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Supplementary Figure S4. A, ACHN cells were transfected with TRIM33 siRNA or control siRNA for 24 h and then stimulated with or without TGF-β1 for 48 h. A wound was then formed by scraping and was measured after 24 h. The results are means {plus minus} S.D. of four independent experiments. *p < 0.05 vs. control siRNA without TGF-β1. White bars indicate 200 μm. B, ACHN cells were transfected with TRIM33 siRNA or control siRNA for 24 h and then stimulated with or without TGF-β1 for 48 h. A cell suspension was added to the upper chamber of matrigel-coated transwell membrane inserts; the lower chamber was filled with the medium and cultured for 12 h. Invasive cells were stained and fluorescence was measured. Values are means {plus minus} S.D. of three independent experiments.

Supplementary Figure S3. A, ACHN cells were transfected with TRIM33 siRNA for 72 h, and then the protein samples were collected and subjected to western blot analysis with anti-TRIM33 antibody. The membrane was re-probed with the anti-β-tubulin antibody. Relative TRIM33 expression is shown as means {plus minus} S.D. of four independent experiments. **p < 0.01 vs. control inhibitor. B, ACHN cells were co-transfected with the reporter construct containing the Smad-binding elements and TRIM33 siRNA or control siRNA, and then were stimulated with TGF-β1 for 24 h. Values are means {plus minus} S.D. of six independent experiments. **p < 0.01; ***p < 0.001 vs. control siRNA.

Supplementary Figure S2. The Caki-2 cells were transfected with TRIM33 siRNA for 72 h, and then protein samples were collected and subjected to western blot analysis by using anti-TRIM33 antibody. The membrane was re-probed with the anti-β-tubulin antibody. Relative TRIM33 expression is shown as means {plus minus} S.D. of four independent experiments. **p < 0.01 vs. control siRNA.

Supplementary Figure S1. A, ACHN cells were transfected with miR-629 mimic or negative control mimic for 72 h. TRIM33 mRNA levels were examined by quantitative real-time PCR. Data are the relative expression normalized to GAPDH mRNA. Values are means {plus minus} S.D. of 3 independent experiments. **p < 0.01 vs. control mimic. B, The expression of miR-629 and TRIM33 mRNAs expression (fold-change: tumor vs. normal) were examined in 41 matched-pair samples of ccRCC by quantitative real-time PCR, and the relationship was evaluated.

Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney, and clear cell RCC (ccRCC) represents its most common histological subtype. To identify a therapeutic target for ccRCC, miRNA expression signatures from ccRCC clinical specimens were analyzed. miRNA microarray and real-time PCR analyses revealed that miR-629 expression was significantly upregulated in human ccRCC compared with adjacent noncancerous renal tissue. Functional inhibition of miR-629 by a hairpin miRNA inhibitor suppressed ccRCC cell motility and invasion. Mechanistically, miR-629 directly targeted tripartite motif-containing 33 (TRIM33), which inhibits the TGFβ/Smad signaling pathway. In clinical ccRCC specimens, downregulation of TRIM33 was observed with the association of both pathologic stages and grades. The miR-629 inhibitor significantly suppressed TGFβ-induced Smad activation by upregulating TRIM33 expression and subsequently inhibited the association of Smad2/3 and Smad4. Moreover, a miR-629 mimic enhanced the effect of TGFβ on the expression of epithelial–mesenchymal transition–related factors as well as on the motility and invasion in ccRCC cells. These findings identify miR-629 as a potent regulator of the TGFβ/Smad signaling pathway via TRIM33 in ccRCC.Implications: This study suggests that miR-629 has biomarker potential through its ability to regulate TGFβ/Smad signaling and accelerate ccRCC cell motility and invasion. Mol Cancer Res; 13(3); 565–74. ©2014 AACR.

