bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5176: Implications of clonality relationship and CD133 expression between cancer synchronous/preceding adenomas and their colorectal adenocarcinoma pairs
Authors: Yang Chih-Yung , Tseng Ju-Yu , Lin Chi-Hung
Published: May 2015

Background:

It is thought that each cell only generates a unifocal lineage that is independent from one another in colorectal adenoma-carcinoma progression. This study compared the genome-wide copy number aberration profiles of adenoma and primary colorectal carcinomas (CRCs) derived from the same patient to trace their clonality relationship. The CD133 positive cells, putative colorectal cancer stem cells, are capable of tumor initiation and metastasis. Therefore, we also investigated the use of CD133 expression as a potential marker for prediction of subsequent risk of colorectal cancer.

Methods:

Microarray-based comparative genomic hybridization (aCGH) experiments were performed on 18 cancer-synchronous polyps (CSP) and 9 cancer-preceding polyps (CPP), together with their corresponding cancers, and 16 cases of incidental polyps (IP). Genome-wide copy number imbalances revealed by aCGH were analyzed to determine the clonal relationship between the paired adenomas and carcinomas. CD133 expression in cancerous tissue was evaluated by immunohistochemistry (IHC) staining.

Results:

There were more CGH lesions in CRC than CSP/CPP, and least in IP. Clonal relationship determined by tissues' aCGH profiles showed that 50 percent of CSP and 67 percent of CPP were clonally related to the concurrent or subsequent cancers, respectively. The expression level of stem cell marker CD133 was significantly higher in CSP/CPP than in IP (P<0.0001), and even higher CD133 staining were observed in CSP/CPP that were clonally related to the corresponding carcinomas than CSP/CPP that were unrelated (P<0.05).

Conclusions:

In the present study more than half of the polyp-carcinoma pairs share highly similar pattern of numerical chromosomal aberrations implying that the pairs might descent from the same monoclonal cell origin. The high expression of stem cell marker CD133 in colorectal adenomas is predictive of high cancer risk for polyp-bearing patients.

Citation Format: Chih-Yung Yang, Ju-Yu Tseng, Chi-Hung Lin. Implications of clonality relationship and CD133 expression between cancer synchronous/preceding adenomas and their colorectal adenocarcinoma pairs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5176. doi:10.1158/1538-7445.AM2014-5176
Abstract 5177: Genome-wide profiling of somatic mutations in liver cancers revealed significantly mutated genes and non-coding regions in liver cancers
Authors: Furuta Mayuko , Fuimoto Akihiro , Shiraishi Yuichi , Miyano Satoru , Tsunoda Tatsuhiko , Nakagawa Hidewaki
Published: May 2015

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Previously we have reported genomic aberrations detected by the whole genome sequencing (WGS) of 27 HCCs and frequently and recurrently mutated genes in this tumor (Nat Genet, 2012). Consistent with recent reports of WGS on human cancers from other groups, large parts of somatic point mutations (98.4%) were detected in non-coding genomic regions in our HCC samples. However, the consequence and significance of somatic mutations in non-coding regions are largely unknown. Here we analyzed 107 hepatitis C virus- (HCV-) associated HCCs by WGS analysis and calculated the somatic mutation rate of all single nucleotide variations (SNVs) and insertion-deletions (indels) within each 500-base bins on entire human genome. Among the whole genomic regions, 67 bins were significantly selected. After removing mutations in self-chained regions or simple repeat regions, which is expected to be false positives due to mapping errors, the bin containing exon 3 of CTNNB1 and multiple bins contains exons of TP53, which are known as driver mutations in HCCs, were highly ranked. In addition, entire coding region of the ALB gene showed high mutation rate compared with surrounding region distinctively. Interestingly, a recurrent short deletion on chromosome 18 was observed in the similar frequency as alterations of CTNNB1 or TP53. This short deletion was somatically detected in 35 of 321 liver cancers (10.9%) by Sanger sequencing. Significant accumulation of somatic mutations on exons of genes such as CTNNB1 and TP53 within entire genome through comparative analyses indicates their importance in hepatocarcinogenesis. Moreover, these results also suggest that novel mutations on non-coding regions, which are somatically and recurrently detected in higher rate in liver cancers, are involved in liver cancer development. These comprehensive approaches using WGS data will help the understanding of functions and alterations in non-coding regions occurred in cancer genome.

Citation Format: Mayuko Furuta, Akihiro Fuimoto, Yuichi Shiraishi, Satoru Miyano, Tatsuhiko Tsunoda, Hidewaki Nakagawa. Genome-wide profiling of somatic mutations in liver cancers revealed significantly mutated genes and non-coding regions in liver cancers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5177. doi:10.1158/1538-7445.AM2014-5177
Abstract 5183: Modulation of miR-29 expression by alpha-fetoprotein is linked to the hepatocellular carcinoma epigenome
Authors: Parpart Sonya T. , Roessler Stephanie , Dong Fei , Rao Vinay , Ji Junfang , Qin Lun–Xiu , Ye Qing–Hai , Tang Zhao–You , Jia Hu–Liang , Wang Xin Wei
Published: May 2015

