Lung metastasis constitutes the main cause of death in osteosarcoma (OS) patients. Survival has not improved in the past ten years. Therefore, new therapeutic strategies are needed. We previously reported that aerosol Gemcitabine (GCB) has a significant therapeutic effect against OS lung metastases. There was however, a subset of cells that fail to respond to GCB treatment resulting in persistent small isolated lung metastases that led to relapse and death. The mechanisms underlying OS resistance to GCB have remained elusive. Autophagy, a catabolic process involved in cellular homeostasis, has emerged as an important mechanism involved in cancer cells response to treatment. However, whether autophagy plays a role in OS resistance to GCB is unknown. Thus, the purpose of this study is to investigate the role of autophagy in OS lung metastases resistance to GCB. In this study, LM7 and CCH-OS-D human OS cells were treated with GCB and induction of autophagy was evaluated. GCB-induced autophagy was demonstrated by formation of acidic vesicular organelles (AVOs), conversion of microtubule-associated light chain 3 (LC3I/LC3II), increase in Beclin 1 (BECN1) and decrease in p62/SQSTM1 protein expression. Additionally, formation of autophagic vacuoles was confirmed by electron microscopy. GCB-induced autophagy also resulted in the inhibition of AKT /mTOR phosphorylation, suggesting this signaling pathway as a possible underlying mechanism responsible for GCB-induced autophagy in OS. To test whether autophagy contributes to OS cells survival to GCB, we determined the impact of autophagy inhibition on the sensitivity of OS cells to GCB. Sensitivity of LM7 cells to GCB was greatly enhanced after autophagy inhibition by the pharmacologic inhibitor hydroxychloroquine (HCQ) and shRNA targeting BECN1, suggesting autophagy as a pro-survival mechanism. Interestingly, inhibition of autophagy in CCH-OS-D cells decreased cell sensitivity to GCB, suggesting that GCB-induced autophagy contributed to cell death. Better understanding of the molecular pathways that govern the process of autophagy will allow identification of new strategies to enhance chemotherapeutic efficacy. Our preliminary work showed that the phosphorylation of Heat Shock protein 27 (HSP27) was significantly higher in GCB-treated LM7 cells when compared to CCH-OS-D GCB-treated cells, indicating a potential role of HSP27 in outlining the role of autophagy in osteosarcoma cells. Taken together, these results suggest that autophagy can be induced by GCB in OS cells and that inhibition of autophagy can lead to either increase or decrease OS cells sensitivity to Gemcitabine.

Citation Format: Janice M. Santiago-O'Farrill, Mario Hollomon, Eugenie Kleinerman, Nancy Gordon. HSP27 as a potential factor to determine the fate of Gemcitabine- induced autophagy in osteosarcoma: Survival vs. death. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2905. doi:10.1158/1538-7445.AM2015-2905