Citations

Abstract Pancreatic ductal adenocarcinoma (PDAC), as with all cancer, is driven by dysregulation of genetic programs due to accumulation of somatic mutations, epigenetic modifications and changes in the micro-environment. Holistic views of perturbations in gene expression networks could illuminate key regulators and pathways for this highly lethal cancer. Toward this end, we performed massively-parallel mRNA-sequencing in normal (n = 10) and tumor (n = 8) derived pancreatic tissue samples as well as pancreatic cancer cell lines (n = 9), and determined differential gene expression (DE) patterns. Sub-network enrichment analyses of all expressed genes based on magnitude of DE identified HNF1A as the regulator of the most significantly and consistently dysregulated expression sub-network in our pancreatic tumor samples (median P = 7.56 x 10-7, median rank = 1, range = 1-25). To explore the effect of HNF1A expression in pancreatic tumor-derived cells we generated stable HNF1A-inducible clones in two pancreatic cancer cell lines. Induction of HNF1A overexpression caused severe growth inhibition (5.3-fold, P = 4.5x10-5 for MIA PaCa-2 clones; 7.2-fold, P = 2.2x10-5 for PANC-1 clones), and G0/G1 cell cycle arrest. This was accompanied by down-regulation of 49 out of 84 assayed cell cycle genes, while only 1 displayed increased expression. These data, combined with HNF1A’s direct regulation of pancreatic development and homeostasis genes (i.e. PDX1, PTF1A and NR5A2), suggest it may be an important tumor suppressor in pancreatic cells. Citation Format: Jason W. Hoskins, Marta Flandez, Jinping Jia, Hemang Parikh, Irene Collins, Mickey Emmanuel, Abdisamad Ibrahim, Wenming Xiao, John Powell, Nuria Malats, Gloria M. Petersen, Fransisco X. Real, Laufey T. Amundadottir. Transcriptome analysis in pancreatic cancer reveals a tumor suppressor function for HNF1A. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B112.

Abstract A genome-wide association study (GWAS) of pancreatic cancer conducted within the NCI Cohort Consortium (PanScan I and II) identified pancreatic cancer susceptibility loci on chromosomes 1q32.1/NR5A2, 5p15.33/CLPTM1L/TERT, 9q34.2/ABO, and 13q22.1. The most significant single-nucleotide polymorphism (SNP) identified on 13q22.1, rs9543325, lies in a 600 kb gene desert; the nearest genes are KLF5, KLF12, PIBF1, DIS3, and BORA (265–586 kb). Imputation using the 1000 Genomes and DCEG reference datasets did not improve the GWAS signal, but produced a set of highly correlated SNPs for functional follow-up. We performed eQTL analysis to test for association between the genotypes of these functional candidate variants and expression of nearby genes. Among 100 normal derived pancreatic tissue samples, DIS3 expression showed the strongest association with a novel 30 bp indel variant in the risk locus (P = 4.0 × 104), indicating risk alleles associate with reduced DIS3 expression. Mutations in DIS3 have previously been identified in acute myeloid leukemia and multiple myeloma, and its expression has been correlated with metastatic potential in colorectal cancer, suggesting DIS3 is relevant to cancer biology. Chromosome conformation capture identified a physical interaction between the indel-containing locus and a region near the DIS3 promoter. Luciferase assay for regulatory function of this indel-containing locus revealed allele-specific silencer activity for the insertion allele. Supershift electromobility shift assay (EMSA) demonstrated binding of LEF1 specifically to the insertion allele of the indel, which contains two in silico predicted LEF1 binding elements. Finally, through immunohistochemical analysis, high DIS3 protein levels associated with better survival for pancreatic cancer patients (hazard ratio = 2.87, 95% CI = 1.49–5.53, P = 0.001). Our results suggest that at least one target gene for the pancreatic cancer risk variants on chr13q22.1 may be DIS3, and that the underlying biology may be mediated by the novel indel through a long-range repressive effect on DIS3 expression. Citation Format: Jason W. Hoskins, Abdisamad Ibrahim, Mickey Emmanuel, Hemang Parikh, Jinping Jia, Irene Collins, Kris Ylaya, Sean F. Altekruse, Stephen M. Hewitt, Gloria M. Petersen, Laufey T. Amundadottir. Functional analysis of the chr13q22.1 pancreatic cancer risk locus suggests allele-specific effects on DIS3 expression with prognostic implications. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B111.

