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Virilizer-like m6A methyltransferase-associated protein (VIRMA) maintains the stability of the m6A writer complex. Although VIRMA is critical for RNA m6A deposition, the impact of aberrant VIRMA expression in human diseases remains unclear. We show that VIRMA is amplified and overexpressed in 15-20% of breast cancers. Of the two known VIRMA isoforms, the nuclear-enriched full-length but not the cytoplasmic-localised N-terminal VIRMA promotes m6A-dependent breast tumourigenesis in vitro and in vivo. Mechanistically, we reveal that VIRMA overexpression upregulates the m6A-modified long non-coding RNA, NEAT1, which contributes to breast cancer cell growth. We also show that VIRMA overexpression enriches m6A on transcripts that regulate the unfolded protein response (UPR) pathway but does not promote their translation to activate the UPR under optimal growth conditions. Under stressful conditions that are often present in tumour microenvironments, VIRMA-overexpressing cells display enhanced UPR and increased susceptibility to death. Our study identifies oncogenic VIRMA overexpression as a vulnerability that may be exploited for cancer therapy.
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Neoplasias de la Mama , Humanos , Femenino , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Respuesta de Proteína Desplegada/genética , ARN/metabolismo , Interferencia de ARN , Microambiente TumoralRESUMEN
MiR-181 expression levels increased in hepatocellular carcinoma (HCC) compared to non-cancerous tissues. MiR-181 has been widely reported as a possible driver of tumourigenesis but also acts as a tumour suppressor. In addition, the miR-181 family regulates the development and function of immune and vascular cells, which play vital roles in the progression of tumours. More complicatedly, many genes have been identified as miR-181 targets to mediate the effects of miR-181. However, the role of miR-181 in the development of primary tumours remains largely unexplored. We aimed to examine the function of miR-181 and its vital mediators in the progression of diethylnitrosamine-induced primary liver cancers in mice. The size of liver tumours was significantly reduced by 90% in global (GKO) or liver-specific (LKO) 181ab1 knockout mice but not in hematopoietic and endothelial lineage-specific knockout mice, compared to WT mice. In addition, the number of tumours was significantly reduced by 50% in GKO mice. Whole-genome RNA-seq analysis and immunohistochemistry showed that epithelial-mesenchymal transition was partially reversed in GKO tumours compared to WT tumours. The expression of CBX7, a confirmed miR-181 target, was up-regulated in GKO compared to WT tumours. Stable CBX7 expression was achieved with an AAV/Transposase Hybrid-Vector System and up-regulated CBX7 expression inhibited liver tumour progression in WT mice. Hepatic CBX7 deletion restored the progression of LKO liver tumours. MiR-181a expression was the lowest and CBX7 expression the highest in iClust2 and 3 subclasses of human HCC compared to iClust1. Gene expression profiles of GKO tumours overlapped with low-proliferative peri-portal-type HCCs. Liver-specific loss of miR-181ab1 inhibited primary liver tumour progression via up-regulating CBX7 expression, but tumour induction requires both hepatic and non-hepatic miR-181. Also, miR-181ab1-deficient liver tumours may resemble low-proliferative periportal-type human HCC. miR-181 was increased with liver tumour growth. More miR-181, darker colour and higher shape. CBX7 was very low in pericentral hepatocytes, increased in early liver tumours, but reduced in advanced liver tumours. Its levels were maintained in miR-181 KO liver tumours. In tumours (T), brown (darker is more) represents miR-181, the blue circle (thicker is more) represents CBX7.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , MicroARNs , Animales , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Ratones , MicroARNs/genética , MicroARNs/metabolismo , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
One of the limitations of immunotherapy is the development of a state referred to as T cell exhaustion (TEx) whereby T cells express inhibitory receptors (IRs) and lose production of effectors involved in killing of their targets. In the present studies we have used the repeated stimulation model with anti CD3 and anti CD28 to understand the factors involved in TEx development and treatments that may reduce changes of TEx. The results show that addition of nicotinamide (NAM) involved in energy supply to cells prevented the development of inhibitory receptors (IRs). This was particularly evident for the IRs CD39, TIM3, and to a lesser extent LAG3 and PD1 expression. NAM also prevented the inhibition of IL-2 and TNFα expression in TEx and induced differentiation of CD4+ and CD8 T cells to effector memory and terminal effector T cells. The present results showed that effects of NAM were linked to regulation of reactive oxygen species (ROS) consistent with previous studies implicating ROS in upregulation of TOX transcription factors that induce TEx. These effects of NAM in reducing changes of TEx and in increasing the differentiation of T cells to effector states appears to have important implications for the use of NAM supplements in immunotherapy against cancers and viral infections and require further exploration in vivo.
