TERT Promoter Revertant Mutation Inhibits Melanoma Growth through Intrinsic Apoptosis.
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ABSTRACT:
SUBMITTER: Wang Y
PROVIDER: S-EPMC8773187 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The aim of this study was exploration of the genetic background of conjunctival melanoma (CM) and correlation with recurrent and metastatic disease. Twenty-eight CM from the Rotterdam Ocular Melanoma Study group were collected and DNA was isolated from the formalin-fixed paraffin embedded tissue. Targeted next-generation sequencing was performed using a panel covering GNAQ, GNA11, EIF1AX, BAP1, BRAF, NRAS, c-KIT, PTEN, SF3B1, and TERT genes. Recurrences and metastasis were present in eight (29%) and nine (32%) CM cases, respectively. TERT promoter mutations were most common (54%), but BRAF (46%), NRAS (21%), BAP1 (18%), PTEN (14%), c-KIT (7%), and SF3B1 (4%) mutations were also observed. No mutations in GNAQ, GNA11, and EIF1AX were found. None of the mutations was significantly associated with recurrent disease. Presence of a TERT promoter mutation was associated with metastatic disease (p-value = 0.008). Based on our molecular findings, CM comprises a separate entity within melanoma, although there are overlapping molecular features with uveal melanoma, such as the presence of BAP1 and SF3B1 mutations. This warrants careful interpretation of molecular data, in the light of clinical findings. About three quarter of CM contain drug-targetable mutations, and TERT promoter mutations are correlated to metastatic disease in CM.
| S-EPMC8198138 | biostudies-literature
Project description:Despite advances in targeted therapies, the treatment of advanced melanoma remains an exercise in disease management, hence a need for biomarkers for identification of at-risk primary melanoma patients. In this study, we aimed to assess the prognostic value of TERT promoter mutations in primary melanomas. Tumors from 300 patients with stage I/II melanoma were sequenced for TERT promoter and BRAF/NRAS mutations. Cumulative curves were drawn for patients with and without mutations with progression-free and melanoma-specific survival as outcomes. Cox proportional hazard regression models were used to determine the effect of the mutations on survivals. Individually, presence of TERT promoter and BRAF/NRAS mutations associated with poor disease-free and melanoma-specific survival with modification of the effect by the rs2853669 polymorphism within the TERT promoter. Hazard ratio (HR) for simultaneous occurrence of TERT promoter and BRAF/NRAS mutations for disease-free survival was 2.3 (95% CI 1.2-4.4) and for melanoma-specific survival 5.8 (95% CI 1.9-18.3). The effect of the mutations on melanoma-specific survival in noncarriers of variant allele of the polymorphism was significant (HR 4.5, 95% CI 1.4-15.2) but could not be calculated for the carriers due to low number of events. The variant allele per se showed association with increased survival (HR 0.3, 95% CI 0.1-0.9). The data in this study provide preliminary evidence that TERT promoter mutations in combination with BRAF/NRAS mutations can be used to identify patients at risk of aggressive disease and the possibility of refinement of the classification with inclusion of the rs2853669 polymorphism within TERT promoter.
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Project description:Although TERT promoter mutations have been associated with a worsened prognosis in melanoma, the relationship between mutation status and downstream telomerase activity and telomere length remains convoluted. Using Sanger sequencing and techniques based on quantitative reverse transcriptase in real time, we evaluated 60 melanoma cell lines for TERT promoter mutational status, copy number, gene expression, and telomere length to provide a comprehensive analysis of the TERT/telomere pathway and establish a classification system whereby the associations between TERT mutations and their downstream molecular manifestations can more easily be ascertained. Mutations at positions -124/125 and -146 were associated with the highest levels of TERT gene expression but had no appreciable impact on absolute telomere length. In contrast, the common variant rs2853669 (at position -245) was significantly associated with longer telomere length via a recessive model in our cohort (P = 0.003). Our results, which are from assays performed on purified melanoma cell lines, suggest that the TERT promoter harbors a more complex mutational landscape than previously thought. Furthermore, the failure of TERT promoter mutations to consistently correlate with TERT expression and telomere length suggests an alternative method whereby tumor cells escape the critical shortening of telomeres.
