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Introduction: Acetyl-CoA synthetase 2 (ACSS2), one of the enzymes that catalyze the conversion of acetate to acetyl-CoA, has been proved to be an oncogene in various cancers. However, the function of ACSS2 is still largely a black box in melanoma. Methods: The ACSS2 expression was detected in melanoma cells and melanocytes at both protein and mRNA levels. Cell viability, apoptosis, migration and invasion were investigated after ACSS2 knockdown. RNA sequencing (RNA-Seq) technology was employed to identify differentially expressed genes caused by ACSS2 knockdown, which were then verified by immunoblotting analysis. Animal experiments were further performed to investigate the influence of ACSS2 on tumor growth and metastasis in vivo. Results: Firstly, we found that ACSS2 was upregulated in most melanoma cell lines compared with melanocytes. In addition, ACSS2 knockdown dramatically suppressed melanoma cell migration and invasion, whereas promoted cell apoptosis in response to endoplasmic reticulum (ER) stress. Furthermore, tumor growth and metastasis were dramatically suppressed by ACSS2 knockdown in vivo. RNA-Seq suggested that the Hippo pathway was activated by ACSS2 knockdown, which was forwardly confirmed by Western blotting and rescue experiments. Taken together, we demonstrated that ACSS2 enables melanoma cell survival and tumor metastasis via the regulation of the Hippo pathway. Discussion: In summary, this study demonstrated that ACSS2 may promote the growth and metastasis of melanoma by negatively regulating the Hippo pathway. Targeting ACSS2 may be a promising target for melanoma treatment.
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Melanoma, arising from the malignant transformation of melanocytes, stands as the most lethal type of skin cancer. While significant strides have been made in targeted therapy and immunotherapy, substantially enhancing therapeutic efficacy, the prognosis for melanoma patients remains unoptimistic. SIRT7, a nuclear-localized deacetylase, plays a pivotal role in maintaining cellular homeostasis and adapting to external stressors in melanoma, with its activity closely tied to intracellular nicotinamide adenine dinucleotide (NAD+). However, its involvement in adaptive resistance to targeted therapy remains unclear. Herein, we unveil that up-regulated SIRT7 promotes mitochondrial biogenesis to render the adaptive resistance to MAPK inhibition in melanoma. Initially, we observed a significant increase of SIRT7 expression in publicly available datasets following targeted therapy within a short duration. In consistent, we found elevated SIRT7 expression in melanoma cells subjected to BRAF or MEK inhibitors in vitro. The up-regulation of SIRT7 expression was also confirmed in xenograft tumors in mice after targeted therapy in vivo. Furthermore, we proved that SIRT7 deficiency led to decreased cell viability upon prolonged exposure to BRAF or MEK inhibitors, accompanied by an increase in cell apoptosis. Mechanistically, SIRT7 deficiency restrained the upregulation of genes associated with mitochondrial biogenesis and intracellular ATP levels in response to targeted therapy treatment in melanoma cells. Ultimately, we proved that SIRT7 deficieny could sensitize BRAF-mutant melanoma cells to MAPK inhibition targeted therapy in vivo. In conclusion, our findings underscore the role of SIRT7 in fostering adaptive resistance to targeted therapy through the facilitation of mitochondrial biogenesis. Targeting SIRT7 emerges as a promising strategy to overcome MAPK inhibitor adaptive resistance in melanoma.
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Resistencia a Medicamentos Antineoplásicos , Melanoma , Biogênese de Organelas , Inibidores de Proteínas Quinases , Sirtuínas , Melanoma/metabolismo , Melanoma/patologia , Melanoma/genética , Melanoma/tratamento farmacológico , Humanos , Sirtuínas/metabolismo , Sirtuínas/genética , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Proteínas Quinases/farmacologia , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/tratamento farmacológico , Camundongos Nus , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidoresRESUMO
BACKGROUND: Vitiligo is a skin disorder with melanocyte destruction caused by complex interplay between multiple genetic and environmental factors. Recent studies have suggested DNA methylation is involved in the melanocyte damage, but the underlying mechanism remains unknown. OBJECTIVE: To explore the abnormal DNA methylation patterns in vitiligo lesional and nonlesional skin, and the mechanism of DNA methylation involved in vitiligo pathogenesis. METHODS: Initially, the genome-wide aberrant DNA methylation profiles in lesional and nonlesional skin of vitiligo were detect via Illumina methylation EPIC 850k Beadchip. Subsequently, a comprehensive analysis was conduct to investigate the genomic characteristics of differentially methylated regions (DMRs). Furthermore, the effects of key aberrant methylated genes on cell apoptosis and function of both melanocytes and keratinocytes were further identified and validated by western bloting, ELISA, and immunofluorescence. RESULTS: Compared with nonlesional skins, we discovered 79 significantly differentially methylated CpG sites in vitiligo lesions. These DMRs were mainly located in the gene body and the TS1500 region. Annexin A2 receptor (ANXA2R), a crucial gene in cell apoptosis, was hypermethylated in vitiligo lesions. Furthermore, we showed that ANXA2R displayed hypermethylation and low expression levels in both keratinocytes and melanocytes of vitiligo patients, and the hypermethylated-triggered downregulation of ANXA2R under oxidative stress induced melanocyte apoptosis, and inhibited the secretion of stem cell factor (SCF) from keratinocytes thus impaired the survival of melanocytes. CONCLUSIONS: Our study illustrates the DNA methylation modiï¬cation in vitiligo, and further demonstrates the molecular mechanism of hypermethylated ANXA2R in the dysfunction of melanocytes under oxidative stress.
