bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4955: TIMP-1 inhibits apoptosis in lung adenocarcinoma cells via Bcl-2 interaction
Authors: Nalluri Srilatha , Gayatri Sitaram , Ohja Juhi , Rojiani Amyn M. , Rojiani Mumtaz V.
Published: August 2012

Tissue inhibitors of matrix metalloproteinases have multifunctional properties, orchestrating diverse biological activities. Previous studies revealed a novel function of TIMP-1 as an inhibitor of apoptosis in mammalian cells. However, the mechanisms by which TIMP-1 exerts its anti-apoptotic effect are not completely understood. The antiapoptotic effect has been correlated with higher levels of Bcl-Xl. Our earlier studies in a CNS model of metastasis, using HB-1, a TIMP-1 overexpressing lung adenocarcinoma cell line (H2009), resulted in more aggressive tumor kinetics and increased vasculature. The present study was undertaken to elucidate the role of TIMP-1 in apoptosis in our model. Utilizing an apoptosis -specific gene array, we found that TIMP-1 overexpression results in 3-fold increased expression of Bcl-2. H2009 and the TIMP1 over-expressing clone - HB1 were treated with staurosporine to induce apoptosis, followed by analysis for poly ADP-ribose polymerase (PARP) cleavage. HB1 showed marked reduction in PARP cleavage in comparison to the controls. Treatment of HB1 cells with ABT737, an inhibitor of Bcl-2, restored apoptosis resulting in PARP cleavage in this clone. To further confirm this mechanism, we induced transient inhibition of Bcl-2 with siRNA, which also restored PARP cleavage in the HB-1 clone. Thus both chemical and biological inhibition resulted in increased apoptosis. Additionally, immunoprecipitation of Bcl-2 from cell lysate co-immunoprecipitated TIMP-1 suggesting a direct interaction between TIMP-1 and Bcl-2. These investigations identify a relationship between TIMP-1 and Bcl-2 in apoptosis inhibition, contributing to aggressive tumor kinetics, utilizing non-MMP dependent 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 4955. doi:1538-7445.AM2012-4955
Abstract 496: Isolation and characterization of a highly invasive subpopulation from MDA-MB-231 breast cancer cells
Authors: Pfeffer Ulrich , Amaro Adriana , Maffei Massimiliano , Mirisola Valentina , Esposito Alessia I. , Astigiano Simonetta , Viale Maurizio , Zunino Annalisa , Giaretti Walter , Angelini Giovanna
Published: August 2012

The acquisition of an invasive phenotype is a pre-requisite for metastasis. We set out to develop cellular systems that can mirror transient and stbale molecular alterations that confer an invasive phenotype to breast cancer cells. We observed that it is possible to isolate invasive subpopulations from moderately invasive cancer cell lines. Enrichment of invasive sub-populations of MDA-MB-231 breast cancer cells in three successive preparative invasion assays in Matrigel covered Boyden chambers yielded a highly invasive cell line. Prolonged cultivation of these cells did not abolish the invasive phenotype although not all phenotypic changes acquired during selection are maintained by long term cultures. Genetic analyses of these cells by cytogenetics and array based comparative genome hybridization revealed many genetic alterations including increased ploidy. The flow cytometric DNA Index (DI) changes from 1.28 to 2.28. Cells with DI 2.28 constitute 1.4% of the parental cell line. Whole genome SNP analysis shows that the two populations are genetically related excluding any cell contamination. The invasive cells proliferate and undergo apoptosis similar to the parental cells. Commitment to apoptosis is increased since invasive cells respond more strongly to curcumin or peroxide induced apoptosis. Invasive cells show relative resistance to the cytotoxic, alkylating agent Doxorubicin and increased sensitivity to the anti-mitotic drugs Vincristine and Taxol. Increased resistance to the topoisomerase II inhibitor Mitoxantrone is observed only transiently in invasive cells and lost in long term cultures. Similarly, the chemokines CXCL1 and -2 are transiently upregulated. Response to the anti-diabetic drug Metformin showed a reduced sensibility for invasive cells in terms of growth inhibition (IC50). Gene expression profiling shows complex alterations in gene expression. Many of the genes that are differentially expressed in highly versus moderately invasive cells are differentially expressed in human breast cancer cases with and without distant metastasis and correlate with disease free survival. The invasive phenotype is not related to stem cell features nor to epithelial mesenchymal transition. These cells constitute a novel model for tumor progression. Tumorigenicity and metastatic potential in vivo are currently being tested.

