Pancreatic Cancer Gene Research Articles

Abstract 2624: Pre-clinical studies of a mutant p53 reactivating drug in pancreatic cancer

Abstract Pancreatic cancer therapy suffers from a lack of effective chemotherapy. TP53 is second only to Kras as the most commonly mutated gene in pancreatic cancer with point mutations occurring in 75% of patients. Recently, our laboratory identified ZMC1 as an allele specific p53 mutant reactivating compound. This compound selectively kills cancer cells that express the p53-R175H mutation by restoring wildtype structure and function to this mutant. It does not reactivate the DNA contact mutant, p53-R273H or p53-R248H. This established ZMC1 as a lead compound for mutant p53 targeted drug development. We are currently investigating the potential of ZMC1 as novel anti-cancer drug through pre-clinical studies in pancreatic cancer. Previously we reported the ZMC1 allele specificity in murine pancreatic cancer cell lines derived from the KPC mouse model expressing mutant KrasG12D and different alleles of TP53 (WT, null, p53R172H, p53R270H). We have investigated the pharmacokinetics (PK) and pharmacodynamics (PD) of ZMC1 in the KPC mouse model. Using LC/MS/MS we developed a sensitive assay for detection of ZMC1 in mouse plasma. We measured drug concentrations in mouse plasma and tumor tissues in different delivery routes and different dosages in the PK studies. We have developed a PD assay for ZMC1 with cleaved caspase-3 immunostaining with which we have tested several doses of ZMC1 and found that we detect activity of ZMC1 in KPCp53-R172H xenograft tumors while at the same time relatively no activity in KPCp53-R270H tumors. These same assays were also performed on KPC autochthanous tumors and again revealed activity of ZMC1 in the KPCp53-R172H mice. Efficacy studies in the KPCp53-R172H xenografts showed potent tumor growth inhibition at both intravenous (1mg/kg) and intraperitoneal (5mg/kg) dosing while no evidence of growth inhibition in KPCp53-R270H. Furthermore, ZMC1 extended the survival days from average 15 days to 26 days in the KPCp53-R172H mice but it was not significantly observed in the KPCp53-R270H mice. The tumor growth rate was also decreased in the KPCp53-R172H but not in the KPCp53-R270H mice. These studies indicated that ZMC1 is an allele-specific p53 mutant reactivating compound and holds promise for future applications in pancreatic cancer. Citation Format: Xin Yu, Ashley T. Tsang, Zhe Li, Oliver S. Eng, Hongxia Lin, Murugesan Gounder, Darren R. Carpizo. Pre-clinical studies of a mutant p53 reactivating drug in pancreatic cancer. [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 2624. doi:10.1158/1538-7445.AM2015-2624

Abstract 293: Therapeutic effects of the bromodomain inhibitors JQ1 and I-BET 762 on pancreatic cancer

Abstract Pancreatic cancer is expected to be the second most deadly cancer by 2030 with very few effective therapeutic options to improve patient survival. Bromodomain inhibitors (JQ1 and I-BET 762) are a new generation of chemotherapeutic drugs that target BET proteins, impairing their ability to bind to acetyl modified lysines and therefore interfering with transcriptional initiation and elongation. BET proteins regulate several genes responsible for cell cycle, apoptosis and inflammation. I-BET 762 is currently in clinical trials for cancer and has the advantage of oral administration. Kras is one of the most common mutated genes in pancreatic cancer (90%); however its protein is a difficult drug target. p-Erk, responsible for several pathways regulating cell survival, is a downstream effector of Kras that has been shown to be essential for the progression and maintenance of pancreatic cancer. LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre (KPC) and LSL-KrasG12D/+;Pdx-1-Cre (KC) mouse models are commonly used to study pancreatic cancer. KPC mice develop the full spectrum of pancreatic cancer by an average age of 20 weeks. The KC mouse model is commonly used to study pancreatic cancer progression after stimulation with a pancreatic inflammatory agent, such as cerulein. JQ1 and I-BET 762 reduce the protein levels of p-ERK 1/2 in cells lines derived from a pancreatic tumor or ascites of KPC animals. JQ1 and I-BET 762 are also effective at inhibiting cell growth in human pancreatic cells harboring Kras mutations. When KC mice are injected with LPS to induce inflammation and pancreatitis, p-ERK 1/2 protein levels are significantly increased. This model is being used to test the effects of JQ1 and I-BET 762 after a single LPS injection, in KC mice. Pancreatic cancer is driven by several cytokines and chemokines, including CCL2 and IL-6, which recruit and regulate the inflammatory cells (macrophages, T cells and MDSC (Myeloid derived suppressor cells) cells) in the tumor. Levels of IL-6 and CCL2 in cell lines developed from KPC mice are reduced when treated with JQ1 and I-BET 762. KC mice stimulated with LPS also have higher levels of CCL2 and IL-6 than mice stimulated with saline. In conclusion this study showed that the bromodomain inhibitors JQ1 and I-BET 762 had positive effects in suppressing targets of inflammation in pancreatic cancer. We thank James Bradner for the generous gift of JQ1. Citation Format: Ana S. Leal, Charlotte R. Williams, Michael B. Sporn, Karen T. Liby. Therapeutic effects of the bromodomain inhibitors JQ1 and I-BET 762 on pancreatic cancer. [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 293. doi:10.1158/1538-7445.AM2015-293

