Abstract 2200: CCN6/WISP3 exerts its tumor suppressor function through regulation of BMP signaling by direct binding to BMP4 in the extracellular environment

Abstract CCN6 is a secreted protein with tumor suppressor function in the most aggressive form of locally advanced breast cancer, inflammatory breast cancer. CCN6 was reported to be lost in 80% cases of inflammatory breast cancer and 19% cases of stage matched non inflammatory breast cancer. It was shown that CCN6 knockdown (KD) in benign breast cells triggered an epithelial to mesenchymal transition with E-cadherin downregulation (Am J Pathol 172(4):893-904, 2008) recently shown to be associated with acquisition of growth factor independent signaling (Cancer Res 70 (8):3340-3350, 2010). The molecular mechanism by which CCN6 regulates extracellular signaling to suppress breast cancer invasion is not known. As a member of the CCN family of matricellular proteins, named after connective tissue growth factor, cysteine-rich 61, and nephroblastoma overexpressed, CCN6 shares the highly conserved protein motifs of the CCN family, which in some cases have been reported to bind to bone morphogenetic protein (BMP) and transforming growth factor beta (TGFβ) (J Cell Sci 119(23):4803-4810, 2006). We hypothesized that the invasive phenotype of CCN6 KD cells may be dependent upon cytoskeletal alterations through modulation of growth factor signaling pathways like TGFβ/BMP. The invasive phenotype of lentivirus mediated control and CCN6 KD cells was assessed by growth on matrigel for 15 days following published protocol. The effect on TGFβ/BMP signaling was examined by a pathway specific PCR array for TGFβ/BMP (SA Biosciences), immunoblotting and immunocytochemistry. To ascertain the tumor suppressor effect of CCN6, we examined if recombinant human CCN6 (rhCCN6) could revert the invasive phenotype by treatment with 500 ng/ml rhCCN6 (PeproTech). Similarly small molecule inhibitors were used to inhibit activated P38 kinase (5 μM SB203580), TGFβ type I receptor I kinase (TβRI) (400 nM SB431542) and TGFβ activated kinase1 (TAK1) (0.3 μM 5Z-7-oxozeaenol). The specific binding of CCN6 with BMP4 was confirmed by co-immunoprecipitation, crosslinking, competitive binding and surface plasmon resonance. We found that CCN6 loss in benign breast cells triggers a highly invasive phenotype with loss of α6 integrin, which was rescued by treatment with rhCCN6. CCN6 loss in HME cells antagonized BMP4-mediated Smad1/5 activation and enhanced the effect of BMP4 on the non-canonical pathway to activate TAK1 and P38 through activation of TβRI. Small molecule inhibition of activated P38 or TAK1 or TβRI, or of BMP4 by blocking antibody reverted the invasive phenotype. CCN6 protein had the opposite effect, potentiation of BMP4-mediated Smad1/5 signaling by direct binding to BMP4. Conclusively, by direct binding to BMP4 CCN6 regulates BMP4 mediated Smad and alternate P38 signaling. Thus, CCN6 in the extracellular space may act as a master switch and regulator of BMP4 to suppress invasion in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2200. doi:10.1158/1538-7445.AM2011-2200