Abstract A25: GLI inhibition as a new strategy to treat lung squamous cell carcinoma.
Authors: Lingling Huang, Vonn Walter, Neil Hayes, Mark Onaitis
Journal: Clinical Cancer Research (2014)

Abstract Purpose: Lung squamous cell carcinoma (LSCC) comprises ~30% of non-small cell lung cancers and currently lacks effective targeted therapies. Although the overexpression of HEDGEHOG (HH)-GLI signaling components has been reported in LSCC patient samples, the requirement for HH-GLI signaling in regulating LSCC cellular survival and proliferation remains unknown. In this study, we investigated the role of HH-GLI signaling in LSCC, and studied the therapeutic potential of HH-GLI signaling inhibition. Experimental Design: Two independent LSCC patient cohorts were analyzed for gene expression to study the activation pattern of HH-GLI signaling. Four representative human LSCC cell lines were examined for expression of HH-GLI signaling components. Cell proliferation and apoptosis were assayed in these cells after blocking the HH-GLI pathway by lentiviral-shRNA knockdown or small molecule inhibitors. A xenograft model in immunodeficient mice was used to determine the in vivo therapeutic efficacy of GLI inhibitors. Results: LSCC has been classified into four distinct mRNA expression subtypes. In both cohorts, the classical subtype (~36% of LSCC) showed a HH-GLI activation pattern with increased expression of positive regulators and decreased expression of negative regulators relative to the other three subtypes. Particularly, GLI2 was consistently highly expressed in the classical subtype, and strong positive correlations between GLI2 and the classical subtype markers (SOX2, TP63 and PIK3CA) were observed, suggesting an important role of GLI2 in this subtype. In three GLI2-positive LSCC cell lines, genetic deletion of GLI2 by lentiviral-shRNAs caused extensive apoptosis and significant growth inhibition, indicating a critical role of GLI2 in regulating cell survival and cell death. Pharmacological inhibition of HH-GLI signaling by GLI inhibitor GANT61 phenocopied the loss of GLI2 in GLI2-positive cells, but had little effect on a GLI-negative cell line. In the xenograft model, GANT61 significantly suppressed tumor progression and reduced final tumor weight for GLI2-positive cell lines in comparison to solvent control, but had no effects on the GLI-negative LSCC tumors, suggesting a specific anti-tumor activity of GANT61. The expression of HH downstream targets was significantly reduced by GANT61 both in vitro and in vivo, demonstrating a successful blockage of the HH-GLI pathway. In contrast to GANT61, GDC-0449, a clinically available SMO inhibitor, only produced limited cytotoxicity in vitro despite the universal expression of SMO, indicating the existence of SMO-independent activation of GLI signaling in these cells. Arsenic trioxide (ATO), an FDA-approved treatment of acute promyelocytic leukemia, has recently been shown to inhibit GLI2 ciliary accumulation and promote its degradation in other cell types. Therefore, we are also investigating the clinical relevance of ATO in treating a selected subset of LSCC that has GLI2 overexpression. Conclusions: Our studies reveal an important role of GLI2 in LSCC cell survival. Different from standard-of-care chemotherapy or inhibiting kinase signaling cascades, we present a potential strategy to treat a particular subset of lung squamous cell carcinoma by targeting the GLI transcriptional network. Citation Format: Lingling Huang, Vonn Walter, Neil Hayes, Mark Onaitis. GLI inhibition as a new strategy to treat lung squamous cell carcinoma. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr A25.