Gemcitabine-resistant MIA PaCa-2 Research Articles

Abstract 3021: The role of survivin splice variants in pancreatic ductal adenocarcinoma Gemcitabine resistance

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is a highly lethal disease with poor prognosis and an increased chemoresistance. Dysfunction in alternative splicing has been associated with chemoresistance in many cancers. Alternative splicing of survivin mRNA generates six splicing isoforms some of which are associated with cancer progression and chemoresistance. Survivin is a member of the inhibitors of apoptosis (IAPs) family and a cell cycle-associated oncoprotein overexpressed in almost all cancers. Interestingly, some of these survivin splice variants are proapoptotic (2α and 2β) while others are antiapoptotic (survivin, Δex3, 3α and 3β). It has been reported that increased levels of survivin 2β and 3β play a role in chemoresistance in ovarian cancers and other tumors, but this has not been studied in pancreatic cancer. Our recent RT-qPCR data also show an overexpression of all survivin splice variants in chemoresistant PDAC cell lines compared to the chemo-sensitive cell line. We, therefore, hypothesize that increased expression of survivin splice variants 2β and 3β contribute to chemo-resistance in PDAC. To test this hypothesis, we repeatedly selected the resistant cells from the normally chemosensitive MiaPaca2 cell line after exposure to gemcitabine. We then assessed the levels of survivin splice variants in the new cell line (gemcitabine-resistant MiaPaca2). Using the Alamar blue assay, we confirmed that Panc1 and gemcitabine-resistant MiaPaca2 are more resistant to gemcitabine compared to MiaPaca2 cells. We also observed a statistically significant overexpression of survivin 2β and 3β in gemcitabine-resistant cell lines compared to the more sensitive MiaPaca2 cells. To further test our hypothesis, we will knockdown survivin 2β and 3β in Panc1 and gemcitabine-resistant MiaPaca2, and/or overexpress these splice variants in MiaPaca2 cells then assess response to gemcitabine. The outcomes of this study might create new biomarkers for predicting the clinical outcomes of gemcitabine treatment and generate new therapeutic targets for pancreatic cancer. Note: This abstract was not presented at the meeting. Citation Format: Janviere Kabagwira, Amber Gonda, Mei Li Kwong. The role of survivin splice variants in pancreatic ductal adenocarcinoma Gemcitabine resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3021.

Abstract 2114: Glycosyltransferase ST6Gal-I protects against chemotherapy induced DNA damage and subsequent apoptosis in pancreatic adenocarcinoma cells

Abstract ST6Gal-I is a sialyltransferase that adds α2-6 linked sialic acids to cell surface proteins as they pass through the trans-Golgi. Sialic acids, being negatively charged, are able to alter the function of selected cell surface receptors, which leads to dysregulation of various downstream cellular pathways. Overexpression of ST6Gal-I has been observed in various cancers including ovarian and pancreatic cancer. We have previously shown that knockdown of ST6Gal-I expression increases ovarian cancer cell susceptibility to the chemotherapeutic drug cisplatin. In the present study we further investigate whether resistance to additional drugs such as gemcitabine, the front line treatment for pancreatic cancer, is affected by ST6Gal-I activity. MiaPaCa-2 and BxPC3 pancreatic cancer cell lines, having high ST6Gal-I expression, were employed for our studies. To elucidate the mechanistic role of ST6Gal-I in gemcitabine resistance we created stable ST6Gal-I knockdown lines of MiaPaCa-2 and BxPC3 cells. Gemcitabine induced cell death was more pronounced in the knockdown cell lines, indicated by heightened activation of caspase-3. Gemcitabine is metabolized to a nucleoside analogue, gemcitabine triphosphate, which induces apoptosis by promoting DNA damage. Increased single stand DNA damage in the knockdown cells was confirmed using alkaline comet assay. Gemcitabine treatment led to greater activation of DNA damage markers and response elements (γH2AX, phospho CHK1, phospho CHK2), along with increased levels of cleaved caspase-3 in the knockdown cells as compared to empty vector control cells. We next developed a stable gemcitabine resistant MiaPaCa-2 cell line by growing parental cells in gemcitabine. We selected for the population of cells that survived and were able to replicate in gemcitabine containing media. ST6Gal-I levels were found to be increased in the stable gemcitabine resistant lines relative to parental cell lines, suggesting that cells with high ST6Gal-I expression selectively survive gemcitabine treatment. By measuring the levels of genes involved in activation over those responsible for inactivation of gemcitabine, we were able to obtain a gemcitabine sensitivity predictive ratio for MiaPaCa-2 and BxPC3 ST6Gal-I knockdown, empty vector, parental and stable gemcitabine resistant cell lines. This ratio has been described previously in literature as a metric for gauging gemcitabine resistance. According to the ratios obtained, ST6Gal-I knockdown enhanced drug sensitivity whereas high expression of ST6Gal-I offered protection against gemcitabine induced apoptosis. Collectively these data indicate that upregulation of ST6Gal-I imparts tumor cell survival through prevention of gemcitabine induced DNA damage. Citation Format: Asmi Chakraborty, Matthew Schultz, Hoa Q. Trummell, James A. Bonner, Susan Bellis. Glycosyltransferase ST6Gal-I protects against chemotherapy induced DNA damage and subsequent apoptosis in pancreatic adenocarcinoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2114.

MiRNA profiling in pancreatic cancer and restoration of chemosensitivity

Pancreatic cancers relapse due to small but distinct population of cancer stem cells (CSCs) which are in turn regulated by miRNAs. The present study identifies a series of miRNAs which were either upregulated (e.g. miR-146) or downregulated (e.g. miRNA-205, miRNA-7) in gemcitabine resistant MIA PaCa-2 cancer cells and clinical metastatic pancreatic cancer tissues. Gemcitabine resistant MIA PaCa-2 cells possessed distinct ALDH-positive CSC fraction expressing stem cell markers OCT3/4 and CD44 and chemoresistance marker class IIIβ-tubulin (TUBB3) which decreases on transfection with miR-205 resulting in the restoration of chemosensitivity to gemcitabine.

Abstract 3549: Synthetic triterpenoid CDDO-Me induces apoptosis in pancreatic cancer cells by inducing oxidative stress

Abstract Pancreatic ductal adenocarcinoma (PDA) is a highly lethal human malignancy with dismal prognosis and few effective therapeutic options. Novel agents that are safe and effective are urgently needed. Oleanolic acid-derived synthetic triterpenoids are potent antitumorigenic agents, but their efficacy for pancreatic cancer has not been investigated. We have found that methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me), a oleanane-derived synthetic triterpenoid exhibits potent anticancer activity against human pancreatic cancer cell lines. CDDO-Me inhibited the growth of both K-ras mutated (MiaPaca2, Panc1 and Capan2) and wild-type K-ras (BxPC3) pancreatic cancer cells at very low concentrations. The growth inhibitory activity of CDDO-Me was due to the induction of apoptosis characterized by increased annexin-V-FITC and cleavage of PARP-1 and procaspases-3, -8 and-9. Killing of PDA cells by CCDO-Me involved the generation of reactive oxygen species (ROS) as determined from the oxidation of H2DCFD by flow cytometry and its inhibition by general purpose antioxidant NAC. CDDO-Me was also highly active against gemcitabine resistant MiaPaCa2 cells expressing markers of tumor-initiating cells (cancer stem-like cells?). An ongoing pilot experiment has shown the antitumor activity of CDDO-Me against BxPC3 xenograft in vivo. Together, these data demonstrated potential of CDDO-Me for treating pancreatic cancer. 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 3549. doi:10.1158/1538-7445.AM2011-3549