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1.
Cancer Res ; 2021 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-34645608

RESUMO

CD271 (NGFR) is a neurotrophin receptor that belongs to the tumor necrosis receptor (TNFR) family. Upon ligand binding, CD271 can mediate either survival or cell death. While the role of CD271 as a marker of tumor-initiating cells is still a matter of debate, its role in melanoma progression has been well documented. Moreover, CD271 has been shown to be upregulated after exposure to both chemotherapy and targeted therapy. In this study, we demonstrate that activation of CD271 by a short ß-amyloid-derived peptide (Aß(25-35)) in combination with either chemotherapy or MAPK inhibitors induces apoptosis in 2D and 3D cultures of 8 melanoma cell lines. This combinatorial treatment significantly reduced metastasis in a zebrafish xenograft model and led to significantly decreased tumor volume in mice. Administration of Aß(25-35) in ex vivo tumors from immunotherapy- and targeted therapy-resistant patients significantly reduced proliferation of melanoma cells, showing that activation of CD271 can overcome drug resistance. Aß(25-35) was specific to CD271-expressing cells and induced CD271 cleavage and phosphorylation of JNK (pJNK). The direct protein-protein interaction of pJNK with CD271 led to PARP1 cleavage, p53 and caspase activation, and pJNK-dependent cell death. Aß(25-35) also mediated mitochondrial reactive oxygen species (mROS) accumulation, which induced CD271 overexpression. Finally, CD271 upregulation inhibited mROS production, revealing the presence of a negative feedback loop in mROS regulation. These results indicate that targeting CD271 can activate cell death pathways to inhibit melanoma progression and potentially overcome resistance to targeted therapy.

2.
Eur J Cancer ; 156: 149-163, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34454317

RESUMO

BACKGROUND: Melanoma brain metastases (MBM) have a poor prognosis. Systemic treatments that have improved outcomes in advanced melanoma have been shown to have an intracranial (IC) effect. We studied the efficacy and outcomes of combined immune checkpoint inhibitor ipilimumab/nivolumab (Combi-ICI) or targeted therapy (Combi-TT) as first-line treatment in MBM. METHODS: MBM patients treated with Combi-ICI or Combi-TT within 3 months after MBM diagnosis. Endpoints were progression-free survival (PFS) and overall survival (OS). RESULTS: 53 patients received Combi-ICI, 32% had symptomatic MBM and 33.9% elevated LDH. 71.7% required local treatment. The disease control rate was 60.3%. IC response rate (RR) was 43.8% at 3-months with durable responses at 6- (46.5%) and 12-months (53.1%). Extracranial (EC) RR was 44.7% at 3-months and 50% at 12-months. Median PFS was 9.6 months (95% CI 3.6-NR) and median overall survival (mOS) 44.8 months (95% CI; 26.2-NR). 63 patients received Combi-TT, 55.6% of patients had symptomatic MBM, 57.2% of patients had elevated LDH and 68.3% of patients required local treatment. The disease control rate was 60.4%. ICRR was 50% at 3-months, but dropped at 6-months (20.9%). ECRR was 69.2% at 3-months and 17.6% at 12-months. Median PFS was 5.8 months (95% CI 4.2-7.6) and mOS 14.2 months (95% CI 8.99-26.8). In BRAFV600 patients, 26.7% of patients received Combi-ICI and 73.3% Combi-TT with OS (p = 0.0053) and mPFS (p = 0.03) in favour to Combi-ICI. CONCLUSION: Combi-ICI showed prolonged mOS with sustainable IC and EC responses. Despite the initially increased efficacy, Combi-TT responses at 12 months were low. Combi-ICI appeared superior to Combi-TT for OS and PFS in BRAFV600 patients. Other clinical factors are determinants for first-line treatment choice.

