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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; Braf\(^{CA}\); Pten\(^{lox/+}\) 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.
Highlights
• The integrated stress response leads to a general ATF4-dependent activation of NRF2
• ATF4 causes a CHAC1-dependent GSH depletion, resulting in NRF2 stabilization
• An elevation of NRF2 transcript levels fosters this effect
• NRF2 supports the ISR/ATF4 pathway by improving cystine and antioxidant supply
Summary
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.
Proteolytic cleavage of the extracellular domain affects signaling of parathyroid hormone 1 receptor
(2022)
Parathyroid hormone 1 receptor (PTH1R) is a member of the class B family of G protein-coupled receptors, which are characterized by a large extracellular domain required for ligand binding. We have previously shown that the extracellular domain of PTH1R is subject to metalloproteinase cleavage in vivo that is regulated by ligand-induced receptor trafficking and leads to impaired stability of PTH1R. In this work, we localize the cleavage site in the first loop of the extracellular domain using amino-terminal protein sequencing of purified receptor and by mutagenesis studies. We further show, that a receptor mutant not susceptible to proteolytic cleavage exhibits reduced signaling to G\(_s\) and increased activation of G\(_q\) compared to wild-type PTH1R. These findings indicate that the extracellular domain modulates PTH1R signaling specificity, and that its cleavage affects receptor signaling.
Mutations in the PRKACA gene are the most frequent cause of cortisol-producing adrenocortical adenomas leading to Cushing’s syndrome. PRKACA encodes for the catalytic subunit α of protein kinase A (PKA). We already showed that PRKACA mutations lead to impairment of regulatory (R) subunit binding. Furthermore, PRKACA mutations are associated with reduced RIIβ protein levels; however, the mechanisms leading to reduced RIIβ levels are presently unknown. Here, we investigate the effects of the most frequent PRKACA mutation, L206R, on regulatory subunit stability. We find that Ser\(^{114}\) phosphorylation of RIIβ is required for its degradation, mediated by caspase 16. Last, we show that the resulting reduction in RIIβ protein levels leads to increased cortisol secretion in adrenocortical cells. These findings reveal the molecular mechanisms and pathophysiological relevance of the R subunit degradation caused by PRKACA mutations, adding another dimension to the deregulation of PKA signaling caused by PRKACA mutations in adrenal Cushing’s syndrome.
Background
Wilms tumor (WT) is the most common renal tumor in childhood. Among others, MYCN copy number gain and MYCN P44L and MAX R60Q mutations have been identified in WT. MYCN encodes a transcription factor that requires dimerization with MAX to activate transcription of numerous target genes. MYCN gain has been associated with adverse prognosis in different childhood tumors including WT. The MYCN P44L and MAX R60Q mutations, located in either the transactivating or basic helix-loop-helix domain, respectively, are predicted to be damaging by different pathogenicity prediction tools, but the functional consequences remain to be characterized.
Methods
We screened a large cohort of unselected WTs for MYCN and MAX alterations. Wild-type and mutant protein function were characterized biochemically, and we analyzed the N-MYC protein interactome by mass spectrometric analysis of N-MYC containing protein complexes.
Results
Mutation screening revealed mutation frequencies of 3% for MYCN P44L and 0.9% for MAX R60Q that are associated with a higher risk of relapse. Biochemical characterization identified a reduced transcriptional activation potential for MAX R60Q, while the MYCN P44L mutation did not change activation potential or protein stability. The protein interactome of N-MYC-P44L was likewise not altered as shown by mass spectrometric analyses of purified N-MYC complexes. Nevertheless, we could identify a number of novel N-MYC partner proteins, e.g. PEG10, YEATS2, FOXK1, CBLL1 and MCRS1, whose expression is correlated with MYCN in WT samples and several of these are known for their own oncogenic potential.
Conclusions
The strongly elevated risk of relapse associated with mutant MYCN and MAX or elevated MYCN expression corroborates their role in WT oncogenesis. Together with the newly identified co-expressed interactors they expand the range of potential biomarkers for WT stratification and targeting, especially for high-risk WT.