Globally, hepatocellular carcinoma (HCC) accounts for 70-85% of primary liver cancers and ranks second in the leading cause of male cancer death. Serum alpha-fetoprotein (AFP), normally highly expressed in the liver only during fetal development, is reactivated in 60% of HCC tumors and associated with poor patient outcome. We hypothesize that AFP+ and AFP- tumors differ biologically. Using microarray-based global microRNA profiling in 223 HCC patients, we found that members of the miR-29 family were the most significantly (p<0.001) down-regulated miRNAs in AFP+ tumors. Consistent with miR-29's role in targeting DNA methyltransferase 3A (DNMT3A), a key enzyme regulating DNA methylation, we found a significant inverse correlation (p<0.001) between miR-29 and DNMT3A gene expression suggesting that they might be functionally antagonistic. Moreover, global DNA methylation array profiling reveals that AFP+ and AFP- HCC tumors have distinct global DNA methylation patterns and that increased DNA methylation is associated with AFP+ HCC. Interestingly, miR-29 family physiological expression is low in mouse embryonic livers but gradually increases after birth. This is in contrary to AFP expression, which dramatically decreases after birth. Experimentally, we demonstrated that increased AFP expression, or conditioned media from AFP expressing HCC cells, inhibits miR-29a expression in AFP- HCC cells. AFP also inhibited transcription of the miR-29a/b-1 locus and this effect is mediated through c-MYC binding to the miR-29a/b-1 promoter. Our findings indicate that tumor biology differs considerably between AFP+ HCC and AFP- HCC and that AFP is a functional antagonist of miR-29, which may contribute to global epigenetic alterations and poor prognosis in HCC.

Citation Format: Sonya T. Parpart, Stephanie Roessler, Fei Dong, Vinay Rao, Junfang Ji, Lun–Xiu Qin, Qing–Hai Ye, Zhao–You Tang, Hu–Liang Jia, Xin Wei Wang. Modulation of miR-29 expression by alpha-fetoprotein is linked to the hepatocellular carcinoma epigenome. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5183. doi:10.1158/1538-7445.AM2014-5183
Abstract 5186: Reduced expression of BRM in hepatocellular carcinoma
Authors: Yasui Kohichiroh , Gen Yasuyuki , Tomie Akira , Kitaichi Tomoko , Itoh Yoshito
Published: May 2015

Background: Hepatocellular carcinoma (HCC) is the fifth most common malignancy in men and the eighth most common in women worldwide. The eukaryotic genome is organized into highly ordered chromatin structures in the nucleus. Chromatin creates a barrier to nuclear processes, such as transcription, by obstructing the access of the transcriptional machinery and gene-specific regulators to recognition sequences within target promoters. The SWI/SNF protein complex, an ATPase-dependent chromatin remodeling enzyme, mobilizes nucleosomes and functions as a master regulator of gene expression and chromatin dynamics. SWI/SNF is a large multiprotein complex that contains either BRG1 or brahma (BRM) as the catalytic ATPase. Each SWI/SNF complex thus incorporates one of two possible ATPases, BRG1 or BRM.

Aims: To examine alterations of BRG1 and BRM in HCC.

Methods: We investigated DNA copy number aberrations in human HCC cell lines using a high-density oligonucleotide microarray. We determined DNA copy numbers and expression levels of BRG1 and BRM genes in primary HCC tumors, and conducted further searches for mutations in BRG1 and BRM genes.

Results: Homozygous deletion of the BRG1 gene was found in HCC cell line SNU398. Copy number losses of BRG1 and BRM genes were observed in 14 (26%) and 7 (13%) of 54 primary HCC tumors respectively. We found four somatic missense mutations in the BRG1 gene in two of 36 primary HCC tumors, but no mutations in BRM gene. Expression of BRM mRNA, but not BRG1 mRNA, was significantly reduced in primary HCC tumors, compared to non-tumor tissue counterparts. Immunohistochemical analyses of non-tumour liver tissues showed that BRM protein was expressed in hepatocytes and bile-duct epithelial cells, whereas BRG1 protein was expressed in bile-duct epithelial cells, but not in hepatocytes. BRM protein expression was lost in nine (22.5%) of 40 HCC tumours. Loss of BRM protein expression was significantly associated with poor overall survival.

Conclusion: Reduced expression of BRM may contribute to the carcinogenesis of HCC. Although deletions and mutations in BRG1 gene were identified, the role of BRG1 in HCC tumourigenesis remains unclear.

Citation Format: Kohichiroh Yasui, Yasuyuki Gen, Akira Tomie, Tomoko Kitaichi, Yoshito Itoh. Reduced expression of BRM in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5186. doi:10.1158/1538-7445.AM2014-5186
Abstract 5188: Integrated exome analysis in childhood hepatoblastoma: Biological approach for next clinical trial designs
Authors: Hiyama Eiso , Kurihara Sho , Onitake Yoshiyuki , Morihara Nagisa , Ikeda Kyoko , Hiyama Keiko
Published: May 2015

