stably exist in human body fluids, including plasma, saliva and urine, although ribonucleases (RNases) also circulate throughout the body. Therefore, miRNAs are excreted in RNase-resistant lipid vesicles, such as exosomes and apoptotic bodies, which can deliver gene-silencing signals between living cells in vitro and in vivo. However, very little is known about the exosome-mediated intra-cellular communication machinery. Here we demonstrate with labeling of exosomes with green fluorescent protein (GFP) linked to CD63, the transfer and incorporation of exosomes into recipient cells via indirect or direct contact with cells in a co-culture system. These findings suggest that secretory exosomes are transfered from cell to cell, transferring genetic information that can affect a wide array of biological events.

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 4151. doi:1538-7445.AM2012-4151
Abstract 4154: Exosomal microRNAs: mediators for cell-cell communications
Authors: Singh Ramesh , Mo Yin Yuan
Published: August 2012

Background: Exosomes are 30-100nm membrane vesicles of endocytic origin, mediating diverse biological functions including tumor-cell invasion, cell-cell communication and antigen presentation through transfer of proteins, mRNAs and microRNAs to neighboring or distant cells. MicroRNAs are small regulatory non-coding RNA molecules that regulate protein-coding genes. Recent evidence suggests that microRNAs can be released through ceramide-dependent secretory machinery regulated by neutral sphingomyelinase 2 (nSMase2) enzyme expressed by smpd3 gene that triggers exosome secretion. In this study, we investigated the exosome-mediated microRNA transfer among various types of cells, the regulation of exosome-mediated microRNA secretion and functional role of these exosomal microRNAs inside the recipient cell after uptake. Methods: Exosomes were isolated from culture media of cells by serial centrifugation, filtration and ultracentrifugation method. Isolated exosomes were stained with PKH26 dye and then observed for cellular uptake in different cells by confocal microscopy after counterstaining with Hoechst stain. In addition, this uptake was also shown by exosome isolated from stable 293T cell line overexpressing the pCDH-CD63-GFP fusion protein. To determine the effect of nSMase2 on exosome secretion, stable cell lines were established with smpd3 gene. Levels of microRNAs secreted in media were determined by real time PCR. To validate the function of exosomal microRNA in the recipient cells, we determined the protein level of microRNA target genes by western blot. Results: We first showed that exosomes can be transferred among different cell lines through direct uptake. We found that miR-10b is highly expressed in metastatic breast cancer MDA-MB-231 cells as compared to non-metastatic breast cells; it is actively secreted into medium. For example, nSMase2 promoted the exosome-mediated miR-10b secretion whereas ceramide inhibitor suppressed this secretion. Importantly, western blot revealed that upon uptake, miR-10b can still reduce the protein level of its target genes such as Bim, indicating its functional significance. Conclusion: We found that the exosomes secreted from cancer cells containing microRNA can be taken up by other cells and have functional role in the recipient cells suggesting the importance of exosome-mediated microRNA transfer in tumor microenvironment. Our results collectively suggest that a set of specific microRNAs may play an important role in modulating tumor microenvironment through exosomes. Thus, a better understanding of this process may aid in the development of novel therapeutic agents.

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 4154. doi:1538-7445.AM2012-4154
Abstract 4156: Interaction between the rabphillin-3A-like gene and phosphorylation of mammalian target of rapamycin (mTOR) in colorectal cancer
Authors: Katkoori Venkat R. , Choudhary Sandeep , Butler Abraham R. , Shanmugam Chandrakumar , Bovell Liselle L. , Samuel Temesgen , Athar Mohammed , Grizzle William E. , Manne Upender
Published: August 2012