Abstract 2187: Pan-cancer identification and prioritization of cancer-associated differentially expressed genes: A biomarker discovery application

Abstract Cancer is fundamentally a disease of disordered gene expression. In fact, reversal or neutralization of the changes in gene expression has been shown to be attractive targets for the development of new anti-cancer drugs and therapeutic strategies. New approaches such as antibody drug conjugates (ADCs) also target differentially expressed genes as a mean to recognize tumor cells to selectively deliver toxins to a tumor. In the past decade, the global analysis of gene expression in human cancers have led to the development of a number of potential new biomarkers. For instance, mesothelin (MSLN) was identified as an over-expressed gene in pancreatic cancer and later was proved to be a useful diagnostic marker and so a therapeutic target. Large-scale gene expression analysis, using techniques such as RNA sequencing, provides a powerful tool to identify genes involved in human cancers. In this study, with the ultimate goal being to identify potential novel targets for cancer immunotherapy, we conducted a pan-cancer differential expression analysis in RNA sequencing data from more than 5,000 patients with 25 different cancer types generated by The Cancer Genome Atlas (TCGA). We identified differentially expressed genes (present in at least 5% of samples in a tumor type) in comparison to a large compendium of normal transcriptomes (more than 650 samples, including 30 tissue types) gathered from Genotype-Tissue Expression (GTEx), illumina BodyMap project 2.0, TCGA, and an in-house database. In total, we identified 892 putative tumor-associated differentially expressed genes. In order to further identify novel candidate genes and rank them based on their antigenic potential, we performed an extensive literature search and systematic review to collect the characteristics of an ideal tumor antigen (TA). We developed an Analytic Hierarchy Process (AHP) model - a multiple-criteria decision-making solution - to depict antigen properties for ranking and prioritizing the tumor-associated differentially expressed genes. Our model recognizes the known tumor antigens (such as CA9, Nectin-4, FN1, MSLN and MUC16, which are currently in clinic or pre-clinical studies) in the top 25 of the ranked list. We are currently validating the top-ranked novel antigens in an orthogonal panel of tumor and normal tissues and cell lines using PCR. Note: This abstract was not presented at the meeting. Citation Format: Daryanaz Dargahi, Richard D. Swayze, Leanna Yee, Peter J. Bergqvist, Bradley J. Hedberg, Alireza Heravi-Moussavi, Edie M. Dullaghan, Ryan Dercho, Christopher Bond, Jianghong An, John S. Babcook, Steven JM Jones. Pan-cancer identification and prioritization of cancer-associated differentially expressed genes: A biomarker discovery application. [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 2187. doi:10.1158/1538-7445.AM2015-2187

Abstract 5148: NR5A2 is essential for pancreas development and affects pancreas carcinogenesis

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers today. A genome-wide association study (GWAS) has implicated the nuclear receptor NR5A2 in modulating risk for pancreatic cancer. While a role for this gene in pancreatic cancer has not been previously recognized, it plays an important role in stem cell pluripotency and metabolism. Using the zebrafish model, we demonstrate that genetic or pharmacologic inhibition of nr5a2 activity results in absence of exocrine pancreas, while leaving endocrine pancreas unaffected, indicating an essential role in pancreas organ formation. Further, we localize nr5a2 expression to the endodermal bud that gives rise to the exocrine pancreas, consistent with a role for nr5a2 in regulating differentiation of pancreas progenitors. In resected human PDAC specimens and in vitro cell lines, we find that NR5A2 expression is altered compared to normal ductal epithelium. Moreover, shRNA knockdown of NR5A2 dramatically alters proliferation of PDAC cell lines, supporting the hypothesis that NR5A2 is involved in pancreatic carcinogenesis. These data support a model that NR5A2 regulates the proliferation and differentiation of exocrine pancreas progenitor cells during development. We postulate that loss-of-function of NR5A2 results in dysregulation of these processes in the adult pancreas by driving acinar cells to acquire properties of progenitor cells, and therefore is an important step in pancreas oncogenesis. Note: This abstract was not presented at the meeting. Citation Format: Sahar Nissim, Olivia Weeks, John Hedgepeth, Julia Wucherpfennig, Xiao-Xu Wang, Alec Kimmelman, Wolfram Goessling. NR5A2 is essential for pancreas development and affects pancreas carcinogenesis. [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 5148. doi:10.1158/1538-7445.AM2015-5148

Abstract 1764: Galeterone and its novel analogs induce profound anti-cancer activities in human pancreatic cancer cell lines: Implications for pancreatic cancer therapy

