title: Downregulation of SAV1 plays a role in pathogenesis of high-grade clear cell renal cell carcinoma.
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.
authors.affiliation: Department of Molecular Pathology, Oita University, Oita, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Matsuura
authors.forename: Chisato
authors.initials: C
authors.lastname: Nakada
authors.forename: Mizuho
authors.initials: M
authors.lastname: Mashio
authors.forename: Takahiro
authors.initials: T
authors.lastname: Narimatsu
authors.forename: Taichiro
authors.initials: T
authors.lastname: Yoshimoto
authors.forename: Masato
authors.initials: M
authors.lastname: Tanigawa
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Tsukamoto
authors.forename: Naoki
authors.initials: N
authors.lastname: Hijiya
authors.forename: Ichiro
authors.initials: I
authors.lastname: Takeuchi
authors.forename: Takeo
authors.initials: T
authors.lastname: Nomura
authors.forename: Fuminori
authors.initials: F
authors.lastname: Sato
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Mimata
authors.forename: Masao
authors.initials: M
authors.lastname: Seto
authors.forename: Masatsugu
authors.initials: M
authors.lastname: Moriyama
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D017209:Apoptosis / Q000502:physiology; D015153:Blotting, Western; D002292:Carcinoma, Renal Cell / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015536:Down-Regulation; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D007680:Kidney Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000503:physiopathology; D010750:Phosphoproteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors; D000091102:YAP-Signaling Proteins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C415864:SAV1 protein, human; D014157:Transcription Factors; D000091102:YAP-Signaling Proteins; C088374:YAP1 protein, human