Long-term smoking-mediated downregulation of Smad3 induces tumorigenicity and carboplatin resistance in non-small cell lung cancer

Abstract

Inactivating mutations in TGF-©¬ receptors and Smad signal transducers that contribute to resistance to TGF-©¬, are associated with only very small number of NSCLC. The Smad dependent pathway is involved in the tumor suppressive functions of TGF-¥â. Epidemiological studies have demonstrated that most cases of lung cancer (85-90%) are directly attributable to cigarette smoking. However, nothing is known how smoking is involved in inhibiting tumor suppressor functions of TGF-©¬ and whether or how the chemo resistance of platinum-based drugs seen in lung cancer patients who are smokers is dependent on this pathway. To address this issue, lung adenocarcinoma A549 and immortalized bronchial epithelial HPL1A cells were chronically treated (300 days) with cigarette smoke condensate (CSC) and Dimethyl sulphoxide (DMSO, as a control) to mimic the conditions of long-term cigarette smoking. Prolonged exposure of these cells to CSC resulted in decreased Smad-mediated TGF-¥â signaling due to reduced expression of Smad3 both in the protein and mRNA level. The decrease in Smad3 is due to histone deacetylation. Long-term CSC treatment reduced apoptosis, increased cell viability, decreased TGF-¥â-mediated growth inhibition, and enhanced tumorigenicity both in vitro and in vivo. Re-expression of Smad3 in the long-term CSC treated cells reversed the cancerous phenotypes observed due to long-term CSC treatment. Looking further into public databases revealed that the expression of Smad3 is lower in lung tumors of current smokers compared to that observed in never-smokers. The long-term CSC treatment also rendered the cells resistant to platinum-based chemotherapy; by up regulating Bcl2. Blocking the effect of Bcl2 both by small molecule inhibitor ABT-737 and siRNA approaches re-sensitized the cells to platinum-based chemotherapy. The re-expression of exogenous Smad3 in the long-term CSC treated cells, decreased the Bcl2 levels and re-sensitized the cells to platinum-based chemotherapy. Thus, Smad3 controls the expression of Bcl2 and sensitivity to platinum based drugs in our model system. Collectively, these data provide evidence that cigarette smoking promotes tumorigenicity and makes the cells resistant to platinum-based chemotherapy by abrogating apoptosis, partly by reducing expression of Smad3.