Abstract Pancreatic cancer is a lethal disease with a 3-5% survival rate. Histologically, 90% of human pancreatic cancer is presented as pancreatic ductal adenocarcinoma (PDAC). The aggressive nature of the disease and its high metastatic potential makes the development of efficacious therapeutic strategies very significant. Current therapeutic strategies are ineffective as the deregulated RAS/MAPK and PI3K/AKT/mTOR pathways to enhance drug resistance. Gemcitabine alone or in combination with erlotinib, the currently used drugs for PDAC, elicit only marginal survival benefits. The modest efficacy of gemcitabine therapy has been attributed to activation of the NF-κB pathway which activates genes involved in gemcitabine resistance. Recent studies have implicated the eIF4F (specifically, eIF4E-Mnk1/2 axis) that control gene expression at the translational level towards PDAC gemcitabine drug resistant. Several groups have also implicated the androgen receptor in PDAC progression/metastasis and have investigated the use anti-androgens in several in vitro and vivo models of PDAC and in a few clinical trials with mixed results. In the course of our studies to develop more potent androgen receptor degrading agents (ARDAs), we discovered that galeterone (gal) and its novel analogs also effectively target oncogenic eukaryotic protein translation. These novel small molecule inhibitors significantly depleted Mnk1/2 protein expression and decreased eIF4E phosphorylation. Interestingly, our studies revealed that the phosphorylation level of Nf-kB (p65) was significantly decreased after exposing pancreatic cancer cells to ARDAs. We note that these targets are implicated in PDAC progression and drug resistance. Here we report for the first time, the efficacy of galeterone and its lead ARDA analogs in inhibiting cell viability of both gemcitabine-naïve and gemcitabine/erlotinib-resistant human pancreatic cancer cells in vitro. Gal/ARDAs alone or in combination with gemcitabine significantly reduced the colony forming potential of gemcitabine-naïve and gemcitabine resistant pancreatic cancer cells. Decreased invasive potential of PDAC cells was shown through wound-healing and matrigel-coated Boyden chambers assays. Gal/ARDAs also caused significant depletion of matrix metalloproteinases (MMPs). Ezh2, a prominent metastatic enhancing gene in pancreatic cancer was also downregulated at the protein level. Immunoblot analysis of treated lysates also revealed induction of apoptosis via caspase 3 and PARP cleavage. Flow cytometric analysis showed that gal/ARDAs did not only deplete cyclins but also inhibited cell cycle progression. Our results highlight the unique ability of gal and its novel analogs to inhibit the growth, colonization and metastatic potential of a variety of PDAC cells through modulation of several oncogenic targets. Citation Format: Andrew K. Kwegyir-Afful, Puranik Purushottamachar, Marlena S. Martin, Vincent C.o. Njar. Galeterone and its novel analogs induce profound anti-cancer activities in human pancreatic cancer cell lines: Implications for pancreatic cancer therapy. [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 1764. doi:10.1158/1538-7445.AM2015-1764

Abstract 4186: p120 catenin: A novel regulator of epithelial cell delamination in early Kras-driven pancreatic cancer

Abstract By 2020, pancreatic cancer is estimated to climb from the 4th to the 2nd most common cause of cancer-related deaths in the United States. This deadly disease has continued to remain largely refractory to chemotherapeutic and treatment regimens, and patients often experience a heavy metastatic burden. A study combining the Sleeping beauty transposon random insertion mutagenesis system with an oncogenic KrasG12D allele in mice as a screen to identify candidate pancreatic cancer genes identified genes enriched in adherens and tight junctions as significantly enriched in Kras-driven neoplasia with Ctnnd1 as a locus commonly mutated in mice developing metastatic progression of pancreatic cancer. Ctnnd1 encodes the adherens junction protein p120 catenin, which is integral in stabilization of cadherin molecules at cell membranes. p120 catenin is misexpressed in an estimated 60% primary pancreatic tumors and low/absent expression levels and predominant cytoplasmic localization of p120 catenin in primary resected pancreatic tumors correlates with worse survival in pancreatic cancer patients. Yet, the mechanisms by which p120 catenin contributes to the pathogenesis of pancreatic cancer are not clear. We have comprehensively examined p120 catenin staining in human Pancreatic Intraepithelial Neoplasia (PanIN) and identified mislocalization of p120 catenin to the cytoplasm as early as PanIN2. 3/5 PanIN3 examined had predominant cytoplasmic staining, which led us to hypothesize that p120 catenin might play a critical role in early pancreatic neoplasia, before the onset of Pancreatic Ductal Adenocarcinoma. To examine the role of p120 catenin during early Kras-driven pancreatic neoplasia, we ablated p120 catenin in a mouse model of preinvasive pancreatic cancer, KCiMist1. KCiMist1p120f/f pancreases display significant acceleration of acinar to ductal metaplasia (ADM) and PanIN formation when compared to KCiMist1p120wt/wt pancreases one month post tamoxifen injection. KCiMist1p120f/f pancreases are significantly larger than KCiMist1p120wt/wt pancreases with 92.89% pancreatic area occupied by Fibrostroma one month post tamoxifen injection. As a result, KCiMist1p120f/f animals have severe exocrine pancreatic insufficiency and die on average 8 weeks earlier than their KCiMist1p120wt/wt controls. Lineage tracing revealed a prominent epithelial cell delamination phenotype in KCiMist1p120f/f pancreases. Quantification of epithelial cells in the extensive stroma revealed a striking 832/7000 CK19+ cells in KCiMist1p120f/f pancreases vs 15/7000 CK19+ cells in KCiMist1p120wt/wt pancreases. Microarray analysis showed >1263 differentially expressed genes! IPA pathway analysis revealed significant gene expression changes in PI3K/AKT and Cdc42 signaling. Taken together, our results suggest a critical role for p120 catenin in regulating epithelial cell delamination in early pancreatic cancer. Citation Format: Audrey M. Hendley, Yue J. Wang, Janivette Alsina, Ishrat Ahmed, Hao Zhang, Samuel Savidge, Hao Ho, Albert Reynolds, Anirban Maitra, Michael Goggins, Christine Iacobuzio-Donahue, Steven D. Leach, Jennifer M. Bailey. p120 catenin: A novel regulator of epithelial cell delamination in early Kras-driven pancreatic cancer. [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 4186. doi:10.1158/1538-7445.AM2015-4186