3.
Nat Commun ; 12(1): 5056, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417458

RESUMO

Melanoma cells rely on developmental programs during tumor initiation and progression. Here we show that the embryonic stem cell (ESC) factor Sall4 is re-expressed in the Tyr::NrasQ61K; Cdkn2a-/- melanoma model and that its expression is necessary for primary melanoma formation. Surprisingly, while Sall4 loss prevents tumor formation, it promotes micrometastases to distant organs in this melanoma-prone mouse model. Transcriptional profiling and in vitro assays using human melanoma cells demonstrate that SALL4 loss induces a phenotype switch and the acquisition of an invasive phenotype. We show that SALL4 negatively regulates invasiveness through interaction with the histone deacetylase (HDAC) 2 and direct co-binding to a set of invasiveness genes. Consequently, SALL4 knock down, as well as HDAC inhibition, promote the expression of an invasive signature, while inhibition of histone acetylation partially reverts the invasiveness program induced by SALL4 loss. Thus, SALL4 appears to regulate phenotype switching in melanoma through an HDAC2-mediated mechanism.


Assuntos
Epigênese Genética , Melanoma/genética , Melanoma/patologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Fator de Células-Tronco/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Animais , Sequência de Bases , Carcinogênese/genética , Carcinogênese/patologia , Adesão Celular/genética , Linhagem Celular Tumoral , Linhagem da Célula , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Histona Desacetilase 2/metabolismo , Histonas/metabolismo , Humanos , Melanócitos/metabolismo , Melanócitos/patologia , Camundongos Nus , Camundongos Transgênicos , Invasividade Neoplásica , Micrometástase de Neoplasia , Ligação Proteica , Carga Tumoral
4.
Eur J Cancer ; 149: 37-48, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33823361

RESUMO

BACKGROUND: Anti-PD1-based immunotherapy is currently used in most patients with advanced melanoma. Despite the remarkable data regarding overall survival, the optimal treatment duration is still unknown. METHODS: We evaluated the outcome of 125 patients with advanced melanoma with and without brain metastases (MBM), treated either with anti-PD1 monotherapy (N = 97) or combined with anti-CTLA4 (N = 28) after elective treatment discontinuation due to complete response (CR) (group A, N = 86), or treatment-limiting toxicity (N = 33) and investigator's decision (ID, N = 6) (group B) with subsequent CR. RESULTS: For group A, median duration of treatment (mDoT) was 22 months (range 5-49) and median time to CR 9 months (range 2-47). Accordingly, mDoT for group B was 3 months (range 0-36) and median time to CR 7 months (range 1-32). Seven patients from group A and three from group B experienced disease recurrence. Off-treatment survival was not reached. Median off-treatment response time (mOTRt) was 19 months (range 0-42) and 25 months (range 0-66), respectively. For MBM, mOTRt was 17 months (range 7-41) and 28 months (range 9-39), respectively. After a median follow-up of 38 months (range 9-70), seven (5.6%) patients had deceased, one (0.8%) due to melanoma. CONCLUSIONS: Treatment discontinuation is feasible also in patients with MBM. Efficacy outcomes seemed to be similar in both groups of patients who achieved CR, regardless of reason for discontinuation. In patients who experienced disease relapse, treatment re-challenge with anti-PD1 resulted in subsequent renewed response.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Inibidores de Checkpoint Imunológico/uso terapêutico , Melanoma/tratamento farmacológico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Neoplasias Cutâneas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/secundário , Intervalo Livre de Doença , Europa (Continente) , Estudos de Viabilidade , Feminino , Humanos , Inibidores de Checkpoint Imunológico/efeitos adversos , Masculino , Melanoma/imunologia , Melanoma/secundário , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/imunologia , Recidiva , Estudos Retrospectivos , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologia , Fatores de Tempo , Adulto Jovem
5.
J Mol Diagn ; 23(6): 691-697, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33775845