Background: Hepatobastoma (HBL) accounts for more than 80% of childhood hepatic malignant tumors. The outcome of the HBL cases mainly depends on the biological characteristics and disease stagings, but the candidate genes correlated with HBL progression have not been identified. Sequence capture enrichment strategies and massively parallel next generation sequencing (NGS) were used for such gene discovery.Methods: Out of approximately 300 HBL case treated according to the JPLT (Japanese study group for Pediatric Liver Tumor)-2 protocol, 48 samples cases were analyzed by a method for whole-exome sequencing coupling Ilumina Nextera whole-exome capture to the Illumina DNA-sequencing platform. In these cases, 10 had lung metastatic cases and 13 of the remaining 38 cases had vascular invasion or multifocal lesions at admission. Consequently, 14 cases had a relapse and 10 cases died of disease. Using conventional technology, we investigated CNNB1 point-mutations and deletions in these cases.Results: Among these 48 HBL cases, mutation and deletion in CNNB1 exon 3 was identified in 40 cases, which had been already detected by conventional techniques. The remaining 8 cases showed the mutation of Wnt signal genes including AXIN1 and FAP genes. Among the cases with metastatic lesion and/or consequently dead cases, stemness-associated and cancer-associated genes showed homozygous deletion (e. g. CDKN2A), a homozygous inversion in the genes including NF1, and an unbalanced translocation. The response rates in the 35 cases that underwent neo-adjuvant chemotherapy revealed that existence of the mutations in several genes associated with proliferation was significantly correlated with chemo-resistance. Therefore, we are now constructing next clinical trial for HBL based on the whole-exome era.Conclusions: Whole-exome sequencing using NGS in hepatoblastoma revealed several candidates mutated or deleted genes except for CNNB1 in Wnt-signaling pathway. NGS can be also used in research and diagnostic settings to screen for mutations and deletion/insertion of the genes associated with malignant grade and chemo-resistance. Further NGS analysis provided important candidates of indicators for diagnostic and therapeutic targets for HBL clinical trials.

Note: This abstract was not presented at the meeting.

Citation Format: Eiso Hiyama, Sho Kurihara, Yoshiyuki Onitake, Nagisa Morihara, Kyoko Ikeda, Keiko Hiyama. Integrated exome analysis in childhood hepatoblastoma: Biological approach for next clinical trial designs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5188. doi:10.1158/1538-7445.AM2014-5188
Abstract 5191: Genome-wide mutational analysis reveals core signaling pathways in mucinous neoplasms of the appendix
Authors: Alakus Hakan , Babicky Michele , Ghosh Pradipta , Yost Shawn , Jepsen Kristen , Dai Yang , Arias Angelo , Samuels Michael , Mose Evangeline , Peterson Michael , Lowy Andrew , Frazer Kelly , Harismendy Olivier
Published: May 2015

Mucinous Neoplasm of the Appendix (MNA) is a rare malignancy often diagnosed after metastasis to the peritoneal surfaces resulting in Mucinous Carcinomatosis Peritonei (MCP). Genetic alterations in MNA are poorly characterized due to its low incidence, the hypo-cellularity of MCPs and a lack of relevant pre-clinical models. As such, application of targeted therapies to this disease is limited to those developed for colorectal cancer (CRC) and not based on molecular rationale. We sequenced the whole exomes of 10 MCPs to identify genome-wide somatic mutations and copy number aberrations and validated significant findings in 19 additional cases using deep sequencing and microdroplet PCR. These studies demonstrate that MNA has a different molecular makeup than CRC. Most tumors have co-existing oncogenic mutations in KRAS (26/29) and GNAS (21/29) and are characterized by cAMP-PKA pathway activation. High-grade tumors are GNAS wild-type (5/6), suggesting they do not progress from low-grade tumors. MNAs do share some genetic alterations with CRC including gain of 1q (5/10), Wnt and TGFβ pathway alteration. In contrast, mutations in TP53 (1/10) and APC (0/10), common in CRC, are rare in MNA. Coincident activation of the Ras-Raf-MEK-ERK and cAMP-PKA signaling pathways appears to be shared with pancreatic intraductal papillary mucinous neoplasm. Thus, MNA genome-wide mutational analysis reveals genetic alterations distinct from CRC, in support its unique pathophysiology and suggests MEK and PKA inhibition as new therapeutic opportunities.

Citation Format: Hakan Alakus, Michele Babicky, Pradipta Ghosh, Shawn Yost, Kristen Jepsen, Yang Dai, Angelo Arias, Michael Samuels, Evangeline Mose, Michael Peterson, Andrew Lowy, Kelly Frazer, Olivier Harismendy. Genome-wide mutational analysis reveals core signaling pathways in mucinous neoplasms of the appendix. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5191. doi:10.1158/1538-7445.AM2014-5191
Abstract 5193: Novel candidate oncogenes with mutation hot spots in microsatellite unstable colorectal cancer
Authors: Gylfe Alexandra E. , Tuupanen Sari , Hänninen Ulrika , Kondelin Johanna , Ristolainen Heikki , Katainen Riku , Pitkänen Esa , Taipale Minna , Taipale Jussi , Andersen Claus Lindbjerg , Renkonen-Sinisalo Laura , Järvinen Heikki , Böhm Jan , Mecklin Jukka-Pekka , Vahteristo Pia , Aaltonen Lauri A.
Published: May 2015

Around 15% of colorectal cancers (CRCs) show microsatellite instability (MSI). MSI CRCs are prone to repeat mutations due to defective mismatch repair. The high background mutation frequency has discouraged systematic mutations screens in this tumor type. However, these tumors might form a sensitive system for generation and selection of oncogenic mutation hot spots. The aim of this study is to identify novel oncogenes with mutation hot spots that drive MSI CRC tumorigenesis.