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 496. doi:1538-7445.AM2012-496
Abstract 4960: The role of YY1 in paclitaxel-induced cytotoxicity in epithelial ovarian cancer
Authors: Ota Tsuyoshi , Ota Takayo , Chien Jeremy , Shridhar Viji , Kurachi Hirohisa
Published: August 2012

The Yin Yang 1 (YY1) transcription factor is a member of Polycomb protein family, with important roles in development, differentiation, replication and cell proliferation. While YY1 expression is correlated with survival in serous ovarian cancer, its role in chemotherapeutic agent-mediated cytotoxicity is not known. Immunoblot analysis revealed that YY1 is highly expressed in ovarian cancer cell lines, but not in immortalized ovarian surface epithelial (OSE) cell lines, suggesting a possible role of YY1 in ovarian carcinogenesis. shRNA mediated stable downregulation of YY1 resulted in inhibition of proliferation by CyQuant assay in SKOV3 and SKOV3ip cells. While YY1 downregulation increased the sensitivity against paclitaxel in both cell lines compared to nontargeted cells (NTC) as determined by MTT and clonogenic assays, it had no effect on sensitivity to cisplatin-mediated cytotoxicity. YY1 downregulated clones treated with paclitaxel resulted in an increase in cleavage of poly (ADP-ribose) polymerase and caspase-3/7 activity compared to NTC clones treated with it in SKOV3 and SKOV3ip1 cells. To determine the mechanism of paclitaxel sensitivity modulated by YY1, we performed cell cycle analysis and determined the expression of microtubules at protein level. Cell cycle analysis showed that downregulation of YY1 decreased the proportion of G0/G1 phase with corresponding increase in cells accumulating both S and G2/M phases in SKOV3 and SKOV3ip1 cells. Using nocodazole which synchronizes cells into mitotic phase, we found that blocking mitotic exit by YY1 knockdown slowed the cyclin B1 proteolysis, thus allowing more time for death initiation by paclitaxel. Immunoblot analysis showed that downregulation of YY1 increased the acetylation of alpha-tubulin and attenuated betaIII-tubulin expression compared to NTC cells. Immunoprecipitation and immunofluorescence assays revealed that YY1 interacts with HDAC6 which deacetylates alpha-tubulin and these proteins co-localized in mitotic phase, suggesting that dysfunction of HDAC6 by downregulation of YY1 could lead to microtubules stabilization, resulting in the paclitaxel-induced apoptosis by blocking mitotic exit. Moreover, nude mice bearing xenografts with stable downregulation of YY1 in SKOV3ip1 were more sensitive to paclitaxel-induced cytotoxicity compared to xenografts of NTC cells. Additionally immunohistchemical staining showed a decrease in betaIII-tubulin expression in tumor of YY1 downregulated clones compared to that of NTC clones. Collectively, these results indicate that YY1 acts as a modulator of microtubules expression and might be therapeutically targeted against epithelial ovarian 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 4960. doi:1538-7445.AM2012-4960
Abstract 4970: Convergence of protein kinase and caspase signaling: A mechanism for pathological rewiring of survival pathways by protein kinase CK2
Authors: Turowec Jacob P. , Duncan James S. , Li Shawn , Gloor Greg , Litchfield David W.
Published: August 2012

Crosstalk between protein kinases and caspases has become evident in light of the observation that phosphorylation within the caspase cleavage motif impairs proteolysis. Given that phosphorylation networks and sensitivity to apoptotic cues are altered in cancer signaling, we sought to investigate if the negative regulation of caspase-mediated degradation by protein phosphorylation is a global mechanism that manifests in changes to cancer cell survival. To systematically investigate the extent to which phosphorylation regulates the caspase degradome, we employed a combination of peptide cleavage assays, database mining, and N-terminomics. These studies revealed that phosphorylation could potentially regulate the cleavage of a significant proportion of caspase substrates. One notable example of phosphorylation-regulated caspase cleavage was the observation that pro-caspase-3 itself is phosphorylated in a manner that prevents subsequent cleavage and activation by upstream initiator caspases. To further examine the role of phosphorylation in regulating the activation of caspase-3, we investigated the phosphorylation of pro-caspase-3-FLAG in HeLa cells transfected with each of the catalytic isoforms of protein kinase CK2 in the presence or absence of the regulatory CK2β subunit. These studies suggest that the activation of caspase-3 could be regulated by CK2 in cells. Coupled with the observations that CK2 is constitutively activity and expressed at elevated levels in a number of different cancers, our observations suggest that increased levels of CK2 could lead to a pathological phosphorylation of pro-caspase-3 to prevent its activation and promote cancer cell survival.

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 4970. doi:1538-7445.AM2012-4970
Abstract 5047: Dysregulated microRNAs in cervical neoplasm
Authors: Wong Yick Fu , Cheung Tak Hong , Man Mimi Kwun Nok , Yu Mei Yun , Yim So Fan , Siu Nelson S.S. , Lo Keith W.K. , Doran Graeme , Wong Raymond R.Y. , Wang Vivian W. , Smith David I. , Worley Michael J. , Chung Tony K.H.
Published: August 2012

