Poster Presentation 25th Lorne Cancer Conference 2013

Targeting of Hedgehog pathway via pharmacologic inhibition of Gli induces apoptosis and autophagy in malignant mesothelioma cells     (#247)

Chuan Bian Lim 1 2 3 , Philip J Thompson 1 2 3 , Svetlana Baltic 1 2 3 , YC Gary Lee 1 2 3 , D Neil Watkins 4 , Cecilia M Prele 1 2 3 , Steven E Mutsaers 1 2 3
  1. Lung Institute of Western Australia, Perth, Western Australia, Australia
  2. Centre for Asthma, Allergy and Respiratory Research, Perth, Western Australia, Australia
  3. School of Medicine and Pharmacology, University of Western Australia, Perth, Western Australia, Australia
  4. Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia

The Gli transcription factors (Gli1, Gli2 and Gli3) of the Hedgehog signaling pathway are key driver of cancer cell survival. In this study, we investigated the in vitro effect of a small molecule inhibitor of Gli, GANT61, on cell proliferation and apoptosis in human malignant mesothelioma (MM) cells. Inhibition of GLI by GANT61 led to a dose-dependent reduction in cell proliferation and clonogenic survival in all ten of the MM cell lines tested. GANT61 induced G1 cell-cycle arrest and apoptosis with increased annexin V staining. The expression of GLI1 and GLI2 mRNA was significantly reduced in GANT61-exposed cells in a time-dependent fashion. Furthermore, we found that GANT61 downregulated the expression of PTCH1 mRNA (direct target of GLI), suggesting that the antitumor action of GANT61 is mediated by Gli1/2. GANT61 triggered the generation of reactive oxygen species (ROS) and quenching of ROS by N-acetyl L-cysteine, a ROS scavenger, resulted in partial protection from GANT61-induced cytotoxicity, supporting ROS-mediated cytotoxic effects of GANT61 in MM cells. Interestingly, we demonstrate that GANT61 exerts a novel autophagic response in MM cells, as shown by vital dye staining of MM cells with acridine orange followed by the accumulation of acidic vesicular organelles in the cytoplasm of cells. This was inhibited by addition of bafilomycin A1, an autophagy inhibitor. Furthermore, combination of GANT61 with bafilomycin A1 potentiated the anticancer effect of GANT61 through induction of apoptosis, unravelling the cytoprotective role of GANT61-induced autophagy in MM cells. Quenching of ROS also partially but significantly reduced autophagy, suggesting that ROS triggers autophagy. Finally, median dose-effect analysis revealed that co-treatment with GANT61 and cisplatin induced synergistic cytotoxic effects against MM cells. Our results offer an initial preclinical proof-of-concept to co-target cytoprotective Gli and autophagy to improve treatment outcomes in MM.