The exomes of 25 MSI tumors and respective healthy tissues were sequenced as the discovery set. The exome data was searched for mutation hot spots, with recurrent somatic missense mutations in at least two tumors. Potential mutation hot spots were observed in 43 genes and among these were the following known oncogenes: BRAF (V600E), CTNNB1 (T41A) and PIK3CA (H1047R). Novel potential mutation hot spots were identified in 40 genes and these were validated by Sanger sequencing. Mutation hot spots in 33 genes were confirmed and these were further screened in a validation set of 254 MSI CRCs. Fourteen genes displayed hotspot mutations also in the validation set with a total hot spot mutation frequency of 1.1-3.6 %. Many of the validated genes encode for known cancer-related proteins and for proteins with molecular and cellular functions relevant to cancer development and progression.

Further work is needed to clarify the functional role of the identified hot spot mutations in MSI CRC tumorigenesis. The novel mutation hotspots may be utilized to develop personalized tumor profiling and therapy.

Citation Format: Alexandra E. Gylfe, Sari Tuupanen, Ulrika Hänninen, Johanna Kondelin, Heikki Ristolainen, Riku Katainen, Esa Pitkänen, Minna Taipale, Jussi Taipale, Claus Lindbjerg Andersen, Laura Renkonen-Sinisalo, Heikki Järvinen, Jan Böhm, Jukka-Pekka Mecklin, Pia Vahteristo, Lauri A. Aaltonen. Novel candidate oncogenes with mutation hot spots in microsatellite unstable colorectal cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5193. doi:10.1158/1538-7445.AM2014-5193
Abstract 5194: MiR-200c expression and methylation status determines epithelial characteristics of NSCLC
Authors: Shien Kazuhiko , Toyooka Shinichi , Soh Junichi , Yamamoto Hiromasa , Hashida Shinsuke , Suzawa Ken , Otsuka Tomoaki , Asano Hiroaki , Tsukuda Kazunori , Miyoshi Shinichiro
Published: May 2015

Background: MicroRNA (miR)-200 family members are frequently silenced in advanced cancer and have been implicated in the process of epithelial-to-mesenchymal transition (EMT) and tumor invasion. In this study, we investigated the roles of miR-200c in non-small cell lung cancer (NSCLC). Materials and Methods: We analyzed miR-200c promoter methylation status by methylation specific PCR and expression by quantitative RT-PCR in 34 NSCLC cell lines and 10 NSCLC clinical samples. To eliminate the effects of contamination of normal lung epithelial cells and mesenchymal cells in clinical samples, we used primary cultured NSCLC cells obtained by surgical resection. We assessed the association of miR-200c status with histology, EMT-related protein expression, and genetic alteration. Results: Mir-200c was methylated in 10 (30%) NSCLC cell lines and partially methylated in 7 (21%) cell lines. Expression of miR-200c was silenced in all methylated cell lines and restored with 5-aza-2′-deoxycytidine treatment. In cell lines with miR-200c methylation and silenced, protein expression of epithelial markers was reduced and mesenchymal markers were increased. Introduction of miR-200c into highly invasive cells with miR-200c silencing inhibited cell invasion. Interestingly, miR-200c was unmethylated and not silenced in many EGFR mutated NSCLC cell lines and clinical samples. In HEK293T cells stably transfected with wild-type EGFR, mutant EGFR, wild-type KRAS, or mutant KRAS, miR-200c expression was up-regulated only in mutant EGFR transfectant. Conclusions: Mir-200c expression was regulated by promoter methylation in NSCLC, and related to epithelial characteristics and cell invasion. In EGFR mutated NSCLC cells, miR-200c was generally unmethylated and expressed, suggesting the existence of mechanism that retain EGFR mutated NSCLC cells to the epithelial state through the miR-200c expression.

Citation Format: Kazuhiko Shien, Shinichi Toyooka, Junichi Soh, Hiromasa Yamamoto, Shinsuke Hashida, Ken Suzawa, Tomoaki Otsuka, Hiroaki Asano, Kazunori Tsukuda, Shinichiro Miyoshi. MiR-200c expression and methylation status determines epithelial characteristics of NSCLC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5194. doi:10.1158/1538-7445.AM2014-5194
Abstract 5196: Different miRNAs expression in leiomyosarcoma and undifferentiated pleomorphic sarcoma
Authors: Pazzaglia Laura , Guled Mohamed , Borze Ioana , Benassi Maria Serena , Picci Piero , Knuutila Sakari
Published: May 2015

