Gankyrin plays an essential role in Ras-induced tumorigenesis through regulation of the RhoA/ROCK pathway in mammalian cells
Jiang-Hong Man, … , Hui-Yan Li, Xue-Min Zhang
Jiang-Hong Man, … , Hui-Yan Li, Xue-Min Zhang
Published July 12, 2010
Citation Information: J Clin Invest. 2010;120(8):2829-2841. https://doi.org/10.1172/JCI42542.
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Research Article Oncology

Gankyrin plays an essential role in Ras-induced tumorigenesis through regulation of the RhoA/ROCK pathway in mammalian cells

Abstract

Activating mutations in Ras proteins are present in about 30% of human cancers. Despite tremendous progress in the study of Ras oncogenes, many aspects of the molecular mechanisms underlying Ras-induced tumorigenesis remain unknown. Through proteomics analysis, we previously found that the protein Gankyrin, a known oncoprotein in hepatocellular carcinoma, was upregulated during Ras-mediated transformation, although the functional consequences of this were not clear. Here we present evidence that Gankyrin plays an essential role in Ras-initiated tumorigenesis in mouse and human cells. We found that the increased Gankyrin present following Ras activation increased the interaction between the RhoA GTPase and its GDP dissociation inhibitor RhoGDI, which resulted in inhibition of the RhoA effector kinase Rho-associated coiled coil–containing protein kinase (ROCK). Importantly, Gankyrin-mediated ROCK inhibition led to prolonged Akt activation, a critical step in activated Ras–induced transformation and tumorigenesis. In addition, we found that Gankyrin is highly expressed in human lung cancers that have Ras mutations and that increased Gankyrin expression is required for the constitutive activation of Akt and tumorigenesis in these lung cancers. Our findings suggest that Gankyrin is a key regulator of Ras-mediated activation of Akt through inhibition of the downstream RhoA/ROCK pathway and thus plays an essential role in Ras-induced tumorigenesis.

Authors

Jiang-Hong Man, Bing Liang, Yue-Xi Gu, Tao Zhou, Ai-Ling Li, Tao Li, Bao-Feng Jin, Bing Bai, Hai-Ying Zhang, Wei-Na Zhang, Wei-Hua Li, Wei-Li Gong, Hui-Yan Li, Xue-Min Zhang

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Figure 2

Knockdown of Gankyrin impedes Akt activation.

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Knockdown of Gankyrin impedes Akt activation. (A) NIH3T3 cells stably ex...
(A) NIH3T3 cells stably expressing Gankyrin shRNA or a scramble control were infected with a retroviral construct encoding ER:Ras G12V. Cells were serum starved overnight followed by induction with 100 nM 4-OHT for the indicated times. Cells were then harvested and probed with the indicated antibodies by immunoblot. ER:Ras G12V, expressing inducible activated Ras, was induced by 4-OHT. (B) Constitutively activated Akt rescues tumorigenicity in Ras-transformed NIH3T3 cells with Gankyrin knocked down. Overexpression of Myr-Akt in Ras-transformed NIH3T3 cells stably expressing Gankyrin shRNA restored the tumorigenesis capability of the cells in the soft agar assay. Data are shown as mean ± SD and are representative of 3 independent experiments. (C) HEK293 cells transfected with control or Gankyrin-specific siRNA were serum starved overnight and then treated with EGF (100 ng/ml) for the indicated times. The cells were then harvested and probed with the indicated antibodies by immunoblot. (D) Complementation of Flag-Gankyrin in knockdown HEK293 cells rescues Akt activation. HEK293 cells cotransfected with Gankyrin-specific siRNAs and the expression construct Flag-Gankyrin (mouse [m]) were serum starved overnight, treated with EGF, and tested for the phosphorylation of Akt.