MicroRNA-587 antagonizes 5-FU-induced apoptosis and confers drug resistance by regulating PPP2R1B expression in colorectal cancer

Drug resistance is one of the major hurdles for cancer treatment. However, the underlying mechanisms are still largely unknown and therapeutic options remain limited. In this study, we show that microRNA (miR)-587 confers resistance to 5-fluorouracil (5-FU)-induced apoptosis in vitro and reduces the potency of 5-FU in the inhibition of tumor growth in a mouse xenograft model in vivo. Further studies indicate that miR-587 modulates drug resistance through downregulation of expression of PPP2R1B, a regulatory subunit of the PP2A complex, which negatively regulates AKT activation. Knockdown of PPP2R1B expression increases AKT phosphorylation, which leads to elevated XIAP expression and enhanced 5-FU resistance; whereas rescue of PPP2R1B expression in miR-587-expressing cells decreases AKT phosphorylation/XIAP expression, re-sensitizing colon cancer cells to 5-FU-induced apoptosis. Moreover, a specific and potent AKT inhibitor, MK2206, reverses miR-587-conferred 5-FU resistance. Importantly, studies of colorectal cancer specimens indicate that the expression of miR-587 and PPP2R1B positively and inversely correlates with chemoresistance, respectively, in colorectal cancer. These findings indicate that the miR-587/PPP2R1B/pAKT/XIAP signaling axis has an important role in mediating response to chemotherapy in colorectal cancer. A major implication of our study is that inhibition of miR-587 or restoration of PPP2R1B expression may have significant therapeutic potential to overcome drug resistance in colorectal cancer patients and that the combined use of an AKT inhibitor with 5-FU may increase efficacy in colorectal cancer treatment.Colorectal cancer is the third most common cancer and the second leading cause of cancer-related mortality in the US. 5-Fluorouracil (5-FU) is one of the chemotherapeutic drugs most widely used alone or combined with other drugs in colorectal cancer treatment.¹ 5-FU primarily interrupts synthesis of the pyrimidine thymidine, a nucleoside required for DNA replication, by blocking the activity of thymidylate synthase.² Consequently, 5-FU induces cell cycle arrest and/or apoptosis in cancer cells. Although adjuvant 5-FU treatment has yielded a good success rate, the failure of treatment in over 90% of patients with metastatic cancer is due to drug resistance.³ Many mechanisms have been suggested to be responsible for drug resistance, including blocking apoptosis.^{2, 4, 5, 6} Although resistance to chemotherapy is one of the biggest obstacles for effective cancer therapy, no significant advance has been made to identify targets overcoming drug resistance.⁷MicroRNAs (miRNAs) are a class of small (about 22 bps) non-coding regulatory RNA molecules, which regulate gene expression primarily by binding to the 3′-UTRs of their target mRNAs to initiate sequence-specific mRNA cleavage or to inhibit translation.⁸ It is estimated that more than one-third of human genes and the majority of genetic pathways are regulated by miRNAs.⁹ MiRNAs have been virtually linked to all known biological processes as well as various pathological diseases including cancer.¹⁰ Alternations in miRNA expression have been associated with many human cancers.^{11, 12} The pleiotropic nature of gene regulation by miRNAs implies that some miRNAs may function as crucial mediators of drug resistance. In fact, miRNA-based anticancer therapies are being developed, either alone or in combination with targeted therapies, with the goal to improve disease response and increase patient survival.¹³The protein kinase B (AKT/PKB) kinases, including AKT1, AKT2 and AKT3, are essential regulators of various signaling pathways and cellular processes.¹⁴ Hyper-activation of AKT kinases have been frequently observed in human cancers.¹⁵ Activation of AKT requires both translocation to the plasma membrane and phosphorylation at Thr308 and Ser473.^{16, 17} Further studies have demonstrated that Thr308 phosphorylation is necessary and sufficient for AKT activation¹⁸ and that dephosphorylation at Thr308 alone leads to deactivation of AKT.^{19, 20} X-linked inhibitor of apoptosis protein (XIAP) is a member of the inhibitor of apoptosis proteins (IAPs) family and has a significant role in cell survival by modulating death-signaling pathways at a post-mitochondrial level.^{21, 22} Studies have shown that AKT activation can enhance the protein stability of XIAP, therefore elevating XIAP expression.^{23, 24, 25} Consequently, AKT has been shown to promote cell survival through the XIAP-mediated anti-apoptotic pathway.²⁶The serine/threonine protein phosphatase 2 A (PP2A) holoenzyme is composed of a catalytic C subunit, a structural A subunit and a regulatory B subunit. PPP2R1B or PP2A A subunit beta isoform (PP2A-Aβ) is a constant regulatory subunit of PP2A required to activate PP2A. PPP2R1B was initially characterized as a tumor suppressor. It is located at a chromosomal region (11q23) frequently deleted in human cancers.²⁷ Its mutations and alterations have been found in colorectal and other cancers.^{28, 29} Cancer-associated mutants of PPP2R1B have been shown to be incompetent to bind the B and/or C subunits in vitro, resulting in PP2A inactivation.^{30, 31} PP2A regulates numerous signaling pathways. Specifically, PP2A has an important role in regulating AKT activity by dephosphorylating AKT at Thr308 and Ser473, leading to AKT inactivation.^{19, 32, 33, 34}In this study, we have discovered a novel miR-587/PPP2R1B (PP2A)/pAKT/XIAP signaling axis that mediates the response of colon cancer cells to 5-FU treatment. Our results show that miR-587 expression is suppressed by 5-FU treatment in the sensitive but not resistant colon cancer cells. MiR-587 confers resistance to 5-FU-induced apoptosis through the inhibition of PPP2R1B expression, which is a direct target of miR-587. Knockdown of PPP2R1B by siRNAs confers 5-FU resistance in colon cancer cells, mimicking miR-587 effect. Inhibition of miR-587 expression or rescue of PPP2R1B expression in colon cancer cells increases their sensitivity to 5-FU treatment. Additionally, an AKT inhibitor, MK-2206, re-sensitizes miR-587-expressing cells to 5-FU treatment. Moreover, experiments in tumor xenograft mouse models reveal that miR-587 significantly reduces the effectiveness of 5-FU in the inhibition of tumor growth in vivo. Importantly, studies of colorectal cancer specimens indicate a positive correlation between miR-587 expression and chemoresistance and an inverse correlation between PPP2R1B expression and drug resistance. Our studies have identified miR-587 as a potential target for drug resistance in colorectal cancer and suggested that modulating the PPP2R1B (PP2A)/pAKT/XIAP axis may have benefits against drug resistance.