Open Access Research article

Downregulation of SAV1 plays a role in pathogenesis of high-grade clear cell renal cell carcinoma

Keiko Matsuura1, Chisato Nakada1, Mizuho Mashio1, Takahiro Narimatsu12, Taichiro Yoshimoto16, Masato Tanigawa3, Yoshiyuki Tsukamoto1, Naoki Hijiya1, Ichiro Takeuchi4, Takeo Nomura2, Fuminori Sato2, Hiromitsu Mimata2, Masao Seto5 and Masatsugu Moriyama17*

Author Affiliations

1 Department of Molecular Pathology, Oita University, Oita, Japan

2 Department of Urology, Faculty of Medicine, Oita University, Oita, Japan

3 Division of Biomolecular Medicine and Medical Imaging, Oita University, Oita, Japan

4 Department of Scientific and Engineering Simulation, Graduate School of Engineering, Nagoya Institute of Technology, Nagoya, Japan

5 Division of Molecular Medicine, Aichi Cancer Center, Nagoya, Japan

6 Department of Pathology and Diagnostic Pathology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan

7 Department of Molecular Pathology, Faculty of Medicine, Oita University, Hasama-machi, Yufu-city, Oita 879-5593, Japan

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BMC Cancer 2011, 11:523  doi:10.1186/1471-2407-11-523

Published: 20 December 2011

Abstract

Background

Clinical outcome of patients with high-grade ccRCC (clear cell renal cell carcinoma) remains still poor despite recent advances in treatment strategies. Molecular mechanism of pathogenesis in developing high-grade ccRCC must be clarified. In the present study, we found that SAV1 was significantly downregulated with copy number loss in high-grade ccRCCs. Therefore, we investigated the SAV1 function on cell proliferation and apoptosis in vitro. Furthermore, we attempted to clarify the downstream signaling which is regulated by SAV1.

Methods

We performed array CGH and gene expression analysis of 8 RCC cell lines (786-O, 769-P, KMRC-1, KMRC-2, KMRC-3, KMRC-20, TUHR4TKB, and Caki-2), and expression level of mRNA was confirmed by quantitative RT-PCR (qRT-PCR) analysis. We next re-expressed SAV1 in 786-O cells, and analyzed its colony-forming activity. Then, we transfected siRNAs of SAV1 into the kidney epithelial cell line HK2 and renal proximal tubule epithelial cells (RPTECs), and analyzed their proliferation and apoptosis. Furthermore, the activity of YAP1, which is a downstream molecule of SAV1, was evaluated by western blot analysis, reporter assay and immunohistochemical analysis.

Results

We found that SAV1, a component of the Hippo pathway, is frequently downregulated in high-grade ccRCC. SAV1 is located on chromosome 14q22.1, where copy number loss had been observed in 7 of 12 high-grade ccRCCs in our previous study, suggesting that gene copy number loss is responsible for the downregulation of SAV1. Colony-forming activity by 786-O cells, which show homozygous loss of SAV1, was significantly reduced when SAV1 was re-introduced exogenously. Knockdown of SAV1 promoted proliferation of HK2 and RPTEC. Although the phosphorylation level of YAP1 was low in 786-O cells, it was elevated in SAV1-transduced 786-O cells. Furthermore, the transcriptional activity of the YAP1 and TEAD3 complex was inhibited in SAV1-transduced 786-O cells. Immunohistochemistry frequently demonstrated nuclear localization of YAP1 in ccRCC cases with SAV1 downregulation, and it was preferentially detected in high-grade ccRCC.

Conclusions

Taken together, downregulation of SAV1 and the consequent YAP1 activation are involved in the pathogenesis of high-grade ccRCC. It is an attractive hypothesis that Hippo signaling could be candidates for new therapeutic target.

Keywords:
Clear cell renal cell carcinoma; SAV1; Hippo pathway