RESUMO

Reliable transportation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) patient samples from a swabbing station to a diagnostics facility is essential for accurate results. Therefore, cooling or freezing the samples is recommended in case of longer transportation times. In this study, SARS-CoV-2 detectability by RT-PCR was assessed after prolonged unfrozen storage or repetitive freeze-thawing of SARS-CoV-2 samples. SARS-CoV-2-positive patient swabs stored in viral transport medium were exposed to different temperatures (4°C, 25°C, and 35°C) and to repetitive freeze-thawing, to assess the effect of storage conditions on RT-PCR detection. SARS-CoV-2 RNA was still reliably detected by RT-PCR after 21 days of storage in viral transport medium, even when the samples had been stored at 35°C. The maximum observed change in cycle threshold value per day was 0.046 (±0.019) at 35°C, and the maximum observed change in cycle threshold value per freeze-thaw cycle per day was 0.197 (±0.06). Compared with storage at 4°C, viral RNA levels deviated little but significantly when stored at 25°C or 35°C, or after repeated freeze-thawing. The results of this study indicate that viral RNA levels are relatively stable at higher temperatures and repetitive freeze-thawing.


Assuntos
Teste de Ácido Nucleico para COVID-19/normas , COVID-19/diagnóstico , RNA Viral/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , SARS-CoV-2/genética , Manejo de Espécimes/métodos , COVID-19/epidemiologia , Teste de Ácido Nucleico para COVID-19/instrumentação , Teste de Ácido Nucleico para COVID-19/métodos , Congelamento , Humanos , Nasofaringe/virologia , Estabilidade de RNA , Suíça/epidemiologia , Temperatura , Fatores de Tempo
6.
Nat Commun ; 12(1): 1434, 2021 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-33664264

RESUMO

Although melanoma is initiated by acquisition of point mutations and limited focal copy number alterations in melanocytes-of-origin, the nature of genetic changes that characterise lethal metastatic disease is poorly understood. Here, we analyze the evolution of human melanoma progressing from early to late disease in 13 patients by sampling their tumours at multiple sites and times. Whole exome and genome sequencing data from 88 tumour samples reveals only limited gain of point mutations generally, with net mutational loss in some metastases. In contrast, melanoma evolution is dominated by whole genome doubling and large-scale aneuploidy, in which widespread loss of heterozygosity sculpts the burden of point mutations, neoantigens and structural variants even in treatment-naïve and primary cutaneous melanomas in some patients. These results imply that dysregulation of genomic integrity is a key driver of selective clonal advantage during melanoma progression.


Assuntos
Aneuploidia , Variações do Número de Cópias de DNA/genética , Genoma Humano/genética , Melanoma/genética , Neoplasias Cutâneas/genética , Progressão da Doença , Exoma/genética , Humanos , Mutação INDEL/genética , Melanócitos/patologia , Mutação Puntual/genética , Polimorfismo de Nucleotídeo Único/genética , Sequenciamento Completo do Exoma , Sequenciamento Completo do Genoma
7.
Elife ; 102021 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-33438577

RESUMO

The microphthalmia-associated transcription factor (MITF) is a critical regulator of melanocyte development and differentiation. It also plays an important role in melanoma where it has been described as a molecular rheostat that, depending on activity levels, allows reversible switching between different cellular states. Here, we show that MITF directly represses the expression of genes associated with the extracellular matrix (ECM) and focal adhesion pathways in human melanoma cells as well as of regulators of epithelial-to-mesenchymal transition (EMT) such as CDH2, thus affecting cell morphology and cell-matrix interactions. Importantly, we show that these effects of MITF are reversible, as expected from the rheostat model. The number of focal adhesion points increased upon MITF knockdown, a feature observed in drug-resistant melanomas. Cells lacking MITF are similar to the cells of minimal residual disease observed in both human and zebrafish melanomas. Our results suggest that MITF plays a critical role as a repressor of gene expression and is actively involved in shaping the microenvironment of melanoma cells in a cell-autonomous manner.

