Abstract
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.
Highlights
Ribonucleotide reductase (RR) is a rate-limiting enzyme for cell replication which catalyzes the reduction of ribonucleotides to deoxyribonucleotides during DNA synthesis
Accumulating evidence suggests that RR acts as a positive determinant for tumor cell proliferation and metastasis as well as the development of chemoresistance to nucleoside analogs used for treating pancreatic cancer [2,3,4,5,6]
Comparable ribonucleotide reductase subunit M2 (RRM2) expressions were identified between most gemcitabine-sensitive cell lines (L3.6pl and Capan-1) and human pancreatic ductal epithelial (HPDE) (Fig. 1A–B)
Summary
Ribonucleotide reductase (RR) is a rate-limiting enzyme for cell replication which catalyzes the reduction of ribonucleotides to deoxyribonucleotides during DNA synthesis. It is overexpressed in a number of solid tumors including pancreatic [1]. Accumulating evidence suggests that RR acts as a positive determinant for tumor cell proliferation and metastasis as well as the development of chemoresistance to nucleoside analogs used for treating pancreatic cancer (e.g., gemcitabine, capecitabine, 5-fluorouracil) [2,3,4,5,6]. RRM2 expression has been shown to be induced in chemoresistant cells by gene amplification, transcriptional activation, and perhaps other unidentified mechanisms [5,9]. Recent studies have shown that exogenous manipulations of RRM2 expression by siRNA or antisense oligonucleotides improve chemosensitivity in pancreatic cancer [10,11]
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