Citations

Abstract Introduction Epigenetic changes are reversible features of the genome that regulate gene transcription and protein expression on several levels including DNA methylation, histone modification or miRNA expression. We investigated the association between inherited variation in genes of key epigenetic processes and risk of multiple cancers within the GAME-ON consortium. Methods We performed a pathway based meta-analysis using genotypes from more than 50,000 cases of breast, lung, prostate, ovarian and colorectal cancer cases and more than 60,000 controls from various genome wide association studies participating in the GAME-ON consortium to estimate associations with cancer risk. Using the 1000GenomeProject database, we selected 505,702 genotyped and imputed single nucleotide polymorphisms in 551 genes (flanking region +/- 250kb) related to DNA methylation, histone modification or chromatin remodeling based on GO and GeneCard databases. In order to allow variants to be associated with only a subset of traits we used subset based meta-analysis. False-discovery rate (FDR) corrected p-values (q-values) lower than 0.05 were considered significant. Results and Discussion 582 SNPs were significantly associated with risk of at least one cancer. We identified nine major regions that showed significant associations with more than one cancer type. Among the most interesting regions was the region around PHC3 (3q36), which showed associations with prostate and colorectal cancer and clear cell ovarian carcinomas. PHC3 is involved in chromatin remodeling and plays a role in epithelial neoplasms. Significant Odds ratios (ORs) ranged from 0.80 to 1.31. The number of risk and protective alleles in this region was similarly distributed (19 and 18, respectively). One of the strongest associations was observed for rs76925190 (intronic in PRKC1), which increased the risk of colorectal and prostate cancer (q-value 4.28*10-10). Variants in this region were previously associated with prostate cancer. Polymorphisms in the region (19q13) around BABAM1 (RISC and BRCA1 A complex member 1), were associated with lung, breast, ovarian and prostate cancer. BABAM1 is associated with the BRCA1-complex. Its function in histone modification and DNA repair emphasizes its importance in carcinogenesis. Significant ORs ranged from 0.88 to 1.14 with similar distribution of risk and protective alleles in this region (19 and 17, respectively). The strongest association was observed for rs4808076 (intronic in ANKLE1), which increased the risk of squamous lung, serous ovarian and ER- -breast cancer (q-value 2.40*10-6). Variants in this region were previously associated with risk of breast and ovarian cancer. Conclusions This study emphasizes the importance of variants in genes of epigenetic processes on cancer risk and further provides insights into novel, pleiotropic epigenetic mechanisms of cancer development. Citation Format: Dominique Scherer, Reka Toth, Linda Kelemen, Angela Risch, Aditi Hazra, Jean Pierre Issa, Victor Moreno, Rosalind A. Eeles, John Quackenbush, Ellen L. Goode, Shuji Ogino, Rayjean Hung, Cornelia M. Ulrich. Genetic variants in epigenetic pathways and risk of multiple cancer types in the GAME-ON consortium. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4612. doi:10.1158/1538-7445.AM2015-4612

