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. 2019 Jun;110(6):2063-2074.
doi: 10.1111/cas.14018. Epub 2019 May 3.

c-Ski Accelerates Renal Cancer Progression by Attenuating Transforming Growth Factor β Signaling

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Free PMC article

c-Ski Accelerates Renal Cancer Progression by Attenuating Transforming Growth Factor β Signaling

Luna Taguchi et al. Cancer Sci. .
Free PMC article

Abstract

Although transforming growth factor beta (TGF-β) is known to be involved in the pathogenesis and progression of many cancers, its role in renal cancer has not been fully investigated. In the present study, we examined the role of TGF-β in clear cell renal carcinoma (ccRCC) progression in vitro and in vivo. First, expression levels of TGF-β signaling pathway components were examined. Microarray and immunohistochemical analyses showed that the expression of c-Ski, a transcriptional corepressor of Smad-dependent TGF-β and bone morphogenetic protein (BMP) signaling, was higher in ccRCC tissues than in normal renal tissues. Next, a functional analysis of c-Ski effects was carried out. Bioluminescence imaging of renal orthotopic tumor models demonstrated that overexpression of c-Ski in human ccRCC cells promoted in vivo tumor formation. Enhancement of tumor formation was also reproduced by the introduction of a dominant-negative mutant TGF-β type II receptor into ccRCC cells. In contrast, introduction of the BMP signaling inhibitor Noggin failed to accelerate tumor formation, suggesting that the tumor-promoting effect of c-Ski depends on the inhibition of TGF-β signaling rather than of BMP signaling. Finally, the molecular mechanism of the tumor-suppressive role of TGF-β was assessed. Although TGF-β signaling did not affect tumor angiogenesis, apoptosis of ccRCC cells was induced by TGF-β. Taken together, these findings suggest that c-Ski suppresses TGF-β signaling in ccRCC cells, which, in turn, attenuates the tumor-suppressive effect of TGF-β.

Keywords: TGF-β; apoptosis; c-Ski; orthotopic tumor model; renal cell cancer.