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Project description:Malignant melanoma is an extremely malignant tumor with a high mortality rate and an increasing incidence with a high mutation load. The frequency of mutations in the TERT promoter exceeds the frequency of any known noncoding mutations in melanoma. A growing number of recent studies suggest that the most common mutations in the TERT promoter (ATG start site -124C>T and -146C>T) are associated with increased TERT mRNA expression, telomerase activity, telomere length, and poor prognosis. Recently, it has been shown that TERT promoter mutations are more correlated with the occurrence, development, invasion, and metastasis of melanoma, as well as emerging approaches such as the therapeutic potential of chemical inhibition of TERT promoter mutations, direct telomerase inhibitors, combined targeted therapy, and immunotherapies. In this review, we describe the latest advances in the role of TERT promoter mutations and telomerase in promoting the occurrence, development, and poor prognosis of melanoma and discuss the clinical significance of the TERT promoter and telomerase in the treatment of melanoma.
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Project description:Grouper iridovirus (GIV) belongs to the genus Ranavirus of the family Iridoviridae; the genomes of such viruses contain an anti-apoptotic caspase recruitment domain (CARD) gene. The GIV-CARD gene encodes a protein of 91 amino acids with a molecular mass of 10,505 Daltons, and shows high similarity to other viral CARD genes and human ICEBERG. In this study, we used Northern blot to demonstrate that GIV-CARD transcription begins at 4 h post-infection; furthermore, we report that its transcription is completely inhibited by cycloheximide but not by aphidicolin, indicating that GIV-CARD is an early gene. GIV-CARD-EGFP and GIV-CARD-FLAG recombinant proteins were observed to translocate from the cytoplasm into the nucleus, but no obvious nuclear localization sequence was observed within GIV-CARD. RNA interference-mediated knockdown of GIV-CARD in GK cells infected with GIV inhibited expression of GIV-CARD and five other viral genes during the early stages of infection, and also reduced GIV infection ability. Immunostaining was performed to show that apoptosis was effectively inhibited in cells expressing GIV-CARD. HeLa cells irradiated with UV or treated with anti-Fas antibody will undergo apoptosis through the intrinsic and extrinsic pathways, respectively. However, over-expression of recombinant GIV-CARD protein in HeLa cells inhibited apoptosis induced by mitochondrial and death receptor signaling. Finally, we report that expression of GIV-CARD in HeLa cells significantly reduced the activities of caspase-8 and -9 following apoptosis triggered by anti-Fas antibody. Taken together, these results demonstrate that GIV-CARD inhibits apoptosis through both intrinsic and extrinsic pathways.
| S-EPMC4457926 | biostudies-literature
Project description:BackgroundUveal melanoma is the most frequent primary tumour of the eye. It is molecularly clearly distinct from cutaneous melanoma and shows a different pattern of driver mutations. The influence of sunlight ultraviolet (UV) exposure on the aetiology of uveal melanoma is a matter of debate. The recent identification of driver mutations in the promoter of the telomerase reverse transcriptase (TERT) gene with UV-induced cytidine-to-thymidine transitions in cutaneous melanoma prompted us to investigate whether these mutations also occur in uveal melanoma.MethodsWe analysed 50 cases of uveal melanoma obtained from enucleation surgery for mutations in the genes GNAQ, GNA11, BAP1, SF3B1, EIFAX1 and TERT, measured gene expression using microarrays and analysed gene copy numbers by SNP arrays.ResultsWe detected a TERT mutation in only one case of a 57-year-old white male patient with clinical and histopathological features typical for uveal melanoma. The tumour showed mutations in GNA11 and EIF1AX that are typical for uveal melanoma and absent from cutaneous melanoma. No mutations were detected in GNAQ, BAP1 and SF3B1 that are frequently mutated in uveal melanoma. Both copies of chromosome 3 were retained. Several tumours among which the one carrying the TERT promoter mutation showed elevated TERT expression. Consistent with previous reports, GNAQ is inversely associated with chromosome 3 monosomy and metastasis. BAP1 mutations are significantly associated with chromosome 3 monosomy but not with relapse.ConclusionThese data indicate that TERT mutations are rare in uveal melanoma. No conclusion can be drawn on their potential influence on tumour progression.
