Mitophagy Induction by ROS-PINK1 Signaling Protects Head and Neck Cancer From Radiotherapy

Abstract

Approximate 25% of patients with local advanced head and neck cell carcinoma (HNSCC) treated with radiation still suffer from local relapse and are considered radiation resistant. We previously determined that radiation induces autophagy, a pro-survival cellular stress response, in HNSCC. In this study, we examine the consequence of autophagy inhibition and investigate the molecular mechanism underlying RT-induced autophagy.Autophagy was assessed using a nano-Luc LC3 reporter assay (Promega), immunoblotting and immunofluorescence for LC3 and acridine orange in multiple HNSCC cell lines. RNAi knockdown of EGFR, LAPTM4B and PINK1 were used to test the involved signaling molecules. CM-H2DCFDA was used as a measure of reactive oxygen species (ROS). Hydrogen peroxide was used to stimulate ROS production. Trolox, a ROS scavenger, was used to determine the correlation between ROS and RT-induced autophagy. Mitophagy determination was completed by using MitoTracker Red (Invitrogen) combined with immunofluorescence staining for LC3 and immunoblotting. Radiation was delivered using a RS225 cabinet irradiator at a dose rate of approximately 3 Gy/min with dose validation by TLD using custom, geometry specific phantoms. SAR405, a VPS34 inhibitor, was used to inhibit autophagy. A flank xenograft model using A253 cells was used to test the combination of autophagy inhibitors and radiotherapy in vivo.RT caused a two-fold increase in autophagy as assessed using the reporter assay and immunoblotting. Knockdown of EGFR and LAPTM4B, two proteins important in growth-factor deprivation induced autophagy, did not influence autophagy in RT-treated cells. RT did increase the accumulation of ROS (∼50%) and the dephosphorylation of mTOR (∼25%). Addition of Trolox to RT contributed to a considerable decrease in both ROS (∼50%) and autophagy (∼50%). RT also resulted in a mRNA elevation of PINK1 (∼1.6 fold), a mitochondrial modulator. Immunoblotting for LC3 in both the cytoplasmic and mitochondrial fraction indicated RT mostly increases mitophagy rather than bulk autophagy, which was confirmed using immunofluorescent staining for LC3 and MitoTracker red. Using a clonogenic survival assay, the combination of SAR405 and RT resulted in complete loss of cell survival suggesting a radiosensitizing effect. In vivo, SAR405 treatment combined with RT improved tumor control when compared to RT or SAR405 alone.RT-induced mitophagy involves ROS-PINK1 pathway and the regulation of mTOR. Inhibition of autophagy resulted in decreased cell survival in vitro and decreased in vivo tumor growth. These results suggest that targeting mitophagy may be a viable approach to sensitize HNSCC to radiation treatment.