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Downregulation of Ral GTPase-activating protein promotes tumor invasion and metastasis of bladder cancer

Abstract

The small GTPase Ral is known to be highly activated in several human cancers, such as bladder, colon and pancreas cancers. It is reported that activated Ral is involved in cell proliferation, migration and metastasis of bladder cancer. This protein is activated by Ral guanine nucleotide exchange factors (RalGEFs) and inactivated by Ral GTPase-activating proteins (RalGAPs), the latter of which consist of heterodimers containing a catalytic α1 or α2 subunit and a common β subunit. In Ras-driven cancers, such as pancreas and colon cancers, constitutively active Ras mutant activates Ral through interaction with RalGEFs, which contain the Ras association domain. However, little is known with regard to the mechanism that governs aberrant activation of Ral in bladder cancer, in which Ras mutations are relatively infrequent. Here, we show that Ral was highly activated in invasive bladder cancer cells due to reduced expression of RalGAPα2, the dominant catalytic subunit in bladder, rather than increased expression of RalGEFs. Exogenous expression of wild-type RalGAPα2 in KU7 bladder cancer cells with invasive phenotype, but not mutant RalGAPα2-N1742K lacking RalGAP activity, resulted in attenuated cell migration in vitro and lung metastasis in vivo. Furthermore, genetic ablation of Ralgapa2 promoted tumor invasion in a chemically-induced murine bladder cancer model. Importantly, immunohistochemical analysis of human bladder cancer specimens revealed that lower expression of RalGAPα2 was associated with advanced clinical stage and poor survival of patients. Collectively, these results are highly indicative that attenuated expression of RalGAPα2 leads to disease progression of bladder cancer through enhancement of Ral activity.

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Acknowledgements

We would like to thank Aaron Mathew Coutts for proofreading of the manuscript. We appreciate all members of the Cancer Research Course for Integrated Research Training in Kyoto University Graduate School of Medicine for their helpful advice and discussion. This study was supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (No. 22591766 to HN and OO, 22890015, 22501009, 20013201 and 23113702 to R Shirakawa and HH) as well as grants from Uehara Memorial Foundation (to T Kobayashi and OO), Takeda Science Foundation, the Suzuken Memorial Foundation, Daiichi-Sankyo Foundation of Life-Science, Kurokawa Cancer Research Foundation (to R Shirakawa), and Novartis Foundation for the Promotion of Science (to HH).

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Correspondence to H Nishiyama or H Horiuchi.

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Saito, R., Shirakawa, R., Nishiyama, H. et al. Downregulation of Ral GTPase-activating protein promotes tumor invasion and metastasis of bladder cancer. Oncogene 32, 894–902 (2013). https://doi.org/10.1038/onc.2012.101

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