Abstract Telomeres are complex structures that cap chromosome ends, protecting them from degradation, double strand breaks and end-to-end fusions. Telomeres are maintained by the enzyme telomerase, which is made up of a reverse transcriptase encoded by TERT, and an RNA template encoded by TERC. The telomere structure itself is composed of proteins encoded by ACD, ACYP2, BICD1, DKC1, DCLRE1B, MPHOSPH6, NAF1, NOLA1, NOLA2, NOLA3, OBFC1, PIK3C3, POT1, RTEL1, TEP1, TERF1, TERF2, TERF21P, TINF2, TNKS/PINX1, TPP1 and ZNF208. Several single nucleotide polymorphisms (SNPs) in the TERT and adjoining CLPTM1L gene region are associated with multiple cancer types, and some are associated with both increased and decreased risks across different cancer types. We systematically characterized the patterns of association between variants in these 25 telomere structure and maintenance genes and risk across five cancer types in the Genetic Association and Mechanisms in Oncology (GAME-ON) consortium. We performed a subset-based meta-analysis (ASSET) of 209,367 directly measured and imputed SNPs, one megabase up- and downstream of these genes, across genome-wide association studies of colorectal (5,100 cases, 4,831 controls), lung (12,160 cases, 16,838 controls), breast (15,748 cases, 18,084 controls), ovarian (4,369 cases, 9,123 controls) and prostate (14,160 cases, 12,724 controls) cancers. Correlations (r2) between SNPs were examined in Haploview using the 1000 Genomes Project CEU population. A total of 87 TERT, 123 TERC and 26 DCLRE1B SNPs were associated with cancer risk at gene-level Bonferroni-corrected p-values of 4.2-7.8×10−6. Patterns of association were similar for prostate and colorectal cancers in DCLRE1B. Much stronger associations were observed in TERT and TERC, with 63 and 24 SNPs reaching genome-wide significance (p<5.0×10−8), respectively. Of these, 9 TERT and 9 TERC SNPs were correlated at r2<0.75. The most strongly associated SNPs in TERT (rs37004, p = 2.6×10−11) and TERC (rs76925190, p = 1.5×10−15) are in regions containing documented risk loci for lung and prostate cancers, respectively. TERT rs37004 (p = 1.2×10−13) and 6 of the other 9 SNPs in TERT were associated only with lung cancer risk. For the other 2 SNPs, there was a suggestion that patterns of risk were opposite for lung and prostate cancers. While TERC rs76925190 was strongly associated with prostate cancer risk (p = 5.4 x10−17), it was also suggestively associated with colorectal cancer risk (p<0.005). This pattern was similar for another 2 of the 9 SNPs, while for the rest, associations were limited to prostate cancer. The complex patterns of association in telomere structure and maintenance genes observed across cancer types may provide insight about the mechanisms through which telomere dysfunction in different tissues influences cancer risk. Citation Format: Sara Karami, Younghun Han, Fredrick R. Schumacher, Zsofia Kote-Jarai, Sara Lindstrom, John S. Witte, Iona Cheng, Shenying Fang, Jiali Han, Peter Kraft, Fengju Song, Rayjean J. Hung, James McKay, Stephen J. Chanock, Mala Pande, Angela Risch, Hongbing Shen, Christopher A. Haiman, Lisa Boardman, Cornelia M. Ulrich, Graham Casey, Ulrike Peters, Nilanjan Chatterjee, Brandon Pierce, Wei Zheng, Christopher I. Amos, Jennifer A. Doherty. Risk loci in telomere structure and maintenance genes across five cancer types: GAME-ON Consortium. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4609. doi:10.1158/1538-7445.AM2015-4609

Abstract Background: The Tumor Growth Factor-β (TGF-β) pathway is a potent tumor suppressor pathway. Its inactivation has been implicated in hepatocarcinogenesis. However, in patients with advanced stage Hepatocellular Carcinoma (HCC), TGF-β1 serum concentrations are elevated and correlated to tumor size, recurrence and shortened survival. The mechanisms mediating TGF-β1 ligand overexpression in HCC are unknown. Material and Methods: Immunohistochemical analysis of human HCC samples was performed using Tissue Micro Array. Established human HCC cell lines were used for in vitro studies. Gene expression was assessed by qRT-PCR, ELISA and immunoblot analysis. shRNA was used for gene silencing. Transcription factor DNA binding was evaluated by chromatin immunoprecipitation. For promoter analysis, transcription factor binding site containing DNA fragments were cloned into the enhancer or promoter position of luciferase expression vectors and subsequent transfected. Results: In up to 68% of advanced stage HCC tumors Stem Cell Factor (SCF) is overexpressed, and SMAD2 and STAT3 are constitutively activated. SCF expression in human HCC tumors is intratumorally and intracellularly correlated to expression of pSer465/467SMAD2 and pTyr705STAT3. In HCC cell lines, SCF stimulation induces TGF-β1 transcription, expression and secretion, all of which are abrogated by genetic silencing of STAT3. TGF-β1 stimulation of human HCC cells induces TGF-β1 transcription, and genetic silencing of SCF prevents TGF-β1 autoregulation. We identified two STAT3 recognition sites within the TGF-β1 gene. In human HCC cells, TGF-β1 stimulation results in STAT3 binding to one of the identified recognition sites. TGF-β1 activates luciferase expression vectors under control of the corresponding STAT3 binding site but not in its absence. TGF-β1-induced STAT3 binding and expression vector activation is abrogated in HCC cells with genetically silenced SCF. TGF-β1 induces SCF expression but SMAD2 knockdown inhibits TGF-β1-induced SCF transcription and expression. We identified 7 putative SMAD2-binding elements (SBE) within the SCF promoter. TGF-β1 stimulation results in SMAD2 binding to the SCF promoter and activation of luciferase expression vectors regulated by the SCF promoter; conversely, genetic silencing of SMAD2 abrogates TGF-β1-induced SCF promoter activation. Importantly, knockdown of SCF restored the antiproliferative functions of TGF-β, and prevented TGF-β-induced tumor cell migration and invasion. Conclusion: In advanced stage HCC, SCF drives TGF-β1 autoregulation through STAT3-mediated regulation of the TGF-β1 promoter. Thereby, SCF and TGF-β1 form a positive feedback loop that functionally switches the TGF-β pathway from a tumor suppressor to a tumor promoter in advanced stage HCC. Citation Format: Pingyu Zhang, Andres Rojas, Ying Wang, Nina M. Munoz, Lianchun Xiao, Jing Wang, Gregory J. Gores, Mien-Chie Hung, Boris R. A. Blechacz. A positive feedback loop between TGF-β and SCF mediates TGF-β1 ligand overexpression in advanced hepatocellular carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3911. doi:10.1158/1538-7445.AM2015-3911

