Murray Korc

Inactivation of Ink4a/Arf leads to deregulated expression of miRNAs in K-Ras transgenic mouse model of pancreatic cancer.

Human pancreatic cancer (PC) is an aggressive disease, which has been recapitulated in transgenic animal model that provides unique opportunity for mechanistic understanding of disease progression and also for testing the efficacy of novel therapeutics. Emerging evidence suggests deregulated expression of microRNAs (miRNAs) in human PC, and thus we investigated the expression of miRNAs in pancreas tissues obtained from transgenic mouse models of K‐Ras (K), Pdx1‐Cre (C), K‐Ras;Pdx1‐Cre (KC), and K‐Ras;Pdx1‐Cre;INK4a/Arf (KCI), initially from pooled RNA samples using miRNA profiling, and further confirmed in individual specimens by quantitative RT‐PCR. We found over‐expression of miR‐21, miR‐221, miR‐27a, miR‐27b, and miR‐155, and down‐regulation of miR‐216a, miR‐216b, miR‐217, and miR‐146a expression in tumors derived from KC and KCI mouse model, which was consistent with data from KCI‐derived RInk‐1 cells. Mechanistic investigations revealed a significant induction of EGFR, K‐Ras, and MT1‐MMP protein expression in tissues from both KC and KCI mouse compared to tissues from K or C, and these results were consistent with similar findings in RInk‐1 cells compared to human MIAPaCa‐2 cells. Furthermore, miR‐155 knock‐down in RInk‐1 cells resulted in the inhibition of cell growth and colony formation consistent with down‐regulation of EGFR, MT1‐MMP, and K‐Ras expression. In addition, miR‐216b which target Ras, and forced re‐expression of miR‐216b in RInk‐1 cells showed inhibition of cell proliferation and colony formation, which was correlated with reduced expression of Ras, EGFR, and MT1‐MMP. These findings suggest that these models would be useful for preclinical evaluation of novel miRNA‐targeted agents for designing personalized therapy for PC. J. Cell. Physiol. 227: 3373–3380, 2012.

Activated K-Ras and INK4a/Arf Deficiency Promote Aggressiveness of Pancreatic Cancer by Induction of EMT Consistent With Cancer Stem Cell Phenotype

Pancreatic ductal adenocarcinoma (PDAC) is one of the most frequently diagnosed cancers and the fourth leading cause of cancer‐related death in the United States, suggesting that there is an urgent need to design novel strategies for achieving better treatment outcome of patients diagnosed with PDAC. Our previous study has shown that activation of Notch and NF‐κB play a critical role in the development of PDAC in the compound K‐RasG12D and Ink4a/Arf deficient transgenic mice. However, the exact molecular mechanism by which mutated K‐Ras and Ink4a/Arf deficiency contribute to progression of PDAC remains largely elusive. In the present study, we used multiple methods, such as real‐time RT‐PCR, Western blotting assay, and immunohistochemistry to gain further mechanistic insight. We found that the deletion of Ink4a/Arf in K‐RasG12D expressing mice led to high expression of PDGF‐D signaling pathway in the tumor and tumor‐derived cell line (RInk‐1 cells). Furthermore, PDGF‐D knock‐down in RInk‐1 cells resulted in the inhibition of pancreatosphere formation and down‐regulation of EZH2, CD44, EpCAM, and vimentin. Moreover, we demonstrated that epithelial–mesenchymal transition (EMT) was induced in the compound mice, which is linked with aggressiveness of PDAC. In addition, we demonstrated that tumors from compound transgenic mice have higher expression of cancer stem cell (CSC) markers. These results suggest that the acquisition of EMT phenotype and induction of CSC characteristics could be linked with the aggressiveness of PDAC mediated in part through the activation of PDGF‐D, signaling. J. Cell. Physiol. 228: 556–562, 2013.

PMTDS: a computational method based on genetic interaction networks for Precision Medicine Target-Drug Selection in cancer

BackgroundPrecision medicine attempts to tailor the right therapy for the right patient. Recent progress in large-scale collection of patents’ tumor molecular profiles in The Cancer Genome Atlas (TCGA) provides a foundation for systematic discovery of potential drug targets specific to different types of cancer. However, we still lack powerful computational methods to effectively integrate multiple omics data and protein-protein interaction network technology for an optimum target and drug recommendation for an individual patient.MethodsIn this study, a computation method, Precision Medicine Target Drug Selection (PMTDS) based on genetic interaction networks is developed to select the optimum targets and associated drugs for precision medicine style treatment of cancer. The PMTDS system includes three parts: a personalized medicine knowledgebase for each cancer type, a genetic interaction network-based algorithm and a single patient molecular profiles. The knowledgebase integrates cancer drugs, drug-target databases and gene biological pathway networks. The molecular profiles of each tumor consists of DNA copy number alteration, gene mutation, and tumor gene expression variation compared to its adjacent normal tissue.ResultsThe novel integrated PMTDS system is applied to select candidate target-drug pairs for 178 TCGA pancreatic adenocarcinoma (PDAC) tumors. The experiment results show known drug targets (EGFR, IGF1R, ERBB2, NR1I2 and AKR1B1) of PDAC treatment are identified, which provides important evidence of the PMTDS algorithm’s accuracy. Other potential targets PTK6, ATF, SYK are, also, recommended for PDAC. Further validation is provided by comparison of selected targets with, both, cell line molecular profiles from the Cancer Cell Line Encyclopedia (CCLE) and drug response data from the Cancer Therapeutics Response Portal (CTRP). Results from experimental analysis of forty six individual pancreatic cancer samples show that drugs selected by PMTDS have more sample-specific efficacy than the current clinical PDAC therapies.ConclusionsA novelty target and drug priority algorithm PMTDS is developed to identify optimum target-drug pairs by integrating the knowledgebase base with a single patient’s genomics. The PMTDS system provides an accurate and reliable source for target and off-label drug selection for precision cancer medicine.

