ABSTRACT
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.
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.