MicroRNAs (miRNAs) play an important role in a variety of physiological processes as well as pathophysiological processes, including cancinogenesis. This study was to identify a distinct miRNA expression signature for cervical intraepithelial neoplasia (CIN) and to reveal individual miRNAs that may be involved in the development of cervical carcinoma. Expression profiling using quantitative real-time RT-PCR of 202 miRNAs was performed on micro-dissected high-grade CIN (CIN 2/3) tissues as compared to normal cervical epithelium. The expression of 12 miRNAs including miR-518a, miR-34b, miR-34c, miR-20b, miR-338, miR-9, miR-512-5p, miR-424, miR-345, miR-10a, miR-193b and miR-203 were significantly different between high-grade CIN and normal epithelium. This miRNA signature was further validated by an independent set of high-grade CIN tissue. The same characteristic signature can also be used to distinguish cervical squamous cell carcinoma (SCC) from normal controls. Target prediction analysis revealed that these dysregulated miRNAs control the apoptosis signaling pathway and cell cycle regulation. These findings contribute to understanding of the pathogenesis of cervical carcinoma at the molecular 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 5047. doi:1538-7445.AM2012-5047
Abstract 5053: MicroRNA expression profile of astrocytoma according to its malignant potential in Puerto Rican brain tumor samples
Authors: Rivera-Díaz Mónica , Quintero Mario , Billoch Jorge , Marcos Maria , Echevarría Ileabett , Vivas-Meji Pablo E.
Published: August 2012

Astrocytoma, the tumor of astrocytic glial cells, is the most common type of central nervous system (CNS) neoplasms, accounting for more than 60% of all primary brain tumors. As histology-based classification is highly subjective, there is a need for more robust histology-independent molecular classifiers. Although different gene expression and genome-wide array-CGH expression profiles have been use for classification, these expression signatures are yet to be translated to utility in clinical settings suggesting that these studies require further characterization and validation. Micro RNAs (miRNAs) are endogenous, short (19-24 nucleotides) non-protein-coding RNAs that regulate gene expression at the post-transcriptional level. Alterations in the expression profiles of many miRNAs have been described in numerous human tumors. Recent evident indicate that the grade types of astrocytoma could be better classified by using a micro miRNA expression profiles compared with histological or gene expression analysis. Thus, the aim of this project was to construct a miRNA expression profile of astrocytoma according to its malignant potential in Puerto Rican brain tumor samples. Archived formalin-fixed paraffin-embedded (FFPE) samples were used in this study. Not shown in previous literature, our preliminary results showed that miR-27b was more abundant in high grade vs. low grade astrocytoma. Although, miRNA profiling appears to be a very promising tool, nonetheless, further validation of these discriminatory miRNAs in a large number of patients and in independent studies is necessary before clinical application becomes realistic.

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 5053. doi:1538-7445.AM2012-5053
Abstract 5056: A glimpse into the somatic mutation landscape of melanoma through exome sequencing of 121 tumor-normal pairs
Authors: Hodis Eran , Watson Ian , Theurillat Jean-Philippe , Zou Lihua , Place Chelsea , Nickerson Elizabeth , Auclair Daniel , Cibulskis Kristian , Sivachenko Andrey , Kryukov Gregory , Stransky Nicolas , Ramos Alex H. , Voet Douglas , Lawrence Michael S. , Stojanov Petar , Saksena Gordon , McKenna Aaron , Carter Scott L. , Pugh Trevor , Noble Michael , Lin Pei , Lichtenstein Lee , Zupko Robert , Sougnez Carrie , Guiducci Candace , Onofrio Robert C. , Ambrogio Lauren , Fennell Timothy , Chong Kelly , Winckler Wendy , Ardlie Kristin , Lander Eric S. , Golub Todd , Meyerson Matthew , Gabriel Stacey B. , Getz Gad , Wagner Stefan , Schadendorf Dirk , Hoon Dave S. B. , Chin Lynda , Garraway Levi A.
Published: August 2012

Melanoma is an aggressive skin cancer of melanocytic origin characterized by high metastatic potential and mutation rate. Affording a survey of the wide breadth of genomic lesions found in melanoma, we present here an analysis of the somatic mutations discovered in the sequenced exomes of 121 melanoma tumor-normal pairs. We identify frequent genomic alterations both in genes previously implicated in melanoma (BRAF, NRAS, TP53, CDKN2A, PTEN) as well as in several genes whose role in melanoma tumorigenesis has not yet been established and thus are of particular interest. To do so we implement a novel method to increase the identification of genes that are significantly recurrently mutated in melanoma in the setting of its exceptionally high mutation rate. A preponderance of C>T transitions (∼85%) in the observed mutational profile reflects a history of DNA damage due to UV radiation, though the majority of somatic mutations in known melanoma genes are not C>T events. Our study broadens understanding of the genomic lesions involved in melanoma tumorigenesis, and we expect our analysis approach to inform future genomic studies of cancer lineages with similarly high mutation rates.