Abstract 2951: DUSP1 is a novel target for enhancing pancreatic cancer cell sensitivity to gemcitabine.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnPancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with a poor prognosis that is characterized by marked chemoresistance to a broad spectrum of chemotherapeutic drugs, as a consequence of intrinsic cell-autonomous factors, and extrinsic stroma-associated factors. Dual specificity protein phosphatase 1 (DUSP1) has been reported to be overexpressed in pancreatic cancer cells in PDAC. Moreover, DUSP1 was shown to be essential for efficient in vitro colony formation and in vivo tumorigenicity, indicating that DUSP1 may contribute to the biological aggressiveness of PDAC. However, it is not known whether DUSP1 overexpression contributes to PDAC chemoresistance. Using BxPC3 and COLO-357 human pancreatic cancer cells, we show that gemcitabine activated c-JUN N-terminal kinase (JNK) and p38 mitogen activated protein kinase (p38 MAPK), key kinases in two major stress-activated signaling pathways. Activation of JNK and p38 MAPK was associated with increased apoptosis, as evidenced by increased cleavage of PARP and Caspase 3. However, gemcitabine also enhanced DUSP1 transcription, as demonstrated by increased DUSP1 mRNA levels, and increased RNA polymerase II loading at both DUSP1 promoter and gene body. Moreover, shRNA-mediated inhibition of DUSP1 enhanced JNK and p38 MAPK activation and sensitized both cell lines to gemcitabine. Taken together, these results suggest that gemcitabine-mediated upregulation of DUSP1 contributes to a negative feedback loop that attenuates its beneficial actions on stress pathways and apoptosis, raising the possibility that targeting DUSP1 in PDAC may have the dual advantage of suppressing proliferation while enhancing gemcitabine chemosensitivity.nnCitation Format: Fang Liu, Murray Korc. DUSP1 is a novel target for enhancing pancreatic cancer cell sensitivity to gemcitabine. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2951. doi:10.1158/1538-7445.AM2013-2951

Abstract 4433: RB mediates TGF-β effects on growth inhibition and invasion in pancreatic cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnPancreatic ductal adenocarcinoma (PDAC) is a deadly malignancy that arises from precursor lesions, termed pancreatic intraepithelial neoplasia (PanIN). PDACs aggressiveness derives from its advanced stage when patients present clinically, the high frequency of driver mutations (KRAS, INK4A, TP53 and SMAD4) and the heterogeneous distribution of low frequency driver mutations. To study the potential role of RB in PDAC pathobiology, we generated a novel mouse model with pancreatic RB deletion and oncogenic Kras expression (Rb/K mice). These mice rapidly developed highly proliferative PanIN that progress to PDAC with high frequency, underscoring RBs tumor suppressive role in the pancreas. RB deletion alone, however, did not alter pancreatic histology. Previously, we have demonstrated that Smad7-induced loss of TGF-β-mediated growth inhibition is due to functional inactivation of RB. To further examine RBs role in TGF-β action, we established pancreatic cancer cell lines from the Rb/K model. These cells were resistant to TGF-β1-mediated growth inhibition, whereas growth of murine cell lines that retained RB was markedly inhibited. RB loss also abolished TGF-β-induced cell invasion, but did not alter Smad-dependent TGF-β1 signaling since TGF-β1 induced Smad2 phosphorylation, Smad3/4 nuclear translocation, and transactivation of TGF-β1-dependent reporters. By contrast, RB loss did not affect TGF-β1-induced epithelial-to-mesenchymal transition. Array analysis revealed marked alterations in the gene expression profile of Rb/K cells. These data suggest that RB loss contributes to the dysregulation of TGF-β actions in PDAC and suggest that delineation of these perturbations may yield novel therapeutic approaches in this malignancy.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4433. doi:1538-7445.AM2012-4433

Abstract 3055: PD-0332991, a selective cyclin-dependent-kinase 4/6 Inhibitor, upregulates multiple genes promoting invasion and metastasis in pancreatic ductal adenocarcinoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, ILnnDeregulation of the Cyclin D-CDK4/6-Rb-E2F signaling pathway is among the most commonly found aberrations in human pancreatic ductal adenocarcinoma (PDAC). PD-0332991 is an orally available, highly specific and reversible inhibitor for Cdk4 and Cdk6, and demonstrates in vitro and in vivo growth inhibitory effects in multiple tumor types. However, the potential usefulness of PD-0332991 in PDAC is currently unknown. In this study, we examined the effects of PD-0332991 on multiple human pancreatic cancer cell lines, and we found that this agent inhibited Rb phosphorylation and E2F target gene expression, and induced G0/G1 cell cycle arrest. However, the growth inhibitory effect was only transient, due to the rapidly emerging compensatory changes of the G1/S machinery components, including upregulation of CyclinD1, Cdk4, and Cdk6, and downregulation of Rb, p107, and p130. Microarray analysis revealed that incubation with PD-0332991 upregulated multiple genes which promote pancreatic cancer invasion, metastasis, angiogenesis, and chemoresistance, including LAMC2, CYR61, SERPINE1, F3, ABCA1, ASNS, and DUSP1. In addition, using matrigel-coated transwell invasion chambers, we demonstrated that incubation with PD-0332991 enhanced the invasiveness of human pancreatic cancer cells. These observations suggest that PD-0332991 may have the potential to cause deleterious effects in PDAC by modulating gene expression in a manner that could promote cancer spread, metastasis, and chemoresistance.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3055. doi:1538-7445.AM2012-3055