Abstract 1466: Smad 7 induces invasion, migration and in vivo metastasis in Non Small Cell Lung Cancer

Abstract

Abstract Lung cancer is one of the most common cancers, 75% of the cases being non small cell lung cancer (NSCLC). Despite recent advances in the cancer treatment, invasion and metastasis remains the major cause of cancer associated mortality. Metastasis is a multi-step process which involves local invasion, intravasation, dissemination, extravasation, and colonization. Transforming growth factor beta (TGF-β) is known to facilitate metastasis during the advanced stages of cancer via activating the canonical Smad signaling pathway. Smad 7 is an inhibitor of TGF-β signaling and there are previous reports that this protein can promote and inhibit metastasis in different cell lines. However, a distinct role of Smad7 in NSCLC metastasis has not been investigated so far. In the present study, we screened for the basal expression of Smad 7 in different solid cancer cell lines (breast, cervical, colorectal, NSCLC), observing a differential Smad7 expression. Using Q-RT-PCR, we detected a low mRNA expression of Smad 7 in the two NSCLC cells A549 and H1299. We investigated the effect of Smad 7 over expression in these two cell lines using an adenovirus vector (AdSmad7), and confirmed Smad 7 protein expression by western blot. Additionally, we performed MTT cell proliferation assays, which revealed no significant change in proliferation in Smad 7 over expressing cells. In vitro invasion assays, upon over expression of Smad7, showed a 3- fold increase of invasion in A549 and a 2-fold increase in H1299 cells compared to control cells transduced with the empty vector (AdCMV) (p<0.01). Consistent with the data obtained with Matrigel-Invasion Assays, wound healing assays also showed an increased migration of Smad 7 over expressing cells in a time dependent manner. In initial mechanistic studies, we found that Smad 7 over expression caused an increased activation of Src signaling. This was paralled by an increase of promoter activity and mRNA expression of the known downstream targets u-PAR, MMP-2, MMP-7 and MMP-9, proved by reporter gene assays and Q-RT-PCR. To study whether Smad7 affects in vivo metastasis, we performed Chicken embryo metastasis (CAM) assays and found a significant increase in lung metastasis in Smad 7 over expressing H1299 cells (p = 0.01). Finally, to support the clinical relevance obtained by the in vivo and in vitro studies, we screened 48 NSCLC patients for the endogenous expression of Smad7. By Q-RTPCR, we found that patients with a low endogenous Smad 7 expression in resected tumors showed a significantly better overall survival (p = 0.01). This study shows for the first time that Smad7 increases invasion and metastasis of NSCLC cells, this being paralled by Src activation and induced u-PAR and MMPs expression, and that Smad 7 might be a novel prognostic marker in NSCLC. 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 1466. doi:10.1158/1538-7445.AM2011-1466