Nonclassical activation of gli1 as a therapeutic target for squamous cell lung cancer

Abstract

Background: The Hedgehog (Hh) signaling pathway is critical for embryonic development and its deregulation is implicated in a number of tumor types. The role of the Hh signaling pathway, however, in the initiation and growth of non-small cell lung cancer is largely unknown. Here, we investigate the role of the Hh pathway transcription factor, GLI1, in lung squamous cell carcinoma (SCC) and as a potential therapeutic target for treatment of lung SCC. Methods: GLI1 expression in human SCC cell lines was evaluated by quantitative PCR and Western Blot. siRNA and shRNA of GLI1 in these cell lines were utilized in vitro and in vivo to test the requirement of GLI1 in tumor growth. Small molecule modulators of GLI1 were tested for their therapeutic potential. Results: GLI1 mRNA expression was significantly elevated in lung SCC compared to normal lung and lung adenocarcinoma patient specimens in several human genomic databases. Importantly, overexpression of GLI1 was correlated with poor overall survival in lung cancer patients. siRNA-mediated knock down of GLI1 in SCC cell lines decreased the expression of GLI1 target genes and caused a significant reduction in colony formation. Stable knock down of GLI1 in SCC cell lines caused a significant reduction in growth of xenograft tumors indicating the critical role of GLI1 in lung SCC progression. Inhibition or activation of SMO, an upstream component of Hh pathway, did not alter GLI1 expression level in lung SCC cell lines. However, inhibition of PI3K/AKT and MAPK signaling pathways down-regulated GLI1 expression, suggesting that GLI1 expression is dependent on PI3K/AKT and MAPK pathway activity rather than Hh ligand. Small molecule inhibition of PI3K/ mTOR pathway or GLI1 significantly reduced GLI1 expression, proliferation, and clonogenicity in SCC cell lines. Combinatorial inhibition of PI3K and GLI1 by BKM120 and arsenic trioxide (ATO), respectively, significantly abrogated the in vivo growth SCC tumors in mice and correlated with decreased tumor GLI1 expression. Conclusion: Our findings demonstrate that GLI1 is essential for lung SCC tumor progression. Furthermore, GLI1 expression in SCC is independent of Hh pathway ligand action and dependent on MAPK and PI3K pathway activity. Direct inhibition of GLI1 by repurposing ATO in combination with a PI3K inhibitor may represent a novel therapeutic strategy for lung SCC.