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 5056. doi:1538-7445.AM2012-5056
Abstract 5084: Functional genomic classification of breast cancer using pooled lentivirus shRNA screens
Authors: Marcotte Richard , Brown Kevin , Sayad Azin , Haider Maliha , Ketela Troy , Moffat Jason , Neel Benjamin G.
Published: August 2012

Targeted therapies for most breast cancers are lacking because our knowledge of essential genes driving tumor proliferation is still primitive for each subtype. Although ongoing intensive efforts to genetically characterize large numbers of breast tumors are providing a plethora of new data on genomic abnormalities (e.g., amplifications, deletions, somatic mutations), it can be difficult to access which of these actively drive pathogenesis. Even if an oncogene or tumor suppressor is identified, these often are not amenable to targeted therapy (eg. KRAS, c-MYC, p53, etc). However, unanticipated gene/pathway dependencies can arise as a consequence of these genetic abnormalities in cancer cells (“synthetic lethality”). The recent development of lentiviral-based shRNA libraries enables genome-wide screening of cultured cancer cells in a pooled format, facilitating the identification of genes necessary for cancer cell proliferation and survival in cell culture as well as potential synthetic lethal interactions. The overall objective of this project was to identify subtype-specific targets for human breast cancer using a genome-wide shRNA screen, as well as to compare “functional genomic” and genomic classification schemes. We screened a panel of 53 breast cancer lines using an 80,000 lentiviral shRNA library targeting 16,000 genes in a pooled format. We identified several classes of gene “dropouts,” including general essential genes, which are required for survival or growth in more than 70% of all cell lines, irrespective of subtype. Most of these were enriched in housekeeping functions, although several genes involved in cellular signal transduction also were identified, including mTORC1, RAPTOR, TGFBR2, DDR1 and DDR2. In addition, we observed several “subtype-specific” genes, whose essentiality is restricted to a defined subtype. Furthermore, unsupervised and supervised clustering of our functional screening results identified potential “drivers” unique to each subtype. These include amplified/overexpressed genes such as ERBB2, ERBB3, FOXA1, SPDEF, TFAP2C, and CCDN1 known to be specific to the luminal and HER2 subtypes, respectively. Finally, integration of gene expression, copy number variation, and functional screening results identified potential synthetic lethal interactions with common genetic changes. Our study represents an extensive functional genetic survey of three major breast cancer subtypes, reveals complexities between genomic and functional genomic results, and uncovers several unexpected gene dependencies and potential novel therapeutic target for each subtype.

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 5084. doi:1538-7445.AM2012-5084
Abstract 5089: Evaluation of BMP4 and BMP7 induced gene expression changes in breast cancer cells reveals time-dependent transcription patterns and a common synexpression group of genes
Authors: Rodriguez-Martinez Alejandra , Alarmo Emma-Leena , Saarinen Lilli , Ketolainen Johanna , Nousiainen Kari , Hautaniemi Sampsa , Kallioniemi Anne H.
Published: August 2012

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta superfamily of growth factors. They are well-known for their key roles in the regulation of various developmental processes and bone formation, but in recent years, evidence has also accumulated of their crucial contribution in tumor biology. In particular, BMP4 and BMP7 have been implicated in breast cancer pathogenesis. However, little is known about BMP target genes in the context of tumor cells. Here, we used whole-genome microarrays to explore the effects of BMP4 and BMP7 treatment on global gene expression levels in seven breast cancer cell lines during a 6-point time series. Several complementary methods, including hierarchical clustering of differentially expressed genes, gene ontology enrichment analyses and model based clustering of temporal data, were used for data analysis. Both BMP ligands had a strong influence on gene expression levels, although the response to BMP4 treatment was more pronounced as compared to BMP7. As might have been expected, the cellular functions most strongly affected by the stimulation of BMP signaling were regulation of transcription and development. The observed transcriptional response, as well as its functional outcome, followed a temporal sequence, where the regulation of gene expression and signal transduction was followed by changes in transcripts involved in metabolism and cell proliferation. Hierarchical clustering analysis revealed distinct differences in the response of individual cell lines to BMPs, but also underlined a common synexpression group of genes for both ligands. Remarkably, the majority of the genes within these synexpression groups were shared by the two ligands, and thus these genes are likely to represent the core molecular responses common to BMP4 and BMP7 signaling pathways. Taken together, our data demonstrate that BMP signaling has a substantial effect on gene transcription in breast cancer cells and that the cellular functions affected follow a logical temporal pattern. Our results also reveal components of the cellular transcriptional response that are common to BMP4 and BMP7. Finally, our study provides a list of putative novel BMP target genes relevant in breast cancer.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5089. doi:1538-7445.AM2012-5089
Abstract 5105: Genetic alterations of the metastatic lesions in ovarian carcinoma
Authors: Malek Joel , Mery Eliane , George Binu , Martinez Alejandra , Thiery Jean-Paul , Huang Ruby , Querleu Denis , Rafii Arash
Published: August 2012