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The development of resistance to treatments of melanoma is commonly associated with an upregulation of the MAPK pathway and the development of an undifferentiated state. Previous studies have suggested that melanoma with these resistance characteristics may be susceptible to innate death mechanisms such as pyroptosis triggered by the activation of inflammasomes. In this study, we have taken cell lines from patients before and after the development of resistance to BRAF V600 inhibitors and exposed the resistant melanoma to temozolomide (a commonly used chemotherapy) with and without chloroquine to inhibit autophagy. It was found that melanoma with an inflammatory undifferentiated state appeared susceptible to this combination when tested in vitro and in vivo against xenografts in nonobese diabetic scid gamma mice. Translation of the latter results into patients would promise durable responses in patients treated by the combination. The inflammasome and death mechanism involved appeared to vary between melanoma and involved either AIM2 or NLRP3 inflammasomes and gasdermin D or E. These preliminary studies have raised questions as to the selectivity for different inflammasomes in different melanoma and their selective targeting by chemotherapy. They also question whether the inflammatory state of melanoma may be used as biomarkers to select patients for inflammasome-targeted therapy.
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Inflamasomas , Melanoma , Animales , Humanos , Inflamasomas/metabolismo , Melanoma/tratamiento farmacológico , Melanoma/metabolismo , Ratones , Ratones Endogámicos NOD , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas B-raf/genética , PiroptosisRESUMEN
Dysregulation of epigenetic modifiers is a frequent event in melanoma and underlies many aspects of melanoma biology, including resistance to targeted therapy and immunotherapies. Here, we report that dual targeting of BET and CDK9 proteins have synergistic effects against melanoma cells in vitro and in vivo. The BET inhibitor (IBET151) and CDK9 inhibitor (CDKI73) synergistically killed melanoma cells in vitro independent of their BRAF or NRAS mutation status. The combination of drugs markedly inhibited the growth of human melanoma C002M cells in vitro in three-dimensional spheroids and in vivo in NOD-SCID gamma mice compared with vehicle control and the individual drugs (P < 0.05). Cell death was associated with mitochondrial depolarization, caspase-dependent apoptosis with cleavage of PARP1, and downregulation of anti-apoptotic proteins BCL2, BCLXL, and MCL1. Gene set enrichment analysis revealed downregulation of hallmark gene sets associated with E2F, G2M checkpoint, and c-MYC. Survival analysis showed worse prognosis with high G2M, E2F, or c-MYC gene signatures, suggesting biomarkers of response of BET and CDK9 inhibitors in melanoma. This combination of epigenetic inhibitors targets multiple downstream genes, leading to cell death of melanoma cells in vitro and in vivo, and warrants further investigation for treatment of melanoma in patients not responding to current therapies.
