bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3979: Ethnic polymorphisms in the GGAA microsatellite response element of key Ewing's sarcoma EWS/FLI-target genes
Authors: Monument Michael J. , Beck Robert , Watkins W. Scott , Jorde Lynn B. , Randall R. Lor , Lessnick Stephen L.
Published: August 2012

Study Rationale: Unlike other cancer models, the risk factors predisposing patients to the development of Ewing's sarcoma and predicting subsequent disease behavior are largely unknown. Interestingly, the incidence of Ewing's sarcoma is highest in populations of European descent, which is 10X higher than the incidence observed in those of African descent. Furthermore, cure rates and overall survival are significantly worse in patients of African descent. Recently, it has been shown that the EWS/FLI fusion product selectively binds to a (GGAA)n microsatellite response element embedded within the promoter region of key EWS/FLI-target oncogenes. An increasing number of these repeats also appears to positively affect DNA binding and gene activation. We hypothesize that differences in polymorphic GGAA microsatellites within the promoter regions of key EWS/FLI-target genes may exist in African and European populations, contributing to observed differences in Ewing's sarcoma incidence and outcomes. Methods: Genomic DNA samples were obtained from 100 subjects each of African and European descent. The microsatellite regions within the promoter elements of two key EWS/FLI target genes (NR0B1 and CAV1) were PCR amplified, cloned and sequenced. Microsatellite sequences were analyzed and the overall length of the microsatellite, the total number of GGAA repeats and the maximal number of consecutive repeats were quantified. Data was statistically interpreted using a comparative two-tailed t-test. Results: Measurements of the CAV1 microsatellite response element were quite similar for both African and European subjects. In sharp contrast, marked inter-ethnic variation was observed for the NR0B1 GGAA microsatellite response element. African samples had a significantly longer GGAA microsatellite, a greater number of total (GGAA)n repeats and longer consecutive (GGAA)n repeats (p<0.0001). Conclusions: These data demonstrate a marked inter-ethnic variation in the genetic constitution of the Ewing's sarcoma EWS/FLI-response element within the NR0B1 promoter. Given the epidemiological differences of Ewing's sarcoma susceptibility and disease outcome in these divergent populations and the importance of NR0B1 dysregulation in Ewing's sarcoma oncogenesis, this novel finding warrants further functional investigation.

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 3979. doi:1538-7445.AM2012-3979
Abstract 398: Tumor microenvironment imposes major alterations and profoundly shapes functional status of macrophages: peritoneal and tumor-associated macrophages from tumor hosts are two very different subpopulations
Authors: Torroella-Kouri Marta , Rodriguez Dayron , Silvera Risset , Nadji Mehrjad , Caso Raul , Rodriguez Gracielena , Gonzalez-Perez Ruben R. , Iragavarapu-Charyulu Vijaya , Carrio Roberto
Published: August 2012

Macrophages are key players in the inflammatory response and exhibit significant roles in the different stages of tumor progression. We have previously reported deficient functions of peritoneal macrophages of D1-DMBA3 mammary tumors bearers (T-PEMs). We showed that T-PEMs are impaired in inflammatory activity due to diminished expression of transcription factors NFκB and C/EBP. We have also demonstrated that T-PEMs display neither M1 nor M2 phenotypes, exhibit deficiencies in the production of several inflammatory cytokines, are less differentiated and show increased apoptosis. Importantly, we showed for the first time that their apoptosis in periphery contributes to increased myelopoiesis. We examined the blood monocytes from these tumor mice and demonstrated that these cells are neither inflammatory nor suppressive and are less differentiated, similar to the T-PEMs they later become. In the present study we investigate tumor-associated macrophages (TAMs) from the same tumor model and compare them with T-PEMs and with peritoneal macrophages from normal mice (N-PEMs). Our results show that TAMs downregulate IL-12p70, but upregulate IL-12p40, IL-23, IL-6 and IL-10 and chemokines and their receptors are very differently expressed in these two macrophages subpopulations. NFκB and C/EBP family members are decreased in TAMs as compared with T-PEMs, however NFκBp50 homodimers, STAT1/pSTAT1 and STAT3/pSTAT3 are all overexpressed. Moreover TAMs block T-cell proliferation and are more prone to apoptosis than T-PEMs due to a higher expression of activated caspase-3 and downregulation of Bcl-X. Our results show that T-PEMs and TAMs have equally impaired phagocytic capacity compared with fully-phagocytic N-PEMs. Moreover, TAMs constitutively express iNOS, produce nitric oxide but no arginase and are Gr-1high and CD11blow, thus are not classic myeloid-derived suppressor cells. Collectively, our results show that the exposure to different locations and tumor factor gradients determine macrophage phenotypes and functions. The tumor microenvironment, as the setting in most intimate contact with tumor cells is the one that most alters macrophage activity. Targeting tumor factors and other cells in the tumor microenvironment may allow reversion of macrophage dysfunction.