Abstract Background: Current systemic agents for advanced Hepatocellular carcinoma (HCC) achieve minimal survival benefits. Tumor Growth Factor-β (TGF-β) is a potent tumor suppressor via its SMAD2/3 signaling axis; its inactivation has been implicated in hepatocarcinogenesis. Paradoxically, TGF-β serum concentrations are elevated in patients with advanced HCC. The TGF-β receptor is currently explored in clinical trials as a therapeutic target. However, the mechanisms mediating the functional switch of the TGF-β pathway to an oncogenic role in advanced HCC are unknown. Material and Methods: Gene expression profiling of human HCC was performed using the Oncomine database. Immunohistochemical analysis of human HCC samples was performed using Tissue Micro Arrays. Established human HCC cell lines were used for in vitro studies. Gene expression was assessed by qRT-PCR, ELISA and immunoblot analyses. shRNA was used for gene silencing. Cell cycle analysis was performed by FACS. Cell proliferation was assessed by microscopic quantification, MTS- and BrdU-assay. Apoptosis was analyzed by TUNEL assay and DAPI. Migration was evaluated by wound assay, and invasion by Boyden Chamber assay. Results: In human HCC, SMAD2 overexpression and decreased expression of the negative TGF-β/SMAD regulator SKI was correlated to shortened survival. Intranuclear pSer465/467-SMAD2 was identified in up to 65% of human HCC tumors, and correlated with TNM-stage, survival and pTyr705STAT3. In 50% of human HCC cell lines, the TGF-β/SMAD2 axis was functional. Exclusively in HCC cells with intact SMAD-signaling, TGF-β induced STAT3 Tyr705-phosporylation and nuclear translocation. We show that TGF-β1 transcriptionally regulates expression of the c-KIT ligand Stem Cell Factor (SCF). SCF is overexpressed in advanced HCC tumors, and correlated to pSMAD2, metastases and decreased survival. In human HCC cells, SCF activates STAT3 via auto- and paracrine stimulation of its cognate receptor c-KIT. TGF-β1-induced activation of STAT3 is inhibited by SCF-knockdown, inhibition of c-KIT or JAK1. The TGF-β1/STAT3 axis neutralizes the cell cycle inhibitory function of TGF-β in human HCC cell lines, and instead promotes EMT, tumor cell migration and invasion. Disruption of the TGF-β/STAT3 signaling axis inhibits its tumor promoting effects and restores TGF-β antiproliferative effects. Conclusion: In advanced HCC, TGF-β/SMAD2-signaling is constitutively activated and correlated to worse outcomes. The functional switch of TGF-β is mediated through aberrant expression of SCF with subsequent stimulation of the c-KIT/STAT3 signaling axis. Disruption of this axis restores TGF-β tumor suppressor function. Disruption of the molecular network between TGF-β1/SMAD2 and SCF/STAT3 constitutes a novel therapeutic strategy aiming at restoration of a dysregulated tumor suppressor. Citation Format: Andres Rojas, Pingyu Zhang, Ying Wang, Nina M. Munoz, Lianchun Xiao, Jing Wang, Gregory J. Gores, Mien-Chie Hung, Boris R. A. Blechacz. TGF-β/SMAD2 signaling drives tumor-progression via c-KIT/STAT3-signaling in advanced HCC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1962. doi:10.1158/1538-7445.AM2015-1962

Abstract Summary: Structural variations (SVs) are large genomic rearrangements that vary significantly in size, making them challenging to detect with the relatively short reads from next-generation sequencing (NGS). Different SV detection methods have been developed; however, each is limited to specific kinds of SVs with varying accuracy and resolution. Previous works have attempted to combine different methods, but they still suffer from poor accuracy particularly for insertions. We propose MetaSV, an integrated SV caller which leverages multiple orthogonal SV signals for high accuracy and resolution. MetaSV proceeds by merging SVs from multiple tools for all types of SVs. It also analyzes soft-clipped reads from alignment to detect insertions accurately since existing tools underestimate insertion SVs. Local assembly in combination with dynamic programming is used to improve breakpoint resolution. Paired-end and coverage information is used to predict SV genotypes. Using simulation and experimental data, we demonstrate the effectiveness of MetaSV across various SV types and sizes. Availability and implementation: Code in Python is at http://bioinform.github.io/metasv/. Contact: rd@bina.com Supplementary information: Supplementary data are available at Bioinformatics online.