Abstract Pancreatic adenocarcinoma (PDAC) is the 4th most common cause of cancer related deaths in the United States and has a dismal 5-year survival rate of 6%. This prognosis is attributed the disease’s fast and asymptomatic progression to metastasis, leading to late diagnosis upon which time there are few treatment options available. The A Disintegrin and Metalloproteinase (ADAM) family are sheddase proteins known to regulate cell adhesion and function. This regulation can allow ADAMs to influence the ability of tumor cells to disseminate from the primary tumor and metastasize to distant organs. ADAM10, which is seen to be up-regulated in both chronic pancreatitis and PDAC, cleaves a variety of substrates including several cell adhesion molecules such as L1-CAM, N-Cadherin, and E-Cadherin. Using a conditional knockout for ADAM10 in the Kras+/G12D;Ptf1a+/Cre (KC) mouse model of pancreatic cancer, we have observed an absence of metastasis when ADAM10 is genetically ablated. To investigate the potential role that ADAM10 plays in this, we used a shRNA approach to knock-down ADAM10 expression in various human pancreatic cancer cells lines. Analysis of these cell lines suggests a partial restoration of a less aggressive, differentiated phenotype, consistent with the increased longevity of the ADAM10 knockout KC mice. Citation Format: Louise V. Peverley, Christopher J. Halbrook, Jason C. Hall, Christine M. Ardito, Howard C. Crawford. Investigating the role of ADAM10 in pancreatic tumor differentiation. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A77.

Aim: Missing data, particularly missing variables, can create serious analytic challenges in observational comparative effectiveness research studies. Statistical linkage of datasets is a potential method for incorporating missing variables. Prior studies have focused upon the bias introduced by imperfect linkage. Methods: This analysis uses a case study of hepatitis C patients to estimate the net effect of statistical linkage on bias, also accounting for the potential reduction in missing variable bias. Results: The results show that statistical linkage can reduce bias while also enabling parameter estimates to be obtained for the formerly missing variables. Conclusion: The usefulness of statistical linkage will vary depending upon the strength of the correlations of the missing variables with the treatment variable, as well as the outcome variable of interest.

Abstract Clinical trials have established the benefit of androgen deprivation therapy (ADT) combined with radiotherapy in prostate cancer. ADT sensitizes prostate cancer to radiotherapy-induced death at least in part through inhibition of DNA repair machinery, but for unknown reasons, adjuvant ADT provides further survival benefits. Here, we show that androgen receptor (AR) expression and activity are durably upregulated following radiotherapy in multiple human prostate cancer models in vitro and in vivo. Moreover, the degree of AR upregulation correlates with survival in vitro and time to tumor progression in animal models. We also provide evidence of AR pathway upregulation, measured by a rise in serum levels of AR-regulated hK2 protein, in nearly 20% of patients after radiotherapy. Furthermore, these men were three-fold more likely to experience subsequent biochemical failure. Collectively, these data demonstrate that radiotherapy can upregulate AR signaling after therapy to an extent that negatively affects disease progression and/or survival. Cancer Res; 75(22); 4688–96. ©2015 AACR.

Aberrant intracellular calcium levels and increased cAMP signaling contribute to the development of polycystic kidney disease (PKD). cAMP can be hydrolyzed by various phosphodiesterases (PDEs). To examine the role of cAMP hydrolysis and the most relevant PDEs in the pathogenesis of PKD, we examined cyst development in Pde1 - or Pde3 -knockout mice on the Pkd2 −/WS25 background (WS25 is an unstable Pkd2 allele). These PDEs were selected because of their importance in cross-talk between calcium and cyclic nucleotide signaling ( PDE1 ), control of cell proliferation and cystic fibrosis transmembrane conductance regulator (CFTR) –driven fluid secretion (PDE3), and response to vasopressin V2 receptor activation (both). In Pkd2 −/WS25 mice, knockout of Pde1a , Pde1c , or Pde3a but not of Pde1b or Pde3b aggravated the development of PKD and was associated with higher levels of protein kinase A–phosphorylated (Ser133) cAMP–responsive binding protein (P-CREB), activating transcription factor-1, and CREB–induced CRE modulator proteins in kidney nuclear preparations. Immunostaining also revealed higher expression of P-CREB in Pkd2−/ WS25 ; Pde1a −/− , Pkd2− /WS25 ; Pde1c −/− , and Pkd2−/ WS25 ; Pde3a −/− kidneys. The cystogenic effect of desmopressin administration was markedly enhanced in Pkd2 −/WS25 ; Pde3a −/− mice, despite PDE3 accounting for only a small fraction of renal cAMP PDE activity. These observations show that calcium- and calmodulin-dependent PDEs ( PDE1A and PDE1C ) and PDE3A modulate the development of PKD, possibly through the regulation of compartmentalized cAMP pools that control cell proliferation and CFTR–driven fluid secretion. Treatments capable of increasing the expression or activity of these PDEs may, therefore, retard the development of PKD.