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Apoptose , Metilação de DNA , Queratinócitos , Melanócitos , Estresse Oxidativo , Vitiligo , Humanos , Vitiligo/genética , Vitiligo/patologia , Melanócitos/metabolismo , Melanócitos/patologia , Apoptose/genética , Queratinócitos/metabolismo , Adulto , Masculino , Feminino , Ilhas de CpG/genética , Pele/patologia , Pele/metabolismo , Adulto Jovem , Estudos de Casos e Controles , Pessoa de Meia-IdadeRESUMO
Vitiligo is a depigmented skin disease due to the destruction of melanocytes. Under oxidative stress, keratinocyte-derived chemokine C-X-C motif ligand 16 (CXCL16) plays a critical role in recruiting CD8+ T cells, which kill melanocytes. Autophagy serves as a protective cell survival mechanism and impairment of autophagy has been linked to increased secretion of the proinflammatory cytokines. However, the role of autophagy in the secretion of CXCL16 under oxidative stress has not been investigated. Herein, we initially found that autophagy was suppressed in both keratinocytes of vitiligo lesions and keratinocytes exposed to oxidative stress in vitro. Autophagy inhibition also promoted CXCL16 secretion. Furthermore, upregulated transient receptor potential cation channel subfamily M member 2 (TRPM2) functioned as an upstream oxidative stress sensor to inhibit autophagy. Moreover, TRPM2-mediated Ca2+ influx activated calpain to shear autophagy related 5 (Atg5) and Atg12-Atg5 conjugate formation was blocked to inhibit autophagy under oxidative stress. More importantly, Atg5 downregulation enhanced the binding of interferon regulatory factor 3 (IRF3) to the CXCL16 promoter region by activating Tank-binding kinase 1 (TBK1), thus promoting CXCL16 secretion. These findings suggested that TRPM2-restrained autophagy promotes CXCL16 secretion via the Atg5-TBK1-IRF3 signaling pathway under oxidative stress. Inhibition of TRPM2 may serve as a potential target for the treatment of vitiligo. © 2024 The Pathological Society of Great Britain and Ireland.
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Canais de Cátion TRPM , Vitiligo , Humanos , Vitiligo/metabolismo , Vitiligo/patologia , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Linfócitos T CD8-Positivos/patologia , Queratinócitos/patologia , Estresse Oxidativo , Autofagia , Quimiocina CXCL16/metabolismoRESUMO
BACKGROUND: Tumor cells frequently suffer from endoplasmic reticulum (ER) stress. Previous studies have extensively elucidated the role of tumorous unfolded protein response in melanoma cells, whereas the effect on tumor immunology and the underlying mechanism remain elusive. METHODS: Bioinformatics, biochemical assays and pre-clinical mice model were employed to demonstrate the role of tumorous inositol-requiring transmembrane kinase/endoribonuclease 1α (IRE1α) in anti-tumor immunity and the underlying mechanism. RESULTS: We firstly found that IRE1α signaling activation was positively associated with the feature of tumor-infiltrating lymphocytes. Then, pharmacological ER stress induction by HA15 exerted prominent anti-tumor effect in immunocompetent mice and was highly dependent on CD8+T cells, paralleled with the reshape of immune cells in tumor microenvironment via tumorous IRE1α-XBP1 signal. Subsequently, tumorous IRE1α facilitated the expression and secretion of multiple chemokines and cytokines via XBP1-NF-κB axis, leading to increased infiltration and anti-tumor capacity of CD8+T cells. Ultimately, pharmacological induction of tumorous ER stress by HA15 brought potentiated therapeutic effect along with anti-PD-1 antibody on melanoma in vivo. CONCLUSIONS: Tumorous IRE1α facilitates CD8+T cells-dependent anti-tumor immunity and improves immunotherapy efficacy by regulating chemokines and cytokines via XBP1-NF-κB axis. The combination of ER stress inducer and anti-PD-1 antibody could be promising for increasing the efficacy of melanoma immunotherapy.