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Antineoplásicos/uso terapéutico , Quinasa 9 Dependiente de la Ciclina/antagonistas & inhibidores , Melanoma/tratamiento farmacológico , Proteínas del Tejido Nervioso/genética , Pirimidinas/uso terapéutico , Receptores de Superficie Celular/genética , Neoplasias Cutáneas/tratamiento farmacológico , Sulfonamidas/uso terapéutico , Animales , Apoptosis , Biomarcadores Farmacológicos , Línea Celular Tumoral , Sinergismo Farmacológico , Quimioterapia Combinada , Epigenómica , Humanos , Ratones , Ratones SCID , Metástasis de la Neoplasia , Estadificación de Neoplasias , Proteínas del Tejido Nervioso/metabolismo , Receptores de Superficie Celular/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Major differences in survival of men and women from infectious diseases and cancers have been highlighted by death rates from COVID-19 infections. In cancer, attention has been focussed on differences in gene expression from X chromosomes in men and women with a preponderance of genes involved in immune responses being expressed in women. Important findings have been that some of the genes are important epigenetic regulators that play fundamental roles in immune responses.
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Infecciones/metabolismo , Neoplasias/mortalidad , Factores Sexuales , COVID-19/mortalidad , COVID-19/virología , Cromosomas Humanos X , Cromosomas Humanos Y , Femenino , Predisposición Genética a la Enfermedad , Humanos , SARS-CoV-2/aislamiento & purificación , Tasa de SupervivenciaRESUMEN
BACKGROUND: Survival from melanoma is strongly related to patient sex, with females having a survival rate almost twice that of males. Many explanations have been proposed but have not withstood critical scrutiny. Prior analysis of different cancers with a sex bias has identified six X-linked genes that escape X chromosome inactivation in females and are, therefore, potentially involved in sex differences in survival. Four of the genes are well-known epigenetic regulators that are known to influence the expression of hundreds of other genes and signaling pathways in cancer. METHODS: Survival and interaction analysis were performed on the skin cutaneous melanoma (SKCM) cohort in The Cancer Genome Atlas (TCGA), comparing high vs. low expression of KDM6A, ATRX, KDM5C, and DDX3X. The Leeds melanoma cohort (LMC) on 678 patients with primary melanoma was used as a validation cohort. RESULTS: Analysis of TCGA data revealed that two of these genes-KDM6A and ATRX-were associated with improved survival from melanoma. Tumoral KDM6A was expressed at higher levels in females and was associated with inferred lymphoid infiltration into melanoma. Gene set analysis of high KDM6A showed strong associations with immune responses and downregulation of genes associated with Myc and other oncogenic pathways. The LMC analysis confirmed the prognostic significance of KDM6A and its interaction with EZH2 but also revealed the expression of KDM5C and DDX3X to be prognostically significant. The analysis also confirmed a partial correlation of KDM6A with immune tumor infiltrates. CONCLUSION: When considered together, the results from these two series are consistent with the involvement of X-linked epigenetic regulators in the improved survival of females from melanoma. The identification of gene signatures associated with their expression presents insights into the development of new treatment initiatives but provides a basis for exploration in future studies.
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The development of changes in T cells, referred to as T cell exhaustion, has been suggested as a cause of primary or acquired resistance to immunotherapy by immune checkpoint blockade (ICB). A limited number of studies, largely performed on tumor infiltrating lymphocytes (TILs), has provided evidence in support of this hypothesis, but whether similar changes occur in circulating blood lymphocytes has received little attention. In the present study, a comprehensive analysis of peripheral blood leukocytes from 42 patients taken over the course of treatment with anti-PD-1 was undertaken. The patients included those grouped as responders (who did not progress), primary non-responders (primary resistance) and those with acquired resistance (who initially responded then subsequently progressed). Analysis included surface markers of exhaustion, production of cytokines following in vitro stimulation, and assessment of transcription factor levels associated with T cell exhaustion. There were differences in innate cell populations between responders and non-responders at baseline and maintained throughout therapy. Frequencies of total and classical CD14+CD16- monocytes were higher and the major subset of NK cells (CD16hiCD56+) was significantly smaller in the primary resistance group compared with responders. However, differences in peripheral blood expression of exhaustion markers were not evident between the treatment groups. T cell exhaustion markers were expressed in practically all patients and the major observation was an increase in CD39 on CD4 T cells during treatment. The results confirm the association of Eomes transcription factor with T cell exhaustion but levels of expression and the ratio with T-bet over Eomes did not differ between the patient groups. Thus, peripheral blood expression of T cell exhaustion markers does not distinguish between responders and non-responders to anti-PD-1 therapy. CD4 T cell expression of IFNγ also differed in pre-treatment samples, indicating that predictors of response unrelated to exhaustion may be present in peripheral blood. The association of response with innate cell populations and CD4 T cell responses requires further study.
