SMAD6 Contributes to Patient Survival in Non–Small Cell Lung Cancer and Its Knockdown Reestablishes TGF-β Homeostasis in Lung Cancer Cells

Abstract

The malignant transformation in several types of cancer, including lung cancer, results in a loss of growth inhibition by transforming growth factor-β (TGF-β). Here, we show that SMAD6 expression is associated with a reduced survival in lung cancer patients. Short hairpin RNA (shRNA)–mediated knockdown of SMAD6 in lung cancer cell lines resulted in reduced cell viability and increased apoptosis as well as inhibition of cell cycle progression. However, these results were not seen in Beas2B, a normal bronchial epithelial cell line. To better understand the mechanism underlying the association of SMAD6 with poor patient survival, we used a lentivirus construct carrying shRNA for SMAD6 to knock down expression of the targeted gene. Through gene expression analysis, we observed that knockdown of SMAD6 led to the activation of TGF-β signaling through up-regulation of plasminogen activator inhibitor-1 and phosphorylation of SMAD2/3. Furthermore, SMAD6 knockdown activated the c-Jun NH2-terminal kinase pathway and reduced phosphorylation of Rb-1, resulting in increased G0-G1 cell arrest and apoptosis in the lung cancer cell line H1299. These results jointly suggest that SMAD6 plays a critical role in supporting lung cancer cell growth and survival. Targeted inactivation of SMAD6 may provide a novel therapeutic strategy for lung cancers expressing this gene.

Prognostic and Predictive Value of Cell Cycle Deregulation in Non-Small-Cell Lung Cancer

“…An in vitro study showed that overexpression of the TGF-β receptor II restored TGF-β sensitivity and reduced tumor growth in lung cancer cells [212]. Data on targeting the TGF-β signaling pathways in lung cancer are sparse; however, a recent study suggests that deactivating the inhibitory downstream molecule SMAD6 may improve patient outcome [213]. Taken together, TGF-β plays a complex and often diverse role in cancer which requires further clarification, particularly with regard to possible therapeutic approaches involving TGF-β pathways.…”

Dephosphorylation of threonine-821 of the retinoblastoma tumor suppressor protein (Rb) is required for apoptosis induced by UV and Cdk inhibition

“…[10][11][12] Further, the observation that Rb is dephosphorylated in apoptosis induced by various stimuli also supports a required role for Rb dephosphorylation in apoptosis. [13][14][15][16][17][18][19] Because it was unclear which phosphorylation sites of Rb were involved in the apoptotic signal, we utilized glutamic acid mutagenesis to individually change each Rb phosphorylation site to glutamic acid, to mimic the phosphorylated state. A previous study utilizing glutamic acid mutagenesis of the Rb protein employed phosphorylation site mutants that contained different combinations of between 1-12 altered sites.…”

“…[10][11][12] Loss of phosphorylation of the Rb protein has been observed during apoptosis. [13][14][15][16][17][18][19] Also, the specific activation of Rb-directed phosphatase activity has been shown to be required for apoptosis to occur. 13,14,16,20,21 These studies suggest that phosphorylated Rb protects against apoptosis, and dephosphorylation of Rb is involved in triggering cell death.…”

TGF-² Signaling and the Role of Inhibitory Smads in Non-small Cell Lung Cancer

“…25 In lung cancer, Smad6 is overexpressed in a portion of the tumors, and high expression of Smad6 is associated with poor survival in patients with non-small cell lung cancer. 26 Knockdown of Smad6 in overexpressed lung cancer cells induced apoptosis and growth arrest at the G1 phase, which contributed to the reestablishment of TGF- β homeostasis in lung cancer cells by reactivating the TGF- β signal pathway. 26 Similarly, overexpression of Smad7 causes malignant conversion in multistage cancer models by cooperating with oncogenic Ras and enhances tumorigenicity in pancreatic cancer.…”