Mucin 1 Regulates Cox-2 Gene in Pancreatic Cancer.

Eighty percent of pancreatic ductal adenocarcinomas (PDAs) overexpress mucin 1 (MUC1), a transmembrane mucin glycoprotein. MUC1(high) PDA patients also express high levels of cyclooxygenase 2 (COX-2) and show poor prognosis. The cytoplasmic tail of MUC1 (MUC1-CT) partakes in oncogenic signaling, resulting in accelerated cancer progression. Our aim was to understand the regulation of Cox-2 expression by MUC1. Levels of COX-2 and MUC1 were determined in MUC1(-/-), MUC1(low), and MUC1(high) PDA cells and tumors using reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemistry. Proliferative and invasive potential was assessed using MTT and Boyden chamber assays. Chromatin immunoprecipitation was performed to evaluate binding of MUC1-CT to the promoter of COX-2 gene. Significantly higher levels of COX-2 mRNA and protein were detected in MUC1(high) versus MUC1(low/null) cells, which were recapitulated in vivo. In addition, deletion of MUC1 gene and transient knockdown of MUC1 led to decreased COX-2 level. Also, MUC1-CT associated with the COX-2 promoter at ∼1000 base pairs upstream of the transcription start site, the same gene locus where nuclear factor κB p65 associates with the COX-2 promoter. Data supports a novel regulation of COX-2 gene by MUC1 in PDA, the intervention of which may lead to a better therapeutic targeting in PDA patients.

Abstract B113: Prevalence of germline BRCA and mismatch repair (MMR) gene mutations in pancreatic cancer

Abstract Background: Germline mutations of BRCA1, BRCA2, MLH1, MSH2, and MSH6 are known to increase risk of pancreatic cancer. However, the prevalence of pathogenic germline mutations of these genes among pancreatic cancer patients is unknown. Accurately characterizing the frequencies of mutations of these genes will inform patient selection for future gene discovery studies and, clinically, foster screening strategies and tailored treatments. Objective: To determine the prevalence of pathogenic germline mutations of BRCA1, BRCA2, MLH1, MSH2, and MSH6 in a cohort of pancreatic cancer patients, and to determine the association between mutation carrier status and personal and family history of cancer. Methods: The Ontario Pancreas Cancer Study (OPCS) enrolls all consenting participants with a diagnosis of pancreatic cancer into a province-wide electronic pathology database. A cross-sectional sample of 300 patients from 715 OPCS probands enrolled between April 2003 and August 2012 were randomly selected from three strata based on a family history of pancreatic, breast, and ovarian cancer. Targeted next-generation sequencing was successfully performed on germline DNA from 291 of the 300 selected probands. Mutations were classified as benign, of unknown significance, or pathogenic by literature and database review. Pathogenic mutations were confirmed with Sanger sequencing. Prevalence estimates representing the whole OPCS were estimated using the Horvitz-Thompson estimator. Univariate analysis determined association between carrier status and clinical covariates, and regression analysis for overall survival. Results: A total of 7 pathogenic germline mutations were identified: 1 BRCA1, 2 BRCA2, 1 MLH1, 2 MSH2, and 1 MSH6. The estimated prevalence of pathogenic mutations in BRCA1 and BRCA2 among probands in this OPCS series was 1.1% (95% confidence interval: 0.1-2.0%); in the mismatch repair genes MLH1, MSH2, and MSH6 it was 1.5% (0.3-2.6%). Both a personal history of colorectal cancer and a first-degree relative with colorectal cancer, breast cancer, or melanoma were significantly associated (p<0.001 and p<0.01, respectively) with MMR mutations. There were no significant differences in tumor location within the pancreas (head/uncinate versus body/tail), clinical nodal status, rates of resection, age at diagnosis, BMI, history of smoking, history of pancreatitis, family history of pancreatic cancer, family history of colorectal cancer, or differences in survival between the MMR or sporadic cohorts. Conclusion: This study is the first to quantify population-based prevalences of germline mutations of BRCA1, BRCA2, MLH1, MSH2, and MSH6 in pancreatic cancer. Surprisingly, the prevalence of pathogenic germline mutations of the MMR genes in pancreatic cancer is higher than expected, and is comparable to that of BRCA1 and BRCA2. The prevalence of the MMR germline mutations is also comparable to the prevalence of MMR germline mutations in colorectal cancer cohorts. Relatives who carry MMR gene mutations can benefit from tailored cancer prevention strategies; thus mutational analysis of MLH1, MSH2, and MSH6 should be included in molecular genetic testing and counseling strategies for pancreatic cancer patients, especially those with a family history of malignancy. Translational studies will follow to determine if stratifying pancreatic cancer risk by familial susceptibility genes, as in colorectal cancer, fosters tailored and cost-effective primary and secondary prevention strategies for carriers. Moreover, unique chemotherapy sensitivity and resistance patterns would be expected for MMR-associated pancreatic cancer, as is emerging for BRCA-associated pancreatic cancer. Citation Format: Iris Selander, Robert Grant, Ashton Connor, Shamini Selvarajah, Ayelet Borgida, Laurent Briollais, Jordan Lerner-Ellis, Spring Holter, Steven Gallinger. Prevalence of germline BRCA and mismatch repair (MMR) gene mutations in pancreatic cancer. [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 B113.

