Date of Graduation
Doctor of Philosophy (PhD)
Eugenie S. Kleinerman, MD
Hesham Amin, MD
Joya Chandra, Ph.D.
Juan Fueyo, MD
Varsha Ghandi, Ph.D.
Despite treatment improvement for osteosarcoma (OS), overall survival has remained unchanged in the last 20 years. Pulmonary metastasis continues to be the main cause of death; novel therapeutic strategies are urgently needed to improve the survival rate of these patients. Previous data in our laboratory has demonstrated that aerosol gemcitabine (GCB) treatment has a significant therapeutic effect on metastatic OS. However, treatment efficacy is decreased due to acquired resistance by a population of tumor cells that fails to respond to treatment. Recent studies have implicated autophagy as a resistance mechanism in various types of cancer. The purpose of this study was to determine whether the process of autophagy plays a role in protecting OS cells from the cytotoxic effects of GCB, allowing them to survive and avoid drug-induced cell death.
We first evaluated the ability of GCB to induce autophagy using a panel of OS cells. Following GCB treatment Acridine orange staining revealed the formation of acidic vesicular organelles (AVOs), a hallmark of autophagy. Western blot analysis showed an increase in several autophagy markers including; increased conversion of microtubule-associated light chain 3 (LC3I/LC3II), increased Beclin expression and a decrease in p62/SQSTM1 protein expression. Formation of autophagic vacuoles was also demonstrated using electron microscopy. These results confirm that autophagy was induced following GCB treatment. Furthermore, western blot analysis and kinase array assays demonstrated that GCB treatment decrease the phosphorylation of AKT, mTOR, p70S6K and PRAS40, suggesting that this signaling pathway is involved in the induction of autophagy by GCB.
To test whether autophagy contributes to the resistance of OS cells to GCB, we determined the impact of autophagy inhibition on the sensitivity of OS cells to GCB. Autophagy inhibition by either the pharmacologic inhibitor Hydroxychloroquine or genetic inhibition of BECN and ATG5 resulted in either increased or decreased sensitivity of OS cells to gemcitabine depending on the cell line tested. Specifically, the sensitivity of LM7 cells to GCB was greatly enhanced after autophagy inhibition, suggesting autophagy as a cytoprotective mechanism. However, inhibition of autophagy in CCH-OS-D and K7M3 cells decreased cell sensitivity to GCB, suggesting that GCB-induced autophagy contributed to cell death.
A more thorough understanding of the molecular pathways that govern the autophagy process may allow the identification of a biologic marker that can predict whether autophagy function to inhibit or promote GCB-induced cytotoxicity. Our preliminary data showed that phosphorylation of Heat Shock Protein 27 (HSP27) correlated with autophagy as a mechanism of cellular resistance in different OS cell lines. Taken together, these results suggest that GCB induces autophagy in OS cells through the AKT/mTOR pathway and that when HSP27 is induced inhibition of autophagy will increase sensitivity to GCB.
Autophagy, Osteosarcoma, Gemcitabine, Hydroxychloroquine, HSP27, ATG5, BECLIN, mTOR, AKT