Background: Ovarian cancer is the most deadly gynecological cancer. The high rate of mortality is due to the large tumor burden with extensive metastatic lesion of the abdominal cavity. There are few studies on genetic alterations and their consequences in peritoneal metastatic tumors when compared to their matched ovarian primary tumors. Our hypothesis is that differences between the metastatic and primary lesions might be the cause of residual disease and, most importantly may have a role in post-chemotherapeutic recurrences. Methods: We conducted integrated genomics analysis on matched primary and metastatic tumors from 9 patients. In the papers presented here we analyze genome-wide Copy Number Variations (CNVs) using SNP Arrays targeting peritoneal metastasis differences, Gene expression differences using Microarrays also targeting peritoneal metastasis differences, and for some patients, Single Nucleotide Polymorphisms (SNPs) in genes through Exome sequencing. Results: Here we show that CNVs vary significantly between primary and metastatic tumors and include genes that have been considered potential chemotherapeutic targets based on primary tumor only data. Gene expression differences, while minor, showed highly statistically significant enrichment of genes in ovarian cancer critical pathways. In agreement with findings in other cancers, exome sequencing data revealed very few SNP differences of which most metastasis enriched SNPs were present at very low levels in the primary tumor. The results presented here should allow better design of therapies to target residual ovarian cancer disease.

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 5105. doi:1538-7445.AM2012-5105
Abstract 5142: Up-regulation of glutamine synthetase attenuates the oncogenicity of wnt/β-catenin signaling in hepatocellular carcinoma
Authors: Sohn Bo Hwa , Kim Sang-Bae , Park Yun-Yong , Park In Young , Jeong Mi-Ra , Lee Ju-Seog
Published: August 2012

Wnt/β-catenin signaling has been known to be involved in many cellular processes including cell proliferation, migration, and survival. Aberrant activation of Wnt/β-catenin signaling has been widely reported in many cancers and associated with poor outcome after treatments. However, activation of Wnt/β-catenin signaling in hepatocellular carcinoma (HCC) has been known to be significantly associated with favorable outcome. In this study, we identified 82 genes as a β-catenin signature by analyzing expression data from tumor tissues of patients with HCC and liver tissues from active β-catenin mutant transgenic mice. HCC patients with the β-catenin signature have significantly better prognosis in both overall survival and recurrence-free survival than those without the signature. Further analysis of β-catenin signature revealed over-expression of glutamine synthetase (GS), liver-specific β-catenin target gene, and shift of glutamine metabolism. Cell growth rate were highly dependent on GS expression levels. Cell proliferation rate was increased by GS knockdown using shRNAs but decreased by GS overexpression in HCC cell lines. Intracellular glutamate concentrations were severely decreased by GS overexpression. GS overexpression in HCC cells also led to decrease intracellular α-ketoglutarate levels impeding TCA cycle, and intracellular glutathione levels accumulating high reactive oxygen species (ROS). In conclusion, we found GS up-regulation in HCC cells inhibited cell growth by glutamate depletion and thereby lower anaplerotic metabolic activity and higher ROS levels. Our results present molecular mechanisms for the favorable outcome in HCC patients with CTNNB1 mutations and importance of glutamine metabolism in cancer 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 5142. doi:1538-7445.AM2012-5142
Abstract 5147: Glutamine addiction: Recognizing and harnessing the pathometabolic potential of Ras-mediated macropinocytosis
Authors: Commisso Cosimo , Soydaner-Azeloglu Rengin G. , Bar-Sagi Dafna
Published: August 2012

Oncogenic mutations in Ras-encoding genes are found in approximately 30% of all human tumors and are most prevalent in carcinomas of the pancreas, colon, lung and bladder. Oncogenic forms of Ras are trapped in a constitutively active state and, as a consequence, promote the persistent stimulation of effector pathways that control cell growth, survival and proliferation. A prominent phenotypic feature associated with oncogenic Ras expression is the stimulation of macropinocytosis, an endocytic process that mediates the uptake of extracellular fluid via large, heterogeneous vesicles known as macropinosomes. While in amoeboid organisms this process has been linked to nutrient internalization, the functional significance of oncogenic Ras-mediated macropinocytosis is unknown. We have found that cancer cells harboring oncogenic Ras utilize macropinocytosis to internalize extracellular albumin, a major constituent of extracellular fluids and a potentially rich source of amino acids. Furthermore, we have established that tumor cells harboring oncogenic Ras mutations display robust macropinocytosis in vivo. Utilizing cell biological approaches and amino acid quantification, we determined that extracellular albumin that is internalized via oncogenic Ras-stimulated macropinosomes is targeted for degradation leading to the intracellular production of albumin-derived amino acids. Cancer cells display a heightened dependence on glutamine due to their need to sustain rapid proliferation, bioenergetics and macromolecular biosynthesis. We have observed that in cells expressing oncogenic Ras, the adverse effects of glutamine deprivation on cell growth can be rescued by macropinocytosis-mediated uptake of extracellular albumin. Moreover, the capacity of macropinocytic inhibition to diminish this growth advantage is dependent on both glutamine and glutamine-derived metabolites. These results indicate that macropinocytosis may serve as a glutamine supply mechanism that is particularly critical under conditions where free glutamine is limiting. Together, these data implicate oncogenic Ras-mediated macropinocytosis in the regulation of cancer cell metabolism and point to the possibility of exploiting this process in the design of anti-cancer therapies.

