Nitroprusside induces melanoma ferroptosis with serum supplementation and prolongs survival under serum depletion or hypoxia

When proliferating tumor cells expand to areas distant from vascular sites, poor diffusion of oxygen and nutrients occur, generating a restrictive hypoxic gradient in which susceptible tumor cells die. The heterogeneous population surviving hypoxia and metabolic starvation include de-differentiated cancer stem cells (CSC), capable of self-renewing tumor-initiating cells (TICs), or those that divide asymmetrically to produce non-tumor-initiating differentiated (NTI-D) cell progeny. Under such restrictive conditions, both populations slowly proliferate, entering quiescence or senescence, when exiting from cell cycle progression. This may drive chemoresistance and tumor recurrence, since most anti-cancer treatments target rapidly proliferating cells. Since persistent or additional stress may increase NTI-D cells conversion to TICs, we investigated whether nutrient depletion or hypoxia influence expression of tyrosinase, a crucial enzyme for melanin synthesis, and B16 melanoma survival, when exposed to iron-dependent cell death oxidative stress produced by the Fenton reaction, resembling ferroptosis. - a) proliferating B16 melanoma with 10% serum-supplementation (10%S) normoxically express hypoxia inducible factor 1α (HIF1α) but lose tyrosinase, in contrast to those transiently exposed to (SF) serum-free medium, in which both HIF1α and tyrosinase are co-expressed; b) in contrast to the resistance to SNP toxicity in (SF) cells with higher tyrosinase expression, those in (10%S) are killed by iron from nitroprusside/ferricyanide (SNP) irrespective of exogenous H 2 O 2 , in a reaction antagonized by the anti-oxidant and MEK inhibitor UO126; c) Moreover, under transient serum depletion, SNP cooperates with hypoxia (1.5% oxygen), prolonging B16 melanoma (SF) survival; d) the hypoxia mimetic CoCl 2 inhibits proliferation-associated cyclin A, irrespective of SNP, in (10%S) cells or in transiently serum-depleted (SF) cells. However, only in the latter cells, CoCl 2 but not SNP, induce loss of HIF1α and apoptosis-associated PARP cleavage; e) longer term adaptation to survive serum depletion, generates ( SS) cells resistant to SNP toxicity, which aerobically co-express HIF1α and tyrosinase. In SS B16 melanoma, exogenous non-toxic 100 μM H 2 O 2 super-induces the ratio of tyrosinase to HIF1α. However, co-treatment of SS-B16 cells with SNP plus exogenous H 2 O 2, partly increases PARP cleavage by reciprocally decreasing tyrosinase expression. SIGNIFICANCE .- These results suggest that a phenotypic plasticity in response to depletion of nutrients and/or oxygen, helps decide whether melanoma cells undergo either death by ferroptosis, or resistance to it, when challenged by the same exogenous oxidative stress (iron ± H 2 O 2 ). • Proliferating melanoma show normoxic HIF1α, tyrosinase loss and undergo ferroptosis. • Serum depletion inhibits ferroptosis and prolongs melanoma survival. • Serum depletion promotes co-expression of tyrosinase and HIF1α.