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 398. doi:1538-7445.AM2012-398
Abstract 3987: Merlin is a negative regulator of human melanoma
Authors: Murray Lucas B. , Lau Ingar , Yu Qin
Published: August 2012

Merlin is a cytoskeletal linker protein encoded by the neurofibromatosis type 2 (NF2) gene. Like the other Band 4.1 protein family members, it serves to connect cell surface proteins to the actin cytoskeleton. Defects in the protein caused by mutations of the NF2 gene give rise to NF2 disease, which is generally characterized by the formation of bilateral vestibular schwannomas and, to a lesser extent, meningiomas and ependymomas. In addition to these tumor types, merlin has been shown to be mutated and/or merlin protein expression lost in other cancers including mesothelioma, thyroid cancers, and melanomas, albeit to a lesser extent. In the current study, we set out to elucidate the role of merlin in the progression of human melanoma. We first show that merlin knockdown enhances melanoma cell migration and invasion in vitro. Furthermore, we show that decreased merlin expression leads to increased subcutaneous melanoma growth in immuno-compromised mice. Tumors derived from merlin knockdown cells display border regions that are generally less well defined, with cases in which tumor cells are visible in the surrounding dermis and adipose tissue, which is consistent with the more invasive phenotype observed in vitro upon merlin knockdown. To compliment these knockdown experiments we overexpressed merlin in a metastatic melanoma cell line. Upon increased merlin expression, the melanoma cells exhibit decreased in vitro migration, invasion, and proliferation. These cells also display a diminished ability to form anchorage independent colonies in soft agar. Additionally, increased merlin expression inhibits in vivo subcutaneous growth of the melanoma cells in our xenograft experiments. Lastly, we surveyed several signaling pathways to identify a mechanism that exerts the negative regulatory effect of merlin on melanoma growth. We found that merlin protein levels in human melanoma cells are correlated with the extent of induced activation of the known tumor suppressors in the Hippo signaling pathway, MST1/2 Ser/Thr kinases, which have been shown to function downstream of merlin in other cell types. Taken together, these results provide evidence for the first time that merlin is a negative regulator of human melanoma progression and that it may exert its inhibitory effects through the activation of the MST1/2 kinases.

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 3987. doi:1538-7445.AM2012-3987
Abstract 399: Deletion of the Mint3 gene in mice abrogates macrophage functions and increases resistance to cancer metastasis
Authors: Hara Toshiro , Sakamoto Takeharu , Seiki Motoharu
Published: August 2012

Hypoxia inducible factor-1 (HIF-1) is the master transcription factor to shift the energy production system from oxidative phosphorylation (OXPHOS) to glycolysis during hypoxia. Although most cells employ OXPHOS for ATP during normoxia, macrophage is unique in using glycolysis constitutively. We have proposed that macrophages use Mint3 to inhibit FIH-1 that suppresses transcriptional activity of HIF-1 during normoxia. To demonstrate physiological function of Mint3 in macrophages, we establish Mint3 deficient mice and analyze macrophage specific functions. The mutant mice have no apparent phenotype but exhibit significant resistance against lipopolysaccaride (LPS)-induced septic shock and cancer metastasis. Macrophages obtained from the mutant mice exhibit 60% levels of ATP content compared to wild type cells, lower levels of glycolysis, cytokine production and motility. Thus, cell type specific role of Mint3 in energy production in macrophages is demonstrated for the first time using genetically manipulated mice. Specific function of Mint3 in macrophages might allow us developing therapeutics to regulate excessive macrophage functions during infection and diseases such as 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 399. doi:1538-7445.AM2012-399
Abstract 3990: SPANXA1 suppresses tumor metastasis through WNT pathway in lung cancer
Authors: Kang Po-Hsiang , Hsu Chia-Ying , Su Kang-Yi , Chen Hsuan-Yu , Yu Sung-Liang
Published: August 2012

SPANX (Sperm protein associated with the nucleus, X-linked) gene family has been found higher expression in many cancers, such as testicular germ tumor, melanoma, glioblastoma and hematologic malignancies. However, in our previous study, the gene expression array analysis showed that the expression of SPANX family member A1 (SPANXA1) was higher in lower invasive lung cancer cells, CL 1-0, than in higher invasive cells, CL 1-5. Real-time PCR was used to confirm that mRNA expression of SPANXA1 was 8 times higher in CL 1-0 than CL 1-5. In vitro assays showed that migration and invasion abilities of CL 1-5 stable transfectants were decreased in a dose-depended manner. Interestingly, cell morphology dramatically changed from mesenchymal-like to epithelial-like in CL 1-5 SPANXA1 stable transfectants, which was consistent to its decreased invasive ability in vitro. Therefore, we demonstrated metastasis ability of CL 1-5 SPANXA1 stable transfectants would be down-regulated in vivo, which was performed in mouse metastasis model by tail-vein injection. Next, we set up CL 1-0 lentiviral-base shRNA knockdown clones, and the cell mobility and invasion ability were increased. Because SPANXA1 could induce the cell morphology change, we evaluate the effect of SPANXA1 on the expression of EMT markers. We found up-regulation of E-cadherin, an epithelial marker, and down-regulation of vimentin and N-cadherin, which were known as mesenchymal markers in presence of SPANXA1. Finally, to identify the underlying mechanism of SPANXA1, we carried out gene expression array and 3 of top 10 pathways are EMT-related. Furthermore, quantitative RT-PCR validation showed WNT pathway is involved in SPANXA1-repressed metastasis indicated that SPANXA1 might provide a new treatment in lung 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 3990. doi:1538-7445.AM2012-3990
Abstract 3992: Tid1, a tumor suppressor, regulating the cancer initiation properties of HNSCC
Authors: Chen Yu-Hsuan , Lo Jeng-Fan
Published: August 2012