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 5147. doi:1538-7445.AM2012-5147
Abstract 5149: CYLD downregulation induces aerobic glycolysis via mTOR pathway in human oral squamous cell carcinoma cells
Authors: Nakamura Takuya , Shinriki Satoru , Hayashi Mitsuhiro , Yamamoto Yutaka , Ibusuki Mutsuko , Sueyoshi Takanao , Ota Tomoko , Shinohara Masanori , Ando Yukio
Published: August 2012

The hypermetabolic nature of cancer cells and their increased reliance on “aerobic glycolysis,” as originally described by Otto Warburg et al, are considered metabolic hallmarks of cancer cells including oral squamous cell carcinoma (OSCC) cells. However, its precise mechanisms remain unknown. Cylindromatosis (CYLD) is recognized as a tumor suppressor gene whereas little is available about its impact on cancer progression. Our unpublished data showed that lower CYLD expression was associated with poor prognosis in OSCC patients. The objective of our study was to address specific contributions of CYLD to the signature metabolic features of cancer cells, including the so-called “Warburg effect”. We investigated effects of CYLD knockdown by siRNA on aerobic glycolysis in human OSCC cell lines. As the result of CYLD knockdown, both glucose consumption and lactate production significantly increased in several OSCC cell lines with increased expression of LDHA which is known to be important for glycolysis. Consistently, culture medium became more acidic after CYLD knockdown compared with the control. Repression of CYLD expression induced Akt phosphorylation. Importantly, the increase in aerobic glycolysis induced by CYLD repression was completely blocked by an mTOR inhibitor, rapamycin as well as a PIK inhibitor, LY294002. Our study indicats that dysfunction of CYLD contributs to the Warburg effect through Akt-mTOR pathway in OSCC. Further investigation on the advantage of such increased glycolysis and the change in metabolome profiling may lead to development of effective therapeutic strategies for OSCC and other malignancies.

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 5149. doi:1538-7445.AM2012-5149
Abstract 516: Obinutuzumab (GA101): A novel type II glycoengineered CD20 antibody exhibits enhanced cell death against Rituximab-resistant and -sensitive cell lines in B-cell non-Hodgkin lymphoma
Authors: Tiwari Aradhana Awasthi , Ayello Janet , ven Carmella Van de , Cairo Mitchell S.
Published: August 2012

Background: Rituximab (RTX), an antibody targeting the B-cell antigen CD20, in combination with chemotherapy, is safe, well tolerated and associated with > 90% EFS in children with advanced mature B-Cell NHL (Cairo M.S. et al, ASCO 2010). However, RTX resistance has evolved as one of the limiting factors of such therapy and the exact cause of this resistance is not well understood. C-terminal deletion mutations of CD20 may be one possible reason such resistance or for relapse (Mishima Y. et al, Blood Cancer Journal 2011). Obinutuzumab (GA101), a novel type II glycoengineered CD20 antibody of the IgG1 isotype, demonstrates superior cell death induction and its glyco-engineered Fc region has shown to cause significant enhanced ADCC (Mössner et al, Blood 2010; Niederfellner G. et al, Blood 2011). The objective of this study was to evaluate the efficacy of GA101 as compared to RTX against RTX resistant cell lines (Raji-2R and Raji-4RH, generously supplied by M. Barth, MD, Roswell Park Cancer Center, Buffalo, NY) in-vitro. Methods: We evaluated the efficacy of GA101 against Raji (Burkitt Lymphoma), a RTX sensitive cell line (RSCL) and RTX resistant cell lines (RRCL), Raji-2R and Raji-4R (Barth M. et al, ASH 2010). All cell lines were cultured in RPMI with 10% FBS and incubated with dose escalation of GA101 (1-100 µg/ml) for 24 hrs. Cell death was evaluated by staining with AnnexinV/7AAD using a standard kit (BD Biosciences) and flow-cytometry. B-Cell Leukemia-Lymphoma (BLL) U-698-M cells (CD20+) (DSMZ, Germany) were used as the positive control; whereas, Loucy cells (CD20−) (T-ALL) (ATCC, Manhass, VA) were used as the negative control. Results: At 100 µg/ml of GA101 when incubated for 24 hrs, Raji and U-698-M demonstrated 26.5±0.42% and 32.4±1.9% cell death, respectively. Cell death of the RRCL, Raji-2R and Raji-4RH, was 16.3±3.4% and 17.5±0.07% respectively, at 100 µg/ml of GA101 at 24 hrs. Significant cell death was demonstrated with RSCL as compared to the RRCL, Raji vs Raji-2R (p<0.05) and Raji vs Raji-4RH (p<0.01). There was no significant change in cell death with any concentration of GA101 with the Loucy cell line. Conclusion: Based on these findings, GA101 is a promising novel CD20 antibody for the treatment of RTX resistant B-Cell Lymphomas and related B-Cell malignancies. Further studies are aimed to identify novel candidates that may increase the efficacy of GA101 against RTX resistant cell lines in-vitro and in-vivo. One such approach is to combine GA101 with activated NK cells or a chemotherapeutic agent.