Background: Recently, we demonstrated that, for colorectal carcinomas (CRCs), genetic alterations in the rabphillin-3A-like (RPH3AL) gene are associated with aggressive tumor phenotypes and poor patient survival. The mammalian target of rapamycin (mTOR) pathway regulates tumor growth by integrating diverse signals of growth factors, nutrients, and energy sufficiency. Therefore, we determined the effects of wild-type (w+) and mutated (67w-, 175w-, 290w-) RPH3AL on the mTOR signaling pathway in CRC tissues and in a line of colon cancer cells (LoVo). Materials and Methods: Ninety five prospectively collected, frozen CRC tissues and their matching control tissues were analyzed for the mutational status of RPH3AL using the cDNA polymerase chain reaction and direct sequencing. Expression constructs of RPH3AL w+ and the mutant variants were developed using eukaryotic expression vectors. LoVo cells were stably transfected with the w+ and w- forms of RPH3AL. Western blot analyses were performed on lysates of CRC tissues and LoVo cells to assess expression of p53, p27, and PCNA and proteins in the mTOR pathway. Results: Mutational analysis of RPH3AL detected four missense mutations: at codon 67 (Val > Met) in 2 CRCs, at codon 175 (Pro > Ser) in 1 CRC, and at codon 290 (Arg > Lys) in 1 CRC. Western blot analyses demonstrated that over-expression of wt RPH3AL in LoVo cells or CRC tissues with wt RPH3AL led to decreases in total mTOR and to phosphorylation of Rictor p-mTOR (S2481), Raptor p-mTOR (S2448), p70S6K1, and p70S6K. In contrast, in CRC tissues or LoVo cells with over-expression of mutant forms of RPH3AL, there were higher levels of total mTOR and greater phosphorylation of the Rictor, raptor, P70S6K1 and p70S6K. Additionally, CRC w+ RPH3AL and LoVo cells that over-expressed w+ RPH3AL demonstrated higher levels of p27, and lower levels of PCNA, and p53. In contrast, the CRC tissues or LoVo cells with over-expression of RPH3AL mutants exhibited lower levels of p27 and higher levels of PCNA. Conclusion: The results demonstrate that the RPH3AL gene has a tumor-suppressing effect involving changes in the mTOR pathway and suggest that these changes inhibit progression of CRCs. This work was supported by grants from the NCI/NIH (2U54-CA118948-06, R01-CA98932, and R03-CA139629).

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 4156. doi:1538-7445.AM2012-4156
Abstract 4159: PTEN nucleo-cytoplasmic protein compartmentalization and gene status are correlated with aggressiveness of pulmonary neuroendocrine tumors
Authors: Collaud Stephane , Tischler Verena , Perren Aurel , Komminoth Paul , Schraml Peter , Moch Holger , Weder Walter , Soltermann Alex
Published: August 2012

Introduction: Lung neuroendocrine tumors (NET) comprise typical (TC) and atypical carcinoid (AC), large cell neuroendocrine carcinoma (LCNEC) and small cell lung carcinoma (SCLC). Phosphatase and tensin homolog (PTEN) protein is a known cytosolic tumor suppressor with recently described novel nuclear functions. We investigated this differential PTEN protein compartmentalization together with its genomic status in pulmonary NETs. Patients and Methods: A retrospective multicenter cohort of 192 patients with lung NET was investigated on a tissue microarray (TMA, surgical resections = 183/autopsies = 9) by immunohistochemistry (IHC, cytoplasm and nucleus) and fluorescence in-situ hybridization (FISH, nucleus). Results were correlated with markers of neuroendocrine differentiation (synaptophysin, chromogranin A), proliferation (mib-1), cell cycle (p27, cyclin D1) and survival data. Results: The histotypes represented were TC in 58 patients, AC in 42, LCNEC in 32 and SCLC in 60. All NETs showed higher PTEN immunoreactivity in the cytoplasm than in the nucleus, whereby the greatest nucleo-cytoplasmic difference was found for SCLC. In both compartments, immunoreactivity was higher in the carcinoids than in LCNEC and SCLC. Loss of cytosolic or nuclear PTEN protein was correlated with increased mitotic rate, with low synaptophysin or chromogranin A and with PTEN genomic deletion. Loss of nuclear PTEN protein correlated with high nuclear TTF1, whereas loss of cytosolic protein correlated with low p27 and low cyclin D1 (all p-values <0.05). Patients with PTEN genomic deletion or low PTEN immunoreactivity had poorer survivals. Conclusion: PTEN genomic deletion or PTEN protein loss is linked to dedifferentiation and poor prognosis of lung NET. Nucleo-cytoplasmic shuttling of PTEN protein could play a role in the different PTEN inactivation mechanisms.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4159. doi:1538-7445.AM2012-4159
Abstract 4165: Identification of flotillin-1 as a regulator of H-Ras activation in lipid raft
Authors: Yong Hae-Young , Son Hwajin , Kim Eun-Sook , Hwang Jin-Sun , Kim Myeong-Ok , Choi Wahn Soo , Noh Dong-Young , Hwang Ki-Tae , Kim Ki-Bum , Lee Jae-Seon , Kim Hyung Joon , Kim Hong-Hee , Choi Yoomi , Kim Eun Joo , Park So Yeon , Kim Hyeong-Reh Choi , Jeon You Rim , Moon Aree
Published: August 2012