Background: Tid1, a DNAJ/Hsp40 family protein, functions as a tumor suppressor in different types of cancer including HNSCC and influences many signaling pathway in cancer cells. Expression of Tid1 protein is negatively associated with the malignancy of HNSCC cells and tumor tissues. In addition, it has been shown that Tid1 interacts with P53, which exerts negative regulation of Nanog, one of the transcriptional factors to maintain the cellular stemness properties. Epithelial-mesenchymal transition (EMT), a process by which epithelial cells lose their polarity and later acquire a migratory mesenchymal phenotype, is one of the crucial processes that induce tumor invasion and metastasis. Further, gain of EMT could promote stem cells (SCs) properties. Accumulating data support the hierarchical model of cancer initiating cells (CICs) or cancer stem cells (CSCs) in that each tumor formation is governed by a rare subpopulation of cells with self-renewal capacity. CICs have been demonstrated to have capacities of promoting tumor growth, tumor regeneration and metastatic progression. Methods: Cancer cells with Tid1 knockdown or overexpression were generated to evaluate the different properties of stemness and tumorigenicity in vitro and in vivo. Further, the differential gene expression profile of EMT signature and stemness of cancer cell with Tid1 upregulation or downregulation were examined. Results: We observed that Tid1 knockdown enhanced expression of EMT-activator, Snail, and stemness marker Grp78, Nanog, and also exhibited higher ability of migration, invasion, anchorage-independent and sphere formation. In addition, Tid1 overexpression cells decreased cell mobility, sphere formation ability and displayed loss of EMT-activator and stemness marker. Conclusion: Overall, we proved that expression of Tid1 is negatively correlated with EMT and cancer initiation properties in HNSCC. Tid1 potentially could be a key regulator involved in differentiation of cancer cell. Further, therapeutic strategies on target Tid1 mediated malignant properties will be an alternative treatment on for HNSCC.

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 3992. doi:1538-7445.AM2012-3992
Abstract 3993: MiRNA-141 and methylation regulation of WNT signal pathway by suppressing SOX17 in esophageal cancer
Authors: Guo Mingzhou , Jia Yan , Yang Yunsheng , Zhan Qimin , Herman James G.
Published: August 2012

Purpose: WNT/β-catenin gene SOX17 was reported frequently methylated in colonic and hepatic cancer. To explore epigenetic changes of SOX17 as esophageal cancer marker and therapeutic target, epigenetic regulation and tumor suppressor function of SOX17 were analyzed in study. Experimental Design: Methylation specific PCR (MSP) was employed to examine SOX17 methylation in 8 esophageal cancer cell lines and 9 normal mucosa, 60 dysplasia and 169 cancer tissue. Semi-quantitative RT-PCR method was used to examine SOX17 expression before and after 5-aza-2′-deoxycytidine treatment of esophageal cancer cells. Expression of SOX17 and β-catenin were detected by immunohistochemistry (IHC) in esophageal cancer and adjacent tissue. Luciferase reporter assay, colony formation, transfection and western blotting were applied to analyze SOX17 function in WNT signal pathway. Prediction software and miRNAs related detection methods were preformed to evaluate miRNA regulation of SOX17. Results: SOX17 was silenced by promoter region methylation and reactivated by 5-aza-2′-deoxycytidine treatment in esophageal cancer cell lines. Loss of SOX17 expression and accumulation of β-catenin in nuclear and cytoplasm were associated with promoter region hypermethylation in esophageal cancer(test, P=0.0359, P=0.0105). Restoration of SOX17 expression inhibited β-catenin/TCF-dependent transcription and colony formation. The presence of SOX17 methylation correlated significantly with alcohol history in esophageal cancer(test, P=0.0057). MiR-141 was also found to down-regulate SOX17 expression and activated the WNT signal pathway. Conclusions: SOX17 is frequently methylated in esophageal cancer, and SOX17 expression is regulated both by miR-141 and promoter region methylation. Loss of SOX17 removes the normal inhibition of WNT signaling and promotes esophageal tumorigenesis.

