BNIP3 modulates the interface between B16-F10 melanoma cells and immune cells

// Erminia Romano 1 , Nicole Rufo 1 , Hannelie Korf 2, 3 , Chantal Mathieu 3 , Abhishek D. Garg 1 and Patrizia Agostinis 1 1 Laboratory for Cell Death Research and Therapy (CDRT), Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium 2 Laboratory of Hepatology, Department of Chronic Diseases, Metabolism and Ageing (CHROMETA), KU Leuven, Leuven, Belgium 3 Laboratory of Clinical and Experimental Endocrinology (CEE), Department of Chronic Diseases, Metabolism and Ageing (CHROMETA), KU Leuven, Leuven, Belgium Correspondence to: Patrizia Agostinis, email: patrizia.agostinis@kuleuven.be Abhishek D. Garg, email: abhishek.garg@kuleuven.be Keywords: cancer; immunogenic cell death; phagocytosis; macrophage; anticancer vaccination Received: December 23, 2017      Accepted: February 27, 2018      Published: April 03, 2018 ABSTRACT The hypoxia responsive protein BNIP3, plays an important role in promoting cell death and/or autophagy, ultimately resulting in a cancer type-dependent, tumour-enhancer or tumour-suppressor activity. We previously reported that in melanoma cells, BNIP3 regulates cellular morphology, mitochondrial clearance, cellular viability and maintains protein expression of CD47, a pro-cancerous, immunosuppressive 'don't eat me' signal. Surface exposed CD47 is often up-regulated by cancer cells to avoid clearance by phagocytes and to suppress immunogenic cell death (ICD) elicited by anticancer therapies. However, whether melanoma-associated BNIP3 modulates CD47-associated immunological effects or ICD has not been explored properly. To this end, we evaluated the impact of the genetic ablation of BNIP3 (i.e. BNIP3 KD ) in melanoma cells, on macrophage-based phagocytosis, polarization and chemotaxis. Additionally, we tested its effects on crucial determinants of chemotherapy-induced ICD (i.e. danger signals), as well as in vivo anticancer vaccination effect. Interestingly, loss of BNIP3 reduced the expression of CD47 both in normoxic and hypoxic conditions while macrophage phagocytosis and chemotaxis were accentuated only when BNIP3 KD melanoma cells were exposed to hypoxia. Moreover, when exposed to the ICD inducer mitoxantrone, the loss of melanoma cell-associated BNIP3 did not alter apoptosis induction, but significantly prevented ATP secretion and reduced phagocytic clearance of dying cells. In line with this, prophylactic vaccination experiments showed that the loss of BNIP3 tends to increase the intrinsic resistance of B16-F10 melanoma cells to ICD-associated anticancer vaccination effect in vivo . Thus, normoxic vs. hypoxic and live vs. dying cell contexts influence the ultimate immunomodulatory roles of melanoma cell-associated BNIP3.