Metastatic spread is the major cause of death from breast cancer. We previously showed that H-Ras, but not N-Ras, induces invasive/migratory phenotypes of MCF10A human breast epithelial cells, while both H-Ras and N-Ras induce proliferation/transformation. Here, we identified flotillin-1 as an H-Ras-induced lipid raft protein through comparative proteome profiling of lipid raft proteins. Flotillin-1 interacts with H-Ras in lipid raft at a higher affinity than with N-Ras. Small interfering RNA(siRNA)-mediated flotillin-1 knockdown significantly reduced H-Ras activation and H-Ras-mediated motility/invasion in MCF10A cells engineered to express active H-Ras as well as in Hs578T breast carcinoma and T24 bladder carcinoma cells that express endogenous active mutant of H-Ras. These results suggest a positive signal amplification loop between flotillin-1 and H-Ras for the invasive signaling program. Flotillin-1 was crucial for the activation of the Ras-GRF1 exchange factor and its interaction with H-Ras in lipid rafts. Using a xenograft tumor model, we show that knockdown of flotillin-1 reduces the tumor growth in vivo. Importantly, tissue microarrays of 289 breast cancer patients revealed that flotillin-1 expression in the plasma membrane positively correlates with HER2/neu expression(p<0.001) and high histologic grade(p=0.014). Although infrequent, the membranous flotillin-1 expression is significantly associated with poor disease-free survival of patients(p=0.005), suggesting the clinical importance of predicting the characteristics of a small subpopulation of malignant breast cancer. Taken together, our findings provide new insight into the molecular basis of the Ras isoform-specific signaling mechanism leading to cell invasion that depends on the lipid-based sorting platforms.

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 4165. doi:1538-7445.AM2012-4165
Abstract 4166: Tumor suppressor activity of the ERK/MAPK pathway by promoting selective protein degradation
Authors: Deschênes-Simard Xavier , Gaumont-Leclerc Marie-France , Bourdeau Véronique , Moiseeva Olga , Forest Valérie , Mallette Frédérick A. , Lessard Frédéric , Saad Fred , Mes-Masson Anne-Marie , Ferbeyre Gerardo
Published: August 2012