Abstract IA5: Translating genetic discoveries to patient care

Abstract Next-generation sequencing technologies have changed the speed at which cancer genetics research can be performed and this research has fundamentally changed our understanding of pancreatic cancer. 1. Families: It has been estimated that 10% of pancreatic cancer has an inherited basis. Whole-exome and whole-genome sequencing studies have led to the discovery of several new familial pancreatic cancer genes including PALB2 and ATM. It is hoped that new multi-institutional collaborative efforts will dramatically increase the pace of discovery. Patients found to harbor a genetic alteration that predisposes them to pancreatic cancer will be the first to benefit from early detection efforts. 2. Cystic neoplasms: Some cystic lesions in the pancreas are harmless benign tumors, while others are precancerous. Lives can be saved if significant precursor lesions can be identified and removed, but there is a risk of over-treatment if the benign harmless cysts are removed. Whole-exome sequencing all of the four major types of cystic neoplasms of the pancreas has revealed a pattern of mutations specific for each cyst type. In the future, cyst fluid can be sampled and cyst type determined by sequencing. 3. Early detection: The genetic alterations in a cancer and in its precursor lesions offer extremely sensitive and highly specific markers for the early detection of pancreatic neoplasia. These genetic alterations can be detected in pancreatic juice collected at endoscopy and even in the plasma. 4. Invasive cancer: Genetic alterations in a cancer can be the basis for individualized therapies. 5. Metastatic cancer: The sequencing of metastatic cancer has helped define the timeline of tumor progression in the pancreas. In sum, and in short, these are exciting times in the war against pancreatic cancer. The translation of discoveries made by sequencing pancreatic tumors to patient care holds the promise for reducing mortality from this terrible disease. Citation Format: Ralph H. Hruban. Translating genetic discoveries to patient care. [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 IA5.

GSH2 promoter methylation in pancreatic cancer analyzed by quantitative methylation-specific polymerase chain reaction.

Tumor suppressor gene silencing via promoter hypermethylation is an important event in pancreatic cancer pathogenesis. Aberrant DNA hypermethylation events are highly tumor specific, and may provide a diagnostic tool for pancreatic cancer patients. The objective of the current study was to identify novel methylation-related genes that may potentially be used to establish novel therapeutic and diagnostic strategies against pancreatic cancer. The methylation status of the GS homeobox 2 (GSH2) gene was analyzed using the sodium bisulfite sequencing method. The GSH2 methylation ratio was examined in primary carcinomas and corresponding normal tissues derived from 47 patients with pancreatic cancer, using quantitative methylation-specific polymerase chain reaction. Methylation ratios were found to be associated with the patient's clinicopathological features. GSH2 gene methylation was detected in 26 (55.3%) of the 47 pancreatic cancer patients, indicating that it occurs frequently in pancreatic cancer. A significant association with methylation was observed for tumor-node-metastasis stage (P=0.031). GSH2 may be a novel methylation-sensitive tumor suppressor gene in pancreatic cancer and may be a tumor-specific biomarker of the disease.

A multistep high-content screening approach to identify novel functionally relevant target genes in pancreatic cancer.

In order to foster the systematic identification of novel genes with important functional roles in pancreatic cancer, we have devised a multi-stage screening strategy to provide a rational basis for the selection of highly relevant novel candidate genes based on the results of functional high-content analyses. The workflow comprised three consecutive stages: 1) serial gene expression profiling analyses of primary human pancreatic tissues as well as a number of in vivo and in vitro models of tumor-relevant characteristics in order to identify genes with conspicuous expression patterns; 2) use of ‘reverse transfection array’ technology for large-scale parallelized functional analyses of potential candidate genes in cell-based assays; and 3) selection of individual candidate genes for further in-depth examination of their cellular roles. A total of 14 genes, among them 8 from “druggable” gene families, were classified as high priority candidates for individual functional characterization. As an example to demonstrate the validity of the approach, comprehensive functional data on candidate gene ADRBK1/GRK2, which has previously not been implicated in pancreatic cancer, is presented.

