Pancreatic ductal adenocarcinoma (PDA) is expected to become the 2nd leading cause of cancer-related mortality in the United States by 2020. One of the reasons PDA remains one of the most lethal malignancies is due to drug resistance mechanisms to both conventional and targeted chemotherapeutic regimens. Previously, it has been shown that PDA tumors exhibit high levels of hypoxia, characterized by low oxygen pressure (pO2) and decreased O2 intracellular perfusion. Importantly, chronic hypoxia is strongly associated with resistance to cytotoxic chemotherapy and chemoradiation in a widely understudied phenomenon known as hypoxia-induced chemoresistance. Recently, the hypoxia-inducible, serine-threonine, pro-oncogenic kinase PIM1 has emerged as a key regulator of hypoxia-induced chemoresistance in PDA and other cancers. While its role in therapeutic resistance has been previously described, the molecular mechanism(s) behind PIM1 overexpression in PDA is unknown. Here, we demonstrate that cis-acting AU-rich elements (ARE) present in the PIM1 mRNA 3′UTR mediate a regulatory interaction with the mRNA-stability factor HuR in the context of tumor hypoxia. We have previously demonstrated that HuR, a predominantly nuclear protein, translocates to the cytoplasm upon acute cancer-associated stress and selectively stabilizes key survival-associated transcripts. Here, we demonstrate by immunofluorescence and western blot analyses of fractionated lysates that PDA cells in hypoxic conditions mobilize HuR to the cytoplasm, where it binds and stabilizes the PIM1 transcript. The functional consequence of this interaction is observed with a 5-fold increase in PIM1 protein expression (p≤0.001). PIM1 imparts its oncogenic role by phosphorylating and inactivating key apoptotic proteins (e.g., BAD) and proteins involved in the DNA repair pathway. In this regard, siRNA-mediated knockdown of HuR abrogates hypoxia-induced PIM1 expression and its downstream phosphorylation of the pro-apoptotic protein BAD. As a result, cells undergo growth arrest and succumb to apoptosis. Most importantly, we demonstrate that HuR's regulation of PIM1 modulates a potent chemoresistance phenotype. HuR-mediated stabilization of PIM1 promoted dramatic resistance to 5-Fluorouracil (5-FU) and Oxaliplatin, two critical components of the promising combination therapy FOLFIRINOX. We employed confocal microscopy to analyze DNA damage foci formation by tracking and quantifying γH2AX foci in response to 5-FU and oxaliplatin. As a result of PIM1 up-regulation in hypoxia, or forced PIM1 overexpression in normoxia, PDA cells exhibit fewer DNA damage foci (p≤0.01) due to PIM1's ability to regulate DNA repair. From a translational standpoint, we demonstrate that disruption of the HuR:PIM1 axis by HuR knockdown results in PDA cell growth inhibition and enhances sensitivity to cytotoxic chemotherapeutic agents (5-FU and oxaliplatin). Similarly, pharmacological inhibition of HuR by MS-444 (Novartis), a small molecule that inhibits HuR homodimerization and its cytoplasmic translocation, abrogates hypoxia-induced PIM1 overexpression and dramatically enhances PDA cell sensitivity to oxaliplatin and 5-FU under physiologic low oxygen conditions. Taken together, these results support the notion that HuR has pro-oncogenic properties in PDA cells by enabling them with selective growth advantages in stressful tumor microenvironment niches. Accordingly, our studies provide evidence that therapeutic disruption of HuR's regulation of PIM1 may be a key strategy in breaking an elusive chemotherapeutic resistance mechanism acquired by PDA cells that reside in a hypoxic tumor microenvironment.
Citation Format: Fernando F. Blanco, Nicole C. Meisner-Kober, Eric Londin, Isidore Rigoutsos, Makarand V. Risbud, Charles J. Yeo, Jordan M. Winter, Jonathan R. Brody. The mRNA-binding protein HuR promotes hypoxia-induced chemoresistance through post-transcriptional regulation of the serine-threonine kinase PIM1. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr A05.