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 3 H-gemcitabine transport was lower in pancreatic cancer cell lines and correlated with cytotoxic IC 50 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); 1825-35. Ó2011 AACR.

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Overexpression of ribonucleotide reductase subunit M2 (RRM2), involved in deoxyribonucleotide synthesis, drives the chemoresistance of pancreatic cancer to nucleoside analogs (e.g., gemcitabine). While silencing RRM2 by synthetic means has shown promise in reducing chemoresistance, targeting endogenous molecules, especially microRNAs (miRNAs), to advance chemotherapeutic outcomes has been poorly explored. Based on computational predictions, we hypothesized that the let-7 tumor suppressor miRNAs will inhibit RRM2-mediated gemcitabine chemoresistance in pancreatic cancer. Reduced expression of the majority of let-7 miRNAs with an inverse relationship to RRM2 expression was identified in innately gemcitabine-resistant pancreatic cancer cell lines. Direct binding of let-7 miRNAs to the 3′ UTR of RRM2 transcripts identified post-transcriptional regulation of RRM2 influencing gemcitabine chemosensitivity. Intriguingly, overexpression of human precursor-let-7 miRNAs led to differential RRM2 expression and chemosensitivity responses in a poorly differentiated pancreatic cancer cell line, MIA PaCa-2. Defective processing of let-7a precursors to mature forms, in part, explained the discrepancies observed with let-7a expressional outcomes. Consistently, the ratios of mature to precursor let-7a were progressively reduced in gemcitabine-sensitive L3.6pl and Capan-1 cell lines induced to acquire gemcitabine resistance. Besides known regulators of let-7 biogenesis (e.g., LIN-28), short hairpin RNA library screening identified several novel RNA binding proteins, including the SET oncoprotein, to differentially impact let-7 biogenesis and chemosensitivity in gemcitabine-sensitive versus -resistant pancreatic cancer cells. Further, LIN-28 and SET knockdown in the cells led to profound reductions in cellular proliferation and colony-formation capacities. Finally, defective processing of let-7a precursors with a positive correlation to RRM2 overexpression was identified in patient-derived pancreatic ductal adenocarcinoma (PDAC) tissues. These data demonstrate an intricate post-transcriptional regulation of RRM2 and chemosensitivity by let-7a and that the manipulation of regulatory proteins involved in let-7a transcription/processing may provide a mechanism for improving chemotherapeutic and/or tumor growth control responses in pancreatic cancer.

Identification of epigenetic reversal agents for use in combination chemotherapies to treat human pancreatic ductal adenocarcinomas (PDAC) remains an unmet clinical need. Pharmacological inhibitors of Enhancer of Zeste Homolog 2 (EZH2) are emerging as potential histone methylation reversal agents for the treatment of various solid tumors and leukemia; however, the surprisingly small set of mRNA targets identified with EZH2 knockdown suggests novel mechanisms contribute to their anti-tumorigenic effects. Here, 3-deazaneplanocin-A (DZNep), an inhibitor of S-adenosyl-L-homocysteine hydrolase and EZH2 histone lysine-N-methyltransferase, significantly reprograms noncoding miRNA (miR) expression and dampens TGFβ1-induced epithelial-to-mesenchymal (EMT) signals in pancreatic cancer. In particular, miR-663a and miR-4787-5p were identified as PDAC-downregulated miRs that were reactivated by DZNep to directly target TGFβ1 for RNA interference. Lentiviral overexpression of miR-663a and miR-4787-5p reduced TGFβ1 synthesis and secretion in PDAC cells and partially phenocopied DZNep’s EMT-resisting effects, whereas locked nucleic acid (LNA) antagomiRs counteracted them. DZNep, miR-663a, and miR-4787-5p reduced tumor burden in vivo and metastases in an orthotopic mouse pancreatic tumor model. Taken together, these findings suggest the epigenetic reprogramming of miRs by synthetic histone methylation reversal agents as a viable approach to attenuate TGFβ1-induced EMT features in human PDAC and uncover putative miR targets involved in the process.

Previous studies in our laboratory identified that 3-deazaneplanocin A (DZNep), a carbocyclic adenosine analog and histone methyl transferase inhibitor, suppresses TGFβ-induced epithelial-to-mesenchymal (EMT) characteristics. In addition, DZNep epigenetically reprograms miRNAs (miRs) to regulate endogenous TGFβ1 levels via miR-663/4787 mediated RNA interference (Mol Cancer Res. 2016 Sep 13. pii: molcanres.0083.2016) (1). While DZNep also attenuates exogenous TGFβ-induced EMT response, the mechanism of this inhibition was unclear. Here, DZNep induced miR-202-5p to target both TGFβ receptors, TGFBR1 and TGFBR2, for RNA interference and thereby contribute to the suppression of exogenous TGFβ-induced EMT in pancreatic cancer cells. Lentiviral overexpression of miR-202 significantly reduced the protein levels of both TGFβ receptors and suppressed TGFβ signaling and EMT phenotypic characteristics of cultured parenchymal pancreatic cancer cells. Consistently, transfection of anti-miRs against miR-202-5p resulted in increased TGFBR1 and TGFBR2 protein expressions and induced EMT characteristics in these cells. In stellate pancreatic cells, miR-202 overexpression slowed growth as well as reduced stromal extracellular membrane matrix (ECM) protein expression. In orthotopic pancreatic cancer mouse models, both immunodeficient and immunocompetent, miR-202 reduced tumor burden and metastasis. Together, these findings demonstrate an alternative mechanism of DZNep in suppressing TGFβ signaling at the receptor level and uncover the EMT suppressing role of miR-202 in pancreatic cancer. Implications These findings support the possibility of combining small molecule- (e.g., DZNep analogs) or large molecule- (e.g., miRs) based epigenetic modifiers with conventional nucleoside analogs (e.g., gemcitabine, capecitabine) to improve the anti-metastatic potential of current pancreatic cancer therapy.