Mutations in the p16 gene in DMBA-induced pancreatic intraepithelial neoplasia and pancreatic cancer in rats

7, 12-dimethylbenzanthracene (DMBA)-induced pancreatic intraepithelial neoplasia (PanIN) and pancreatic cancer in rats provide a classic model for uncovering the molecular mechanisms underlying pancreatic cancer. However, this model has not been characterized genetically, and in particular, the major genetic alterations in the p16 gene are unknown. Lesions of PanIN and pancreatic cancer were induced with DMBA implantation in 40 rats, and control pancreatic tissue was obtained from 10 age-matched rats without exposure to DMBA. Pancreatic tissue was harvested three months after DMBA implantation and DNA was extracted. Homozygous deletions and point mutations of the p16 (exons 1 and 2) gene were detected by PCR amplification and direct sequencing. DMBA implantation in the 40 rats induced 26 PanINs and 9 carcinomas. The overall frequency of p16 alterations in the pancreatic tissue of these rats was 42.86% (15/35), and the changes were point mutations, not homozygous deletions. p16 mutations were present in 30.77% (8/26) of the rats with PanIN and 77.78% (7/9) of the rats with carcinoma (P<0.05). The increasing incidence of p16 alterations was detected in 20.00% (1/5) of PanIN-1, 28.57% (2/7) of PanIN-2 and 35.71% (5/14) of PanIN-3 lesions. Our findings indicated that p16 alteration is a common event in the carcinogenesis of this model and that the mutation pattern is analogous to that of human lesions.

Novel agents in early phase clinical studies on refractory pancreatic cancer.

The standard current treatment options in advanced pancreatic cancer have demonstrated minimal or modest only efficacy for the majority of patients. Unfortunately, the mortality and morbidity remain high crying out for better treatments and results. With the exception of erlotinib, which received approval by the Food and Drug Administration of the United States in 2005, no other novel agents have since been added in our treatment quiver. Therefore, the search for novel approaches continuous at the laboratory and clinical level. At the 2012 American Society of Clinical Oncology Gastrointestinal Symposium, results of some interesting early phases clinical studies were presented. First, in Abstract #198, toxicity and efficacy results from the phase I/II study of cixutumumab, an insulin growth factor-1 receptor (IGF-1R) antibody combined with the standard gemcitabine and erlotinib treatment were presented, but the outcomes suggest no real clinical benefit. Second, the early safety and clinical data from the novel monoclonal antibody (ensituximab) against the mucin epitope NPC-1C in pancreatic and colon cancer patients were presented (Abstract #233) and again no particular efficacy was observed. Finally, interesting results which definitely deserve further exploration were presented in Abstract #211, which tested the combination of ipilimumab, an antibody against the cytotoxic T-lymphocyte antigen 4 (CTLA-4), with a cell-based vaccine transfected with the granulocyte macrophage colony-stimulating factor (GM-CSF) gene in advanced refractory pancreatic cancer. Though, it seems we have not yet found the culprit and the solution of this devastating disease, a small step forward might have been achieved.

Endocrine gland‑derived vascular endothelial growth factor modulates proliferation, apoptosis and migration in pancreatic cancer cells.

Endocrine gland‑derived vascular endothelial growth factor(EG‑VEGF) is a newly cloned factor that selectively acts on the endothelium of endocrine gland cells. EG‑VEGF was previously identified as an important cytokine, involved in the modulation of apoptosis in pancreatic cancer cell lines. The present study examined the effects of EG‑VEGF proliferation and migration, in pancreatic cancer cells. To determine the potential for EG‑VEGF as a therapeutic target for pancreatic cancer, the expression of EG‑VEGF were measured in pancreatic cancer tissue, and the association between its expression and the clinicopathological characteristics of the pancreatic cancer patients was determined. The results of the present study suggest that EG‑VEGF may act as a novel tumor gene in pancreatic cancer. EG‑VEGF was rarely expressed in the normal pancreatic tissue, but was highly expressed in the pancreatic cancer tissue. These data suggest that EG‑VEGF may be a cancer‑specific, and possibly tissue‑specific, survival factor in the pancreas. In the Mia PaCa‑2 pancreatic cancer cell line, EG‑VEGF was shown to promote proliferation and cellular invasion, and modulate the phosphorylation of mitogen‑activated protein kinase, a modulator for the malignant phenotype.