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Melanoma , Animais , Camundongos , Linfócitos T CD8-Positivos/patologia , Quimiocinas , Citocinas , Endorribonucleases , Melanoma/patologia , NF-kappa B , Proteínas Serina-Treonina Quinases/metabolismo , Linfócitos T/metabolismo , Microambiente TumoralRESUMO
Melanoma is the most lethal skin cancer originating from the malignant transformation of epidermal melanocyte. The dysregulation of cellular metabolism is a hallmark of cancer, including in melanoma. Aberrant branched-chain amino acids (BCAA) metabolism and related enzymes has been greatly implicated in the progression of multiple types of cancer, whereas remains far from understood in melanoma. Herein, we reported that the critical BCAA metabolism enzyme branched-chain amino acid transaminase 2 (BCAT2) is an oncogenic factor in melanoma by activating lipogenesis via the epigenetic regulation of fatty acid synthase (FASN) and ATP-citrate lyase (ACLY) expressions. Firstly, we found that BCAT2 expression was prominently increased in melanoma, and highly associated with clinical stage. Then, it was proved that the deficiency of BCAT2 led to impaired tumor cell proliferation, invasion and migration in vitro, and tumor growth and metastasis in vivo. Further, RNA sequencing technology and a panel of biochemical assays demonstrated that BCAT2 regulated de novo lipogenesis via the regulation of the expressions of both FASN and ACLY. Mechanistically, the inhibition of BCAT2 suppressed the generation of intracellular acetyl-CoA, mitigating P300-dependent histone acetylation at the promoter of FASN and ACLY, and thereby their transcription. Ultimately, zinc finger E-box binding homeobox 1 (ZEB1) was identified as the upstream transcriptional factor responsible for BCAT2 up-regulation in melanoma. Our results demonstrate that BCAT2 promotes melanoma progression by epigenetically regulating FASN and ACLY expressions via P300-dependent histone acetylation. Targeting BCAT2 could be exploited as a promising strategy to restrain tumor progression in melanoma.
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Melanoma , Proteínas da Gravidez , Humanos , Lipogênese/genética , ATP Citrato (pro-S)-Liase/genética , ATP Citrato (pro-S)-Liase/metabolismo , Histonas/metabolismo , Epigênese Genética , Melanoma/genética , Transaminases/genética , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Antígenos de Histocompatibilidade Menor/metabolismo , Ácido Graxo Sintase Tipo I/genéticaRESUMO
BACKGROUND: The activation of CD8+ T cells and their trafficking to the skin through JAK-STAT signaling play a central role in the development of vitiligo. Thus, targeting this key disease pathway with innovative drugs is an effective strategy for treating vitiligo. Natural products isolated from medicinal herbs are a useful source of novel therapeutics. Demethylzeylasteral (T-96), extracted from Tripterygium wilfordii Hook F, possesses immunosuppressive and anti-inflammatory properties. METHODS: The efficacy of T-96 was tested in our mouse model of vitiligo, and the numbers of CD8+ T cells infiltration and melanocytes remaining in the epidermis were quantified using whole-mount tail staining. Immune regulation of T-96 in CD8+ T cells was evaluated using flow cytometry. Pull-down assay, mass spectrum analysis, molecular docking, knockdown and overexpression approaches were utilized to identify the target proteins of T-96 in CD8+ T cells and keratinocytes. RESULTS: Here, we found that T-96 reduced CD8+ T cell infiltration in the epidermis using whole-mount tail staining and alleviated the extent of depigmentation to a comparable degree of tofacitinib (Tofa) in our vitiligo mouse model. In vitro, T-96 decreased the proliferation, CD69 membrane expression, and IFN-γ, granzyme B, (GzmB), and perforin (PRF) levels in CD8+ T cells isolated from patients with vitiligo. Pull-down assays combined with mass spectrum analysis and molecular docking showed that T-96 interacted with JAK3 in CD8+ T cell lysates. Furthermore, T-96 reduced JAK3 and STAT5 phosphorylation following IL-2 treatment. T-96 could not further reduce IFN-γ, GzmB and PRF expression following JAK3 knockdown or inhibit increased immune effectors expression upon JAK3 overexpression. Additionally, T-96 interacted with JAK2 in IFN-γ-stimulated keratinocytes, inhibiting the activation of JAK2, decreasing the total and phosphorylated protein levels of STAT1, and reducing the production and secretion of CXCL9 and CXCL10. T-96 did not significantly inhibit STAT1 and CXCL9/10 expression following JAK2 knockdown, nor did it suppress upregulated STAT1-CXCL9/10 signaling upon JAK2 overexpression. Finally, T-96 reduced the membrane expression of CXCR3, and the culture supernatants pretreated with T-96 under IFN-γ stressed keratinocytes markedly blocked the migration of CXCR3+CD8+ T cells, similarly to Tofa in vitro. CONCLUSION: Our findings demonstrated that T-96 might have positive therapeutic responses to vitiligo by pharmacologically inhibiting the effector functions and skin trafficking of CD8+ T cells through JAK-STAT signaling.