Figures

Figure 1
Figure 1
Upregulation of c‐Ski expression in clear cell renal carcinoma (ccRCC) tissues. A, Comprehensive analysis of gene expression data from the NCBI Gene Expression Omnibus (GEO) database (GSE53757) shows expression levels of transforming growth factor beta (TGF‐β) signaling pathway components in ccRCC tissues (n = 72) and matched normal renal tissues (n = 72). Expression values are shown by dot plots. Data represent the mean ± SD. ***< .001. B, Images of immunohistochemical staining with an anti‐c‐Ski antibody and H&E staining of ccRCC tissues and corresponding normal renal tissues from the same patient. c‐Ski staining in boxed region is shown at high magnification. Scale bars are 100 μm. ENG, endoglin
Figure 2
Figure 2
Enhanced tumor formation by c‐Ski in OSRC‐2 cells. A, OSRC‐2 cells were infected with lentiviral vectors encoding GFP (OSRC‐2‐GFP) or c‐Ski (OSRC‐2‐c‐Ski) and analyzed by qRTPCR for SKI expression. Data represent the mean ± SD. **< .01. B, Immunoblots of lysates of OSRC‐2‐GFP and OSRC‐2‐c‐Ski cells with the indicated antibodies. C, qRTPCR analysis of SERPINE1 expression. OSRC‐2‐GFP and OSRC‐2‐c‐Ski cells were stimulated with transforming growth factor beta (TGF‐β) for 2 h and analyzed by qRTPCR. Data represent the mean ± SD. **< .01. D, Tumor‐forming ability of OSRC‐2 cells. BALB/c nu/nu male mice received renal orthotopic injection of OSRC‐2‐GFP (n = 6) or OSRC‐2‐c‐Ski (n = 5) cells (1 × 105 cells per mouse). Upper panels: representative photographs of in vivo bioluminescence imaging of tumor‐bearing mice 13 d after the injection. Lower panel: overall luminescence signal intensity; data represent the mean ± SE. ***< .001. E, Tumor‐forming ability of OSRC‐2 cells. Tumor formation in mice in (D) was examined 3 wks after the injection. Upper panels: representative photographs of brightfield and ex vivo bioluminescence imaging of extracted kidneys. Lower panel: overall luminescence signal intensity; data represent the mean ± SE. ***< .001
Figure 3
Figure 3
Enhanced tumor formation by c‐Ski in Caci‐1 cells. A, Caki‐1 cells were infected with lentiviral vectors encoding GFP (Caki‐1‐GFP) or c‐Ski (Caki‐1‐c‐Ski) and analyzed by qRTPCR for SKI expression. Data represent the mean ± SD. ***< .001. B, Immunoblots of lysates of Caki‐1‐GFP and Caki‐1‐c‐Ski cells with the indicated antibodies. C, qRTPCR analysis of SERPINE1 expression. Caki‐1‐GFP and Caki‐1‐c‐Ski cells were stimulated with transforming growth factor beta (TGF‐β) for 2 h and analyzed by qRTPCR. Data represent the mean ± SD. *< .05, **< .01. D, Tumor‐forming ability of Caki‐1 cells. BALB/c nu/nu male mice received renal orthotopic injection of Caki‐1‐GFP (n = 6) or Caki‐1‐c‐Ski (n = 5) cells. Upper panels: representative photographs of in vivo bioluminescence imaging of tumor‐bearing mice 3 wks after the injection. Lower panel: luminescence‐positive area in all mice; data represent the mean ± SE. *< .05. E, Tumor‐forming ability of Caki‐1 cells. Tumor formation in mice in (D) was examined 5 wks after the injection. Upper panels: representative photographs of brightfield and ex vivo bioluminescence imaging of extracted kidneys. Lower panel: luminescence‐positive area; data represent the mean ± SE. **< .01
Figure 4
Figure 4
Enhanced tumor formation by dnTβRII in OSRC‐2 cells. A, Immunoblots of cell lysates with the indicated antibodies. OSRC‐2 cells were infected with lentiviral vectors encoding GFP (OSRC‐2‐GFP) or HA‐tagged dnTβRII (OSRC‐2‐dnTβRII). Cells were stimulated with transforming growth factor beta (TGF‐β) for 2 h. B, qRTPCR analysis of SERPINE1 expression. Cells were stimulated with TGF‐β for 2 h and analyzed by qRTPCR. Data represent the mean ± SD. *< .05. C, Tumor‐forming ability of OSRC‐2 cells. BALB/c nu/nu male mice received renal orthotopic injection of OSRC‐2‐GFP (n = 8) or OSRC‐2‐dnTβRII (n = 8) cells. Representative photographs of in vivo bioluminescence imaging of tumor‐bearing mice 3 wks after the injection. D, Tumor‐forming ability of OSRC‐2 cells. Tumor formation in mice in (C) was examined 3 wks after the injection. Upper panels: representative photographs of ex vivo bioluminescence imaging of extracted kidneys. Lower panel: overall luminescence signal intensity; data represent the mean ± SE. *< .05. E, Metastatic ability of OSRC‐2 cells. Metastatic lung tumor in mice shown in (C) was examined 3 wks after the injection. Left panels: representative photographs of ex vivo bioluminescence imaging of extracted lungs. Right panel: overall luminescence signal intensity; data represent the mean ± SE
Figure 5
Figure 5
Enhanced tumor formation by dnTβRII in Caci‐1 cells. A, Immunoblots of cell lysates with the indicated antibodies. Caki‐1 cells were infected with lentiviral vectors encoding GFP (Caki‐1‐GFP) or HA‐tagged dnTβRII (Caki‐1‐dnTβRII). Cells were stimulated with transforming growth factor beta (TGF‐β) for 2 h. B, qRTPCR analysis of SERPINE1 expression. Cells were stimulated with TGF‐β for 2 h and analyzed by qRTPCR. Data represent the mean ± SD. **< .01. C, Tumor‐forming ability of Caki‐1 cells. BALB/c nu/nu male mice received renal orthotopic injection of Caki‐1‐GFP (n = 5) or Caki‐1‐dnTβRII (n = 4) cells. Representative photographs of in vivo bioluminescence imaging of tumor‐bearing mice 7 wks after the injection. D, Tumor‐forming ability of Caki‐1 cells. Tumor formation in mice shown in (C) was examined 8 wks after the injection. Upper panels: representative photographs of ex vivo bioluminescence imaging of extracted kidney. Lower panel: luminescence‐positive area; data represent the mean ± SE. *< .05
Figure 6
Figure 6
Induction of apoptosis of clear cell renal carcinoma (ccRCC) cells by transforming growth factor beta (TGF‐β). A, Apoptosis in primary tumor tissues dissected from mice shown in Figure 4D, which were killed 3 wks after the transplantation. Left panels: tumor tissues were subjected to TUNEL staining. Representative images were captured for tissue from each mouse. Right panel: apoptosis was quantified by counting the number of TUNEL‐positive cells in three independent fields; data represent the mean ± SD. *< .05. Scale bar is 50 μm. B, Colony formation of indicated cells in soft agar. Cells were stimulated with or without TGF‐β for 14 d. Left panels: Representative photographs. Right panel: colony number was counted in three independent fields; data represent the mean ± SD. ***< .001. Scale bar is 200 μm. C, Colony formation of indicated cells in detached culture. Cells were stimulated with or without TGF‐β for 2 d. Representative photographs are shown. Scale bar is 200 μm

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