We report that ERK/MAPK signaling mediates tumor suppression. Oncogenic forms of ras are found in approximately 25% of all human cancers and are generally associated with uncontrolled proliferation of cancer cells. However, expression of oncogenic ras in normal cells is associated with cellular senescence. In primary cells expressing oncogenic ras, ERK knockdown by shRNA prevented the activation of the DNA damage response, p53, p16INK4a/Rb and senescence and cooperated with hTERT in transformation. In human prostate neoplasms, high levels of phosphorylated-ERK were found in benign lesions while in malignant lesions lower levels correlated with a higher Gleason score and earlier disease relapse after treatment. Mechanistically, the antitumor effects of ERK/MAPK signaling included the selective proteasome-dependent degradation of proteins required for cell cycle progression, mitochondrial functions and cell signaling and the inhibition of the phosphoinositide-3-kinase/AKT (PI3K/AKT) signaling pathway. We thus show for the first time that the outcome of ERK/MAPK signaling can be controlled at the level of ERK1/2, the final members of the pathway. A quantitative control at the end of the pathway provides a mechanism for specific modulation of ERK/MAPK signaling since controlling the input may also affect the numerous signaling pathways activated by membrane receptors. The data also indicate that the levels of the ERK kinases control a binary switch between cell proliferation and senescence and explain the maintenance of the senescence phenotype despite the presence of constitutively activated growth factor signaling pathways. Since inhibitors of the ERK pathway are under consideration for clinical use, we caution that they may trigger the escape of dormant senescent cells leading to malignant tumors.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4166. doi:1538-7445.AM2012-4166
Abstract 4167: KRas induces a Src/PEAK1/ErbB2 kinase amplification loop to drive pancreatic cancer growth, metastasis and therapy resistance
Authors: Kelber Jonathan A. , Reno Theresa , Kaushal Sharmeela , Metildi Cristina , Stoletov Konstantin , Weems Jessica , Park Fred D. , Wright Tracy , Mose Evangeline , Hoffman Robert M. , Lowy Andrew M. , Bouvet Michael M. , Klemke Richard L.
Published: August 2012

Early biomarkers and effective therapeutic strategies are desperately needed to treat pancreatic ductal adenocarcinoma (PDAC), which has a dismal 5-year patient survival rate. We recently identified PEAK1 (pseudopodium-enriched atypical kinase one, SGK269) as a catalytically active tyrosine kinase that associates with the cytoskeleton and regulates cell migration and proliferation. Tyrosine kinases are highly sought after candidates as biomarkers and therapeutic targets because they induce specific phoshorylation signatures and because their catalytic activity and downstream signals can be inhibited by small molecules. The goal of this current study was to assess the role of PEAK1 expression as a diagnostic/prognostic biomarker and potential therapeutic target in human malignancies. Here, we report that the novel tyrosine kinase PEAK1 is upregulated in human malignancies, including human PDAC and pancreatic intraepithelial neoplasia (PanIN). Oncogenic KRas induced a PEAK1-dependent kinase amplification loop between Src, PEAK1 and ErbB2 to drive PDAC tumor growth and metastasis in vivo. Suprisingly, blockade of ErbB2 increased Src-dependent PEAK1 expression, PEAK1-dependent Src activation and tumor growth in vivo, suggesting a mechanism for the resistance of PDAC to therapeutic intervention. Indeed, PEAK1 mediates multiple therapy resistance phenotypes that present major hurdles in the clinical treatment of this disease. Thus, PEAK1 is a novel biomarker, critical signaling hub, and new therapeutic target in PDAC.

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 4167. doi:1538-7445.AM2012-4167
Abstract 4170: Mutation and chemosensitivity profiling of 18 human melanoma cell lines
Authors: Kelter Gerhard , Krumbach Rebekka , Maier Armin , Giesemann Torsten , Vuaroqueaux Vincent , Foucault Frédéric , Metz Thomas , Fiebig Heinz-Herbert
Published: August 2012