MiR-183 induces cell proliferation, migration, and invasion by regulating PDCD4 expression in the SW1990 pancreatic cancer cell line

The aim of this study was to investigate the function of miR-183 in the SW1990 cancer cell line, and the mechanisms regulating these processes. miRNAs are known to play important roles in cancer cell development. However, the pattern and biological role of miR-183 in pancreatic cancer remain largely unknown. Here, we have reported the reduction in pancreatic cancer cell growth in vitro by miR-183 intervention, by inducing apoptosis and decreasing the Bcl-2 expression. Moreover, miR-183 was observed to enhance pancreatic cancer cell migration and invasion, whereas inhibition of miR-183 caused an opposite effect. miR-183 inhibition was shown to increase E-cadherin expression and decrease N-cadherin expression. These regulatory actions play an important role in the cancer epithelial-mesenchymal transition (EMT). Mechanistically, we demonstrated that the overexpression of miR-183 decreased the expression of PDCD4 (programmed cell death 4) mRNA and protein, and vice versa. This helped to identify PDCD4 as the target genes in pancreatic cancer. In conclusion, our analyses indicated miR-183 to be an important contributor to cell migration. This could also be used as a potential therapeutic target for pancreatic cancer treatment.

Biodegradable nanoparticle-mediated K-ras down regulation for pancreatic cancer gene therapy.

RNA interference (RNAi) targeting the K-ras oncogene mutation in pancreatic cancer mediated by small interfering RNA (siRNA) transfection is a very promising treatment. However, the rapid degradation and negative charge of naked siRNAs restrict their direct delivery into cells. In this contribution, we propose a safe and effective transmembrane transport nanocarrier formulation based on a newly developed biodegradable charged polyester-based vector (BCPV) for K-ras siRNA delivery into pancreatic cancer cells. Our results have shown that these biodegradable and biocompatible vectors are able to transfect siRNAs targeting mutant K-ras into MiaPaCa-2 cells with high transfection and knockdown efficiency. More importantly, the RNAi process initiated a cascade gene regulation of the downstream proteins of K-ras associated with cell proliferation, migration, invasion and apoptosis. We observed that after the mutant K-ras siRNA transfection, the growth, migration and invasion of the MiaPaCa-2 cells were significantly reduced; also, the apoptosis of the pancreatic cancer cells was promoted. Although in vivo testing data are limited, we propose that the BCPV based nanoparticle formulation could be a promising candidate as non-viral vectors for gene therapy in clinical settings.

Lentiviral Vector-based Insertional Mutagenesis Identifies Genes Involved in the Resistance to Targeted Anticancer Therapies

The high transduction efficiency of lentiviral vectors in a wide variety of cells makes them an ideal tool for forward genetics screenings addressing issues of cancer research. Although molecular targeted therapies have provided significant advances in tumor treatment, relapses often occur by the expansion of tumor cell clones carrying mutations that confer resistance. Identification of the culprits of anticancer drug resistance is fundamental for the achievement of long-term response. Here, we developed a new lentiviral vector-based insertional mutagenesis screening to identify genes that confer resistance to clinically relevant targeted anticancer therapies. By applying this genome-wide approach to cell lines representing two subtypes of HER2(+) breast cancer, we identified 62 candidate lapatinib resistance genes. We validated the top ranking genes, i.e., PIK3CA and PIK3CB, by showing that their forced expression confers resistance to lapatinib in vitro and found that their mutation/overexpression is associated to poor prognosis in human breast tumors. Then, we successfully applied this approach to the identification of erlotinib resistance genes in pancreatic cancer, thus showing the intrinsic versatility of the approach. The acquired knowledge can help identifying combinations of targeted drugs to overcome the occurrence of resistance, thus opening new horizons for more effective treatment of tumors.

Identification of dysregulated pathways associated with pancreatic cancer by survival analysis.

In order to identify the dysregulated pathways associated with pancreatic cancer, the fourth leading cause of cancer mortality in the United States, tumor and non-tumor samples were systematically analyzed in the present study. Initially, dysregulated genes in pancreatic cancer were identified using paired t-test. Subsequently, dysregulated biological pathways involved in the development of pancreatic cancer were identified by enrichment analysis. Finally, individual survival analysis of the significantly dysregulated functions was conducted at the pathway level. Our results indicated that the pathway named ̔Pathways in cancer was significantly correlated with survival time. In addition, the mean survival time of individual and genetic variation demonstrated a significantly negative correlation, that is, the lower the genetic variation, the longer the survival time. Furthermore, detailed analysis of genes on the pathway named ̔Pathways in cancer denoted that this pathway involved multiple cancer hallmark signals and several dysregulated cancer genes, including tumor protein p53, myelocytomatosis, Kirsten rat sarcoma, phosphatidylinositol 3-kinase, v-raf murine sarcoma viral oncogene homolog B1 and cyclin-dependent kinase inhibitor 2A. According to the DrugBank database, certain oncogenes have been validated to be the targets of drugs, including Sorafenib, Trastuzumab, Imatinib and Paclitaxel or were under investigation. An improved understanding of the pathophysiology of pancreatic cancer has been achieved based on our results and the present study aimed to provide guidance for the development of drugs to treat pancreatic cancer.

