Pancreatic ductal adenocarcinoma (PDA) remains a lethal malignancy despite tremendous progress in its molecular characterization. Indeed, PDA tumors harbor four signature somatic mutations1–4, and a plethora of lower frequency genetic events of uncertain significance5. Here, we used Sleeping Beauty (SB) transposon-mediated insertional mutagenesis6,7 in a mouse model of pancreatic ductal preneoplasia8 to identify genes that cooperate with oncogenic KrasG12D to accelerate tumorigenesis and promote progression. Our screen revealed new candidates and confirmed the importance of many genes and pathways previously implicated in human PDA. Interestingly, the most commonly mutated gene was the X-linked deubiquitinase Usp9x, which was inactivated in over 50% of the tumors. Although prior work had attributed a pro-survival role to USP9X in human neoplasia9, we found instead that loss of Usp9x enhances transformation and protects pancreatic cancer cells from anoikis. Clinically, low USP9X protein and mRNA expression in PDA correlates with poor survival following surgery, and USP9X levels are inversely associated with metastatic burden in advanced disease. Furthermore, chromatin modulation with trichostatin A or 5-aza-2′-deoxycytidine elevates USP9X expression in human PDA cell lines to suggest a clinical approach for certain patients. The conditional deletion of Usp9x cooperated with KrasG12D to rapidly accelerate pancreatic tumorigenesis in mice, validating their genetic interaction. Therefore, we propose USP9X as a major new tumor suppressor gene with prognostic and therapeutic relevance in PDA.

Purpose Genetic alterations of KRAS, CDKN2A, TP53 and SMAD4 are the most frequent events in pancreatic cancer. We determined the extent to which these four alterations are coexistent in the same carcinoma, and their impact on patient outcome. Experimental Design Pancreatic cancer patients who underwent an autopsy were studied (n=79). Matched primary and metastasis tissues were evaluated for intragenic mutations in KRAS, CDKN2A and TP53 and immunolabeled for CDKN2A, TP53 and SMAD4 protein products. The number of altered driver genes in each carcinoma was correlated to clinicopathologic features. Kaplan-Meier estimates were used to determine median disease free and overall survival, and a Cox proportional hazards model used to compare risk factors. Results The number of genetically altered driver genes in a carcinoma was variable, with only 29 patients (37%) having an alteration in all four genes analyzed. The number of altered driver genes was significantly correlated with disease free survival (p=0.008), overall survival (p=0.041) and metastatic burden at autopsy (p=0.002). On multivariate analysis, the number of driver gene alterations in a pancreatic carcinoma remained independently associated with overall survival (p=0.046). Carcinomas with only one to two driver alterations were enriched for those patients with the longest survival (median 23 months, range 1–53). Conclusions Determinations of the status of the four major driver genes in pancreatic cancer, and specifically the extent to which they are coexistent in an individual patients cancer, provides distinct information regarding disease progression and survival that is independent of clinical stage and treatment status.

Purpose Pancreatic cancer (PC) is the fourth cause of death from cancer in the western world. Majority of patients present with advanced unresectable disease responding poorly to most chemotherapeutic agents. Chemotherapy for PC might be improved by adjusting it to individual genetic profiles. We attempt to identify genetic predictors of chemosensitivity to broad classes of anticancer drugs. Experimental Design Using a panel of genetically defined human PC cell lines, we tested gemcitabine (anti-metabolite), docetaxel (anti-microtubule), mitomycin C (alkylating), irinotecan (topoisomerase I inhibitor), cisplatin (crosslinking), KU0058948 (Parp1 inhibitor), triptolide (terpenoid drug) and artemisinin (control). Results All PC cell lines were sensitive to triptolide and docetaxel. Most PC cells were also sensitive to gemcitabine and MMC. The vast majority of PC cell lines were insensitive to cisplatin, irinotecan, and a Parp1 inhibitor. However, individual cell lines were often sensitive to these compounds in unique ways. We found that DPC4/SMAD4 inactivation sensitized PC cells to cisplatin and irinotecan by 2–4 fold, but they were modestly less sensitive to gemcitabine. PC cells were all sensitive to triptolide and 18% were sensitive to the Parp1 inhibitor. P16/CDKN2A inactivated PC cells were 3–4 fold less sensitive to gemcitabine and MMC. Conclusions Chemosensitivity of PC cells correlated with some specific genetic profiles. These results support the hypothesis that genetic subsets of pancreatic cancer exist, and these genetic backgrounds may permit one to personalize the chemotherapy of PC in the future. Further work will need to confirm these responses and determine their magnitude in vivo.