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 3993. doi:1538-7445.AM2012-3993
Abstract 3996: Identification of DEC1-interacting protein DNAJB6 and its role in esophageal squamous cell carcinoma
Authors: Yu Zhuoyou , Wong Victor Chun Lam , Chan Kwok Wah , Lung Maria Li
Published: August 2012

Esophageal squamous cell carcinoma (ESCC) has an especially high incidence in China, with five-year survival rates in Hong Kong being of ∼14%. Previously, a critical region at 9q33-34 was narrowed down and identified to be associated with tumor-suppressive function in ESCC. Within this region, Deleted in Esophageal Cancer 1 (DEC1), was identified to suppress anchorage-independent growth and tumorigenesis. In this study, we searched for DEC1-interacting partners by yeast two-hybrid screening. DNAJ (Hsp40) homologue subfamily B member 6 (DNAJB6) was identified as a DEC1-interacting protein and its interaction was further confirmed by the GST pull-down assay and co-localization studies. A putative DEC1-interacting sequence was mapped to the C-terminal end of DNAJB6 coding sequence. Using a tissue microarray constructed with tissues from Hong Kong, significantly higher survival in distant metastasis-free (M0) patients was found to be associated with higher DNAJB6 nuclear staining. DNAJB6 is down-regulated in 12 out of 14 (85.7%) ESCC cell lines, when compared to expression in an immortalized normal esophageal epithelial cell line. The two isoforms of DNAJB6 have distinct cellular localizations. DNAJB6a is mainly localized to the nucleus, while DNAJB6b shows diffuse staining in both the nucleus and cytoplasm. Through migration/invasion assay, we showed that expressions of both isoforms were inversely associated with migration/invasion ability of ESCC cell lines. These results suggest DNAJB6 plays a role in ESCC migration/invasion and metastasis. This gene may play a critical role in ESCC metastasis in an isoform-specific manner, which will be the focus of future studies.

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 3996. doi:1538-7445.AM2012-3996
Abstract 3999: Experimental study of tumor suppressor gene RBM5 in non-small cell lung cancer
Authors: Wang Ke , Shao Chen , Zhao Lijing
Published: August 2012

Introduction: Deletion within the lung cancer tumour suppressor region at 3p21.3 constitutes the earliest premalignant chromosomal aberration in human lung cancers. Nineteen genes map to this region. RNA Binding Motif 5(RBM5) maps to one end of this 19-gene deletion breakpoint, and is not included in a slightly smaller 17-gene deletion that is common to breast and renal tumours. This suggests a critical role of RBM5 in lung cancers. To further explore the role of RBM5 in lung cancer, we examine the RBM5 expression in primary tissues and the associated function in vitro. Method: Paired non-tumor and tumor samples were obtained from 30 adenocarcinomas. The mRNA and protein expression of RBM5 were examined by semi-quantitative reverse transcription-polymerase chain reaction and Western blot. A549 cell line was used to determine the apoptotic function of RBM5 in vitro. A549 cells were transient transfected with RBM5, the AnnexinV, PAPP and cell cycle analysis were performed by flow cytometry and Western blot respectively. Results: We found that RBM5 mRNA and protein expression levels were reduced in tumor tissue compared the non-tumor tissue (P<0.05). Overexpression of RBM5 in A549 cells resulted in an increase of PAPP expression, phosphatidyl serine (PS) flip to the outer cellular membrane and G arrest in cell cycle. Conclusion: Decreased expression of RBM5 in adenocarcinomas suggests that RBM5 may play a crutial role in the carcinogenesis. Enhanced apoptotic activity induced by RBM5 overexpression in A549 cell line might be one of the essential mechanisms by which RBM5 function as a tumour 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 3999. doi:1538-7445.AM2012-3999
Abstract 4: Gene amplification of EGFR, HER2, HER3 and HER4 in esophageal squamous cell carcinoma
Authors: Kato Hiroaki , Arao Tokuzo , Matsumoto Kazuko , Fujita Yoshihiko , Kimura Hideharu , Nishiki Kouhei , Shiraishi Osamu , Yasuda Atsushi , Peng Ying-Feng , Shinkai Masayuki , Imano Motohiro , Imamoto Haruhiko , Yasuda Takushi , Okuno Kiyotaka , Shiozaki Hitoshi , Nishio Kazuto
Published: August 2012

Overexpression of epidermal growth factor receptor (EGFR) and its family members HER2, HER3 and HER4 are found in a variety of human malignancies and their roles in cancer development and progression has been widely recognized for long time. Molecular drugs targeting EGFR family, such as small-molecule inhibitors and specific monoclonal antibodies, are now under intensive investigation in clinical setting. In esophageal squamous cell carcinoma (ESCC), EGFR and HER2 are frequently overexpressed and their poor prognostic impacts have been reported to date. However, there is no study evaluating gene amplification of EGFR, HER2, HER3 and HER4 simultaneously in ESCC in a large cohort. We examined gene amplification of EGFR, HER2, HER3 and HER4 using realtime PCR-based copy number assay on 196 surgical specimens of formalin-fixed, paraffin-embedded (FFPE) samples. Gene amplification of EGFR and HER2 (copy number > 4) was observed in 7 % and 11%, respectively. Although the copy numbers were relatively low, we found gene amplification of HER3 and HER4 to be observed in 8% and 7%, respectively. Gene amplification at least one or more genes was observed in 23 % (45/196) of ESCC samples. Overlapping gene amplification was identified in 8 % (15/196) of samples. In relation with clinical characteristics, gene amplification of HER2 and HER4 was significantly involved in shorter relapse-free survivals in stage III ESCC. In conclusion, gene amplification of EGFR family was frequently observed in ESCC and PCR-based copy number assay could be a powerful tool for detecting gene amplification using FFPE samples. Our results strongly encourage the development of EGFR family-targeted therapy for ESCC with gene amplification.

