Therapeutics, Targets, and Chemical Biology CNT1 Expression Influences Proliferation and Chemosensitivity in Drug-Resistant Pancreatic Cancer Cells

Overcoming the inherent chemoresistance of pancreatic cancers remains a major goal of therapeutic investigations in this disease. In this study, we discovered a role for the human concentrative nucleoside transporter-1 (hCNT1; SLC28A1), a high-affinity pyrimidine nucleoside transporter, in determining the chemosensitivity of human pancreatic cancer cells to gemcitabine, the drug used presently as a standard of care. Compared with normal pancreas and pancreatic ductal epithelial cells, hCNT1 expression was frequently reduced in pancreatic tumors and tumor cell lines. In addition, hCNT1-mediated H-gemcitabine transport was lower in pancreatic cancer cell lines and correlated with cytotoxic IC50 estimations of gemcitabine. In contrast to gemcitabine-sensitive pancreatic cancer cell lines, MIA PaCa-2, a gemcitabine-resistant pancreatic cancer cell line, exhibited relatively restrictive, cell cycle-dependent hCNT1 expression and transport. hCNT1 translation was suppressed in the late G1-enriched MIA PaCa-2 cell population possibly in an miRNA-dependent manner, which corresponded with the lowest hCNT1-mediated gemcitabine transport during this phase. Although hCNT1 protein was induced during G1/S transition, increased hCNT1 trafficking resulted in maximal cell surface recruitment and transport-overshoot in the G2/M phase-enriched cell population. hCNT1 protein was directed predominantly to proteasomal or lysosomal degradation in S or G2/M phase MIA PaCa-2 cells, respectively. Pharmacological inhibition of hCNT1 degradation moderately increased cell surface hCNT1 expression and cellular gemcitabine transport in MIA PaCa-2 cells. Constitutive hCNT1 expression reduced clonogenic survival of MIA PaCa-2 cells and steeply augmented gemcitabine transport and chemosensitization. In addition to supporting a putative tumor suppressor role for hCNT1, our findings identify hCNT1 as a potential candidate to render drug-resistant pancreatic cancer cells amenable to chemotherapy. Cancer Res; 71(5); 1–11. 2011 AACR. Introduction 20,20-Difluoro-20-deoxycytidine (dFdC; gemcitabine) is currently used as a first-line treatment against locally advanced and metastatic adenocarcinoma of the pancreas (1, 2). Gemcitabine is phosphorylated intracellularly to its active diphosphate (dFdC-DP) and triphosphate (dFdC-TP) forms that inhibit DNA and RNA replication (3, 4, 5). In order for hydrophilic gemcitabine (log P 1⁄4 1.1 to -2.0) to enter cells, membrane-bound nucleoside transporters are prerequisites (as cells deficient in nucleoside transporter are resistant to gemcitabine cytotoxicity (6)). Studies to date suggest 2 transporters from each of the human concentrative nucleoside transporter family (hCNT1, hCNT3) and the equilibrative nucleoside transporter family (hENT1, hENT2) are capable of translocating gemcitabine across the cell surface (3, 6, 7– 10). Despite the expression of one or more of the aforementioned transporters in most cells types, hENT1 is generally considered to be predominantly involved in gemcitabine transport in tumors as its expression correlates with cellular proliferation (8, 11). Clinical evidence also shows pancreatic tumor cells with high hENT1 expression exhibiting increased gemcitabine chemosensitivity (6, 12–15). Unlike hENT1, the roles of CNTs, especially hCNT1, in governing gemcitabine cytotoxicity in tumors are not well understood. hCNT1 is normally expressed at the apical surfaces of well-differentiated epithelial tissues such as intestinal villi and hepatocytes (16, 17). hCNT1mRNA and/or protein are also detected in the pancreas, kidneys, skeletal muscles, uterus corpus, salivary glands, and placenta (16, 18). Consistent with hCNT1 expression in differentiated epithelial tissues, the rat ortholog of hCNT1, rCNT1, is regulated by differentiationpromoting agents (19–23). Although the degree of tumor cell differentiation often correlates with the clinical response to chemotherapy, it is not clear whether the expression of hCNT1 itself is altered in tumors, and if so, whether such alterations influence tumor cell proliferation and drug sensitization. Earlier studies detected hCNT1 expression in solid (e.g., breast) tumors, predominantly localized in the cytoplasm and nucleus (24). Evidence for the reduction or complete loss Authors' Affiliations: Departments of Phamaceutical and Biomedical Sciences, Biology, and Biochemistry, University of Georgia, Athens, Georgia Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org). Corresponding Author: Rajgopal Govindarajan, 234A, Wilson Pharmacy, UGA, Athens, GA 30602. Phone: 706-542-5759; Fax: 706-542-5358; E-mail: [email protected] doi: 10.1158/0008-5472.CAN-10-2736 2011 American Association for Cancer Research. Cancer Research www.aacrjournals.org OF1 Research. on April 13, 2017. © 2011 American Association for Cancer cancerres.aacrjournals.org Downloaded from Published OnlineFirst February 22, 2011; DOI: 10.1158/0008-5472.CAN-10-2736