Rare and highly aggressive adult soft tissue sarcomas, leiomyosarcoma (LMS) and undifferentiated pleomorphic sarcoma (UPS), contain complex genomics characterized by a multitude of rearrangements, amplifications and deletions. Since differential diagnosis between the two sarcoma histotypes continues to remain a challenge, we attempted to identify a specific signature useful for improving the differential diagnosis, and we performed miRNA profiling on series of LMS and UPS samples stored at Rizzoli biobank. First, we investigated the different miRNA expression profile in 20 primary sarcoma samples, 10 high-grade LMS and 10 UPS, MFH-like, using miRNA array technique. The top 5 differentially expressed (p<0.01) miRNAs (miR-199b-5p, miR-320a, miR-199a-3p, miR-126, miR-22) were validated by RT-PCR on a series of further 27 UPS and 21 LMS. Confirming our microarray data, an higher expression of miR-199-5p and a lower expression of miR-320a was seen in UPS when compared to LMS. To evaluate the potential role of the above selected miRNAs, the gene targets recognized by three different target prediction databases (miRBase, TargetScan , miRanda) were combined with a list of LMS- and UPS-associated genes reported in literature. From an integrated analysis, IMP3, ROR2, MDM2, CDK4, and UPA genes were identified. By immunohistochemistry analysis, most of them showed strong protein expression in LMS group. Our data suggest a potential role of miR-320a and miR-199-5p in LMS and UPS development and expand our knowledge about these complex sarcoma histotypes. Association analysis between expression and chromosomal locations of miRNAs are ongoing to identify chromosomal regions involved in these tumors.

Citation Format: Laura Pazzaglia, Mohamed Guled, Ioana Borze, Maria Serena Benassi, Piero Picci, Sakari Knuutila. Different miRNAs expression in leiomyosarcoma and undifferentiated pleomorphic sarcoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5196. doi:10.1158/1538-7445.AM2014-5196
Abstract 520: Functional genomic screens identify microRNA regulators of the oncogenic fusion transcription factor EWS-FLI1
Authors: Kim Suntae , Grohar Patrick J. , Klumpp Carleen , Lal Ashish , Martin Scott E. , Helman Lee J. , Caplen Natasha J.
Published: May 2015

Ewing sarcoma (ES) is a highly aggressive malignant pediatric cancer of the bone and soft tissue with a poor prognosis. The genetic hallmark of ES is the t(11:22)(q24:q12) translocation that generates a fusion transcript containing the 5’ end of EWSR1 and the 3’ end of FLI1 referred to as EWS-FLI1. This fusion generates an oncogenic transcription factor, EWS-FLI1 that is responsible for malignant transformation and is necessary for ES cell survival. To enhance our understanding of the molecular mechanisms underlying ES, with the long-term aim of enhanced treatment for this cancer, we are applying RNAi functional genomic approaches. MicroRNAs (miRNAs) are critical regulators of gene expression. There is evidence that many transcription factors and miRNAs act in concert to regulate gene expression. To identify miRNAs that directly or indirectly modulate EWS-FLI1 activity we conducted parallel screens of mimics or inhibitors of the human miRome using a cell-based reporter assay. The reporter assay consists of two isogenic reporter cell lines generated in the EWS-FLI1 expressing TC32 cell line. One line harbors a luciferase reporter driven by the promoter of an established EWS-FLI1 target NR0B1, while the second line expresses luciferase from a constitutive CMV promoter. A comparison of the effects of a miRNA on these two reporter constructs can identify proteins that specifically modulate EWS-FLI1 activity. We first identified those miRNAs for which a differential effect was seen when the NR0B1- and CMV-luciferase signals were compared and prioritized those where the mimic and inhibitor corresponding to a specific miRNA exhibited opposite effects. Twenty-eight miRNAs were identified, including miR-145, an established regulator of EWS-FLI1. The screen also identified miRNAs that have not been linked to EWS biology. One such candidate is miR-200b, a known regulator of epithelial-mesenchymal transition and tumor progression. We have confirmed that biotinylated-miR-200b captures the EWS-FLI1 mRNA when transfected into TC32 and ∼300 other mRNAs that includes EWS-FLI1 target genes. This functional genomic approach gives us an opportunity to investigate the co-regulatory activity of an oncogenic transcription factor and miRNAs in a systematic fashion that could be extended to investigate the co-regulation of other transcription factors and miRNAs.

Citation Format: Suntae Kim, Patrick J. Grohar, Carleen Klumpp, Ashish Lal, Scott E. Martin, Lee J. Helman, Natasha J. Caplen. Functional genomic screens identify microRNA regulators of the oncogenic fusion transcription factor EWS-FLI1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 520. doi:10.1158/1538-7445.AM2014-520
Abstract 5200: MicroRNA-634 induces caspase-dependent apoptosis by targeting anti-apopotic and mitochondria-related genes in human cancer cells
Authors: Fujiwara Naoto , Inoue Jun , Kawano Tatsuyuki , Kozaki Ken-ichi , Inazawa Johji
Published: May 2015

MicroRNAs (miRNAs) are endogenous small non-coding RNAs composed of 20∼25 base pairs that can negatively regulate gene expression by interfering with the translation of target transcripts. Since it has been known that some miRNAs function as tumor suppressors (TS) and their expression levels are often down-regulated in cancer, TS-miRNAs are expected to be therapeutic agents for cancer therapy. Thus, we conducted function-based screening using a miRNA library, and identified several TS-miRNAs, including miR-152, miR-218, miR-596.

Recently, we identified miR-634 as a new TS-miRNA, which induced cell death with mitochondrial dysfunction and caspase activation. The expression analysis and the target-prediction program revealed miR-634 might directly target genes related with anti-apoptosis and mitochondrial function.

Importantly, this miRNA was frequently down-regulated in primary tumor samples of esophageal squamous cell carcinoma (ESCC), compared with that in corresponding normal tissues. Furthermore, the combination therapy of miR-634 and cisplatin was effective to ESCC cell line in vitro and in vivo. Thus, these findings suggest that miR-634 may be useful as a potential therapeutic agent of ESCC.