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 4. doi:1538-7445.AM2012-4
Abstract 4001: SOX7 is a potential tumor suppressor in breast cancer
Authors: Stovall Daniel B. , Wan Meimei , Zhang Qiang , Liu Gaifang , Cao Paul , Miller Lance D. , Liu Wennuan , Xu Jianfeng , Sui Guangchao
Published: August 2012

Among U.S. women, breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer-related deaths. Both epigenetic silencing and genetic deletion of tumor suppressors are critical for the development and progression of breast cancer. The SOX (Sex-determining region Y-box) genes encode a family of over 20 transcription factors, including SOX7. The SOX7 gene is located on chromosome arm 8p and is deleted in a number of human cancers, including breast cancer. Previously, SOX7 has been demonstrated to be down-regulated in human tissues of prostate and colon cancers and its ectopic expression inhibits proliferation and increase apoptosis in prostate and colon cancer cell lines. However, the functional role of SOX7 in breast cancer has not yet been reported. Our studies revealed a significant correlation between SOX7 transcript levels and clinical outcome in gene array data from an international meta-cohort of 674 breast cancer patients. Consistently, we observed that SOX7 mRNA levels and protein expression are down-regulated in multiple breast cancer tissues and breast cancer cell lines compared to adjacent normal tissue and non-tumorigenic cells, respectively. Further, we have observed that the SOX7 promoter is hyper-methylated in breast cancer cell lines compared to non-tumorigenic breast cells, and that inhibition of SOX7 methylation results in an increase in SOX7 mRNA. Importantly, shRNA-mediated silencing of SOX7 conferred a growth advantage to non-tumorigenic breast cells, while induced ectopic SOX7 expression inhibited proliferation and migration of breast cancer cells. Overall, our data suggest that SOX7 may act as a novel tumor suppressor 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 4001. doi:1538-7445.AM2012-4001
Abstract 4004: P21-activated kinases (PAKs) are novel therapeutic targets in human breast cancer
Authors: Ong Christy , Jubb Adrian , Haverty Peter , Zhou Wei , Lyle Karen , Harris Adrian , Belvin Marcia , Friedman Lori , Koeppen Hartmut , Hoeflich Klaus P.
Published: August 2012

P21-activated kinases (PAKs) are important mediators of Rac and Cdc42 GTPase function as well as pathways required for Ras-driven tumorigenesis. PAKs have been implicated in signaling by growth factor receptors and morphogenetic processes that control cell polarity, invasion and actin cytoskeleton organization. To better understand the role of PAK1 and PAK4 in breast tumorigenesis, genomic copy number, mRNA and protein expression were determined for a panel of 291 human breast adenocarcinomas and loss-of function was assessed in cell lines using genetic and pharmacologic approaches. Interestingly, PAK1 genomic amplification at 11q13 was prevalent in luminal breast cancer and PAK1 levels in early ductal carcinoma in situ (DCIS) samples was similar to that of primary adenocarcinoma suggesting that PAK1 dysregulation could be an early event in some breast cancers. PAK1 protein expression in adenocarcinoma was associated with lymph node invasion and further increased expression was observed in metastatic tumors. In addition, primary and secondary (28 local recurrences, 56 metastases) adenocarcinomas from a total of 86 patients (81 with matching samples) were evaluated for PAK1 and PAK4 expression to determine modulation of these kinases following adjuvant hormone therapy. Breast cancer cell lines with PAK1 genomic amplification rapidly underwent caspase activation and apoptosis following inhibition of this kinase. Together, our results provide evidence for dysregulation of PAK1 in breast cancer and a role for PAK1 in cellular survival and proliferation in this indication.

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 4004. doi:1538-7445.AM2012-4004
Abstract 402: Plasticity of tumor associated macrophages in a metastatic melanoma model in the mouse
Authors: Gal Annamaria , Tapmeier Thomas T. , Muschel Ruth J.
Published: August 2012

Macrophages acquire different functional traits, with the pro-inflammatory, classically activated M1 and the tissue-repair, alternatively activated M2 phenotypes the best characterized to date. Tumor-associated macrophages (TAMs) have been described as alternatively activated and exert pro-tumoral activity. We investigated TAMs isolated from subcutaneous tumors and experimental lung metastases at an early (72 h) and late (3 wk) stage within a B16F10 mouse melanoma model. The macrophages were characterized according to their expression of genes, surface markers and secreted factors. Interestingly, the mRNA profile revealed both M1- and M2-associated gene expression in macrophages derived from s.c. melanoma, including Cxcl9, Cxcl11, Il-1a, Nos2, and Retnla (FIZZ-1), Arg-1 and Fn1, respectively. In early pulmonary metastases, we found that macrophages also expressed both pro-inflammatory (Il1b, Il12b, Ccl2), and anti-inflammatory (Ccl17 and Ccl22) cytokines and chemokines and other M1- and M2-associated genes, such as Cd40, Cd86, and Il27ra and Itgax (Cdd11c), respectively. Macrophages from late stage lung metastases displayed predominantly alternatively activated characteristics with Arg1, Retnla and Fn1 expression, but pro-inflammatory genes, such as Ccl2, Il1b and Cxcl10 were also induced/upregulated. We therefore suggest an intermediate phenotype of TAMs which does not fit the orthodox M1/M2 paradigm. To describe the mechanism by which macrophages are recruited to pulmonary metastases, we investigated chemokine receptor expression of macrophages infiltrating the lung. While Ccr2 was expressed at high level in macrophages associated with both the control and B16F10 metastases bearing lung, we found that Ccr1 and Ccr5 expression was induced after tumor cell challenge. The secretion profile of B16F10 cells revealed CCL5 (RANTES), a ligand for these receptors, secreted at high level, which suggests that CCL5 attracts macrophages to lung metastases via CCR1 and CCR5. The effect of anti-CCL5 antibody treatment, and s.c melanoma growth and pulmonary metastasis development in CCR1-/- and CCR5-/- mice will be discussed.

