A Kinome-Wide siRNA Screen Identifies Multiple Roles for Protein Kinases in Hypoxic Stress Adaptation, Including Roles for IRAK4 and GAK in Protection against Apoptosis in VHL–/– Renal Carcinoma Cells, Despite Activation of the NF-κB Pathway

Pan, Jing; Zhang, Jing; Hill, Andrew; LaPan, Peter; Berasi, Steve; Bates, Brian; Miller, Christopher C.J.; Haney, Steven

doi:10.1177/1087057113484803

Cited by 8 publications

(3 citation statements)

References 31 publications

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“…). Interestingly, others have reported that stable short hairpin RNAs against Mirk(dyrk1B) prevented the A598 renal carcinoma cell line from forming spheroid cultures (26), consistent with our observations of the loss of spheroid structure with Mirk kinase inhibition. The Mirk/dyrk1B inhibitor EHT5372 caused a progressive loss in tumor cell numbers: 50% after 6 days, 60% after 8 days, 80% after 15 days, and 92% after 21 days (Fig 3A…”

Section: Resultssupporting

confidence: 92%

Mirk kinase inhibition targets ovarian cancer ascites

Deng

Cunningham

et al. 2014

Genes Cancer

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The Mirk/dyrk1B gene is commonly amplified or upregulated in ovarian cancers, and Mirk is an active kinase in these cancers. Mirk mediates cancer cell survival by decreasing toxic ROS levels through maintaining expression of a series of antioxidant genes, possibly through its transcriptional activator functions. Mirk has the unusual property of being most active in quiescent cancer cells because of marked transcriptional downregulation by Akt/mTOR signaling and by MEK/erk signaling in cycling cells. Metastatic ovarian cancer cells form ascites, non-adherent multicellular aggregates floating within the peritoneal fluid. Most ascites cancer cells are in a reversible quiescent, dormant state, suggesting that Mirk might be expressed in these quiescent cells and thus a therapeutic target. The current studies show that ovarian cancer cell line spheroids that mimic ascites cancer spheroids were largely quiescent in G0/G1, and enriched in Mirk and the quiescence proteins, p130/Rb2 and the CDKI p27. Mirk kinase inhibition in spheroids made from established cell lines and in patient-derived ascites cancer cell spheroids reduced spheroid volume, disrupted spheroid structure to single cells, increased apoptosis, and decreased cell numbers. Earlier studies had shown that the mTOR inhibitor RAD001 increased transcription of the Mirk/dyrk1B gene, so treatments combined RAD001 with the most active Mirk kinase inhibitor. The number of ascites cells from 9 patients was reduced a similar amount by cisplatin, Mirk kinase inhibition or RAD001, but reduced substantially more, about 90%, by concurrent treatment with both the Mirk kinase inhibitor EHT5372 and RAD001. Addition of RAD001 increased the amount of toxic ROS induced by Mirk kinase inhibition. Two ascites samples taken one month apart gave similar drug responses, showing reproducibility of the techniques. Thus Mirk/dyrk1B kinase may be a therapeutic target in ovarian cancer ascites.

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Section: Resultssupporting

confidence: 92%

Mirk kinase inhibition targets ovarian cancer ascites

Deng

Cunningham

et al. 2014

Genes Cancer

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“…Thus the Mirk kinase inhibitor reduced the number of Panc1 cells in the spheroids, consistent with the cells in the outside layers dying of apoptosis and reducing spheroid volume. Interestingly, others have reported that stable short hairpin RNAs against Mirk/dyrk1B prevented the A498 renal carcinoma cell line from forming spheroid cultures [ 26 ]. Disruption of spheroids to single cells is a step in their dissolution.…”

Section: Resultsmentioning

confidence: 99%

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

Deng

Friedman

2014

Genes Cancer

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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 αSMA-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.

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“…Functional genetic screens provide an unbiased and powerful means of identifying genes responsible for any phenotype that can be measured experimentally. Unbiased RNAi screens have identified genes that influence the survival of organisms and cells under various stresses [ 33 – 36 ]. These studies provide a foundation for using RNAi screens to uncover genes involved in TME-relevant stress responses, but so far have not been applied genome-wide to identify genes that modulate the survival of cancer cells under hypoxia or lactic acidosis.…”

Section: Introductionmentioning

confidence: 99%

ACLY and ACC1 Regulate Hypoxia-Induced Apoptosis by Modulating ETV4 via α-ketoglutarate

et al. 2015

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In order to propagate a solid tumor, cancer cells must adapt to and survive under various tumor microenvironment (TME) stresses, such as hypoxia or lactic acidosis. To systematically identify genes that modulate cancer cell survival under stresses, we performed genome-wide shRNA screens under hypoxia or lactic acidosis. We discovered that genetic depletion of acetyl-CoA carboxylase (ACACA or ACC1) or ATP citrate lyase (ACLY) protected cancer cells from hypoxia-induced apoptosis. Additionally, the loss of ACLY or ACC1 reduced levels and activities of the oncogenic transcription factor ETV4. Silencing ETV4 also protected cells from hypoxia-induced apoptosis and led to remarkably similar transcriptional responses as with silenced ACLY or ACC1, including an anti-apoptotic program. Metabolomic analysis found that while α-ketoglutarate levels decrease under hypoxia in control cells, α-ketoglutarate is paradoxically increased under hypoxia when ACC1 or ACLY are depleted. Supplementation with α-ketoglutarate rescued the hypoxia-induced apoptosis and recapitulated the decreased expression and activity of ETV4, likely via an epigenetic mechanism. Therefore, ACC1 and ACLY regulate the levels of ETV4 under hypoxia via increased α-ketoglutarate. These results reveal that the ACC1/ACLY-α-ketoglutarate-ETV4 axis is a novel means by which metabolic states regulate transcriptional output for life vs. death decisions under hypoxia. Since many lipogenic inhibitors are under investigation as cancer therapeutics, our findings suggest that the use of these inhibitors will need to be carefully considered with respect to oncogenic drivers, tumor hypoxia, progression and dormancy. More broadly, our screen provides a framework for studying additional tumor cell stress-adaption mechanisms in the future.

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A Kinome-Wide siRNA Screen Identifies Multiple Roles for Protein Kinases in Hypoxic Stress Adaptation, Including Roles for IRAK4 and GAK in Protection against Apoptosis in VHL–/– Renal Carcinoma Cells, Despite Activation of the NF-κB Pathway

Cited by 8 publications

References 31 publications

Mirk kinase inhibition targets ovarian cancer ascites

Mirk kinase inhibition targets ovarian cancer ascites

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

ACLY and ACC1 Regulate Hypoxia-Induced Apoptosis by Modulating ETV4 via α-ketoglutarate

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