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Project description:Systematic sequencing of human cancer genomes has identified many recurrent mutations in the protein-coding regions of genes but rarely in gene regulatory regions. Here, we describe two independent mutations within the core promoter of telomerase reverse transcriptase (TERT), the gene coding for the catalytic subunit of telomerase, which collectively occur in 50 of 70 (71%) melanomas examined. These mutations generate de novo consensus binding motifs for E-twenty-six (ETS) transcription factors, and in reporter assays, the mutations increased transcriptional activity from the TERT promoter by two- to fourfold. Examination of 150 cancer cell lines derived from diverse tumor types revealed the same mutations in 24 cases (16%), with preliminary evidence of elevated frequency in bladder and hepatocellular cancer cells. Thus, somatic mutations in regulatory regions of the genome may represent an important tumorigenic mechanism.
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Project description:Metformin is the most widely used antidiabetic drug because of its proven efficacy and limited secondary effects. Interestingly, recent studies have reported that metformin can block the growth of different tumor types. Here, we show that metformin exerts antiproliferative effects on melanoma cells, whereas normal human melanocytes are resistant to these metformin-induced effects. To better understand the basis of this antiproliferative effect of metformin in melanoma, we characterized the sequence of events underlying metformin action. We showed that 24?h metformin treatment induced a cell cycle arrest in G0/G1 phases, while after 72?h, melanoma cells underwent autophagy as demonstrated by electron microscopy, immunochemistry, and by quantification of the autolysosome-associated LC3 and Beclin1 proteins. In addition, 96?h post metformin treatment we observed robust apoptosis of melanoma cells. Interestingly, inhibition of autophagy by knocking down LC3 or ATG5 decreased the extent of apoptosis, and suppressed the antiproliferative effect of metformin on melanoma cells, suggesting that apoptosis is a consequence of autophagy. The relevance of these observations were confirmed in vivo, as we showed that metformin treatment impaired the melanoma tumor growth in mice, and induced autophagy and apoptosis markers. Taken together, our data suggest that metformin has an important impact on melanoma growth, and may therefore be beneficial in patients with melanoma.
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Project description:Although melanoma therapy is improved by novel molecular targeted reagents, including vemurafenib, aberrant proliferation and early metastasis remain obstacles for melanoma; therefore, novel target molecules for melanoma need to be identified. In this study, we focused on deubiquitinating enzymes, which regulate protein stability through ubiquitin-proteasome systems, and identified 26S proteasome non-ATPase regulatory subunit 14 (PSMD14) as a molecule related to melanoma growth using siRNA library screening. Similar to a previous report, PSMD14 knockdown strongly induced p21 expression and inhibited RB phosphorylation in melanoma. After in silico analysis, TGF-β signaling was identified as a negatively correlated gene set with PSMD14 expression. Although TGF-β signaling is also related to the invasive phenotype of melanoma, PSMD14 knockdown suppressed melanoma migration and reduced SLUG expression, suggesting that targeting PSMD14 suppresses both growth and migration. Furthermore, SMAD3 expression increased in nucleus and SMAD3 degradation was delayed after PSMD14 knockdown. Thus, our present study suggests that targeting PSMD14 can inhibit melanoma growth and migration through either SMAD3 accumulation or SLUG reduction, respectively.
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Project description:The mechanism of telomerase re-activation in cancer had remained elusive until the discovery of frequent mutations in the promoter of the TERT gene that encodes the catalytic reverse transcriptase subunit of telomerase. We investigated the regulation of TERT expression in melanoma cell lines and our results show that promoter mutations render TERT expression dependent on MAPK activation due to oncogenic BRAF or NRAS mutations. Mutations in the TERT promoter create binding sites for ETS transcription factors. ETS1, expressed in melanoma cell lines, undergoes activating phosphorylation by ERK at Thr38 residue as a consequence of constitutively activated MAPK pathway. We demonstrate that ETS1 binds on the mutated TERT promoter leading to the re-expression of the gene. The inhibition of ETS1 resulted in reduced TERT expression. We provide evidence that the TERT promoter mutations provide a direct link between TERT expression and MAPK pathway activation due to BRAF or NRAS mutations via the transcription factor ETS1.
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