Abstract 14: CCN6 knockdown-induced epithelial to mesenchymal transition and invasion is mediated through upregulation of TGFβ type III receptor

Abstract

Abstract Epithelial to mesenchymal transition (EMT) is an important event during tumor invasion and metastasis. EMT is regulated by extracellular matrix components and soluble growth factors (reviewed in Nat Rev Mol Cell Biol 7:131-42, 2006). Secreted protein CCN6 is a multimodular matricellular regulatory protein, reported to be lost in 80% cases of highly aggressive inflammatory breast cancer subtype and 60% cases of all invasive breast carcinomas. Our previous studies showed that CCN6 regulates epithelial differentiation and its loss triggers EMT in human mammary epithelial cells (HME) through loss of E-cadherin by inducing Snail and Zeb1 (Am J Pathol 172(4):893-04, 2008). However the mechanism is not known. TGFβ signaling is known to cause EMT and invasion at advance stages of tumor progression by inducing Zeb1 (reviewed in J Mammary Gland Biol Neoplasia 15(2):169-90, 2010). Since, the structure of CCN6 predicts binding sites for TGFβ, we hypothesized that CCN6 loss induced EMT and invasion is mediated through deregulation of TGFβ signaling. HME cells with lentivirus mediated knockdown (KD) of CCN6 were characterized by 3 dimensional cultures. TGFβ-Smad response was examined in serum starved cells in a dose and time dependent manner. Growth, invasion, EMT and Smad-response were tested in control and CCN6 KD cells with siRNA or lentivirus mediated inhibition of TGFβ type III receptor (TβR III); and in CCN6 overexpressing human metastatic MDAMB231 cells (which have low protein levels of CCN6) in vitro and in vivo. We found that the CCN6 KD cells present a very malignant phenotype with loss of alpha 6 integrin. Associated with this, we observed an upregulaton of transcript (40 fold, *) and protein levels of TβR III, and enhanced activation of Smad2 in response to TGFβ1/TGFβ2/TGFβ3. Inhibition of upregulated TβR III blunted invasion and growth by reducing pSmad2 levels. The CCN6/TβR III double KD cells showed mesenchymal to epithelial transition with re-expression of E-cadherin and reversion of the malignant phenotype into polarized acini. Strengthening our findings CCN6 overexpression in MDAMB231 cells reduced invasion and growth by reducing pSmad2 levels. In vivo, CCN6 overexpressing mammary tumors showed dramatic decrease in tumor volume (70% at 7 weeks of injection, *, n=15 mice/group) and prolonged metastasis free survival with the primary tumors and mets showing more epithelial phenotype, unlike the EMT-like phenotype of controls. Further, treating CCN6 KD cells with 500 ng/ml of recombinant human CCN6 reduced transcript levels of TβR III while no effect was observed on control cells. Conclusively, CCN6 loss induced EMT is mediated through upregulation of TβR III. CCN6 can maintain the epithelial phenotype by regulating E-cadherin through modulation of TβR III. Thus, CCN6 emerges as a therapeutic target that can suppress tumor progression by modulating TGFβ signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 14. doi:1538-7445.AM2012-14