The mRNA-binding protein HuR promotes hypoxia-induced chemoresistance through posttranscriptional regulation of the proto-oncogene PIM1 in pancreatic cancer cells

Abstract

Previously, it has been shown that pancreatic ductal adenocarcinoma (PDA) tumors exhibit high levels of hypoxia, characterized by low oxygen pressure (pO2) and decreased O2 intracellular perfusion. Chronic hypoxia is strongly associated with resistance to cytotoxic chemotherapy and chemoradiation in an understudied phenomenon known as hypoxia-induced chemoresistance. The hypoxia-inducible, pro-oncogenic, serine-threonine kinase PIM1 (Proviral Integration site for Moloney murine leukemia virus 1) has emerged as a key regulator of hypoxia-induced chemoresistance in PDA and other cancers. Although its role in therapeutic resistance has been described previously, the molecular mechanism behind PIM1 overexpression in PDA is unknown. Here, we demonstrate that cis-acting AU-rich elements (ARE) present within a 38-base pair region of the PIM1 mRNA 3'-untranslated region mediate a regulatory interaction with the mRNA stability factor HuR (Hu antigen R) in the context of tumor hypoxia. Predominantly expressed in the nucleus in PDA cells, HuR translocates to the cytoplasm in response to hypoxic stress and stabilizes the PIM1 mRNA transcript, resulting in PIM1 protein overexpression. A reverse-phase protein array revealed that HuR-mediated regulation of PIM1 protects cells from hypoxic stress through phosphorylation and inactivation of the apoptotic effector BAD and activation of MEK1/2. Importantly, pharmacological inhibition of HuR by MS-444 inhibits HuR homodimerization and its cytoplasmic translocation, abrogates hypoxia-induced PIM1 overexpression and markedly enhances PDA cell sensitivity to oxaliplatin and 5-fluorouracil under physiologic low oxygen conditions. Taken together, these results support the notion that HuR has prosurvival properties in PDA cells by enabling them with growth advantages in stressful tumor microenvironment niches. Accordingly, these 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 hypoxic PDA microenvironments.

HuR Contributes to TRAIL Resistance by Restricting Death Receptor 4 Expression in Pancreatic Cancer Cells

“…Various cancer-associated stressors can induce this HuR cytoplasmic translocation including hypoxia (29), glucose deprivation (18), DNA damaging agents (33), anti-DR5 mAb (6) and gemcitabine (34). To determine whether TRAIL treatment of PDA cells can induce HuR translocation from the nucleus to the cytoplasm, we performed immunoblot analysis of cytoplasmic extracts from PDA cells treated with increasing doses of sTRAIL and discovered a more than 2-fold increase in cytoplasmic HuR levels in BxPc-3 cells treated for 3 h at 40 ng/ml of sTRAIL, compared to untreated samples (Fig.…”

“…To authenticate a direct association between DR4 mRNA and HuR, and to localize the site of interaction, constructs containing the 5′- or 3′-UTR of DR4 subcloned immediately upstream or downstream, respectively, of the Renilla reporter gene of psiCHECK2 vector. These reporter constructs were then transfected into Panc-1 cells, as previously described (6, 29, 33). We determined by RT-qPCR that the total input RNA for the RNP-IP contained equivalent DR4 and PIM1 sequences (Supplemental Fig.…”

“…Plasmids used for determining HuR binding were constructed by generating PCR fragments corresponding to either the 5′UTR (forward: 5′-CGTACCTCGAGCACTCCGAATGCGAAGTTCTG -3′; reverse: 5′-GTCTAGCGGCCGCACCTGCCAGGTCAATCCAAGAAGCAG -3′) or 3′UTR (forward: 5′-CGTACCTCGAGAGACTCTTTTTACCAGAGGTTTCCT -3′; reverse: 5′-GTCTAGCGGCCGCAACCAAATCTTTGCATAGGTACCAA -3′) of DR4 and subcloning them into the Xho I and Not I sites of psiCHECK2 (Promega, Madison, WI, USA). Cloning of the Pim-1 3′-UTR into psiCHECK2 was described (29). …”

Hypoxia-induced oxidative stress promotes MUC4 degradation via autophagy to enhance pancreatic cancer cells survival

“…We also observed an increase in p21 expression in hypoxia-treated T3M4 and CD18/HPAF cells, suggestive of growth arrest of PC cells (Supplementary Figure 3A), which was corroborated by our growth kinetics analysis in hypoxia-treated and untreated CD18/HPAF cells (Figure 5b) and by a recent study where hypoxia has shown to cause growth inhibition in PC cell lines. 30 To assess the role of ROS on pAkt reduction, we analyzed its expression in the presence and absence of NAC. Interestingly, hypoxia-mediated downregulation of pAkt in CD18/HPAF cells was abolished upon NAC administration, further emphasizing that the reduction in pAkt levels under hypoxia is ROS dependent (Figure 5c).…”

A Feed-Forward Regulatory Loop between HuR and the Long Noncoding RNA HOTAIR Promotes Head and Neck Squamous Cell Carcinoma Progression and Metastasis

“…HuR, an RBP, has already been shown to mediate post-transcriptional regulation in various cancers [13,14] and is known to interact with a substantial body of RNA transcripts [15,16]. Subsequent studies reported the collaborative [17] or suppressive [18] interplay between HuR and miRNA in many cancers.…”