Citation Format: Naoto Fujiwara, Jun Inoue, Tatsuyuki Kawano, Ken-ichi Kozaki, Johji Inazawa. MicroRNA-634 induces caspase-dependent apoptosis by targeting anti-apopotic and mitochondria-related genes in human cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5200. doi:10.1158/1538-7445.AM2014-5200
Abstract 5202: Yin Yang 1 is a target of miR-34 family and contributes to gastric carcinogenesis
Authors: Wang An-Ming , Huang Tzu-Ting , Hsu Kai-Wen , Chi Chin-Wen , Yeh Tien-Shun
Published: May 2015

Gastric cancer is one of the most common cancers and the second leading cause of cancer-related death worldwide. Transcription factor Yin Yang 1 (YY1) is a multi-functional protein and plays either oncogenic or tumor-suppressive roles in tumor development and progression. Herein, we investigated the role of YY1 in tumorigenesis of gastric cancer cells. Results showed that the forced expression of YY1 contributed to gastric carcinogenesis of SC-M1 cells. Whereas, the knockdown of endogenous YY1 suppressed tumorigenesis of SC-M1 cells. Additionally, the 3′-untranslated region (3′-UTR) of YY1 mRNA was the target of microRNA-34 (miR-34) family consisting of miR-34a, miR-34b, and miR-34c. The ectopic expression of miR-34 family down-regulated YY1 levels in gastric cancer cells. Endogenous YY1 expression was negatively correlated with miR-34b and miR-34c levels in gastric cancer cells. The overexpression of miR-34 family suppressed gastric carcinogenesis through down-regulation of YY1 in gastric cancer cells. Alternatively, the knockdown of endogenous miR-34 family promoted gastric carcinogenesis via up-regulation of YY1 in gastric cancer cells. The miR-34 family also regulated the xenografted tumor growth and lung metastasis of SC-M1 cells through YY1 in vivo. Taken together, these results suggest that miR-34 family-YY1 axis plays an important role in the control of gastric carcinogenesis.

Citation Format: An-Ming Wang, Tzu-Ting Huang, Kai-Wen Hsu, Chin-Wen Chi, Tien-Shun Yeh. Yin Yang 1 is a target of miR-34 family and contributes to gastric carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5202. doi:10.1158/1538-7445.AM2014-5202
Abstract 5209: MicroRNA-193a-5p suppresses tumor proliferation and enhances radio-sensitivity through ERBB2 in esophageal squamous cell carcinoma
Authors: Lu Pei-Jung
Published: May 2015

Esophageal cancer is the ninth leading cause of cancer death in Taiwan. Esophageal squamous cell carcinoma (ESCC) is usually managed with combined modalities, but the prognosis for ESCC patients is still dismal. MicroRNAs (miRNAs) have been implicated in a variety of human cancers progression, and their expression signatures can provide insight into the diagnosis and prognosis of human cancers. However, the dysregulated miRNAs promoted tumor progression in ESCC remain unclear. To address this goal, 1097 miRNAs were analyzed in nine pairs of ESCC and corresponding normal parts with microarray. Tumor suppressive miRNA, miR-193a-5p, was characterized and inversely correlated with poor prognosis of ESCC patients. Manipulating miR-193a-5p in ESCC cell lines and mouse tumor model, the cell proliferation was decreased and the radio-sensitivity was increased. We further used reporter assay to identify ERBB2 as the target of miR-193a-5p. Restored the ERBB2 expression abolished the effects of miR-193a-5p on cell proliferation and radio-sensitivity. These results indicated that miR-193a-5p played a tumor suppressive role through suppressing ERBB2, expression in esophageal squamous cell carcinoma.

Key words: miR-193a-5p, esophageal squamous cell carcinoma, radiotherapy

Note: This abstract was not presented at the meeting.

Citation Format: Pei-Jung Lu. MicroRNA-193a-5p suppresses tumor proliferation and enhances radio-sensitivity through ERBB2 in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5209. doi:10.1158/1538-7445.AM2014-5209
Abstract 521: Glioma-associated IDH mutation induces miR-34a repression and stem cell-like physiology through enhanced PDGF signaling
Authors: Silber Joachim , Harinath Girish , Parthasarathy Prasanna Tamarapu , Fabius Armida W.M. , Turcan Sevin , Chan Timothy A. , Huse Jason T.
Published: May 2015

