Abstract A03: Autophagy and HSP27: A potential link to define autophagy fate in osteosarcoma

Abstract

Survival in osteosarcoma (OS) has not improved in over a decade, thus the need for new therapeutic strategies. Autophagy, a catabolic process used by cells to survive under stress, has been implicated in resistance to chemotherapy. We previously demonstrated aerosol gemcitabine (GCB) efficacy against OS lung metastasis and showed GCB to induce autophagy through the Akt/mTOR signaling pathway. Whether autophagy contributes to tumor response or resistance to GCB has been the focus of our studies. What determines whether chemotherapy-induced autophagy will lead to survival or death is unknown. We found phosphorylated heat shock protein 27 (pHSP27) to potentially define the outcome of chemotherapy-induced autophagy in OS. Small heat shock proteins, specifically HSP27, are upregulated in many cancers, including OS, and associated with treatment resistance. The purpose of this study is to investigate if induction of pHSP27 predicts the role of chemotherapy-induced autophagy in OS lung metastasis. We hypothesize that when pHSP27 is induced, blocking autophagy will lead to increased sensitivity to GCB. Furthermore, inhibition of pHSP27 may revert the effect of GCB-induced autophagy in OS cells by decreasing sensitivity to GCB. We showed in the LM7 and CCH-OS-D human metastatic OS cells in vitro, that GCB induces autophagy as determined by the conversion of microtubule-associated light chain 3 one to two (LC3I/LC3II), increase in Beclin 1, and decrease in p62 protein expression. Sensitivity of LM7 cells to GCB was enhanced after autophagy inhibition by hydroxychloroquine (HCQ), a pharmacologic inhibitor and shRNA targeting Beclin 1, suggesting autophagy as a prosurvival mechanism whereas in CCH-OS-D cells inhibition of autophagy decreased cell sensitivity to GCB, concluding here that induction of autophagy leads to cell death. We demonstrated that pHSP27 is significantly higher in GCB-treated LM7 cells as compared to CCH-OS-D cells. We also showed that inhibition of HSP27 by shRNA targeting HSP27 has no effect on GCB-induced autophagy in LM7 cells. Treatment of shHSP27LM7 with GCB resulted in an increase in LC3I/LC3II conversion and a decrease in p62. We further revealed this in vivo by conducting a pilot study in which shHSP27LM7 cells were injected i.v. into nude mice. 6 weeks later, lung metastases were confirmed. Animals were divided into two groups: untreated and aerosol GCB treated (1mg/mL three times a week). Mice were sacrificed after 2 weeks of therapy. Transmitted electron microscopy demonstrated an increase in the number of autophagosomes in lung metastasis from mice treated with GCB, confirming induction of autophagy in the absence of HSP27. Lastly, in order to demonstrate that inhibition of HSP27 will change GCB-induced autophagy response in LM7 cells, we treated shHSP27LM7 cells with the combination GCB+HCQ and showed increased viability compared to the LM7 GipZ control cells, confirming preliminarily our hypothesis that induction of pHSP27 will determine chemotherapy-induced autophagy response in OS. Citation Format: Grace Nehme, Kumar Felix, Andrew Wahba, Diana M. Fandino, Nancy Gordon. Autophagy and HSP27: A potential link to define autophagy fate in osteosarcoma [abstract]. In: Proceedings of the AACR Special Conference on Targeting PI3K/mTOR Signaling; 2018 Nov 30-Dec 8; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(10_Suppl):Abstract nr A03.