Supplementary Figure S5. A, Caki-2 cells were transfected with the miR-629 inhibitor or control miRNA inhibitor for 72 h, and then protein samples were collected and subjected to western blot analysis with anti-HNF4α antibody. The membrane was re-probed with the anti-β-tubulin antibody. The relative HNF4α expression is shown as mean {plus minus} S.D. of three independent experiments. B, Protein samples from ccRCC cell lines were collected and subjected to western blot analysis for HNF4α. The relative HNF4α expression is shown as means {plus minus} S.D. of three independent experiments. (C, D) The expression of HNF4α in ccRCC specimens was examined by immunohistochemical staining. One normal kidney tissue and 17 ccRCC samples were examined.

Human AlkB homolog 8 (ALKBH8) is highly expressed in high-grade, superficially and deeply invasive bladder cancer. Moreover, ALKBH8 knockdown induces apoptosis in bladder cancer cells. However, the underlying anti-apoptotic mechanism of ALKBH8 in bladder cancer cells has thus far remained unclear. Moreover, there is no direct evidence that highly expressed ALKBH8 is involved in tumor progression in vivo. We here show that ALKBH8 knockdown induced apoptosis via downregulating the protein expression of survivin, an anti-apoptotic factor also exhibiting increased levels in bladder cancer. We also clarify that ALKBH8 transgenic mice showed an accelerated rate of bladder tumor mass and invasiveness in an N-butyl-N-(4-hydroxybutyl)-nitrosamine-induced bladder cancer model. These findings suggest that the high expression of ALKBH8 is critical for the growth and progression of bladder cancer.

Renal cell carcinoma (RCC) is the most common neoplasm of the adult kidney, and clear cell RCC (ccRCC) represents its most common histological subtype. To identify a therapeutic target for ccRCC, miRNA expression signatures from ccRCC clinical specimens were analyzed. miRNA microarray and real-time PCR analyses revealed that miR-629 expression was significantly upregulated in human ccRCC compared with adjacent noncancerous renal tissue. Functional inhibition of miR-629 by a hairpin miRNA inhibitor suppressed ccRCC cell motility and invasion. Mechanistically, miR-629 directly targeted tripartite motif-containing 33 (TRIM33), which inhibits the TGFβ/Smad signaling pathway. In clinical ccRCC specimens, downregulation of TRIM33 was observed with the association of both pathologic stages and grades. The miR-629 inhibitor significantly suppressed TGFβ-induced Smad activation by upregulating TRIM33 expression and subsequently inhibited the association of Smad2/3 and Smad4. Moreover, a miR-629 mimic enhanced the effect of TGFβ on the expression of epithelial–mesenchymal transition–related factors as well as on the motility and invasion in ccRCC cells. These findings identify miR-629 as a potent regulator of the TGFβ/Smad signaling pathway via TRIM33 in ccRCC. Implications: This study suggests that miR-629 has biomarker potential through its ability to regulate TGFβ/Smad signaling and accelerate ccRCC cell motility and invasion. Mol Cancer Res; 13(3); 565–74. ©2014 AACR.

Clear cell renal cell carcinoma (ccRCC) is the most common histologically defined subtype of renal cell carcinoma (RCC). To define the molecular mechanism in the progression of ccRCC, we focused on LOX-like protein 2 (LOXL2), which is critical for the first step in collagen and elastin cross-linking. Using exon array analysis and quantitative validation, LOXL2 was shown to be significantly upregulated in clinical specimens of human ccRCC tumor tissues, compared with adjacent noncancerous renal tissues, and this elevated expression correlated with the pathologic stages of ccRCC. RNAi-mediated knockdown of LOXL2 resulted in marked suppression of stress-fiber and focal adhesion formation in ccRCC cells. Moreover, LOXL2 siRNA knockdown significantly inhibited cell growth, migration, and invasion. Mechanistically, LOXL2 regulated the degradation of both integrins α5 (ITGAV5) and β1 (ITGB1) via protease- and proteasome-dependent systems. In clinical ccRCC specimens, the expression levels of LOXL2 and integrin α5 correlated with the pathologic tumor grades. In conclusion, LOXL2 is a potent regulator of integrin α5 and integrin β1 protein levels and functions in a tumor-promoting capacity in ccRCC. Implications: This is the first report demonstrating that LOXL2 is highly expressed and involved in ccRCC progression by regulating the levels of integrins α5 and β1. Mol Cancer Res; 12(12); 1807–17. ©2014 AACR.