Point mutations in isocitrate dehydrogenase enzymes (IDH1 and IDH2) have been identified in 70-90% of lower-grade gliomas (LGGs; WHO grade II and III) as foundational genomic alterations. Glioma-associated IDH mutations occur in active site arginine residues and confer a neomorphic activity resulting in overproduction of the oncometabolite R(-)-2-hydroxyglutarate (2HG). The affects of 2HG on cellular physiology are widespread and include global shifts in epigenomic profiles. LGGs have also been repeatedly linked with dysregulated platelet-derived growth factor (PDGF) signaling. We have recently demonstrated that miR-34a is downregulated in gliomas in response to dysregulated PDGF signaling and that miR-34a directly targets the PDGF receptor (PDGFRA) in high-grade gliomas. Analyzing a panel of diffuse gliomas, we find that miR-34a is specifically downregulated in LGG in response to IDH mutation. Similarly, in multiple isogenic cell line systems, IDH mutation induces repression of miR-34a. Also in these contexts, IDH mutation induces neurosphere formation and the expression of stem cell-associated genes. miR-34a has been reported to target pluripotency factors such as LIN28, OCT4 and NANOG. Accordingly, we find that murine neuroglial progenitor cells expressing IDH mutant isoform express higher levels of these factors than isogenic controls. The stem cell-like phenotype of IDH mutant-expressing cells is morphologically reversed by restoring miR-34a expression in the cells. Despite findings that IDH mutation correlates with hypermethylation at the MIR34a locus in LGG tumors, we have not been able to demonstrate the functional relevance of this. Instead, we find that IDH mutation is associated with enhanced PDGF signaling, and we demonstrate that inhibition or stimulation of PDGF signaling in IDH mutant cells increases or decreases miR-34a expression, respectively. Our studies support a model in which glioma-associated IDH mutation induces miR-34a repression and stem cell-like physiology through enhanced PDGF signaling.

Citation Format: Joachim Silber, Girish Harinath, Prasanna Tamarapu Parthasarathy, Armida W.M. Fabius, Sevin Turcan, Timothy A. Chan, Jason T. Huse. Glioma-associated IDH mutation induces miR-34a repression and stem cell-like physiology through enhanced PDGF signaling. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 521. doi:10.1158/1538-7445.AM2014-521
Abstract 5211: Regulation of SRC kinases by microRNA-3607 located in a frequently deleted locus in prostate cancer
Authors: Saini Sharanjot , Arora Sumit , Majid Shahana , Shahryari Varahram , Tabatabai Laura Z. , Yamamura Soichiro , Tanaka Yuichiro , Dahiya Rajvir , Deng Guoren
Published: May 2015

Genomic studies suggest that deletions at chromosome (chr) 5q region (particularly chr5q14-q23) are frequent in prostate cancer, implicating this region in prostate carcinogenesis. However, the genes within this region are largely unknown. Here we report for the first time, the widespread attenuation of miR-3607 - a microRNA gene located at chr5q14 region- in prostate cancer. Expression analyses of miR-3607 in a clinical cohort of prostate cancer (PCa) specimens showed that miR-3607 is significantly attenuated and low miR-3607 partment of Urology, UCSF School of Mediacinehip and guidance. his guidance. expression is correlated with tumor progression and poor survival outcome in prostate cancer. Our analyses suggest that miR-3607 expression may be a clinically significant parameter with an associated diagnostic potential. We examined the functional significance of miR-3607 in prostate cancer cell lines and found that miR-3607 overexpression led to significantly decreased proliferation, apoptosis induction and decreased invasiveness. Further, our results suggest that miR-3607 directly represses oncogenic SRC family kinases, LYN and SRC, in prostate cancer. In view of our results, we propose that miR-3607 plays a tumor suppressive role in prostate cancer by regulating SRC kinases that in turn regulates prostate carcinogenesis. To our knowledge this is the first report that: (i) identifies a novel role for miR-3607 located in a frequently deleted region of prostate cancer and (ii) defines novel miRNA mediated regulation of SRC kinases in prostate cancer. Since SRC kinases play a central role in prostate cancer progression and metastasis and are attractive targets, this study has potential implications in the design of better therapeutic modalities for prostate cancer management.

Citation Format: Sharanjot Saini, Sumit Arora, Shahana Majid, Varahram Shahryari, Laura Z. Tabatabai, Soichiro Yamamura, Yuichiro Tanaka, Rajvir Dahiya, Guoren Deng. Regulation of SRC kinases by microRNA-3607 located in a frequently deleted locus in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5211. doi:10.1158/1538-7445.AM2014-5211
Abstract 5212: Regulation of androgen receptor expression by andro-miRs in prostate cancer
Authors: Ebron Jey Sabith , Sikand Kavleen , Singh Jagjit , Shukla Girish C.
Published: May 2015

Androgen receptor (AR) is a member of the steroid receptor family which plays a significant role in the regulation of both prostate cell proliferation and growth suppression. The targeting of androgen- and AR- signaling axis remains the primary therapeutic option for prostate cancer (PCa) treatment. AR mRNA has a long 3′UTR of 6.8 kb length and is predicted to be a target of many miRNAs. The existence of long 3′UTR proposes a complex mechanism of regulation of AR gene expression through miRNAs and other factors including RNA binding proteins and polyadenylation signals. Differential expression of various miRNAs has been reported to be associated with the progression of the disease to harmone-independence and castration-resistant prostact cancer (CRPC). However, the direct regulation of AR transcriptional activity through miRNAs is not clearly understood. Our lab has discovered that AR is a target of andro-miRs (miR-488*, miR-644, miR-149, miR-512-5p). These andro-miRs bind to target sequences in the 3′UTR of AR and downregulates luciferase expression in the target-validation validation experiments as well as endogeneous AR expression. Our ongoing study of andro-miRs also indicates that, in addition to downregulation of AR gene expression by the action of andro-miRs alone and synergistically, these miRNAs can regulate AR signaling leading to androgen-independence and resistance. This post transcriptional regulation of AR gene expression through andro-miRs makes them an interesting candidate for miR-based adjunctive treatment in CRPC treatment.

Note: This abstract was not presented at the meeting.