Malignant melanomas account for a substantial proportion of cancers worldwide and are largely resistant to conventional therapy. The heterogeneity and the invasiveness of melanomas present notable challenges for anti-cancer treatment. Recently, several mutations in RAF, RAS, and PTEN were identified that allow for prognosis of cancer development and progression. Therefore, inhibitors of the RAF-MEK-ERK pathway form attractive candidates for molecular-based targeted treatment of melanoma cancer. Here, we present the molecular characterization and chemosensitivity profiling of 18 human melanoma cell lines. Eight of these were established by Oncotest from human tumor xenografts originally derived from primary patient material. The molecular profiling included mutational analysis for BRAF, PIK3CA, KRAS, NRAS, TP53, and PTEN by sequencing. The chemosensitivity profiles were determined in vitro using a fluorescence-based cytotoxicity assay and included commonly used chemotherapeutic agents for treatment of melanoma, targeted agents (sorafenib and bortezomib) and various BRAF (PLX-4032, PLX-4720, GDC-0879) and MEK (CI-1040, PD0325901) inhibitors. The clinically relevant BRAF V600E substitution was the most prevalent mutation and was found in more than 50% of the melanoma cell lines. The chemosensitivity profiling revealed diverse patterns of selectivity and potency with mean IC50 values between 0.4 nM (vinblastine) and 8.3 µM (carboplatin). Each of the BRAF inhibitors tested was clearly more active in melanoma cell lines carrying a BRAF mutation. On average, IC50 values of BRAF inhibitors were more than 20-fold lower for the BRAF V600E mutated cell lines compared to the wild type cell lines. However, the BRAF mutated cell line MEXF 462NL was resistant towards BRAF inhibitors and it is warranted to compare its gene expression profile and mutational status with BRAF mutated cell lines responding to BRAF inhibitors. By using an ex vivo clonogenic assay, similar activity patterns for BRAF inhibitors were found, with mutated BRAF tumor xenografts showing markedly higher sensitivity. Potential synergistic activity of BRAF inhibitors with other inhibitors of the RAF-MEK-ERK pathway or with standard chemotherapeutic agents will be presented. The mutational status of selected cancer related genes, as well as responsiveness towards many cytotoxic and targeted agents was analyzed for 18 melanoma cell lines. This extensive genotypic and phenotypic characterization makes this melanoma cell line panel a valuable tool for in vitro profiling of novel compounds, in particular inhibitors of the RAF-MEK-ERK pathway.

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 4170. doi:1538-7445.AM2012-4170
Abstract 4173: IKBKE/IKKα is a prognostic marker and is required for KRAS-driven tumorigenecity in non-small cell lung cancer
Authors: Kim Donghwa , Guo Jian-Ping , Gao Jingchun , Yu Catherine , Park John , Coppola Domenico , Haura Eric , Cheng Jin Q.
Published: August 2012

IKBKE (IKKα) is a serine/threonine protein kinase that belongs to the IKK kinase family. Here we demonstrated that IKKα is frequently overexpressed and activated in NSCLC. Elevated protein levels of IKKα were detected in 16/30 (52.6%) cell lines and 91/176 (51.7%) primary tumors. Alterations of IKBKE are associated with overall survival but not tumor stage and grade. Enforcing expression of IKBKE decreases whereas knockdown of IKBKE increases the apoptosis induced by chemotherapeutic agents. Notably, suppression of IKBKE selectively induced cell death and inhibited tumor growth in NSCLC cell lines that depend on oncogenic K-Ras expression. Mechanistically, knockdown of IKBKE largely abrogated activating mutation of K-Ras-induced NFκB activation. These data suggest that IKBKE could be a poor prognostic marker and mediates K-Ras oncogenic function and signaling to NFκB.

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 4173. doi:1538-7445.AM2012-4173
Abstract 4175: Reactive oxygen species-dependent ERK and JNK activation is important to docasahexaenoic acid-induced cell cytotoxicity in human ovarian cancer cells
Authors: Jeong Soyeon , Jing Kaipeng , Shin Soyeon , Kim Nayeong , Oh Hye-Rim , Cho Eun-Ah , Heo Jun-Young , Park Ji-Hoon , Seo Kang-Sik , Han Jeongsu , Park Jong-Il , Park Seung-kiel , Kweon Ki-Ryang , Yoon Wan-Hee , Hwang Byung-Doo , Lim Kyu
Published: August 2012