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 402. doi:1538-7445.AM2012-402
Abstract 4027: Regulation of p16INK4A and TGF-beta by DNA hydroxymethylation in glioblastoma multiforme
Authors: Popkie Anthony P. , deCarvalho Ana C. , Scott Stephanie B. , Eugster Emily E. , Monsma David , Davidson Paula J. , Dawna Dylewski , Mooney Marie R. , Monks Noel R. , Mikkelsen Tom , Webb Craig P.
Published: August 2012

Glioblastoma multiforme (GBM) is the most prevalent form of primary brain cancer in adults. Cancer stem cells (CSCs) are thought to drive the growth and metastasis of tumors, and are readily isolated from GBM patient tumors. These GBM CSCs are maintained under serum-free conditions in neurosphere media, and form tumors upon orthotopic injection in mice. However, when grown in the presence of serum, GBM CSCs undergo morphological changes, have reduced proliferation, exhibit loss of stem cell markers, and have reduced tumorigenicity following orthotopic implantation into the brains of immune compromised mice. Upon return to serum-free neurosphere media, these GBM CSCs revert to neurospheres. We set out to define the transcriptional incongruities between these culture condition-dependent states, and the underlying epigenetic changes which regulate these differences in gene expression. Although genetic alterations in cancer are irreversible, epigenetic changes are inherently reversible, and we hypothesize that the plasticity between culture condition-dependent states is mediated by epigenetic regulation of genes relevant to the ability of these cells to form tumors. Recently, a novel epigenetic mark, 5-hydroxymethylcytosine (5hmC), has been shown to be enriched at regulatory elements within polycomb target genes in embryonic stem cells, and is frequently found at genes that are involved in the maintenance of stem cells. We found that 5hmC levels of several polycomb target genes, including p16INK4A and several components of the TGF-beta pathway involved in epithelial to mesenchymal transition (EMT), are altered in neurospheres compared to CSCs cultured in serum. Loss of p16INK4A expression has recently been associated with increased CSC abundance in breast cancer, and our results suggest that a similar trend exists in GBM. Furthermore, our results suggest that 5hmC is involved in the regulation of polycomb target genes in CSCs, including p16INK4A and components of the TGF-beta pathway. These findings implicate 5hmC in the maintenance of CSCs and regulation of EMT.

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 4027. doi:1538-7445.AM2012-4027
Abstract 4029: Enhanced chemosensitivity of estrogen receptor-α (ERα)-negative breast cancer through soybean genistein-induced epigenetic reactivation of ERα
Authors: Li Yuanyuan , Tollefsbol Trygve
Published: August 2012

Estrogen receptor-α (ERα)-negative breast cancer is clinically aggressive and has a poor prognosis due to the lack of estrogen target-directed therapies. The soybean isoflavone, genistein (GE), has been shown to prevent and inhibit breast cancer and recent studies have suggested that GE can enhance the anticancer capacity of an estrogen antagonist, tamoxifen (TAM), especially in ERα-positive breast cancer cells. However, the role of GE in ERα-negative breast cancer remains unknown. In our current studies, we found that GE can reactivate ERα expression and this effect was synergistically enhanced when combined with a histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), in ERα-negative MDA-MB-231 breast cancer cells. GE treatment also re-sensitized ERα-dependent cellular responses to activator 17β-estradiol (E2) and antagonist TAM. Further studies revealed that GE can lead to remodeling of the chromatin structure in the ERα promoter thereby contributing to ERα reactivation. The global enzymatic activities for HDACs and DNA methyltransferases (DNMTs) mediated by GE were altered to favor ERα up-regulation. Consistently, dietary GE significantly reduced the growth of ERα-negative mouse xenograft tumors due at least in part to ERα reactivation. The combination of GE with TAM had more prominent effects on delaying tumor growth than either treatment alone. Collectively, our studies suggest that soybean genistein can epigenetically restore ERα expression, which in turn increases TAM-dependent anti-estrogen chemosensitivity. The results from our studies revealed a novel chemotherapeutic combination approach using bioactive soybean product and anti-hormone therapy in refractory ERα-negative breast cancer which will provide more effective options in breast 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 4029. doi:1538-7445.AM2012-4029
Abstract 4034: The role of CYP33 in MLL target gene repression
Authors: Poppen Steven , Diaz Manuel
Published: August 2012