Citation Format: Jey Sabith Ebron, Kavleen Sikand, Jagjit Singh, Girish C. Shukla. Regulation of androgen receptor expression by andro-miRs in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5212. doi:10.1158/1538-7445.AM2014-5212
Abstract 5213: microRNA-mediated loss of KDM5B expression leads to suppression of the malignant bladder cancer phenotype
Authors: Veerakumarasivam Abhimanyu , Kodiappan Radha , Chan Soon Choy , Razack Azad Hassan Abdul , Ong Teng Aik
Published: May 2015

We recently identified KDM5B as a putative oncogene. The expression of KDM5B correlated with increased cellular proliferative capacity and was found to be overexpressed in many cancers including bladder cancer (BC). KDM5B is a histone demethylase enzyme that modifies chromatin structure to specify cellular transcriptional states. It is a cancer/testis antigen; whereby in normal conditions, expression is limited to early embryonic stages and the testis in adults. Thus, KDM5B is an excellent therapeutic target. Since the expression of KDM5B in embryonic stem cells is orchestrated by miRNAs and that the expression of miRNAs are altered in BC, we hypothesized that miRNAs may be the switch that can abate the expression of KDM5B and thus affect the malignant phenotype of BC cells. This study was aimed at identifying and modulating the expression of miRNAs that are able to regulate KDM5B expression and BC phenotypes. By coupling in silico screens with RT-QPCR analysis of in vitro expression of BC cells of different malignant potential, we predicted several miRNAs as potential regulators of KDM5B transcripts. The functional effects of specific miRNAs on KDM5B expression, cell cycle, proliferation and invasion of BC cells was determined by modulating the expression of these miRNAs using mimics (upregulation) and antagomirs (downregulation). Expression of miR-137 in BC cells was found to be inversely correlated with KDM5B expression. Ectopic overexpression of miR-137 led to the repression of KDM5B expression, reduced cell growth and invasion as well as increased apoptosis of BC cells. In contrast, the downregulation of miR-137 expression led to reverse effects. The expression modulation of miR-137 also affected the expression of other BC-associated genes such as JDP2 and EGR2. While our results suggest that miR-137 can mitigate the KDM5B-associated BC phenotype, further studies on understanding the effect on aberrant histone methylation patterns are warranted.

Citation Format: Abhimanyu Veerakumarasivam, Radha Kodiappan, Soon Choy Chan, Azad Hassan Abdul Razack, Teng Aik Ong. microRNA-mediated loss of KDM5B expression leads to suppression of the malignant bladder cancer phenotype. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5213. doi:10.1158/1538-7445.AM2014-5213
Abstract 5215: miR-200b/c/429 sub-family negatively regulating Rho/ROCK signaling pathway and suppresses HCC metastasis
Authors: Wong Chun-Ming , Wei Lai , Au Sandy Leung-Kuen , Au Sandy Leung-Kuen , Fan Dorothy Ngo-Yin , Lee Joyce Man-Fong , Wong Carmen Chak-Lui , Ng Irene Oi-Lin
Published: May 2015

MicroRNAs (miRNAs) are a class of small (∼22 nt) noncoding regulatory RNA molecules that can regulate gene expression by either cleaving or repressing the translation of their mRNA targets. Deregulation of miRNA expression is common in human cancers, including hepatocellular carcinoma (HCC). Recent studies have indicated that the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141 and miR-429) negatively regulates epithelial-mesenchymal transition (EMT) and cell motility by targeting E-cadherin repressors, ZEB1 and ZEB2. However, the roles of the miR-200 family in HCC remain elusive. In this study, we found that miR-200 family was frequently down-regulated in human HCC, which was contributed by the frequently CpG hypermethylation of miR-200 promoters. Functionally, although all miR-200 members suppressed ZEB1/2 expression in HCC, only overexpression of miR-200b/c/429 sub-family impeded HCC cell migration. These findings implied that, in addition to ZEB1/2, other yet unidentified miR-200b/c/429 specific targets may play an important role in HCC metastasis. In silico analyses revealed that miR-200b/c/429 sub-family may have a profound role in modulating cell cytoskeletal reorganization. Along with this notion, we further identified RhoA and ROCK2 as down-stream targets of miR-200b/c/429 sub-family. By using immunofluorescence staining, scanning electron microscope and live cell imaging, we demonstrated that overexpression of miR-200b/c/429 subfamily but not miR-200a/141 sub-family suppressed Rho/ROCK signaling mediated stress fiber and focal adhesion formations, which in turn led to a dramatic cell morphology change and loss of cell migration ability. Furthermore, by employed an orthotopic liver implantation model, we showed that overexpression of miR-200b was sufficient to suppress HCC growth and metastasis in nude mice. Our results together suggest that miR-200b/c/429 sub-family functions as a metastatic suppressor in human HCC by negatively regulating Rho/ROCK signaling pathway.

Citation Format: Chun-Ming Wong, Lai Wei, Sandy Leung-Kuen Au, Sandy Leung-Kuen Au, Dorothy Ngo-Yin Fan, Joyce Man-Fong Lee, Carmen Chak-Lui Wong, Irene Oi-Lin Ng. miR-200b/c/429 sub-family negatively regulating Rho/ROCK signaling pathway and suppresses HCC metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5215. doi:10.1158/1538-7445.AM2014-5215