Abstract Purpose: Esophageal cancer is an aggressive malignancy and often resistant to therapy. Overexpression of EGFR has been associated with poor prognosis of patients with esophageal cancer. However, clinical trials using EGFR inhibitors have not provided benefit for patients with esophageal cancer. Failure of EGFR inhibition may be due to crosstalk with other oncogenic pathways. Experimental Design: In this study, expression of YAP1 and EGFR were examined in EAC-resistant tumor tissues versus sensitive tissues by IHC. Western blot analysis, immunofluorescence, real-time PCR, promoter analysis, site-directed mutagenesis, and in vitro and in vivo functional assays were performed to elucidate the YAP1-mediated EGFR expression and transcription and the relationship with chemoresistance in esophageal cancer. Results: We demonstrate that Hippo pathway coactivator YAP1 can induce EGFR expression and transcription in multiple cell systems. Both YAP1 and EGFR are overexpressed in resistant esophageal cancer tissues compared with sensitive esophageal cancer tissues. Furthermore, we found that YAP1 increases EGFR expression at the level of transcription requiring an intact TEAD-binding site in the EGFR promoter. Most importantly, exogenous induction of YAP1 induces resistance to 5-fluorouracil and docetaxcel, whereas knockdown of YAP1 sensitizes esophageal cancer cells to these cytotoxics. Verteporfin, a YAP1 inhibitor, effectively inhibits both YAP1 and EGFR expression and sensitizes cells to cytotoxics. Conclusions: Our data provide evidence that YAP1 upregulation of EGFR plays an important role in conferring therapy resistance in esophageal cancer cells. Targeting YAP1-EGFR axis may be more efficacious than targeting EGFR alone in esophageal cancer. Clin Cancer Res; 21(11); 2580–90. ©2015 AACR.

Abstract Inflammation is a facilitating process for multiple cancer types. It is believed to affect cancer development and progression through several etiologic pathways, including increased levels of DNA adduct formation, increased angiogenesis, and altered antiapoptotic signaling. This review highlights the application of inflammatory biomarkers in epidemiologic studies and discusses the various cellular mediators of inflammation characterizing the innate immune system response to infection and chronic insult from environmental factors. Included is a review of six classes of inflammation-related biomarkers: cytokines/chemokines, immune-related effectors, acute-phase proteins, reactive oxygen and nitrogen species, prostaglandins and cyclooxygenase-related factors, and mediators such as transcription factors and growth factors. For each of these biomarkers, we provide a brief overview of the etiologic role in the inflammation response and how they have been related to cancer etiology and progression within the literature. We provide a discussion of the common techniques available for quantification of each marker, including strengths, weaknesses, and potential pitfalls. Subsequently, we highlight a few under-studied measures to characterize the inflammatory response and their potential utility in epidemiologic studies of cancer. Finally, we suggest integrative methods for future studies to apply multifaceted approaches to examine the relationship between inflammatory markers and their roles in cancer development. Cancer Epidemiol Biomarkers Prev; 23(9); 1729–51. ©2014 AACR.

Severely comminuted fractures of the metatarsal bones with significant bone and soft-tissue loss have commonly subjected patients to proximal amputation procedures. We describe two patients who experienced high-energy traumatic injuries to their limbs that resulted in significant destruction of their first and second metatarsal bones with overlying soft-tissue trauma not amenable to local coverage. In both cases, a vascularized free fibular osteocutaneous flap was used to reconstruct the metatarsal bone defect and traumatized soft tissues so that a proximal amputation was avoided. At an average of 14 months of follow-up, both patients had recovered well and regained independent ambulation, with one patient being able to play soccer. We show that the free fibular osteoseptocutaneous flap is useful in reconstructing significant metatarsal bone defects and in avoiding amputations in this patient population. The skin component of the flap may be used to fill soft-tissue losses, and the fibula bone may be osteotomized so that more than one ray may be reconstructed. (J Am Podiatr Med Assoc 101(6): 531–536, 2011)