8.
Pigment Cell Melanoma Res ; 34(2): 150-162, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32910840

RESUMO

Oncogenic BRAF and NRAS mutations drive human melanoma initiation. We used transgenic zebrafish to model NRAS-mutant melanoma, and the rapid tumor onset allowed us to study candidate tumor suppressors. We identified P38α-MAPK14 as a potential tumor suppressor in The Cancer Genome Atlas melanoma cohort of NRAS-mutant melanomas, and overexpression significantly increased the time to tumor onset in transgenic zebrafish with NRAS-driven melanoma. Pharmacological activation of P38α-MAPK14 using anisomycin reduced in vitro viability of melanoma cultures, which we confirmed by stable overexpression of p38α. We observed that the viability of MEK inhibitor resistant melanoma cells could be reduced by combined treatment of anisomycin and MEK inhibition. Our study demonstrates that activating the p38α-MAPK14 pathway in the presence of oncogenic NRAS abrogates melanoma in vitro and in vivo. SIGNIFICANCE: The significance of our study is in the accountability of NRAS mutations in melanoma. We demonstrate here that activation of p38α-MAPK14 pathway can abrogate NRAS-mutant melanoma which is contrary to the previously published role of p38α-MAPK14 pathway in BRAF mutant melanoma. These results implicate that BRAF and NRAS-mutant melanoma may not be identical biologically. We also demonstrate the translational benefit of our study by using a small molecule compound-anisomycin (already in use for other diseases in clinical trials) to activate p38α-MAPK14 pathway.

9.
Nat Commun ; 11(1): 5117, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33037203

RESUMO

Exposure of gastric epithelial cells to the bacterial carcinogen Helicobacter pylori causes DNA double strand breaks. Here, we show that H. pylori-induced DNA damage occurs co-transcriptionally in S-phase cells that activate NF-κB signaling upon innate immune recognition of the lipopolysaccharide biosynthetic intermediate ß-ADP-heptose by the ALPK1/TIFA signaling pathway. DNA damage depends on the bi-functional RfaE enzyme and the Cag pathogenicity island of H. pylori, is accompanied by replication fork stalling and can be observed also in primary cells derived from gastric organoids. Importantly, H. pylori-induced replication stress and DNA damage depend on the presence of co-transcriptional RNA/DNA hybrids (R-loops) that form in infected cells during S-phase as a consequence of ß-ADP-heptose/ ALPK1/TIFA/NF-κB signaling. H. pylori resides in close proximity to S-phase cells in the gastric mucosa of gastritis patients. Taken together, our results link bacterial infection and NF-κB-driven innate immune responses to R-loop-dependent replication stress and DNA damage.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Helicobacter pylori/patogenicidade , NF-kappa B/metabolismo , Proteínas Quinases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , DNA/química , DNA/genética , Dano ao DNA , Replicação do DNA/efeitos dos fármacos , Floxuridina , Glicosiltransferases/metabolismo , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/microbiologia , Helicobacter pylori/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Lipopolissacarídeos/metabolismo , Mutação , NF-kappa B/genética , Proteínas Quinases/genética , Espécies Reativas de Oxigênio/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia
10.
Cancer Immunol Res ; 8(9): 1114-1121, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32661093

RESUMO

Concurrent MEK and CDK4/6 inhibition shows promise in clinical trials for patients with advanced-stage mutant BRAF/NRAS solid tumors. The effects of CDK4/6 inhibitor (CDK4/6i) in combination with BRAF/MEK-targeting agents on the tumor immune microenvironment are unclear, especially in melanoma, for which immune checkpoint inhibitors are effective in approximately 50% of patients. Here, we show that patients progressing on CDK4/6i/MEK pathway inhibitor combinations exhibit T-cell exclusion. We found that MEK and CDK4/6 targeting was more effective at delaying regrowth of mutant BRAF melanoma in immunocompetent versus immune-deficient mice. Although MEK inhibitor (MEKi) treatment increased tumor immunogenicity and intratumoral recruitment of CD8+ T cells, the main effect of CDK4/6i alone and in combination with MEKi was increased expression of CD137L, a T-cell costimulatory molecule on immune cells. Depletion of CD8+ T cells or blockade of the CD137 ligand-receptor interaction reduced time to regrowth of melanomas in the context of treatment with CDK4/6i plus MEKi treatment in vivo Together, our data outline an antitumor immune-based mechanism and show the efficacy of targeting both the MEK pathway and CDK4/6.

