WIF1, a Wnt pathway inhibitor, regulates SKP2 and c-myc expression leading to G1 arrest and growth inhibition of human invasive urinary bladder cancer cells

Abstract

Epigenetic silencing of secreted wingless-type (Wnt) antagonists through hypermethylation is associated with tobacco smoking and with invasive bladder cancer. The secreted Wnt inhibitory factor-1 (WIF1) has shown consistent growth-inhibitory effect on various cancer cell lines. Therefore, we assessed the mechanisms of action of WIF1 by either restoring WIF1 expression in invasive bladder cancer cell lines (T24 and TSU-PR1) or using a recombinant protein containing functional WIF1 domain. Both ectopic expression of WIF1 and treatment with WIF1 domain protein resulted in cell growth inhibition via G 1 arrest. The G 1 arrest induced by WIF1 is associated with down-regulation of SKP2 and c-myc and up-regulation of p21/WAF1 and p27/Kip1. Conversely, reexpression of SKP2 in WIF1-overexpressing TSU-PR1 cells attenuated the WIF1-induced G 1 arrest. Furthermore, inhibition of nuclear Wnt signaling by either dominant-negative LEF1 or short hairpin RNA of TCF4 also reduced SKP2 expression. The human SKP2 gene contains two TCF/LEF1 consensus binding sites within the promoter. Chromatin immunoprecipitation/real-time PCR analysis revealed that both WIF1 and dominant-negative LEF1 expression decreased the in vivo binding of TCF4 and B-catenin to the SKP2 promoter. Together, our results suggest that mechanisms of WIF1-induced G 1 arrest include (a) SKP2 downregulation leading to p27/Kip1 accumulation and (b) c-myc down-regulation releasing p21/WAF1 transcription. Additionally, we show that WIF1 inhibits in vivo bladder tumor growth in nude mice. These observations suggest a mechanism for transformation of bladder epithelium on loss of WIF1 function and provide new targets such as SKP2 for intervention in WIF1-deficient bladder cancer.

Wnt inhibitory factor 1 induces apoptosis and inhibits cervical cancer growth, invasion and angiogenesis in vivo

“…This inhibition of cell proliferation is consistent with the in vitro inhibition of cell growth by WIF1 as previously shown for other cancer cell lines (Ai et al, 2006;Kim et al, 2007;Kansara et al, 2009;Kawakami et al, 2009;Tang et al, 2009). However, in previous studies, WIF1 overexpression was shown to induce G 1 arrest in HPVimmortalized osteoblasts and osteosarcoma cell lines containing null or wild-type p53 (Kansara et al, 2009), as well as in bladder cancer cell lines containing mutant p53 (Tang et al, 2009). In contrast, our data show that in cervical cancer cells, which express very low levels of wild-type p53, WIF1 re-expression led to a significant increase in p53 expression, and induced cell-cycle arrest at G 2 /M and extensive apoptosis.…”

“…Re-expression of WIF1 resulted in a significant reduction in the total number of viable cells at all time points (Figure 3a), indicating the potent growth-inhibitory effects of WIF1. In bladder carcinoma and osteosarcoma cell lines, WIF1 has been reported to induce G 1 arrest and growth inhibition (Kansara et al, 2009;Tang et al, 2009). However, consistent with the G 2 /M arrest observed in cervical cancer cells after DAC treatment, we observed a significant increase in the G 2 /M phase of the cell cycle in WIF1-transfected cells (Figure 3b), suggesting a cellspecific role for WIF1.…”

“…WIF1 inhibits cervical tumor growth in vivo Stably transfected WIF1 tumor cells have been used to study WIF1 biological function and to establish mice tumor xenograft models (Kawakami et al, 2009;Tang et al, 2009). These models have shown a slower tumor growth rate for WIF1-expressing cells.…”

“…Other in vivo studies have also evaluated the growth-inhibitory role of WIF1 in melanoma, lung, bladder, renal and prostate cancers (Kim et al, 2007;Lin et al, 2007;Kawakami et al, 2009;Tang et al, 2009;Yee et al, 2010). Most of these studies used tumor cells stably expressing the WIF1 transgene to show that WIF1 overexpression significantly attenuates tumor growth in mouse models.…”

Androgen activates β-catenin signaling in bladder cancer cells

“…These observations suggest that Wnt signaling is active in advanced urothelial tumors. Of note, the application of various small molecules that target the Wnt/b-catenin signaling pathway led to inhibition of bladder cancer cell proliferation (Urakami et al 2006, Tang et al 2009, Hirata et al 2012.…”