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

ABSTRACT: 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.

SUBMITTER: Meinert M

PROVIDER: S-EPMC10825660 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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

Meinert Madlen M Jessen Christina C Hufnagel Anita A Kreß Julia Katharina Charlotte JKC Burnworth Mychal M Däubler Theo T Gallasch Till T Xavier da Silva Thamara Nishida TN Dos Santos Ancély Ferreira AF Ade Carsten Patrick CP Schmitz Werner W Kneitz Susanne S Friedmann Angeli José Pedro JP Meierjohann Svenja S

Redox biology 20231227

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 i ...[more]

PMID: 38219574

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Project description:Activating transcription factor 4 (ATF4) is a critical mediator of metabolic and oxidative homeostasis and cell survival. ATF4 is elevated in response to diverse microenvironmental stresses, including starvation, ER stress damages and exposure to toxic factors. Here we show that ATF4 expression fosters the malignancy of primary brain tumors (WHO grade III and IV gliomas) and increases proliferation and tumor angiogenesis. Hence, ATF4 expression promotes cell migration and anchorage-independent cell growth, whereas siRNA-mediated knockdown of ATF4 attenuates these features of malignancy in human gliomas. Further experiments revealed that ATF4-dependent tumor promoting effects are mediated by transcriptional targeting the glutamate antiporter xCT/SCL7A11 (also known as system Xc-). Thus, xCT is elevated as a consequence of ATF4 activation. We further found evidence that ATF4-induced proliferation can be attenuated by pharmacological or genetic xCT inhibition and ferroptosis inducers such as sorafenib, erastin and GPx4 inhibitor RSL3. Further, fostered xCT expression promotes cell survival and growth in ATF4 knockdown cells. Moreover, increased xCT levels ameliorate sorafenib and erastin-induced ferroptosis. Conversely, ATF4 knockdown renders cells susceptible for erastin, sorafenib and RSL3-induced ferroptosis. We further identified that ATF4 promotes tumor-mediated neuronal cell death which can be alleviated by xCT inhibition. Moreover, elevated ATF4 expression in gliomas promotes tumor angiogenesis. Noteworthy, ATF4-induced angiogenesis could be diminished by ferroptosis inducers erastin and by GPx4 inhibitor RSL3. Our data provide proof-of-principle evidence that ATF4 fosters proliferation and induces a toxic microenvironmental niche. Furthermore, ATF4 increases tumor angiogenesis and shapes the vascular architecture in a xCT-dependent manner. Thus, inhibition of ATF4 is a valid target for diminishing tumor growth and vasculature via sensitizing tumor cells for ferroptosis.

Project description:Ammonium tetrathiomolybdate (TTM) is a copper chelator in clinical trials for treatment of Wilson's disease, tumors and other diseases. In the current study, we innovatively discovered that TTM is a novel NRF2 activator and illustrated that autophagy contributed to TTM-induced NRF2 activation. We showed that TTM treatment promoted NRF2 nuclear translocation and upregulated transcription level of NRF2 target genes including HMOX1, GCLM, and SLC7A11 in vascular endothelial cells (HUVECs). Moreover, NRF2 deficiency directly hindered TTM-mediated antioxidative effects. Followingly, we revealed that overexpression of KEAP1, a negative regulator of NRF2, significantly repressed NRF2 activation induced by TTM. Further mutation analysis revealed that KEAP1 Cys151 is a major sensor responsible for TTM-initiated NRF2 signaling, suggesting that KEAP1 is involved in TTM-mediated NRF2 activation. Notably, we found that TTM can trigger autophagy as evidenced by accumulation of autophagosomes, elevation of LC3BI-II/I, increase of LC3 puncta and activation of AMPK/mTOR/ULK1 pathway. Autophagic flux assay indicated that TTM significantly enhanced autophagic flux in HUVECs. Inhibition of autophagy with knockout of autophagy key gene ATG5 resulted in suppression of TTM-induced NRF2 activation. TTM also induced phosphorylation of autophagy receptor SQSTM1 at Ser349, while SQSTM1-deficiency inhibited KEAP1 degradation and blocked NRF2 signaling pathway, suggesting that TTM-induced NRF2 activation is autophagy dependent. As the novel NRF2 activator, TTM protected against sodium arsenite (NaAsO2)-induced oxidative stress and cell death, while NRF2 deficiency weakened TTM antioxidative effects. Finally, we showed that autophagy-dependent NRF2 activation contributed to the protective effects of TTM against NaAsO2-induced oxidative injury, because of ATG5 or SQSTM1 knockout aggravated NaAsO2-induced elevation of HMOX1, cleaved PARP and γH2AX. Taken together, our findings highlight copper chelator TTM is a novel autophagy-dependent NRF2 activator and shed a new light on the cure for oxidative damage-related diseases.

Project description:BackgroundOxidative stress and neuroinflammation are considered the major central events in the process of Parkinson's disease (PD). Nrf2 is a key regulator of endogenous defense systems. New finds have contacted activation of Nrf2 signaling with anti-inflammatory activities. Therefore, the outstanding inhibition of neuroinflammation or potent Nrf2 signaling activation holds a promising strategy for PD treatment. Icariin (ICA), a natural compound derived from Herba Epimedii, presents a number of pharmacological properties, including anti-oxidation, anti-aging and anti-inflammatory actions. Recent studies have confirmed ICA exerted neuroprotection against neurodegenerative disorders. However, the underlying mechanisms were not fully elucidated.MethodsIn the present study, mouse nigral stereotaxic injection of 6-hydroxydopamine (6-OHDA)-induced PD model was performed to investigate ICA-conferred dopamine (DA) neuroprotection. In addition, adult Nrf2 knockout mice and primary rat midbrain neuron-glia co-culture was applied to elucidate whether ICA-exerted neuroprotection was through an Nrf2-dependent mechanism.ResultsResults indicated that ICA attenuated 6-OHDA-induced DA neurotoxicity and glial cells-mediated neuroinflammatory response. Furtherly, activation of Nrf2 signaling pathway in glial cells participated in ICA-produced neuroprotection, as revealed by the following observations. First, ICA enhanced Nrf2 signaling activation in 6-OHDA-induced mouse PD model. Second, ICA failed to generate DA neuroprotection and suppress glial cells-mediated pro-inflammatory factors production in Nrf2 knockout mice. Third, ICA exhibited neuroprotection in primary neuron-glia co-cultures but not in neuron-enriched cultures (without glial cells presence). Either, ICA-mediated neuroprotection was not discerned after Nrf2 siRNA treatment in neuron-glia co-cultures.ConclusionsOur findings identify that ICA attenuated glial cells-mediated neuroinflammation and evoked DA neuroprotection via an Nrf2-dependent manner.

Dataset Information

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

Publications

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

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