Thiol starvation triggers melanoma state switching in an ATF4 and NRF2-dependent manner. Homo sapiens strain:M14, SK-MEL-28

The cystine/glutamate antiporter xCT is an important source of cysteine for cancer cells. Once taken up, cystine is reduced to cysteine and serves as a building block for the synthesis of glutathione, which efficiently protects cells from oxidative damage and prevents ferroptosis. As melanomas are particularly exposed to several sources of oxidative stress, we investigated the biological role of cysteine and glutathione supply by xCT in melanoma. xCT activity was abolished by genetic depletion in the Tyr::CreER; BrafCA; Ptenlox/+ melanoma model and by acute cystine withdrawal in melanoma cell lines. Both interventions profoundly impacted melanoma glutathione levels, but were surprisingly well tolerated by murine melanomas in vivo and by most human melanoma cell lines in vitro. RNA sequencing of human melanoma cells revealed a strong adaptive upregulation of NRF2 and ATF4 pathways, which orchestrated the compensatory upregulation of genes involved in antioxidant defence and de novo cysteine biosynthesis. In addition, the joint activation of ATF4 and NRF2 triggered a phenotypic switch characterized by a reduction of differentiation genes and induction of pro-invasive features, which was also observed after erastin treatment or the inhibition of glutathione synthesis. NRF2 alone was capable of inducing the phenotypic switch in a transient manner. Together, our data show that cystine or glutathione levels regulate the phenotypic plasticity of melanoma cells by elevating ATF4 and NRF2.

胱氨酸/谷氨酸反向转运蛋白xCT（cystine/glutamate antiporter xCT）是癌细胞获取半胱氨酸的重要来源。细胞摄取胱氨酸后，其会被还原为半胱氨酸，作为谷胱甘肽合成的前体物质，而谷胱甘肽可有效保护细胞免受氧化损伤并抑制铁死亡（ferroptosis）。由于黑色素瘤特别易受多种氧化应激源的影响，我们探究了xCT介导的半胱氨酸与谷胱甘肽供给在黑色素瘤中的生物学功能。在Tyr::CreER; BrafCA; Ptenlox/+黑色素瘤小鼠模型中通过基因敲除，以及在黑色素瘤细胞系中采用急性胱氨酸剥夺法，均可阻断xCT的活性。上述两种干预手段均显著降低了黑色素瘤的谷胱甘肽水平，但令人意外的是，小鼠体内的黑色素瘤以及体外培养的多数人源黑色素瘤细胞系均表现出良好的耐受性。对人源黑色素瘤细胞进行RNA测序（RNA sequencing）分析后发现，核因子E2相关因子2（NRF2）与激活转录因子4（ATF4）通路出现显著的适应性上调，这两条通路协同调控了抗氧化防御及从头半胱氨酸生物合成相关基因的补偿性上调表达。此外，ATF4与NRF2的联合激活还触发了一种表型转换，表现为分化相关基因表达下调以及促侵袭特征的诱导；该现象在艾拉司他丁（erastin）处理或抑制谷胱甘肽合成后同样可被观察到。仅NRF2即可短暂诱导该表型转换。综上，本研究数据表明，胱氨酸或谷胱甘肽水平可通过上调ATF4与NRF2的表达，调控黑色素瘤细胞的表型可塑性。