This study further elucidates the role of CYP33 in gene transcription by studying the effects of CYP33 overexpression and siRNA-mediated down-regulation on the expression of MLL target genes such as c-MYC, p21, p27, and HOXA9. CYP33 is a cis-trans prolyl isomerase (PPIase) which mediates repression of HOX gene expression after binding to the 3rd PHD finger of MLL through its own RRM domain. Upon binding to MLL, CYP33 increases recruitment of HDAC to the MLL repression domain which in turn leads to deacetylation of histone H3. This ultimately causes repression of MLL target genes (Xia et al. 2003). Chromosomal translocations involving the MLL gene (Mixed Lineage Leukemia) can lead to the production of fusion proteins with any of more than 60 different partner proteins and is implicated in the initiation of leukemia. Expression of MLL fusion proteins leads to increased expression of MLL target genes such as HOXA7, HOXA9 and MEIS1 (Hess et al., 2010). MLL-fusion proteins lack the 3rd PHD finger and therefore, lose their ability to bind CYP33; in the presence of these fusion proteins CYP33 can no longer repress MLL target genes. A mutation in CYP33's PPIase domain, leads to an enzymatically inactive CYP33 which can still bind to MLL. Upon overexpression of MLL and CYP33, HDAC is recruited to the MLL complex, as demonstrated by co-immunopreciptiation. However, when the CYP33 PPIase mutant is overexpressed, the lack of PPIase activity prevents the recruitment of HDAC to MLL. As a consequence the chromatin retains the acetylation mark on histone H3 and, although the CYP33 PPIase mutant binds MLL, no repression of MLL target genes is observed. Conversely, upon knockdown of wild-type CYP33, MLL target gene expression and H3K27Ac are increased. This study aids in the understanding of how MLL and CYP33 modulate gene function and helps to identify some of the mechanisms involved in hematopoietic development and leukemia.

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 4034. doi:1538-7445.AM2012-4034
Abstract 4036: Frequent downregulation of the transcription factor Foxa2 in lung cancer through epigenetic silencing
Authors: Basseres Daniela S. , D'Alò Francesco , Yeap Beow Y. , Gonzalez David A. , Yasuda Hiroyuki , Meyerson Matthew , Kobayashi Susumu , Tenen Daniel G. , Halmos Balazs , Costa Daniel B.
Published: August 2012

PURPOSE: We sought to determine the mechanisms of downregulation of the airway transcription factor Foxa2 in lung cancer and the expression status of Foxa2 in non-small-cell lung cancer (NSCLC). METHODS: A series of 25 lung cancer cell lines were evaluated for Foxa2 protein expression, FOXA2 mRNA levels, FOXA2 mutations, FOXA2 copy number changes and for evidence of FOXA2 promoter hypermethylation. In addition, 32 NSCLCs were sequenced for FOXA2 mutations and 173 primary NSCLC tumors evaluated for Foxa2 expression using an immunohistochemical assay. RESULTS: Out of the 25 cell lines, 13 (52%) had undetectable FOXA2 mRNA. The expression of FOXA2 mRNA and Foxa2 protein were congruent in 19/22 cells (p=0.001). FOXA2 mutations were not identified in primary NSCLCs and were infrequent in cell lines. Focal or broad chromosomal deletions involving FOXA2 were not present. The promoter region of FOXA2 had evidence of hypermethylation, with an inverse correlation between FOXA2 mRNA expression and presence of CpG dinucleotide methylation (p<0.0001). In primary NSCLC tumor specimens, there was a high frequency of either absence (42/173, 24.2%) or no/low expression (96/173, 55.4%) of Foxa2. In 130 patients with stage I NSCLC there was a trend towards decreased survival in tumors with no/low expression of Foxa2 (HR of 1.6, 95%CI 0.9-3.1; p=0.122).CONCLUSIONS: Loss of expression of Foxa2 is frequent in lung cancer cell lines and NSCLCs. The main mechanism of downregulation of Foxa2 is epigenetic silencing through promoter hypermethylation. Further elucidation of the involvement of Foxa2 and other airway transcription factors in the pathogenesis of lung cancer may identify novel therapeutic targets.

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 4036. doi:1538-7445.AM2012-4036
Abstract 4040: Silence of miR-203 by promoter methylation in endometrial cancer
Authors: Huang Yi-Wen , Rader Janet S. , Stoner Gary D. , Goodfellow Paul J. , Huang Tim H-M.
Published: August 2012

