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Mirk kinase inhibition blocks the in vivo growth of pancreatic cancer cells.

ABSTRACT: The Mirk/dyrk1B gene is upregulated and sometimes amplified in pancreatic ductal carcinomas. In poor microenvironmental conditions Mirk mediates cell survival by maintaining cancer cells in a largely quiescent, noncycling state and by decreasing toxic ROS levels through maintaining expression of a series of antioxidant genes. Premature entry into cycle, increased ROS levels, DNA damage, and apoptosis follow Mirk kinase depletion or inhibition. Mirk kinase inhibitor EHT5372 treated Panc1 spheroids lost quiescence markers coincident with an increase in cyclin A showing entry into cycle, and exhibited DNA damage, apoptosis and smaller size. EHT5372 treatment in vivo led to an increased fraction of Ki67 positive, cycling cells in Panc1 xenografts whose size was reduced. Pdx-1-cre LSL/KrasG12D/Ink4a/Arf null B6 mice always develop pancreatic cancer, allowing only 30% survival by 8 weeks, while each of the Mirk kinase inhibitor treated mice survived 8 weeks. Mirk inhibition led to a roughly four-fold increase in tumor ?SMA-positive fibroblasts and large stromal collagen-rich infiltrates in the pancreas that can restrain tumor growth. The mTOR inhibitor RAD001 alone, or together with EHT5372, reduced pancreatic cancer size 30-fold, while the drug combination reduced the number of microscopic tumor foci 2-fold compared to RAD001 alone.

SUBMITTER: Deng X

PROVIDER: S-EPMC4209603 | biostudies-literature | 2014 Sep

REPOSITORIES: biostudies-literature

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Mirk kinase inhibition blocks the in vivo growth of pancreatic cancer cells.

Deng Xiaobing X Friedman Eileen E

Genes & cancer 20140901 9-10

The Mirk/dyrk1B gene is upregulated and sometimes amplified in pancreatic ductal carcinomas. In poor microenvironmental conditions Mirk mediates cell survival by maintaining cancer cells in a largely quiescent, noncycling state and by decreasing toxic ROS levels through maintaining expression of a series of antioxidant genes. Premature entry into cycle, increased ROS levels, DNA damage, and apoptosis follow Mirk kinase depletion or inhibition. Mirk kinase inhibitor EHT5372 treated Panc1 spheroid ...[more]

PMID: 25352950

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Project description:Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers and known for its extensive genetic heterogeneity, high therapeutic resistance, and strong variation in intrinsic radiosensitivity. To understand the molecular mechanisms underlying radioresistance, we screened the phenotypic response of 38 PDAC cell lines to ionizing radiation. Subsequent phosphoproteomic analysis of two representative sensitive and resistant lines led to the reproducible identification of 7,800 proteins and 13,000 phosphorylation sites (p-sites). Approximately 700 p-sites on 400 proteins showed abundance changes after radiation in all cell lines regardless of their phenotypic sensitivity. Apart from recapitulating known radiation response phosphorylation markers such as on proteins involved in DNA damage repair, the analysis uncovered many novel members of a radiation-responsive signaling network that was apparent only at the level of protein phosphorylation. These regulated p-sites were enriched in potential ATM substrates and in vitro kinase assays corroborated 10 of these. Comparing the proteomes and phosphoproteomes of radiosensitive and -resistant cells pointed to additional tractable radioresistance mechanisms involving apoptotic proteins. For instance, elevated NADPH quinine oxidoreductase 1 (NQO1) expression in radioresistant cells may aid in clearing harmful reactive oxygen species. Resistant cells also showed elevated phosphorylation levels of proteins involved in cytoskeleton organization including actin dynamics and focal adhesion kinase (FAK) activity and one resistant cell line showed a strong migration phenotype. Pharmacological inhibition of the kinases FAK by Defactinib and of CHEK1 by Rabusertib showed a statistically significant sensitization to radiation in radioresistant PDAC cells. Together, the presented data map a comprehensive molecular network of radiation-induced signaling, improves the understanding of radioresistance and provides avenues for developing radiotherapeutic strategies.

Project description:Pancreatic ductal adenocarcinoma is one of the most aggressive cancer types and is well-known for its general low intrinsic radiosensitivity. In order to resolve mechanisms of how PDAC cells carry out radioresistance, a combination of proteomics and phosphoproteomics of two radiosensitive as well as radioresistant murine PDAC cell lines upon exposition to radiation was performed. A high depth of (phospho)proteomic identifications with > 13000 confidently identified phosphorylation sites and > 7800 proteins was achieved. Measuring the response of the phosphoproteome upon radiation revealed >700 phosphorylation sites on >400 proteins which were regulated in all four cell lines independently of the sensitivity status. This analysis validated already known radiomarkers but also uncovered novel members of the radiation-dependent signaling network in PDAC cells that were shown to be exclusively based on protein phosphorylation but not protein expression. Among those radioresponsive phosphoproteins were ten novel ATM substrates, which were validated by in vitro kinase assays. Comparison of radiosensitive and -resistant cells revealed a complex regulation of apoptotic processes, while changes in expression of DNA repair proteins seemed to not play a pivotal role. Especially increased expression of NQO1 was found to be a potential mechanism enabling resistance by clearing harmful reactive oxygen species from the PDAC cells. Further analysis of the radioresistance-associated-phosphoproteome, which was especially enriched in phosphoproteins with cytoskeleton organizational function, uncovered increased Actin dynamics and FAK activity in radioresistant cells. Both likely lead to increased survival, migrational capacity and perturbations in chromatin condensation which could oppose radiation. Based on the displayed adaptions in cellular protein expression and signaling in resistant PDAC cells, pharmacological inhibition of NQO1 and FAK could be suggested to sensitize towards radiation.

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Mirk kinase inhibition blocks the in vivo growth of pancreatic cancer cells.

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Mirk kinase inhibition blocks the in vivo growth of pancreatic cancer cells.

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