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 516. doi:1538-7445.AM2012-516
Abstract 5162: COX-2 downregulation protects SUM-149 cells following targeting of choline phospholipid metabolism
Authors: Mori Noriko , Stasinopoulos Ioannis , Wildes Flonne , Bhujwalla Zaver M.
Published: August 2012

Enzymes in the choline phospholipid metabolism pathway such as choline kinase (Chk) and phopholipase D1 (PLD1) are being evaluated as novel targets in cancer therapy since these enzymes are overexpressed in aggressive cancers including breast cancer. We have previously shown that single and double downregulation of Chk and PLD1 significantly reduced cell proliferation in breast cancer cells. We also observed a close association between Chk and cyclooxygenase 2 (COX-2), an inducible enzyme that mediates the inflammatory response and plays a role in cancer invasion, angiogenesis and metastasis. COX-2 inhibition is also being considered as a strategy for cancer prevention or treatment. Here we investigated the effect of the COX-2 downregulation combined with PLD1 and Chk downregulation using siRNA, on cell proliferation to achieve improved control and minimize compensatory responses that allow the cell to survive or adapt. The triple negative inflammatory breast cancer cell line, SUM149, was used in this study. Cells were transfected with 25 nM siRNA against COX-2 (siRNA-cox2), PLD1 (siRNA-pld1) or Chk (siRNA-chk) either singly or combined for 48 h. Untreated cells and the transfection reagent (DharmaFECT) treatment were used as negative controls. Immunoblot analysis was done after 48 h transfection using antibodies specific to COX-2, PLD1, Chk-α, and GAPDH (as a loading control). Proliferation/viability of cells was determined using the CCK-8 assay (cell counting kit-8, Dojindo) at day 2 and day 5 after transfection. After 48 h transfection with siRNA-pld1 or siRNA-chk, PLD1 or Chk-α protein were downregulated significantly and COX-2 protein was upregulated. When we combined downregulation of Chk or PLD1 with COX-2 dowregulation, we established significantly lower COX-2 protein levels compared to single knockdown of Chk and PLD1. COX-2 downregulation alone did not significantly reduce proliferation. Downregulation of PLD1 or Chk, however, significantly reduced proliferation compared to untreated cells (PLD1: –75%, Chk: –68%). When we combined COX-2 downregulation with PLD1 or Chk downregulation, cell survival was significantly higher than with just PLD1 or Chk alone (PLD1: –46%. Chk: –28%). These results suggest that COX-2 downregulation rescues cells following downregulation of PLD1 or Chk in SUM149 cells, identifying a different facet of COX-2 in the choline phospholipid pathway. This work supported by R01 CA82337

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 5162. doi:1538-7445.AM2012-5162
Abstract 5173: The human homologue of frog (Xenopus laevis) anterior gradient protein promotes metastasis via regulation of cellular adhesion
Authors: Chanda Diptiman , Sawant Anandi , Hensel Jonathan Adam , Reilly Stephanie D. , Siegal Gene P. , Smith Claire , Isayeva Tatyana , Ponnazhagan Selvarangan
Published: August 2012

Our recent studies indicated a significant up-regulation of anterior gradient protein-2 (AGR-2) in bone metastatic human prostate cancer cells, PC3, following growth in bone marrow conditioned medium. Evidence indicates that AGR2 belongs to the family of protein disulfide isomerases and has been linked to intestinal mucus production, tumor growth and metastasis of various adenocarcinomas and poor survival of patients with breast and prostate cancers. Few recent reports have also reported the regulatory elements of AGR2 function, but its specific role(s) in tumorigenesis and metastasis is still unclear. In the present study, we have analyzed the role of AGR2 in cancer progression and metastasis using prostate cancer as a model. Human prostate cancer tissue microarrays and autopsy tissues, obtained from University of Alabama at Birmingham consortiums were immunostained for AGR-2 expression. Results were also confirmed in a spontaneously developing transgenic adenocarcinoma of mouse prostate (TRAMP) model. Further characterization of AGR-2 function was determined in PC3 cells with targeted silencing of AGR-2 expressions for tumor growth and metastases in vitro and in vivo. Results of our study found AGR-2 expression mainly in the prostate luminar epithelial cells, but no difference was observed between normal prostate gland, BPH and early stages of prostate cancer in both human and TRAMP mouse. Significant reduction in AGR-2 expression was observed in grade IV & V prostate cancers. Interestingly, although declining AGR-2 was evident in higher grade prostate tumors, significantly elevated AGR-2 expressions were observed in the metastatic sites of the same individuals. AGR-2-silenced PC3 cells formed tumors in SCID mice similar to control cells whereas their ability to remain attached to various ECM proteins were greatly compromised. Several key integrins, which mediates ECM binding, were also found to be down-regulated in the AGR-2 silenced PC3 cells, suggesting loss of AGR-2 may be associated with their detachment from the primary tumor and initiation of metastasis. AGR-2 is further up-regulated in metastatic sites indicating its necessity for restoration of integrins for attachment in a new microenvironment.