Nucleoside analogs are used as chemotherapeutic options for the treatment of platinum-resistant ovarian cancers. Human concentrative nucleoside transporter 1 (hCNT1) is implicated in sensitizing solid tumors to nucleoside analogs although its role in determining drug efficacy in ovarian cancers remains unclear. Here we examined the functional expression of hCNT1 and compared its contributions towards gemcitabine efficacy in histological subtypes of ovarian cancer. Radioactivity analysis identified hCNT1-mediated 3H-gemcitabine transport in ovarian cancer cells to be significantly reduced compared with that of normal ovarian surface epithelial cells. Biochemical and immunocytochemical analysis identified that unlike normal ovarian cells which expressed high levels of hCNT1 at the apical cell surface, the transporter was either diminished in expression and/or mislocalized in cell lines of various subtypes of ovarian cancer. Retroviral expression of hCNT1 selectively rescued gemcitabine transport in cell lines representing serous, teratocarcinoma, and endometrioid subtypes, but not clear cell carcinoma (CCC). In addition, exogenous hCNT1 predominantly accumulated in intracytoplasmic vesicles in CCC suggesting defective cellular trafficking of hCNT1 as a contributing factor to transport deficiency. Despite diminution of hCNT1 transport in the majority of ovarian cancers and apparent trafficking defects with CCC, the chemotherapeutic efficacy of gemcitabine was broadly enhanced in all subtypes when delivered via engineered nanoparticles (NPs). Additionally, by bypassing the transport requirement, the delivery of a gemcitabine-cisplatin combination in NP formulation increased their synergistic interactions. These findings uncover hCNT1 as a putative determinant for nucleoside analog chemoresistance in ovarian cancer and may help rationalize drug selection and delivery strategies for various histological subtypes of ovarian cancer.

Pancreatic cancer has a devastating prognosis due to 80-90% of diagnostic cases occurring when metastasis has already presented. Activation of the epithelial-mesenchymal transition (EMT) is a prerequisite for metastasis because it allows for the dissemination of tumor cells to blood stream and secondary organs. Here, we sought to determine the role of SET oncoprotein, an endogenous inhibitor of PP2A, in EMT and pancreatic tumor progression. Among the two major isoforms of SET (isoform 1 and isoform 2), higher protein levels of SET isoform 2 were identified in aggressive pancreatic cancer cell lines. Overexpressing SET isoform 2, and to a lesser extent SET isoform 1, in epithelial cell lines promoted EMT-like features by inducing mesenchymal characteristics and promoting cellular proliferation, migration, invasion, and colony formation. Consistently, knockdown of SET isoforms in the mesenchymal cell line partially resisted these characteristics and promoted epithelial features. SET-induced EMT was likely facilitated by increased N-cadherin overexpression, decreased PP2A activity and/or increased expression of key EMT-driving transcription factors. Additionally, SET overexpression activated the Rac1/JNK/c-Jun signaling pathway that induced transcriptional activation of N-cadherin expression. In vivo, SET isoform 2 overexpression significantly correlated with increased N-cadherin in human PDAC and to tumor burden and metastatic ability in an orthotopic mouse tumor model. These findings identify a new role for SET in cancer and have implications for the design and targeting of SET for intervening pancreatic tumor progression.

We evaluated the potential of an investigational histone methylation reversal agent, 3-deazaneplanocin A (DZNep), in improving the chemosensitivity of pancreatic cancer to nucleoside analogs (i.e., gemcitabine). DZNep brought delayed but selective cytotoxicity to pancreatic cancer cells without affecting normal human pancreatic ductal epithelial (HPDE) cells. Co-exposure of DZNep and gemcitabine induced cytotoxic additivity or synergism in both well- and poorly-differentiated pancreatic cell lines by increased apoptosis. In contrast, DZNep exerted antagonism with gemcitabine against HPDE cells with significant reduction in cytotoxicity compared with the gemcitabine-alone regimen. DZNep marginally depended on purine nucleoside transporters for its cytotoxicity, but the transport dependence was circumvented by acyl derivatization. Drug exposure studies revealed that a short priming with DZNep followed by gemcitabine treatment rather than co-treatment of both agents to produce a maximal chemosensitization response in both gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cells. DZNep rapidly and reversibly decreased trimethylation of histone H3 lysine 27 but increased trimethylation of lysine 9 in an EZH2- and JMJD1A/2C-dependent manner, respectively. However, DZNep potentiation of nucleoside analog chemosensitization was found to be temporally coupled to trimethylation changes in lysine 27 and not lysine 9. Polymeric nanoparticles engineered to chronologically release DZNep followed by gemcitabine produced pronounced chemosensitization and dose-lowering effects. Together, our results identify that an optimized DZNep exposure can presensitize pancreatic cancer cells to anticancer nucleoside analogs through the reversal of histone methylation, emphasizing the promising clinical utilities of epigenetic reversal agents in future pancreatic cancer combination therapies.