Abstract A007: Hsp27 Mediates Sensitivity to Gemcitabine through Mitophagy Regulation in Osteosarcoma

Abstract

Osteosarcoma (OS) is the most common primary malignant bone tumor in children, comprising approximately 3% of all childhood cancers. Metastatic OS remains a clinical challenge largely due to pulmonary metastases, the main cause of death in these patients. Gemcitabine (GCB) alone or in combination with docetaxel, has been recommended as a second line therapy for relapsed OS, though its efficacy remains modest. We have previously demonstrated phosphorylated Heat Shock Protein 27 (HSP27) as a potential biomarker of chemotherapy-induced autophagy response in OS. In addition, HSP27 has been implicated in the regulation of mitophagy, the selective autophagic process targeting mitochondria. Thus, the purpose of our studies is to evaluate the role of Hsp27 in regulating mitophagy in osteosarcoma (OS) and to determine whether it influences cellular sensitivity to gemcitabine. Methods: Human OS cell lines LM7 and CCH-OS-D were used for the in-vitro studies. Mitophagy activity under basal conditions or following GCB treatment was assessed by immunoblot analysis of PINK1 and Parkin expression. In parallel, total Hsp27 and phosphorylated Hsp27 (Ser82) levels were assessed by immunoblotting. Additionally, fluorescence-based mitophagy detection assay was conducted in the OS cells under basal conditions and following GCB treatment. To determine whether mitophagy activation depends on Hsp27, LM7 cells with stable Hsp27 knockdown (LM7 Hsp27 KD) were generated via shRNA transfection. Knockdown efficiency was confirmed by immunoblot analysis of Hsp27 expression. Mitophagy was then evaluated by measuring PINK1 and Parkin levels by immunoblotting. To determine the impact of HSP27 depletion on GCB efficacy, LM7 Hsp27 KD cells were treated with GCB, and the apoptotic signaling was assessed by immunoblotting for cleaved PARP1 and cleaved caspase 3, while cell viability was monitored using the Incucyte system. Results: LM7 cells showed higher basal mitophagy and Hsp27 activity compared to CCH-OS-D as evidenced by elevated PINK1 and Parkin levels and an increased Phospho-Hsp27 ratio. In CCH-OS-D these levels were low to undetectable, limiting assessment of HSP27-dependent effect in this model. GCB further induced mitophagy and Hsp27 activation in LM7 cells, whereas no comparable effect was observed in CCH-OS-D cells, as confirmed by immunoblotting and fluorescent-based mitophagy assay. Knocking down Hsp27 in LM7 reduced both total and Phospho-Hsp27 levels and was accompanied by decreased PINK1 and Parkin expression. Importantly, Hsp27 depletion increased GCB sensitivity, as demonstrated by increased cell death in Incucyte assay and augmented apoptotic signaling, reflected by higher levels of cleaved PARP1 and cleaved caspase 3 on immunoblotting. Conclusion: Hsp27 expression modulates both basal and GCB-induced mitophagy in OS. Hsp27-mediated mitophagy is associated with reduced GCB efficacy, suggesting that targeting Hsp27-regulated mitophagy may enhance therapeutic response. Yan Zheng, Nancy Gordon, Ariana Anjer, Eugenie Kleinerman, Heping Wang. Hsp27 Mediates Sensitivity to Gemcitabine through Mitophagy Regulation in Osteosarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Breaking Barriers in the Fight against Rare Cancers; 2026 Jul 18-20; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(14_Suppl):Abstract nr A007.