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 5173. doi:1538-7445.AM2012-5173
Abstract 5262: Disruption of alternative and classical NF-kappaB signaling patterns: Connections to branching morphogenesis and mammary tumorigenesis
Authors: Onishko Halina , Barham Whitney , Chen Lianyi , Cheng Dongsheng , Tikhomirov Oleg , Connelly Linda , Blackwell Timothy , Yull Fiona E.
Published: August 2012

NF-kappaB transcription factors including p65 (RelA), RelB, p105/p50, and p100/p52 play roles in mammary development and tumorigenesis. While most studies have focused on classical NF-kappaB signaling (p65/p50), alternative NF-kappaB signaling is elevated in human primary breast tumors and in human breast cancer cell lines. In addition, we have reported that overexpression of p100 within the mouse mammary epithelium during pregnancy and lactation led to ductal thickening and hyperplastic foci. To determine the contribution of alternative NF-kappaB signaling to mammary development and tumorigenesis, we have developed murine transgenic models that enable us to inducibly express activators or inhibitors of classical or alternative NF-kappaB signaling in the mammary epithelium by adding doxycycline to the drinking water of mice. Modulation of both the classical and alternative pathways will enable us to determine the roles that each arm plays independently or synergistically. Using immunoflourescent staining we have identified distinct patterns of expression of the alternative and classical pathways in mammary epithelium, a previously undescribed phenomenon. Classical p65 signaling was found within the luminal cell population and p100/p52 signaling was observed almost exclusively within the basal cell layer. Upon induction of constitutively activated IKK2 or a dominant negative IKBalpha, two regulators of the classical NF-κB pathway, alterations in branching morphogenesis were observed within the developing virgin gland. These branching phenotypes were associated with a break down in the previously observed classical/alternative signaling pattern, as luminal cells were now expressing low levels of p100/p52. Furthermore, this patterning is disrupted during the development of polyoma middle T oncogene induced tumors in which we also observe alternative signaling no longer confined to the basal layer. Ongoing experiments with our newest transgenic model are designed to determine the effects of inducing alternative signaling, through overexpression of p52, within the mammary epithelium. Our models may reveal novel roles for the alternative NF-kappaB pathway in maintaining the basal/luminal structure of mammary epithelial ducts and connect aberrant p100/p52 signaling to tumor progression, potentially identifying a novel therapeutic target.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5262. doi:1538-7445.AM2012-5262
Abstract 5263: The APC tumor suppressor controls epithelial polarization and morphogenesis
Authors: Prosperi Jenifer R. , Schwertner Adam , Yang Frank F. , Goss Kathleen H.
Published: August 2012

Adenomatous polyposis coli (APC) is a multifunctional tumor suppressor that is frequently mutated or lost in most epithelial-derived tumor types, including colorectal, breast and renal cancer. We have previously demonstrated that germline heterozygous Apc mutation disrupts mammary epithelial integrity. In addition, Apc mutation advances polyoma middle T antigen (PyMT)-mediated mammary tumorigenesis. Interestingly, in both of these models, the effect of Apc mutation was independent of Wnt/β-catenin pathway activation. The objective of the present study was to extend these observations to in vitro models and identify the molecular mechanism by which APC exerts its tumor suppressive function through regulation of epithelial polarity and morphogenesis. Using lentiviral-mediated RNA interference, APC was stably suppressed in non-transformed HC11 mammary and MDCK kidney epithelial cells. Although the APC-knockdown cells did not show any significant morphological or growth differences compared to control vector-only or scrambled shRNA-infected cells on plastic, they showed a dramatic multi-layering phenotype when cultured on permeable supports. Moreover, the polarized localization of basolateral markers, such as Dlg and Scrib tumor suppressor proteins, and apical proteins, including MUC1, was severely disrupted. When the cells were cultured under three-dimensional conditions and compared to controls, the APC-knockdown epithelial cells formed enlarged, non-spherical acini and cysts with filled-in lumens, many of which were invasive into the surrounding matrix. Remarkably, in the MDCK APC-knockdown cells, inverted polarity was observed with the apical marker gp135 localized to the periphery of the cysts. Similar to our previous in vivo studies, these polarization defects associated with APC loss did not appear to require activation of the Wnt signaling pathway but likely involve dysregulation of APC-associated cytoskeletal and polarity complex components. These data indicate that APC is a potent regulator of epithelial polarization and morphogenesis and suggest that one mechanism by which APC loss promotes tumorigenesis is by disrupting epithelial architecture to subsequently drive tumor cell growth, survival and invasion.

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 5263. doi:1538-7445.AM2012-5263