Although abundant experimental evidences show that the Omega-3 polyunsaturated fatty acids (≥3-PUFAs) prevent carcinogenesis, the exact molecular mechanisms of the anti-cancer actions of α3-PUFAs in ovarian cancer remain incompletely understood. In the present study, the effectiveness of docosahexaenoic acid (DHA), a α3-PUFA, against ovarian cancer cells was investigated. We found that DHA induced cell cytotoxicity in three ovarian cancer cells including PA-1, MDAH2774 and ID8. DHA treatment inhibited the cell proliferation of PA-1 cells in a dose- and time-dependent manner. Meanwhile, DHA-treated ovarian cancer cells showed increased levels of caspase-3 activity, Annexin-V staining positive cells, TUNEL-positive cells and the portion of sub-G1 cells, suggesting that DHA-induced cell death is mainly associated with apoptosis. Western blot and immunocytochemistry assays revealed that DHA also remarkably increased the levels of phospho-ERK and phospho-JNK in both cytosol and nucleus. Moreover, knockdown ERK and JNK by small interfering RNAs partially attenuated the apoptosis induced by DHA, indicating that ERK and JNK activation is responsible for the apoptosis in DHA-treated ovarian cancer cells. In addition, we determined that the activation of ERK and JNK was associated with the reactive oxygen species (ROS) production induced by DHA. ROS scavenger, N-acetyl-L-cysteine (NAC), almost completely blocked the ERK and JNK phosphorylation as well as the apoptosis triggered by DHA. Together, these results indicate that DHA induces ROS and the ROS-dependent ERK and JNK activation is important to DHA-induced cell cytotoxicity in human ovarian cancer cells. [This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-0006232 and 2011-0003060)].

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 4175. doi:1538-7445.AM2012-4175
Abstract 4179: Loss of Hsulf-1 promotes chemoresistance and tumorigenecity in ovarian cancer
Authors: He Xiaoping , Khurana Ashwani , Kaufmann Scott , Shridhar Viji
Published: August 2012

Human Sulfatase1 enzyme (HSulf-1) is down-regulated in a majority of ovarian cancer cell lines and primary tumors, but the functional consequence of this downregulation remains unclear. Here, we show that down-regulation of HSulf-1 by RNA interference resulted in an elevated level of phosphorylated Erk and phosphorqylated Akt as well as decreased Bim level in OV202 ovarian cancer cells. Treatment with PD98059 (p-Erk inhibitor) but not LY90024 (PI3K inhibitor) resulted in increased Bim level in Hsulf-1 knockdown cells, suggesting that increased Erk pathway plays a more important role in modulating Bim level after loss of HSulf-1. Consistently, the results of real-time PCR confirmed that there was no significant change in the mRNA level of Bim after knockdown of HSulf-1 expression. When treated with CDDP, OV202 cells with knockdown of HSulf-1 displayed more resistance to drug-induced apoptosis, with IC50 value 3-fold higher than cells with non-targeting shRNA (NTC), while SKOV3 cells with overexpression of HSulf-1 were more sensitive to CDDP-induced apoptosis compared to the control cells. Moreover, HSulf-1 downregulated OV202 cells showed remarkable growth of colonies in soft agar compared to no colony growing with NTC cells. When injected subcutaneously, HSulf-1 ShRNA clones but not NTC cells formed xenogrfts in nude mice. Importantly, rescuing Bim expression in HSulf-1 shRNA cells not only endowed cells sensitivity to CDDP treatment, but also significantly decreased numbers of colony formation in soft agar and significantly retarded tumor growth in nude mice. Collectively, these results strongly suggest that down-regulation of HSulf-1 promotes chemoresistance and tumorigenicity in ovarian cancer cells through post-translational downregulation of Bim level.

