Thiol starvation triggers melanoma state switching in an ATF4 and NRF2-dependent manner.

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 they 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.

The integrated stress response effector ATF4 is an obligatory metabolic activator of NRF2.

The redox regulator NRF2 becomes activated upon oxidative and electrophilic stress and orchestrates a response program associated with redox regulation, metabolism, tumor therapy resistance, and immune suppression. Here, we describe an unrecognized link between the integrated stress response (ISR) and NRF2 mediated by the ISR effector ATF4. The ISR is commonly activated after starvation or ER stress and plays a central role in tissue homeostasis and cancer plasticity. ATF4 increases NRF2 transcription and induces the glutathione-degrading enzyme CHAC1, which we now show to be critically important for maintaining NRF2 activation. In-depth analyses reveal that NRF2 supports ATF4-induced cells by increasing cystine uptake via the glutamate-cystine antiporter xCT. In addition, NRF2 upregulates genes mediating thioredoxin usage and regeneration, thus balancing the glutathione decrease. In conclusion, we demonstrate that the NRF2 response serves as second layer of the ISR, an observation highly relevant for the understanding of cellular resilience in health and disease.

NRF2 Enables EGFR Signaling in Melanoma Cells.

Receptor tyrosine kinases (RTK) are rarely mutated in cutaneous melanoma, but the expression and activation of several RTK family members are associated with a proinvasive phenotype and therapy resistance. Epidermal growth factor receptor (EGFR) is a member of the RTK family and is only expressed in a subgroup of melanomas with poor prognosis. The insight into regulators of EGFR expression and activation is important for the understanding of the development of this malignant melanoma phenotype. Here, we describe that the transcription factor NRF2, the master regulator of the oxidative and electrophilic stress response, mediates the expression and activation of EGFR in melanoma by elevating the levels of EGFR as well as its ligands EGF and TGFα. ChIP sequencing data show that NRF2 directly binds to the promoter of EGF, which contains a canonical antioxidant response element. Accordingly, EGF is induced by oxidative stress and is also increased in lung adenocarcinoma and head and neck carcinoma with mutationally activated NRF2. In contrast, regulation of EGFR and TGFA occurs by an indirect mechanism, which is enabled by the ability of NRF2 to block the activity of the melanocytic lineage factor MITF in melanoma. MITF effectively suppresses EGFR and TGFA expression and therefore serves as link between NRF2 and EGFR. As EGFR was previously described to stimulate NRF2 activity, the mutual activation of NRF2 and EGFR pathways was investigated. The presence of NRF2 was necessary for full EGFR pathway activation, as NRF2-knockout cells showed reduced AKT activation in response to EGF stimulation compared to controls. Conversely, EGF led to the nuclear localization and activation of NRF2, thereby demonstrating that NRF2 and EGFR are connected in a positive feedback loop in melanoma. In summary, our data show that the EGFR-positive melanoma phenotype is strongly supported by NRF2, thus revealing a novel maintenance mechanism for this clinically challenging melanoma subpopulation.