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Antineoplásicos/uso terapéutico , Linfocitos T CD4-Positivos/inmunología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Inmunoterapia/métodos , Células Asesinas Naturales/inmunología , Melanoma/terapia , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores Farmacológicos , Células Cultivadas , Femenino , Humanos , Tolerancia Inmunológica , Inmunidad Innata , Interferón gamma/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Activación de Linfocitos , Masculino , Melanoma/inmunología , Persona de Mediana Edad , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Escape del TumorRESUMEN
Methylation of DNA at CpG sites is the most common and stable of epigenetic changes in cancer. Hypermethylation acts to limit immune checkpoint blockade immunotherapy by inhibiting endogenous interferon responses needed for recognition of cancer cells. By contrast, global hypomethylation results in the expression of programmed death ligand 1 (PD-L1) and inhibitory cytokines, accompanied by epithelial-mesenchymal changes that can contribute to immunosuppression. The drivers of these contrasting methylation states are not well understood. DNA methylation also plays a key role in cytotoxic T cell 'exhaustion' associated with tumor progression. We present an updated exploratory analysis of how DNA methylation may define patient subgroups and can be targeted to develop tailored treatment combinations to help improve patient outcomes.
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Metilación de ADN/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Inmunoterapia , Melanoma/inmunología , Melanoma/terapia , Antígeno B7-H1/inmunología , Citocinas/inmunología , Resistencia a Antineoplásicos/inmunología , Humanos , Melanoma/patologíaRESUMEN
Mutations in BRAF activate oncogenic MAPK signalling in almost half of cutaneous melanomas. Inhibitors of BRAF (BRAFi) and its target MEK are widely used to treat melanoma patients with BRAF mutations but unfortunately acquired resistance occurs in the majority of patients. Resistance results from mutations or non-genomic changes that either reactivate MAPK signalling or activate other pathways that provide alternate survival and growth signalling. Here, we show the histone deacetylase inhibitor (HDACi) panobinostat overcomes BRAFi resistance in melanoma, but this is dependent on the resistant cells showing a partial response to BRAFi treatment. Using patient- and in vivo-derived melanoma cell lines with acquired BRAFi resistance, we show that combined treatment with the BRAFi encorafenib and HDACi panobinostat in 2D and 3D culture systems synergistically induced caspase-dependent apoptotic cell death. Key changes induced by HDAC inhibition included decreased PI3K pathway activity associated with a reduction in the protein level of a number of receptor tyrosine kinases, and cell line dependent upregulation of pro-apoptotic BIM or NOXA together with reduced expression of anti-apoptotic proteins. Independent of these changes, panobinostat reduced c-Myc and pre-treatment of cells with siRNA against c-Myc reduced BRAFi/HDACi drug-induced cell death. These results suggest that a combination of HDAC and MAPK inhibitors may play a role in treatment of melanoma where the resistance mechanisms are due to activation of MAPK-independent pathways.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Inhibidores de Histona Desacetilasas/farmacología , Melanoma/tratamiento farmacológico , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Sinergismo Farmacológico , Xenoinjertos , Inhibidores de Histona Desacetilasas/administración & dosificación , Humanos , Melanoma/enzimología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Inhibidores de Proteínas Quinasas/administración & dosificación , Distribución Aleatoria , Transducción de Señal/efectos de los fármacosRESUMEN
Direct treatments of cancer such as chemotherapy, radiotherapy and targeted therapy have been shown to depend on recruitment of the immune system for their effectiveness. Recent studies have shown that development of resistance to direct therapies such as BRAF inhibitors in melanoma is associated with suppression of immune responses. We point to emerging data that implicate activation of the polycomb repressive complex 2 (PRC2) and its catalytic component-enhancer of zeste homolog 2 (EZH2)-in progression of melanoma and suppression of immune responses. EZH2 appears to have an important role in differentiation of CD4 T cells and particularly in the function of T regulatory cells, which suppress immune responses to melanoma. We review mechanisms of EZH2 activation at the genomic level and from activation of the MAP kinase, E2F or NF-kB2 pathways. These studies are consistent with activation of EZH2 as a common mechanism for induction of immune suppression in patients failing direct therapies and suggest EZH2 inhibitors may have a role in combination with immunotherapy and targeted therapies to prevent development of immunosuppression.