Abstract 3692: Preclinical studies of a mutant p53 reactivating drug in pancreatic cancer

Abstract Pancreatic cancer therapy suffers from a lack of effective chemotherapy. TP53 is second only to Kras as the most commonly mutated gene in pancreatic cancer with point mutations occurring in 75% of patients. Recently, our laboratory identified NSC319726 (726) as an allele specific p53 mutant reactivating compound (Yu, et al. 2012, Cancer Cell). This compound selectively kills cancer cells that express the p53-R175H mutation by restoring wildtype structure and function to this mutant. It does not reactivate the DNA contact mutant, p53-R273H or p53-R248H. This established 726 as a lead compound for mutant p53 targeted drug development. We are currently investigating the potential of 726 as novel anti-cancer drug through pre-clinical studies in pancreatic cancer. We have investigated the pharmacokinetics (PK) and pharmacodynamics (PD) of 726 in murine pancreatic cancer cell lines derived from the KPC mouse model expressing mutant KrasG12D and different alleles of TP53 (WT, null, p53R172H, p53R270H). Using LC/MS/MS we developed a sensitive assay for detection of 726 in mouse plasma. PK studies indicate a half-life of approximately 4 hours with an AUC of 199.78 ng/ml/h for a single 1mg/kg dose given Intravenously. Treatment of KPC cell lines with 726 indicates that the murine p53R172H protein is reactivated as indicated by loss of mutant conformation and induction of p53 target gene transcription. Cell growth inhibition assays reveal that the cell line KPCp53-R172H is sensitive to 726 while the KPCp53-R270H is not. We have developed a PD assay for 726 consisting of cleaved caspase-3 immunostaining and measurement of transcriptional levels of p53 targets PUMA, Noxa and p21. Using these assays we have tested several doses of 726 and found that we detect activity of 726 in KPCp53-R172H xenograft tumors while at the same time relatively no activity in KPCp53-R270H tumors. These same assays were also performed on KPC autochthanous tumors and again reveal activity of 726 in the KPCp53-R172H mice. Efficacy studies in the KPCp53-R172H xenografts reveal potent tumor growth inhibition at both IV (1mg/kg) and IP (5mg/kg) dosing while no evidence of growth inhibition in KPCp53-R270H. These studies confirm that 726 is an allele-specific p53 mutant reactivating compound and holds promise for future applications in pancreatic cancer. Citation Format: Xin Yu, Zhe Li, Oliver S. Eng, Ashley T. Tsang, Hongxia Lin, Murugesan Gounder, Darren R. Carpizo. Preclinical studies of a mutant p53 reactivating drug in pancreatic 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 3692. doi:10.1158/1538-7445.AM2014-3692

Abstract 5271: Homozygous deletion of the activin A receptor, type IB gene is associated with an aggressive cancer phenotype in pancreatic cancer

Abstract Background: Transforming growth factor, beta (TGFB) signal is considered to be a tumor suppressive pathway based on the frequent genomic deletion of the SMAD4 gene in pancreatic cancer (PC); however; the role of the activin signal, which also belongs to the TGFB superfamily, remains largely unclear. Methods and Results: We found a homozygous deletion of the activin A receptor, type IB (ACVR1B) gene in 2 out of 8 PC cell lines using array-comparative genomic hybridization, and the absence of ACVR1B mRNA and protein expression was confirmed in these 2 cell lines. Activin A stimulation inhibited cellular growth and increased the phosphorylation level of SMAD2 and the expression level of p21CIP1/WAF1 in the Sui66 cell line (wild-type ACVR1B and SMAD4 genes) but not in the Sui68 cell line (homozygous deletion of ACVR1B gene). Stable ACVR1B-knockdown using short hairpin RNA cancelled the effects of activin A on the cellular growth of the PC cell lines. In addition, ACVR1B-knockdown significantly enhanced the colony formation abilities and cellular growth, compared with controls. In a xenograft study, ACVR1B-knockdown resulted in a significantly elevated level of tumorigenesis and a larger tumor volume, compared with the control. Furthermore, in clinical samples, 6 of the 29 PC samples (20.7%) carried a deletion of the ACVR1B gene, while 10 of the 29 samples (34.5%) carried a deletion of the SMAD4 gene. Of note, 5 of the 6 samples with a deletion of the ACVR1B gene also had a deletion of the SMAD4 gene. Conclusion: We identified a homozygous deletion of the ACVR1B gene in PC cell lines and clinical samples and proposed that the deletion of the ACVR1B gene may mediate an aggressive cancer phenotype in PC. Our findings provide novel insight into the role of the activin signal in PC. Citation Format: Yosuke Togashi, Hiroki Sakamoto, Hidetoshi Hayashi, Masato Terashima, Marco A. de Velasco, Yoshihiko Fujita, Yasuo Kodera, Kazuko Sakai, Shuta Tomida, Masayuki Kitano, Masatoshi Kudo, Kazuto Nishio. Homozygous deletion of the activin A receptor, type IB gene is associated with an aggressive cancer phenotype in pancreatic 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 5271. doi:10.1158/1538-7445.AM2014-5271