MicroRNAs (miRNAs), a class of small non-coding RNAs, play important roles in cancer development by perturbing the expression of target genes. DNA methylation, a well-studied epigenetic event, also influences gene expression and aberrant methylation is frequently observed in cancer. We previously reported a new paradigm in which hypermethylation-mediated silencing of a miRNA de-repressed its oncogenic target in cancer cells. SOX4, an oncogene, was found to be overexpressed in endometrial cancer, partly because of hypermethylation of miR-129-2. Herein, thirteen additional miRNAs targeting on the 3′-end of untranslated regions of SOX4, and encompassing CpG islands, were identified using computational analysis. Hypermethylation of eight of the thirteen miRNAs was detected in at least two of seven endometrial cancer cell lines. miR-203 was selected for further analysis because this locus was predominantly hypermethylated in endometrial cancer cell lines, but not in normal endometrium. miR-203 expression can be reactivated by pharmacologic induction of DNA demethylation and histone acetylation, indicating that the loss of miR-203 expression is associated with promoter hypermethylation in endometrial cancer. Restoration of miR-203 by cell transfection decreased SOX4 expression and reduced proliferation of endometrial cancer cells. To confirm these in vitro findings in primary endometrial tumors, specimens from two cohorts were analyzed for miR-203 methylation by quantitative pyrosequencing or MassARRAY. In one cohort, miR-203 was significantly hypermethylated in endometrial tumors (n=31), but not in adjacent or un-related normal (n=18) specimens. Methylation of this locus is related to lymphatic and vascular invasion. In the other cohort, increased methylation was observed in primary tumors (n=114), but not in normal controls (n=22). Hypermethylation of miR-203 was positively associated with MLH1 methylation and MSI status. In conclusion, we present an emerging phenomenon that epigenetic silencing of multiple miRNAs could lead to overexpression of a single oncogene in endometrial cancer. This work is supported, in part, by NIH grants U54 CA113001 (THH) and P50 CA134254 (PJG, THH).

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 4040. doi:1538-7445.AM2012-4040
Abstract 4041: Identification of GNG7 as an epigenetically silenced gene in head and neck cancer by gene expression profiling
Authors: Demokan Semra , Chuang Alice Y. , Chang Xiaofei , Khan Tanbir , Smith Ian M. , Pattani Kavita M. , Dasgupta Santanu , Begum Shahnaz , Khan Zubair , Liegeois Nanette J. , Westra William H. , Sidransky David , Koch Wayne , Califano Joseph A.
Published: August 2012

Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. Aberrant hypermethylation in the promoter region of some known or putative tumor suppressor genes occurs frequently during the development of various cancers including head and neck squamous cell carcinoma (HNSCC). In this study we used an expanded mRNA expression profiling approach followed by microarray expression analysis to identify epigenetically inactivated genes in HNSCC. Two HNSCC cell lines were treated with 5-aza-2′-deoxycytidine followed by microarray analysis to identify epigenetically silenced genes in HNSCC. 1960, 614, and 427 genes were upregulated in HNSCC cell lines JHU-012, JHU-011 and the combination of both cell lines, respectively. HNSCC tumor and normal mucosal samples were used for gene profiling by a 47K mRNA gene expression array and we found, 7140 genes were downregulated in HNSCC tumors compared to normal mucosa as determined by microarray analysis and were integrated with cell line data. Integrative analysis defined 126 candidate genes, of which only seven genes showed differentially methylation in tumors and no methylation in normal mucosa after bisulfite sequencing. After validation by QMSP, one gene, GNG7, was confirmed as being highly methylated in tumors and unmethylated in normal mucosal and salivary rinse samples demonstrating cancer-specific methylation in HNSCC tissues. TXNIP and TUSC2 were partially methylated in tumors and normal salivary rinses but unmethylated in normal mucosa. We concluded GNG7 as a highly specific promoter methylated gene associated with HNSCC. In addition, TXNIP and TUSC2 are also potential biomarkers for HNSCC.

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 4041. doi:1538-7445.AM2012-4041
Abstract 4043: Loss of imprinting of KCNK9 in breast cancer
Authors: Skaar David A. , Seewaldt Victoria L. , Gould Michael N. , Jirtle Randy L.
Published: August 2012

KCNK9 (TASK-3) encodes a two-pore potassium channel that functions in tumor growth and survival. The potassium channel function is necessary for tumor proliferation and survival, by enhancing resistance to hypoxia and apoptosis. KCNK9 overexpression is observed in melanoma, colorectal cancer, and breast and brain tumors, but with no associated mutations found. Rather, the overexpression is often the apparent result of gene amplification of KCNK9, but any other mechanism that increases expression would presumably have a similar effect on tumorigenesis. This importance of gene dosage and expression levels is particularly relevant, as KCNK9 has been demonstrated to be a maternally expressed imprinted gene, which, in effect, reduces copy number. Monoallelic expression of KCNK9 was originally found in brain tissue, but our work has found that it is also imprinted in breast tissue. Thus, we hypothesized that loss of imprint control would be similar to a gene duplication, increasing the effective copy number, and therefore KCNK9 gene expression. To study the regulation of KCNK9 imprinting in tumors, we have screened for differentially methylated regions that may function as imprint control regions (ICRs), and examined the expression status of KCNK9 in breast tumor samples relative to the methylation state of the putative ICRs. Approximately 40-50% of breast tumor samples analyzed show loss of imprinting, seen as expression of both alleles of an exonic polymorphism. Additionally, loss of methylation at a putative ICR upstream of the transcription start site is observed in combination with loss of imprint regulation. This correlated loss of methylation and imprint control is seen most commonly in tumors of the “triple negative” subtype - tumors negative for expression of estrogen receptor, progesterone receptor, and HER2/neu. These novel findings regarding KCNK9 imprint regulation and its disruption in a subset of tumors may lead to new approaches in diagnosis and targeted therapy of 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 4043. doi:1538-7445.AM2012-4043