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 4179. doi:1538-7445.AM2012-4179
Abstract 4180: The sterile 20-like kinase Tao-1 controls tissue growth by regulating the Hippo tumor suppressor pathway
Authors: Poon Carole LC , Lin Jane I. , Zhang Xiaomeng , Harvey Kieran F.
Published: August 2012

The Hippo tumour suppressor pathway is a complex signalling network that controls developmental tissue growth and is frequently deregulated in different human cancers. A key question in Hippo signalling is how the Hippo kinase (MST1/2 in mammals) is activated by upstream regulatory inputs to limit tissue growth. Using a genetic screen in Drosophila melanogaster, we identified the sterile 20-like kinase, Tao-1, as a novel Hippo pathway member that activates Hippo. Tao-1 controls various biological phenomena including microtubule dynamics, animal behaviour and brain development. Here, we describe a previously unappreciated role for Tao-1 as a regulator of epithelial tissue growth by modulating activity of the core Hippo pathway kinase cassette. Tao-1 functions together with Hippo to activate Warts-mediated repression of Yorkie. Tao-1's ability to control SWH pathway activity is evolutionarily conserved as human TAO1 can suppress activity of the Yorkie orthologue, YAP. Human TAO1 controls SWH pathway activity by phosphorylating, and activating, the Hippo orthologue, MST2. Importantly, given that Hippo pathway activity is subverted in many human cancers, our findings identify human TAO kinases (TAO1, TAO2 and TAO3) as candidate tumor suppressor genes.

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 4180. doi:1538-7445.AM2012-4180
Abstract 4181: Absence and/or functional loss of the tumor suppressor Tnk1 is associated with various human neoplasms
Authors: Hoare Kishalay , Flagg Tammy , Zamora Bernadetta M. , Reinhard Mary K. , Hoare Sarasija , May W.Stratford
Published: August 2012

The protein tyrosine kinase activity of Tnk1 (Thirty-eight negative kinase) is required for cell growth suppression due to the fact that the kinase-dead Tnk1 triggers aberrant cell growth, including anchorage independent growth with hyperactivation of Ras (Hoare et. al. 2003; Azoitie et.al. 2007). Mechanistically, the cell growth suppression is associated with the inhibition of Ras activity and the down-stream Raf1-MAPK growth pathway (Hoare et.al. 2003) resulting in G2-M phase cell cycle arrest. A tumor suppressor role of Tnk1 was subsequently confirmed by developing the Tnk1 knockout mouse model by homologous recombination. The TNK1 knockout mice develop many types of tumors upon aging, with B-cell lymphomas, hepatomas, hepatocellular carcinomas, lung adenomas and adenocarcinomas being the most frequent malignancies (Hoare et.al. 2008; May et. al. 2010). Significantly, 40% of Tnk1-/-mice and 26% of Tnk1+/− mice had more than one tumor type develop, likely reflecting Tnk1's broad tumor suppressor function. To confirm the tumor suppressor function of the 72 kDa Tnk1 in human tumors and cell lines, we screened for Tnk1's expression and analyzed its intrinsic/auto kinase activity and in vitro kinase activity. Results indicate that in tumors and cell lines, Tnk1 is either expressed or absent. In B-/T-lymphoma and lung adenocarcinoma cell lines, to name a few, such as Ramos, Jurkat, REH and A549, absence of Tnk1 is associated with aberrant Ras activity, suggesting that loss of Tnk1 expression may contribute to tumor development. In patient specimens and cell lines namely, K562, T47D, MCF7, NCI H82, NCI H387, HEPG2 and HUH, the expressed Tnk1 is found to lack auto-kinase activity and is therefore, inactive in vivo. This data indicates that tumor growth depends on the maintenance of Tnk1 in its inactive form through interaction(s) with scaffolding protein(s) and/or due to SNPs found in its kinase domain. Strikingly, the Hodgkin lymphoma cell line L540 is found to express a tyrosine phosphorylated 60 kDa truncated Tnk-(C17ORF61) fusion gene product (termed tTnk1) raising doubt that it is an activated form of Tnk1 (Gu et. al. 2010). Interestingly, we found that t-Tnk1 is kinase-dead, as it fails to Y-phosphorylate Grb2, a Tnk1 substrate. This supports the concept that t-Tnk1 and/or functionally inactive Tnk1 expressed in tumor cells may function in a dominant negative manner to maintain tumor growth. In conclusion, Tnk1, in human, also functions as a tumor suppressor.

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 4181. doi:1538-7445.AM2012-4181