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Vitiligo , Animais , Camundongos , Vitiligo/tratamento farmacológico , Vitiligo/metabolismo , Linfócitos T CD8-Positivos , Simulação de Acoplamento Molecular , Pele/metabolismoRESUMO
The dysregulation of branched-chain amino acid (BCAA) metabolism and related enzymes has been greatly implicated in the progression of multiple types of cancer, whereas remains far from understood in melanoma. Here, we explored the role of the BCAA metabolism enzyme BCKDHA in melanoma pathogenesis and elucidated the underlying mechanisms. In vitro cell biology experiments and in vivo pre-clinical mice model experiments were performed to investigate the role of BCKDHA in melanoma progression. RNA sequencing, immunohistochemical/immunofluorescence staining and bioinformatics analysis were used to examine the underlying mechanism. BCKDHA expression was prominently increased in both melanoma tissues and cell lines. The up-regulation of BCKDHA promoted long-term tumour cell proliferation, invasion and migration in vitro and tumour growth in vivo. Through RNA-sequencing technology, it was found that BCKDHA regulated the expressions of lipogenic fatty acid synthase (FASN) and ATP-citrate lyase (ACLY), which was thereafter proved to mediate the oncogenic role of BCKDHA in melanoma. Our results demonstrate that BCKDHA promotes melanoma progression by regulating FASN and ACLY expressions. Targeting BCKDHA could be exploited as a promising strategy to restrain tumour progression in melanoma.
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ATP Citrato (pro-S)-Liase , Melanoma , Animais , Camundongos , ATP Citrato (pro-S)-Liase/genética , ATP Citrato (pro-S)-Liase/metabolismo , Linhagem Celular , Proliferação de Células , Lipogênese , Melanoma/genéticaRESUMO
Melanoma is the most lethal type of skin cancer, originating from the malignant transformation of melanocyte. While the development of targeted therapy and immunotherapy has gained revolutionary advances in potentiating the therapeutic effect, the prognosis of patients with melanoma is still suboptimal. During tumor progression, melanoma frequently encounters stress from both endogenous and exogenous sources in tumor microenvironment. SIRT7 is a nuclear-localized deacetylase of which the activity is highly dependent on intracellular nicotinamide adenine dinucleotide (NAD+), with versatile biological functions in maintaining cell homeostasis. Nevertheless, whether SIRT7 regulates tumor cell biology and tumor immunology in melanoma under stressful tumor microenvironment remains elusive. Herein, we reported that SIRT7 orchestrates melanoma progression by simultaneously promoting tumor cell survival and immune evasion via the activation of unfolded protein response. We first identified that SIRT7 expression was the most significantly increased one in sirtuins family upon stress. Then, we proved that the deficiency of SIRT7 potentiated tumor cell death under stress in vitro and suppressed melanoma growth in vivo. Mechanistically, SIRT7 selectively activated the IRE1α-XBP1 axis to potentiate the pro-survival ERK signal pathway and the secretion of tumor-promoting cytokines. SIRT7 directly de-acetylated SMAD4 to antagonize the TGF-ß-SMAD4 signal, which relieved the transcriptional repression on IRE1α and induced the activation of the IRE1α-XBP1 axis. Moreover, SIRT7 up-regulation eradicated anti-tumor immunity by promoting PD-L1 expression via the IRE1α-XBP1 axis. Additionally, the synergized therapeutic effect of SIRT7 suppression and anti-PD-1 immune checkpoint blockade was also investigated. Taken together, SIRT7 can be employed as a promising target to restrain tumor growth and increase the effect of melanoma immunotherapy.
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Melanoma , Sirtuínas , Humanos , Endorribonucleases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Sobrevivência Celular/genética , Evasão da Resposta Imune , Linhagem Celular Tumoral , Melanoma/genética , Microambiente Tumoral , Sirtuínas/genéticaRESUMO
Melanoma is the most lethal form of skin cancer, resulting from the malignant transformation of epidermal melanocytes. Recent revolutionary progress in targeted therapy and immunotherapy has prominently improved the treatment outcome, but the survival of melanoma patients remains suboptimal. Ferroptosis is greatly involved in cancer pathogenesis and can execute the outcome of immunotherapy. However, the detailed regulatory mechanisms of melanoma cell ferroptosis remain elusive. Herein, we report that Wnt/ß-catenin signaling regulates ferroptosis and melanoma immunotherapy efficacy via the regulation of MITF. First of all, we found that Wnt/ß-catenin signaling was prominently suppressed in melanoma cell ferroptosis. Then, we proved that targeting ß-catenin exacerbated melanoma cell ferroptosis by promoting the generation of lipid peroxidation both in vitro and in vivo. Subsequent mechanistic studies revealed that MITF mediated the effect of Wnt/ß-catenin signaling on melanoma cell ferroptosis, and PGC1α and SCD1 were documented as two main effectors downstream of Wnt/ß-catenin-MITF pathway. Ultimately, pharmacological inhibition of ß-catenin or MITF increased the efficacy of anti-PD-1 immunotherapy in preclinical xenograft tumor model by promoting ferroptosis. Taken together, Wnt/ß-catenin signaling deficiency exacerbates ferroptosis in melanoma via the regulation of MITF. Targeting Wnt/ß-catenin-MITF pathway could be a promising strategy to potentiate ferroptosis and increase the efficacy of anti-PD-1 immunotherapy.