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Resistencia a Antineoplásicos/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , Epigénesis Genética , Melanoma/patología , Humanos , Melanoma/tratamiento farmacológico , Melanoma/genéticaRESUMEN
The treatment of melanoma has been revolutionized by new therapies targeting MAPK signaling or the immune system. Unfortunately these therapies are hindered by either primary resistance or the development of acquired resistance. Resistance mechanisms involving somatic mutations in genes associated with resistance have been identified in some cases of melanoma, however, the cause of resistance remains largely unexplained in other cases. The importance of epigenetic factors targeting histones and histone modifiers in driving the behavior of melanoma is only starting to be unraveled and provides significant opportunity to combat the problems of therapy resistance. There is also an increasing ability to target these epigenetic changes with new drugs that inhibit these modifications to either prevent or overcome resistance to both MAPK inhibitors and immunotherapy. This review focuses on changes in histones, histone reader proteins and histone positioning, which can mediate resistance to new therapeutics and that can be targeted for future therapies.
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The epigenetic modifier EZH2 is part of the polycomb repressive complex that suppresses gene expression via histone methylation. Activating mutations in EZH2 are found in a subset of melanoma that contributes to disease progression by inactivating tumor suppressor genes. In this study we have targeted EZH2 with a specific inhibitor (GSK126) or depleted EZH2 protein by stable shRNA knockdown. We show that inhibition of EZH2 has potent effects on the growth of both wild-type and EZH2 mutant human melanoma in vitro particularly in cell lines harboring the EZH2Y646 activating mutation. This was associated with cell cycle arrest, reduced proliferative capacity in both 2D and 3D culture systems, and induction of apoptosis. The latter was caspase independent and mediated by the release of apoptosis inducing factor (AIFM1) from mitochondria. Gene expression arrays showed that several well characterized tumor suppressor genes were reactivated by EZH2 inhibition. This included activating transcription factor 3 (ATF3) that was validated as an EZH2 target gene by ChIP-qPCR. These results emphasize a critical role for EZH2 in the proliferation and viability of melanoma and highlight the potential for targeted therapy against EZH2 in treatment of patients with melanoma.
