We previously established that hepatocyte cell adhesion molecule (hepaCAM), a typical structure of immunoglobulin (Ig)-like adhesion molecules, inhibited the proliferation and the progression of cultured human bladder cancer cells. As increasing evidence reveals that aberrant activation of canonical Wnt pathway is involved in the pathogenesis of bladder cancer, and β-catenin serves as a pivotal molecule of Wnt pathway. Then, we explored whether the anti-proliferation effect of hepaCAM was associated with Wnt/β-catenin pathway in human bladder cancer cells. The negative correlation between hepaCAM and β-catenin in transitional cell carcinoma of bladder (TCCB) was found. Follow by, studied the effect of hepaCAM on the key elements of Wnt pathway. Here, Our researches showed that hepaCAM played a central role in modulating the Wnt/β-catenin signaling pathway by interfering nuclear protein levels of β-catenin, leading to down-regulate transcriptional activity of LEF/TCF and its target genes c-Myc and cyclinD1. Mechanistically, we demonstrated that hepaCAM-activated GSK3β led to elevate the phosphorylation of β-catenin, contributing to the aberrant translocation of β-catenin. In addition, Anti-proliferation and associated molecular mechanisms of hepaCAM were demonstrated by using vivo experiment. In conclusion, our reports uncover that expression of hepaCAM suppresses the proliferation of bladder cancer cells through a Wnt/β-catenin-dependent signaling pathway in vitro and in vivo.

We explored the correlation between hepaCAM and activated p-mTOR in bladder transitional cell carcinoma. We also determined whether the antiproliferation effect of hepaCAM is associated with the AMPK/mTOR pathway. We performed quantitative reverse transcriptase-polymerase chain reaction to determine hepaCAM mRNA expression as well as Western blot to measure hepaCAM and p-mTOR protein levels in 25 men and 5 women. Disease was Ta-T1 in 7 patients, T2-T4 in 23, grade 1 in 13, grade 2 in 9, grade 3 in 8, primary in 13 and recurrent in 17. The WST-8 assay was used to study the effect of hepaCAM on cellular proliferation. p-AMPK, p-mTOR, total AMPK, total mTOR, c-Myc and cyclin D1 were also determined by Western blot. hepaCAM mRNA and protein levels were significantly decreased, while p-mTOR protein was remarkably increased in bladder transitional cell carcinoma compared to adjacent tissues (each p<0.01). Spearman correlation analysis revealed that the hepaCAM decrease was associated with an increase in p-mTOR (r=-0.533, p=0.002). Also, hepaCAM inhibited the proliferation of human bladder transitional cell carcinoma cells. hepaCAM over expression activated AMPK and down-regulated p-mTOR, and its targets c-Myc and cyclin D1. Treatment with the AMPK inhibitor compound C prevented the antiproliferation effect of hepaCAM. Compound C completely blocked hepaCAM induced activation of AMPK and down-regulation of p-mTOR and its targets c-Myc and cyclin D1. Results suggest an important correlation between hepaCAM and p-mTOR. hepaCAM can inhibit bladder cancer cell proliferation through an AMPK/mTOR dependent pathway.