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Ferroptose , Melanoma , Humanos , beta Catenina/metabolismo , Proteínas Wnt/metabolismo , Melanoma/patologia , Via de Sinalização Wnt , Imunoterapia , Fator de Transcrição Associado à Microftalmia/metabolismoRESUMO
Background: Melanoma is a type of skin cancer, which originates from the malignant transformation of epidermal melanocytes, with extremely high lethality. Ferroptosis has been documented to be highly related to cancer pathogenesis and the effect of immunotherapy. In addition, the dysregulation of lncRNAs is greatly implicated in melanoma progression and ferroptosis regulation. However, the significance of ferroptosis-related lncRNA in melanoma treatment and the prognosis of melanoma patients remains elusive. Methods: Via Least Absolute Shrinkage Selection Operator (LASSO) regression analysis in the TCGA SKCM database, a cutaneous melanoma risk model was established based on differentially-expressed ferroptosis-related lncRNAs (DEfrlncRNAs). The nomogram, receiver operating characteristic (ROC) curves, and calibration plots were conducted to examine the predictive performance of this model. Sequentially, we continued to analyze the differences between the high- and low-risk groups, in terms of clinical characteristics, immune cell infiltration, immune-related functions, and chemotherapy drug sensitivity. Moreover, the expressions of DEfrlncRNAs, PD-L1, and CD8 were also examined by qRT-PCR and immunohistochemical staining in melanoma tissues to further confirm the potential clinical implication of DEfrlncRNAs in melanoma immunotherapy. Results: 16 DEfrlncRNAs were identified, and a representative risk score for patient survival was constructed based on these 16 genes. The risk score was found to be an independent prognostic factor for the survival of melanoma patients. In addition, the low-risk group of patients had higher immune cell infiltration in the melanoma lesions, higher sensitivity to chemotherapeutic agents, and a better survival prognosis. Besides, the high expression of the identified 5 DEfrlncRNA in the low-risk group might suggest a higher possibility to benefit from immune checkpoint blockade therapy in the treatment of melanoma. Conclusion: The DEfrlncRNA risk prediction model related to ferroptosis genes can independently predict the prognosis of patients with melanoma and provide a basis for evaluating the response of clinical treatment in melanoma.
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Ferroptose , Melanoma , RNA Longo não Codificante , Neoplasias Cutâneas , Antígeno B7-H1 , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Ferroptose/genética , Humanos , Inibidores de Checkpoint Imunológico , Melanoma/genética , Prognóstico , RNA Longo não Codificante/metabolismo , Neoplasias Cutâneas/genética , Melanoma Maligno CutâneoRESUMO
Melanoma is the most malignant skin cancer, which originates from epidermal melanocytes, with increasing worldwide incidence. The escape of immune surveillance is a hallmark of the tumor, which is manifested by the imbalance between the enhanced immune evasion of tumor cells and the impaired antitumor capacity of infiltrating immune cells. According to this notion, the invigoration of the exhausted immune cells by immune checkpoint blockades has gained encouraging outcomes in eliminating tumor cells and significantly prolonged the survival of patients, particularly in melanoma. Epigenetics is a pivotal non-genomic modulatory paradigm referring to heritable changes in gene expression without altering genome sequence, including DNA methylation, histone modification, non-coding RNAs, and m6A RNA methylation. Accumulating evidence has demonstrated how the dysregulation of epigenetics regulates multiple biological behaviors of tumor cells and contributes to carcinogenesis and tumor progression in melanoma. Nevertheless, the linkage between epigenetics and antitumor immunity, as well as its implication in melanoma immunotherapy, remains elusive. In this review, we first introduce the epidemiology, clinical characteristics, and therapeutic innovations of melanoma. Then, the tumor microenvironment and the functions of different types of infiltrating immune cells are discussed, with an emphasis on their involvement in antitumor immunity in melanoma. Subsequently, we systemically summarize the linkage between epigenetics and antitumor immunity in melanoma, from the perspective of distinct paradigms of epigenetics. Ultimately, the progression of the clinical trials regarding epigenetics-based melanoma immunotherapy is introduced.