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Genes Supresores de Tumor , Melanoma/genética , Melanoma/metabolismo , Complejo Represivo Polycomb 2/genética , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Factor de Transcripción Activador 3/genética , Apoptosis , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Progresión de la Enfermedad , Proteína Potenciadora del Homólogo Zeste 2 , Epigenómica , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Genotipo , Histonas/química , Humanos , Indoles/química , Concentración 50 Inhibidora , Mutación , Piridonas/química , Regulación hacia ArribaRESUMEN
Histone acetylation marks have an important role in controlling gene expression and are removed by histone deacetylases (HDACs). These marks are read by bromodomain and extra-terminal (BET) proteins and novel inhibitiors of these proteins are currently in clinical development. Inhibitors of HDAC and BET proteins have individually been shown to cause apoptosis and reduce growth of melanoma cells. Here we show that combining the HDAC inhibitor LBH589 and BET inhibitor I-BET151 synergistically induce apoptosis of melanoma cells but not of melanocytes. Induction of apoptosis proceeded through the mitochondrial pathway, was caspase dependent and involved upregulation of the BH3 pro-apoptotic protein BIM. Analysis of signal pathways in melanoma cell lines resistant to BRAF inhibitors revealed that treatment with the combination strongly downregulated anti-apoptotic proteins and proteins in the AKT and Hippo/YAP signaling pathways. Xenograft studies showed that the combination of inhibitors was more effective than single drug treatment and confirmed upregulation of BIM and downregulation of XIAP as seen in vitro. These results support the combination of these two classes of epigenetic regulators in treatment of melanoma including those resistant to BRAF inhibitors.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Inhibidores de Histona Desacetilasas/administración & dosificación , Melanoma/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Neoplasias Cutáneas/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Epigénesis Genética , Femenino , Compuestos Heterocíclicos de 4 o más Anillos/química , Histona Desacetilasas/metabolismo , Humanos , Ácidos Hidroxámicos/química , Inmunohistoquímica , Indoles/química , Melanocitos/metabolismo , Melanoma/tratamiento farmacológico , Ratones , Ratones Endogámicos NOD , Ratones SCID , Mitocondrias/metabolismo , Trasplante de Neoplasias , Panobinostat , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Neoplasias Cutáneas/tratamiento farmacológico , Factores de Transcripción , Proteínas Señalizadoras YAPRESUMEN
Epigenetic changes are widespread in melanoma and contribute to the pathogenic biology of this disease. In the present study, we show that I-BET151, which belongs to a new class of drugs that target the BET family of epigenetic "reader" proteins, inhibits melanoma growth in vivo and induced variable degrees of apoptosis in a panel of melanoma cells. Apoptosis was caspase dependent and associated with G1 cell cycle arrest. All melanoma cells tested had increased levels of the BH3 proapoptotic protein BIM, which appeared to be regulated by the BRD2 BET protein and to some extent by BRD3. In contrast, knockdown experiments indicated that inhibition of BRD4 was associated with decreased levels of BIM. Apoptosis was dependent on BIM in some but not all cell lines, indicating that other factors were determinants of apoptosis, such as downregulation of antiapoptotic proteins revealed in gene expression arrays. G1 cell cycle arrest appeared to be mediated by p21 and resulted from inhibition of the BRD4 protein. The activity of BET protein inhibitors appears independent of the BRAF and NRAS mutational status of melanoma, and further studies to assess their therapeutic role in melanoma are warranted.
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Proteínas Reguladoras de la Apoptosis/metabolismo , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Compuestos Heterocíclicos de 4 o más Anillos/química , Melanoma/genética , Melanoma/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Apoptosis , Proteína 11 Similar a Bcl2 , Caspasas/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular , Línea Celular , Línea Celular Tumoral , Supervivencia Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Análisis Mutacional de ADN , Femenino , GTP Fosfohidrolasas/metabolismo , Humanos , Melanoma/tratamiento farmacológico , Ratones , Ratones Endogámicos NOD , Proteínas de Unión al GTP Monoméricas/metabolismo , Trasplante de Neoplasias , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas B-raf/metabolismo , Factores de Transcripción/metabolismo , TranscriptomaRESUMEN
Deleterious germline variants in CDKN2A account for around 40% of familial melanoma cases, and rare variants in CDK4, BRCA2, BAP1 and the promoter of TERT have also been linked to the disease. Here we set out to identify new high-penetrance susceptibility genes by sequencing 184 melanoma cases from 105 pedigrees recruited in the UK, The Netherlands and Australia that were negative for variants in known predisposition genes. We identified families where melanoma cosegregates with loss-of-function variants in the protection of telomeres 1 gene (POT1), with a proportion of family members presenting with an early age of onset and multiple primary tumors. We show that these variants either affect POT1 mRNA splicing or alter key residues in the highly conserved oligonucleotide/oligosaccharide-binding (OB) domains of POT1, disrupting protein-telomere binding and leading to increased telomere length. These findings suggest that POT1 variants predispose to melanoma formation via a direct effect on telomeres.
