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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.

SUBMITTER: Blanco FF

PROVIDER: S-EPMC6818728 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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

Blanco F F FF Jimbo M M Wulfkuhle J J Gallagher I I Deng J J Enyenihi L L Meisner-Kober N N Londin E E Rigoutsos I I Sawicki J A JA Risbud M V MV Witkiewicz A K AK McCue P A PA Jiang W W Rui H H Yeo C J CJ Petricoin E E Winter J M JM Brody J R JR

Oncogene 20150921 19

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) ...[more]

PMID: 26387536

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Project description:The Cancer associated Sm-like (CaSm) oncogene is overexpressed in 87% of human pancreatic tumor samples and CaSm knockdown has demonstrated therapeutic efficacy in murine models of pancreatic cancer. Evidence indicates that CaSm modulates mRNA degradation; however, its target genes and the mechanisms by which CaSm promotes pancreatic cancer remain largely unknown. Here, we demonstrate that the CaSm overexpression alters several hallmarks of cancer â including transformation, proliferation, chemoresistance, and metastasis. Doxycycline-induced CaSm expression enhanced proliferation and both anchorage-dependent and -independent growth of the human Panc-1 cells in vitro. CaSm induction decreased gemcitabine-induced cytotoxicity and altered the expression of apoptotic regulation genes, including Bad, E2F1, and Bcl-XL. CaSm-overexpressing Panc-1 cells were 2-fold more migratory and 4-fold more invasive than the driver controls and demonstrated characteristics of epithelial-to-mesenchymal transition such as morphological changes and decreased E-cadherin expression. CaSm induction resulted in changes in RNA expression of metastasis-associated genes such as MMP1, SerpinB5, uPAR, and Slug. Using a murine model of metastatic pancreatic cancer, injection of CaSm-induced Panc-1 cells resulted in a higher abundance of hepatic metastatic lesions. Overall, CaSm overexpression contributed to a more aggressive cancer phenotype in Panc-1 cells, further supporting the use of CaSm as a therapeutic target against pancreatic cancer. Tet-on driver and tet-on CaSm Panc-1 cells were grown with and without doxycycline (1 Î¼g/mL) for 4 weeks. Levels of CaSm expression were confirmed using RT-PCR (Supplementary Figure 2a). RNA isolation was performed using RNeasy Plus kit (Qiagen, Valencia, CA) following the manufacturerâs protocols. The Hollings Cancer Center Tissue Biorepository core facility confirmed RNA quality using the 18S/28S Ratio Integrity Number using Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA); RNA with a RIN of 10 was used for further analysis (Supplementary Figure 2b). Complement DNA synthesis was performed using RT2 First Strand kit and RT2 SYBR Green qPCR master mix was used for the microarray master mix reagent according to the manufacturerâs manual (SABiosciences). The Cancer PathwayFinder PCR array (SABiosciences) was run on the Roche 480 Lightcycler (Roche, Indianapolis, IN) with 2 plates run for each tet-on driver + doxycycline, tet-on CaSm, and tet-on CaSm + doxycycline. The plates included controls for genomic DNA contamination, reverse transcription, and positive controls. Analysis was performed using SABioscienceâs Free PCR Array Data Analysis Software (ÎÎCt method) with B2M, HPRT1, and GAPDH as pooled housekeeping genes.

Dataset Information

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

Publications

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

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