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Melanoma , Neoplasias Cutâneas , Metilação de DNA , Epigênese Genética , Humanos , Imunoterapia , Melanoma/patologia , Microambiente Tumoral/genéticaRESUMO
BACKGROUND: Although anti-programmed cell death protein 1 (PD-1) immunotherapy is greatly effective in melanoma treatment, low response rate and treatment resistance significantly hinder its efficacy. Tumor cell ferroptosis triggered by interferon (IFN)-γ that is derived from tumor-infiltrating CD8+ T cells greatly contributes to the effect of immunotherapy. However, the molecular mechanism underlying IFN-γ-mediated ferroptosis and related potentially promising therapeutic strategy warrant further clarification. MicroRNAs (miRNAs) participate in ferroptosis execution and can be delivered systemically by multiple carriers, which have manifested obvious therapeutic effects on cancer. METHODS: MiRNAs expression profile in IFN-γ-driven ferroptosis was obtained by RNA sequencing. Biochemical assays were used to clarify the role of miR-21-3p in IFN-γ-driven ferroptosis and the underlying mechanism. MiR-21-3p-loaded gold nanoparticles were constructed and systemically applied to analyze the role of miR-21-3p in anti-PD-1 immunotherapy in preclinical transplanted tumor model. RESULTS: MiRNAs expression profile of melanoma cells in IFN-γ-driven ferroptosis was first obtained. Then, upregulated miR-21-3p was proved to facilitate IFN-γ-mediated ferroptosis by potentiating lipid peroxidation. miR-21-3p increased the ferroptosis sensitivity by directly targeting thioredoxin reductase 1 (TXNRD1) to enhance lipid reactive oxygen species (ROS) generation. Furthermore, miR-21-3p overexpression in tumor synergized with anti-PD-1 antibody by promoting tumor cell ferroptosis. More importantly, miR-21-3p-loaded gold nanoparticles were constructed, and the systemic delivery of them increased the efficacy of anti-PD-1 antibody without prominent side effects in preclinical mice model. Ultimately, ATF3 was found to promote miR-21-3p transcription in IFN-γ-driven ferroptosis. CONCLUSIONS: MiR-21-3 p upregulation contributes to IFN-γ-driven ferroptosis and synergizes with anti-PD-1 antibody. Nanoparticle delivery of miR-21-3 p is a promising therapeutic approach to increase immunotherapy efficacy without obvious systemic side effects.
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Ferroptose , Melanoma , Nanopartículas Metálicas , MicroRNAs , Animais , Linfócitos T CD8-Positivos , Linhagem Celular Tumoral , Ouro , Humanos , Imunoterapia , Melanoma/tratamento farmacológico , Melanoma/genética , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
Vitiligo is a depigmented skin disorder caused by a variety of factors, including autoimmune, metabolic disturbance or their combined effect, etc. Non-targeted metabolomic analyses have denoted that dysregulated fatty acids metabolic pathways are involved in the pathogenesis of vitiligo. However, the exact category of fatty acids that participate in vitiligo development and how they functionally affect CD8+ T cells remain undefined. We aimed to determine the difference in specific fatty acids among vitiligo patients and healthy individuals and to investigate their association with clinical features in patients with vitiligo. Serum levels of fatty acids in 48 vitiligo patients and 28 healthy individuals were quantified by performing ultra-performance liquid chromatography-tandem mass spectrometry. Univariate and multivariate analyses were carried out to evaluate the significance of differences. Moreover, flow cytometry was used to explore the effect of indicated fatty acids on the function of CD8+ T cells derived from patients with vitiligo. We demonstrated that serological level of alpha-linolenic acid (ALA) was markedly upregulated, while that of arachidonic acid (ARA), arachidic acid (AA) and behenic acid were significantly downregulated in patients with vitiligo. Moreover, ALA levels were positively associated with vitiligo area scoring index (VASI) and ARA was a probable biomarker for vitiligo. We also revealed that supplementation with ARA or nordihydroguaiaretic acid (NDGA) could suppress the function of CD8+ T cells. Our results showed that vitiligo serum has disorder-specific phenotype profiles of fatty acids described by dysregulated metabolism of polyunsaturated fatty acids. Supplementation with ARA or NDGA might promote vitiligo treatment. These findings provide novel insights into vitiligo pathogenesis that might add to therapeutic options.