Abstract Introduction: Small cell carcinoma of the ovary of hypercalcemic type (SCCOHT) is arguably the most aggressive ovarian cancer. Most patients are diagnosed at an advanced stage, do not respond to chemotherapy, and die of disease within 1-2 years. It affects children and young women and is reported to occur in families. The cause of the disease is poorly understood. Therefore, we used next generation sequencing technology to identify the genetic basis of the disease. Method: We performed whole genome, whole exome sequencing or targeted sequencing on tumours and germline samples from 17 SCCOHT patients and on the SCCOHT cell line BIN-67 and SCCOHT-1. Immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded tumors from 23 patients and on a tissue microarray of 485 primary ovarian tumours of other subtypes. BIN-67 cells harbouring biallelic inactivation of SMARCA4 were transduced with a lentivirus expressing wild type SMARCA4. Result: We identified inactivating germline and somatic mutations in the SWI/SNF chromatin-remodeling gene SMARCA4 in 79% (11/14) of SCCOHT patients, 2 of whom bore germline mutations, and in both cell lines. SMARCA4 protein was lost in 87% (20/23) of SCCOHT tumours but in only 0.4% (2/485) of other primary ovarian tumours. Reintroduction of wild-type SMARCA4 into BIN-67 cells resulted in altered cell morphology and growth arrest. Conclusions: The mutation spectrum, IHC profile and cell culture phenotype implicate SMARCA4 as a critical tumour suppressor in SCCOHT pathogenesis. Citation Format: Pilar Ramos, Anthony N. Karnezis, David W. Craig, Aleksandar Sekulic, Megan L. Russell, William P.D. Hendricks, Jason J. Corneveaux, Michael T. Barrett, Karey Shumansky, Yidong Yang, Sohrab P. Shah, Leah M. Prentice, Marco A. Marra, Jeffrey Kiefer, Victoria L. Zismann, Troy A. McEachron, Bodour Salhia, Jaime Prat, Blaise A. Clarke, Joseph G. Pressey, John H. Farley, Stephen P. Anthony, Richard B.S. Roden, Heather E. Cunliffe, David G. Huntsman, Jeffrey M. Trent. Small cell carcinoma of the ovary, hypercalcemic type displays frequent inactivating germline and somatic mutations in SMARCA4 [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1327.

Abstract Genome wide association studies (GWAS) have mapped multiple independent cancer risk loci (n = 6) to a small region on chr5p15.33 for at least ten distinct cancers, including bladder, breast, glioma, lung, melanoma, non-melanoma skin, ovarian, pancreas, prostate, and testicular germ cell cancer. This region harbors two plausible target genes, TERT which encodes the catalytic subunit of telomerase reverse transcriptase which maintains chromosome ends by adding telomeres repeats, and CLPTM1L which encodes the cleft lip and palate transmembrane protein 1-like protein which promotes cancer cell growth, protect cells from apoptosis, and can induce abnormal cytokinesis. The most significant pancreatic cancer GWAS SNP on chr5p15.33 was rs401681 (P = 3.7×10−7, OR = 1.19), located in the 13th intron of the CLPTM1L gene and within the independent multi-cancer risk locus in this region most distant from TERT. Imputation and fine mapping in pancreatic cancer resolved this signal by three orders of magnitude (P = 1.4×10−10, OR = 1.30) to a set of seven highly correlated SNPs. Electrophoretic mobility shift assays (EMSA) and luciferase assays across these SNPs led us to a single SNP, rs36115365, that showed allele-specific effects in both assays and overlapped with prominent ENCODE marks indicating regulatory potential. Examining this SNP across cell lines derived from multiple types of cancer linked to this locus, specifically pancreatic, testicular, lung, and melanoma, established a consistent pattern of a specific protein binding and enhanced regulatory activity for the risk allele across eight cancer cell lines (two cell lines per cancer type). siRNA-based targeting of the putative gene regulatory-region harboring rs36115365 resulted in a strong inhibition of TERT expression (average 60%, range 45-85%) but no effect on CLPTM1L expression. This inhibition of TERT expression was stronger from the pancreatic cancer risk as compared to the protective allele. Quantitative mass spectrometry identified Zinc finger protein 148 (ZNF148) as the protein specifically binding the risk allele, further supported by direct and allele-specific binding of purified recombinant ZNF148 in EMSA assays. Lastly, siRNA-mediated knockdown of ZNF148 resulted in significant reduction of TERT but not CLPTM1L expression across multiple cancer cell lines. Our results indicate that the pancreatic cancer GWAS signal at the single independent chr5p15.33 multi-cancer risk locus most distant from TERT may be explained by a single SNP that influences TERT expression via ZNF148. Citation Format: Kevin M. Brown, Jun Fang, Jinping Jia, Zhaoming Wang, Matthew Makowski, Tongwu Zhang, Jason Hoskins, Jiyeon Choi, Younghun Han, Mingfeng Zhang, Mai Xu, Peter Kanetsky, Andresson Thorkell, Gloria M. Petersen, Katherine L. Nathanson, Christopher I. Amos, Maria T. Landi, Stephen J. Chanock, Michiel Vermeulen, Laufey T. Amundadottir. Functional characterization of a multicancer risk locus on chr5p15.33 reveals regulation of TERT by ZNF148. [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 4610. doi:10.1158/1538-7445.AM2015-4610