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Cancer is a disease caused by the accumulation of genetic alterations in association with successive waves of clonal expansion. Mapping of the human genome sequence, in conjunction with technical advances in the ability to sequence entire genomes, have provided new insight into the mutational spectra and genetic events associated with clonal evolution of cancer. Moving forward, a clearer understanding of those alterations that undergo positive and negative selection throughout carcinogenesis and leading to metastatic dissemination would provide a boon not only to our understanding of cancer evolution, but to the development of potential targets for therapeutic intervention as well.

Pancreatic cancer is a highly lethal tumor type for which there are few viable therapeutic options. It is also caused by the accumulation of mutations in a variety of genes. These genetic alterations can be grouped into those that accumulate during pancreatic intraepithelial neoplasia (precursor lesions) and thus are present in all cells of the infiltrating carcinoma, and those that accumulate specifically within the infiltrating carcinoma during subclonal evolution, resulting in genetic heterogeneity. Despite this heterogeneity there are nonetheless commonly altered cellular functions, such as pathways controlling the cell cycle, DNA damage repair, intracellular signaling and development, which could provide for a variety of drug targets. This review aims to summarize current knowledge of the genetics and genomics of pancreatic cancer from its inception to metastatic colonization, and to provide examples of how this information can be translated into the clinical setting for therapeutic benefit and personalized medicine.

Introduction: Pancreatic cancer remains a largely fatal disease, with an estimated 95% of patients succumbing within five years of initial diagnosis, many by metastatic spread. We have previously shown that genetic inactivation of DPC4, a central mediator of TGF-β signaling, is strongly associated with widespread metastasis in pancreatic cancer patients at autopsy. However, because some patients with metastatic pancreatic cancer do not have genetic inactivation of DPC4 or other TGF-β members, we investigated the role of alternative mechanisms of TGF-β pathway inactivation in promoting pancreatic cancer metastasis. Methods: Eighteen cell lines that underwent whole exome sequencing in association with the pancreatic cancer genome project or directed sequencing of all members of the TGF-β pathway were used. TGF-β signaling levels were analyzed in each cell line using a luciferase reporter system under the control of a Smad Binding Element (SBE). Quantitative real-time PCR was performed using cDNA from representative cell lines to assess expression levels of nine known TGF-β pathway antagonists; data were analyzed using the ΔΔCt method with normalization to β-actin. Immunohistochemistry was performed on pancreatic cancer tissue microarrays that included 27 matched pairs of primary and metastatic cancer. Staining intensity and protein localization were analyzed in relation to DPC4 status as well as clinicopathologic data. Results: Functional TGF-β signaling was completely abolished in cell lines with known DPC4 inactivation compared to normal controls, and was also decreased in cell lines with an intact TGF-β pathway, to a lesser extent. Quantitative RT-PCR of known TGF-β antagonists in cell lines with genetically intact TGF-β pathways revealed 50-fold overexpression of BMP and Activin Membrane-Bound Inhibitor (BAMBI) in two of seven cell lines (29%). Immunolabeling for BAMBI in human pancreatic cancer tissues indicated that it is highly expressed in a subset (26%) of pancreatic cancers, consistent with the cell line data. BAMBI overexpression was not associated with clinicopathologic features at diagnosis, nor was there a difference in BAMBI expression among DPC4 wild type versus DPC4 inactive carcinomas. However, specifically, BAMBI overexpression was significantly more frequent in metastases from patients with wild type DPC4 compared to those with inactivated DPC4 (p=0.01). Conclusion: Overexpression of BAMBI occurs at an appreciable level in pancreatic cancer cell lines and tissues, and may promote metastasis in the presence of a genetically intact TGF-β pathway. Mechanistic studies are ongoing to clarify the role of BAMBI in pancreatic cancer metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2438. doi:10.1158/1538-7445.AM2011-2438

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