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Vitiligo , Ácido Araquidônico/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Ácidos Graxos , Ácidos Graxos Insaturados/metabolismo , Humanos , MetabolômicaRESUMO
Vitiligo is a cutaneous depigmenting autoimmune disease caused by the extensive destruction of epidermal melanocytes. Convincing data has defined a critical role for oxidative stress in the pathogenesis of vitiligo. Oxeiptosis is a caspase-independent cell death modality that was reportedly triggered by oxidative stress and operative in pathogen clearance. However, whether oxeiptosis exists in oxidative stress-induced melanocytes demise in vitiligo remains undetermined. In the present study, we initially found that other cell death modalities might exist in addition to the well-recognized apoptosis and necroptosis in H2O2-treated melanocytes. Furthermore, AIFM1 was found to be dephosphorylated at Ser116 in oxidative stress-induced melanocytes death, which was specific to oxeiptosis. Moreover, KEAP1 and PGAM5, upstream of the AIFM1 in oxeiptosis, were found to operate in melanocytic death. Subsequently, the KEAP1-PGAM5-AIFM1 signaling pathway was proved to be involved in oxidative stress-triggered melanocytes demise through the depletion of KEAP1 and PGAM5. Altogether, our study indicated that oxeiptosis might occur in melanocytes death under oxidative stress and contribute to the pathogenesis of vitiligo.
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Autophagy plays a protective role in oxidative stressâinduced melanocyte death. Dysregulated autophagy increases the sensitivity of melanocytes in response to oxidative damage and promotes melanocyte degeneration in vitiligo. However, the molecular mechanism underlying this process is not fully understood. In this study, using RNA-sequencing technology, we compared the transcriptome change between normal and vitiligo melanocytes with or without treatment of oxidative stress. We found that ATG5 and ATG12, the critical components for autophagosome formation, were significantly reduced in vitiligo melanocytes under oxidative stress. Mechanistically, HSF1 is the prime transcription factor for both ATG5 and ATG12, accounting for the reduced level of ATG5 and ATG12 in vitiligo melanocytes. Deficiency of HSF1 led to accumulation of intracellular ROS, imbalance of mitochondrion membrane potential, and apoptosis in melanocytes exposure to oxidative stress. Furthermore, overexpression of HSF1 could ameliorate oxidative stressâinduced melanocytes death through the activation of autophagy by upregulating ATG5 and ATG12. These findings suggested that targeting HSF1-ATG5/12 axis could prevent oxidative stressâinduced melanocyte death and may be used as a therapeutic strategy for vitiligo treatment.
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Hipopigmentação , Vitiligo , Autofagia , Humanos , Melanócitos , Estresse OxidativoRESUMO
Melanoma is the most lethal skin cancer caused by the malignant transformation of epidermal melanocytes. Recent progress in targeted therapy and immunotherapy has significantly improved the treatment outcome, but the survival of patients with advanced melanoma remains suboptimal. Ferroptosis, a cell death modality triggered by iron-dependent lipid peroxidation, reportedly participates in cancer pathogenesis and can mediate the effect of anti-PD-1 immunotherapy in melanoma. However, the detailed regulatory mechanism of ferroptosis remains far from being understood. In this study, we report that CAMKK2 defines the ferroptosis sensitivity of melanoma cells by regulating the AMPKâNRF2 pathway. We first found that CAMKK2 was prominently activated in ferroptosis. Then we proved that CAMKK2 negatively regulated ferroptosis through the activation of NRF2 and the suppression of lipid peroxidation. Subsequent mechanistic studies revealed that AMPK connected CAMKK2 upregulation to NRF2-dependent antioxidative machinery in ferroptosis. In addition, the suppression of CAMKK2 increased the efficacy of ferroptosis inducer and anti-PD-1 immunotherapy in the preclinical xenograft tumor model by inhibiting the AMPKâNRF2 pathway and promoting ferroptosis. Taken together, CAMKK2 plays a protective role in ferroptosis by activating the AMPKâNRF2 pathway. Targeting CAMKK2 could be a potential approach to increase the efficacy of ferroptosis inducers and immunotherapy for melanoma treatment.
