The 3p14.2 tumour suppressor ADAMTS 9 is inactivated by promoter CpG methylation and inhibits tumour cell growth in breast cancer

Chromosome region 3p12‐14 is an important tumour suppressor gene ( TSG ) locus for multiple cancers. ADAMTS 9 , a member of the metalloprotease large family, has been identified as a candidate 3p14.2 TSG inactivated by aberrant promoter CpG methylation in several carcinomas, but little known about its expression and function in breast cancer. In this report, ADAMTS 9 expression and methylation was analysed in breast cancer cell lines and tissue samples. ADAMTS 9 RNA was significantly down‐regulated in breast cancer cell lines (6/8). After treating the cells with demethylation agent Aza and TSA , ADAMTS 9 expression was dramatically increased. Bisulphite genomic sequencing and methylation‐specific PCR detected promoter methylation, which was associated with decreased ADAMTS 9 expression. Hypermethylation was also detected in 130/219 (59.4%) of primary tumours but only in 4.5% (2/44) of paired surgical margin tissues. Ectopic expression of ADAMTS 9 in tumor cells induced significant growth suppression, cell cycle arrest at the G0/G1 phase, enhanced apoptosis and reduced cell migration and invasion. Conditioned culture medium from ADAMTS 9 ‐transfected BT 549 cells markedly disrupted tube formation ability of human umbilical vein endothelial cell ( HUVEC ) in Matrigel. Furthermore, ADAMTS 9 inhibited AKT signaling and its downstream targets ( MDM 2, p53, p21, p27, E‐cadherin, VIM , SNAIL , VEGFA , NF κB‐p65 and MMP 2). In addition, we demonstrated, for the first time, that ADAMTS 9 inhibits AKT signaling, through suppressing its upstream activators EGFR and TGF β1/TβR(I/ II ) in breast cancer cells. Our results suggest that ADAMTS 9 is a TSG epigenetically inactivated in breast cancer, which functions through blocking EGFR ‐ and TGF β1/TβR(I/ II )‐activated AKT signaling.