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Predisposición Genética a la Enfermedad/genética , Melanoma/genética , Modelos Moleculares , Proteínas de Unión a Telómeros/genética , Telómero/metabolismo , Secuencia de Aminoácidos , Australia , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Países Bajos , Linaje , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN , Complejo Shelterina , Neoplasias Cutáneas , Telómero/química , Telómero/genética , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/metabolismo , Reino Unido , Melanoma Cutáneo MalignoRESUMEN
Amino acids, especially leucine and glutamine, are important for tumor cell growth, survival and metabolism. A range of different transporters deliver each specific amino acid into cells, some of which are increased in cancer. These amino acids consequently activate the mTORC1 pathway and drive cell cycle progression. The leucine transporter LAT1/4F2hc heterodimer assembles as part of a large complex with the glutamine transporter ASCT2 to transport amino acids. In this study, we show that the expression of LAT1 and ASCT2 is significantly increased in human melanoma samples and is present in both BRAF(WT) (C8161 and WM852) and BRAF(V600E) mutant (1205Lu and 451Lu) melanoma cell lines. While inhibition of LAT1 by BCH did not suppress melanoma cell growth, the ASCT2 inhibitor BenSer significantly reduced both leucine and glutamine transport in melanoma cells, leading to inhibition of mTORC1 signaling. Cell proliferation and cell cycle progression were significantly reduced in the presence of BenSer in melanoma cells in 2D and 3D cell culture. This included reduced expression of the cell cycle regulators CDK1 and UBE2C. The importance of ASCT2 expression in melanoma was confirmed by shRNA knockdown, which inhibited glutamine uptake, mTORC1 signaling and cell proliferation. Taken together, our study demonstrates that ASCT2-mediated glutamine transport is a potential therapeutic target for both BRAF(WT) and BRAF(V600E) melanoma.
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Sistema de Transporte de Aminoácidos ASC/biosíntesis , Glutamina/metabolismo , Transportador de Aminoácidos Neutros Grandes 1/biosíntesis , Melanoma/patología , Complejos Multiproteicos/antagonistas & inhibidores , Neoplasias Cutáneas/patología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Sistema de Transporte de Aminoácidos ASC/antagonistas & inhibidores , Sistema de Transporte de Aminoácidos ASC/genética , Aminoácidos Cíclicos/farmacología , Compuestos de Bencilo/farmacología , Transporte Biológico , Proteína Quinasa CDC2/biosíntesis , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Humanos , Leucina/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Melanoma/metabolismo , Antígenos de Histocompatibilidad Menor , Complejos Multiproteicos/genética , Proteínas Proto-Oncogénicas B-raf/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Serina/análogos & derivados , Serina/farmacología , Transducción de Señal , Neoplasias Cutáneas/metabolismo , Esferoides Celulares , Serina-Treonina Quinasas TOR/genética , Células Tumorales Cultivadas , Enzimas Ubiquitina-Conjugadoras/biosíntesisRESUMEN
A major challenge in cancer genetics is to determine which low-frequency somatic mutations are drivers of tumorigenesis. Here we interrogate the genomes of 7,651 diverse human cancers and find inactivating mutations in the homeodomain transcription factor gene CUX1 (cut-like homeobox 1) in ~1-5% of various tumors. Meta-analysis of CUX1 mutational status in 2,519 cases of myeloid malignancies reveals disruptive mutations associated with poor survival, highlighting the clinical significance of CUX1 loss. In parallel, we validate CUX1 as a bona fide tumor suppressor using mouse transposon-mediated insertional mutagenesis and Drosophila cancer models. We demonstrate that CUX1 deficiency activates phosphoinositide 3-kinase (PI3K) signaling through direct transcriptional downregulation of the PI3K inhibitor PIK3IP1 (phosphoinositide-3-kinase interacting protein 1), leading to increased tumor growth and susceptibility to PI3K-AKT inhibition. Thus, our complementary approaches identify CUX1 as a pan-driver of tumorigenesis and uncover a potential strategy for treating CUX1-mutant tumors.