Assuntos
Quinases Proteína-Quinases Ativadas por AMP/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Imunoterapia/métodos , Melanoma/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias Cutâneas/metabolismo , Antineoplásicos Imunológicos/uso terapêutico , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Linhagem Celular Tumoral , Ferroptose , Humanos , Peroxidação de Lipídeos , Melanoma/tratamento farmacológico , Receptor de Morte Celular Programada 1/imunologia , Transdução de Sinais , Neoplasias Cutâneas/tratamento farmacológico , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Oxidative stress has a vital role in the early stages of vitiligo. Autoantigens released from apoptotic melanocytes (MC) under oxidative stress are involved in the presentation and recognition of antigens. However, the transport of autoantigens to the cell surface and their release to the extracellular environment are still unclear. Apoptotic bodies (ABs) have always been considered as a key source of immunomodulators and autoantigens. Yet, the role of ABs in the immune mechanism of vitiligo is still unknown. PURPOSE: To explore whether MC's autoantigens translocate into ABs during oxidative stress-induced apoptosis and study the molecular mechanisms underlying autoantigen migration and AB formation. METHODS: PIG3V (an immortalized human vitiligo melanocyte cell line) were treated with H2O2, and ABs were separated. Transmission electron microscopy, flow cytometry, Western blot, mass spectrometry, and other methods were used to determine the relocation of specific antigens in PIG3V cells to ABs. After pretreatment with specific inhibitors (Rho kinase (Y-27632), myosin light chain kinase (MLCK, ML-9), pan-caspase (zVAD-FMK), and JNK (SP600125)), the pathway of autoantigen translocation into ABs and the formation of apoptotic bodies were determined. RESULTS: When treated with 0.8 mM H2O2, ABs were released from these cells. Autoantigens such as tyrosinase-related protein 1 (TYRP-1) and cleavage nuclear membrane antigen Lamin A/C (Asp230) were concentrated in ABs. The expression of autoantigens and the formation of ABs increased in a time- and dose-dependent manner after treatment with H2O2, while the application of specific inhibitors inhibited the formation of apoptotic bodies, i.e., the expression of antigens. CONCLUSION: Vitiligo autoantigens translocate into ABs in the process of apoptosis induced by oxidative stress. The cytoskeletal protein activation pathway and the JNK-related apoptosis pathway are involved in the transport of autoantigens and the formation of ABs. ABs may be the key bridge between MC cell apoptosis and cellular immunity.
Assuntos
Apoptose , Autoantígenos/metabolismo , Vesículas Extracelulares/patologia , Melanócitos/patologia , Estresse Oxidativo , Vitiligo/patologia , Caspases/metabolismo , Proteínas do Citoesqueleto/metabolismo , Humanos , MAP Quinase Quinase 4/metabolismo , Vitiligo/etiologia , Vitiligo/metabolismoRESUMO
Vitiligo is a common depigmentation disease characterized by melanocyte death, which is attributed to various mechanisms such as apoptosis and autoimmune destruction. However, whether necroptosis, a newly discovered way of cell death, plays a key role in the pathogenesis of vitiligo is still elusive and has not been well-studied. In this study, we found that necroptosis markers, including phosphorylated RIP3 and phosphorylated-MLKL, were positive in melanocytes from vitiligo perilesional skin, which supported the existence of necroptosis in vitiligo. Furthermore, the expression of RIP1 was remarkably upregulated in melanocytes treated with hydrogen peroxide. Then, RIP1 intervention suppression and MLKL deficiency could significantly enhance the resistance of melanocytes to hydrogen peroxideâinduced necroptosis. Mechanistically, we confirmed that RIP1 and RIP3 could form necrosomes under oxidative stress and further trigger phosphorylated MLKL translocation to the cell membrane, which led to the destruction of melanocytes. Finally, we showed that RIP1-mediated generation of mitochondrial ROS contributed to necrosome formation in melanocytes. Collectively, our study confirms that necroptosis significantly facilitates oxidative stressâinduced melanocyte death through the RIP1 signaling pathway, offering insight into vitiligo.
Assuntos
Melanócitos/patologia , Necroptose/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/fisiologia , Estresse Oxidativo/fisiologia , Proteínas de Ligação a RNA/fisiologia , Vitiligo/etiologia , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases/fisiologia , Proteína Serina-Treonina Quinases de Interação com Receptores/fisiologia , Transdução de Sinais/fisiologia , Vitiligo/patologiaRESUMO
Melanoma cells are relatively resistant to endoplasmic reticulum (ER) stress, which contributes to tumor progression under stressful conditions and renders tolerance to ER stressâinducing therapeutic agents. Mitochondria are tightly interconnected with ER. However, whether mitochondria play a role in regulating ER stress resistance in melanoma remains elusive. In this study, we reported that the XBP1âMARCH5âMFN2 axis conferred ER stress resistance by coordinating mitochondrial fission and mitophagy in melanoma. Our integrative bioinformatics first revealed that the downregulation of mitochondrial genes was highly correlated with unfolded protein response activation in melanoma. Then we proved that mitochondrial fission and mitophagy were prominently induced to contribute to ER stress resistance both in vitro and in vivo by maintaining mitochondrial function. Mechanistically, the activation of IRE1α/ATF6-XBP1 branches of unfolded protein response promoted the transcription of E3 ligase MARCH5 to facilitate the ubiquitination and degradation of MFN2, which thereby triggered mitochondrial fission and mitophagy under ER stress. Together, our findings show a regulatory axis that links mitochondrial fission and mitophagy to the resistance to ER stress. Targeting mitochondrial quality control machinery can be exploited as an approach to reinforce the efficacy of ER stressâinducing agents against cancer.