Endocrine Abstracts

JOINT1180

Introduction: Adrenocortical carcinoma (ACC) is a rare, aggressive cancer with limited treatment options and frequent resistance to chemotherapy, highlighting the need for improved therapies. Hyperthermia is used to treat ACC metastases primarily through radiofrequency ablation to control disease burden. Incomplete tumour ablation risks exposure to sub-lethal hyperthermia in the transitional zone of heating with the potential for development of thermotolerance. Few studies have examined the biological effects of hyperthermia on ACC cell survival and function. This study explored the thermotolerance in ACC, where cells resist subsequent thermal stress after initial sublethal heat exposure. We hypothesise that sublethal hyperthermia induces thermotolerance to subsequent exposure at 48°C or 50°C, compared to “naïve” (non-hyperthermia exposed) cells, mediated by the heat shock response and TMEM16F, a calcium-dependent scramblase involved in cellular repair.

Methods: ACC primary cell lines, H295R and HAC15, and the metastatic cell line MUC-1, were pre-treated at 45°C using heat-controlled water baths. Cells were rechallenged at 48°C or 50°C after 24 hours or 7 days and compared to naïve cells. Cell death was assessed via Sytox Blue staining via flow cytometry, and protein expression of HSP70, HSP90, P-HSP27, and TMEM16F was analysed using Western Blot.

Results: Surviving ACC cells previously exposed to hyperthermia demonstrated evidence of heat stress following hyperthermia but did not develop thermotolerance. There was no difference in viability between naïve and pre-treated cells at 48°C or 50°C, though MUC-1 showed higher resistance at higher temperatures (≥48°C) compared to H295R and HAC15. While heat stress was evident in all cells following hyperthermia, there was no significant difference in HSP70 or HSP90 expression post-rechallenge between naïve and pre-exposed cells. A marked reduction in P-HSP27, was observed in both naïve and rechallenged cells at 50°C and this reappeared 24 hours post-rechallenge. Additionally, TMEM16F expression was lost immediately at both 48°C and 50°C but reappeared 24 hours post-rechallenge at 48°C only.

Conclusion: Hyperthermia of 45°C did not confer thermotolerance in H295R and HAC15 cells. The marked reduction in P-HSP27 and transient loss of TMEM16F suggest a diminished heat shock response at higher temperatures. Their reappearance 24 hours later indicates a delayed role in recovery but this is insufficient to confer protection against subsequent thermal stress. These findings emphasise the complexity of ACC cell responses to hyperthermia but are reassuring clinically, indicating that thermotolerance may not develop in transitional zones of cancers incompletely ablated whereby they remain sensitive to further ablation challenges.