11.
Cancer Res ; 80(14): 2983-2995, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32503808

RESUMO

Epithelial-to-mesenchymal transition (EMT)-inducing transcription factors (TF) are well known for their ability to induce mesenchymal states associated with increased migratory and invasive properties. Unexpectedly, nuclear expression of the EMT-TF ZEB2 in human primary melanoma has been shown to correlate with reduced invasion. We report here that ZEB2 is required for outgrowth for primary melanomas and metastases at secondary sites. Ablation of Zeb2 hampered outgrowth of primary melanomas in vivo, whereas ectopic expression enhanced proliferation and growth at both primary and secondary sites. Gain of Zeb2 expression in pulmonary-residing melanoma cells promoted the development of macroscopic lesions. In vivo fate mapping made clear that melanoma cells undergo a conversion in state where ZEB2 expression is replaced by ZEB1 expression associated with gain of an invasive phenotype. These findings suggest that reversible switching of the ZEB2/ZEB1 ratio enhances melanoma metastatic dissemination. SIGNIFICANCE: ZEB2 function exerts opposing behaviors in melanoma by promoting proliferation and expansion and conversely inhibiting invasiveness, which could be of future clinical relevance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/14/2983/F1.large.jpg.


Assuntos
Proliferação de Células , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/secundário , Melanoma/patologia , Fatores de Transcrição/metabolismo , Homeobox 2 de Ligação a E-box com Dedos de Zinco/metabolismo , Animais , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Melanoma/genética , Melanoma/metabolismo , Camundongos , Invasividade Neoplásica , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Homeobox 2 de Ligação a E-box com Dedos de Zinco/genética
12.
J Invest Dermatol ; 140(11): 2242-2252.e7, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32389536

RESUMO

Melanomas frequently harbor activating NRAS mutations leading to activation of MAPK kinase (MEK) and extracellular signal-regulated kinase 1/2 signaling; however, the clinical efficacy of inhibitors to this pathway is limited by resistance. Tumors rewire metabolic pathways in response to stress signals such as targeted inhibitors and drug resistance, but most therapy-resistant preclinical models are generated in conditions that lack physiological metabolism. We generated human NRAS-mutant melanoma xenografts that were resistant to the MEK inhibitor (MEKi) PD0325901 in vivo. MEKi-resistant cells showed cross-resistance to the structurally distinct MEKi trametinib and elevated extracellular signal-regulated kinase 1/2 phosphorylation and downstream signaling. Additionally, we observed upregulation of the serine synthesis pathway and PHGDH, a key enzyme in this pathway. Suppressing PHGDH in MEKi-resistant cells together with MEKi treatment decreased oxidative stress tolerance and cell proliferation. Together, our data suggest targeting PHGDH as a potential strategy in overcoming MEKi resistance.


Assuntos
GTP Fosfo-Hidrolases/genética , Glutationa/metabolismo , Melanoma/tratamento farmacológico , Proteínas de Membrana/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Mutação , Fosfoglicerato Desidrogenase/antagonistas & inibidores , Animais , Resistencia a Medicamentos Antineoplásicos , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Feminino , Humanos , Melanoma/genética , Melanoma/metabolismo , Camundongos , Serina/biossíntese , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Acta Biomater ; 106: 136-144, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32044460

RESUMO

After skin tissue injury or pathological removal, vascularization timing is paramount in graft survival. As full thickness skin grafts often fail to become perfused over larger surfaces, split-thickness grafts are preferred and can be used together with biomaterials, which themselves are non-angiogenic. One way of promoting vascular ingrowth is to "pre-vascularize" an engineered substitute by introducing endothelial cells (ECs). Since it has been previously demonstrated that surface structured biomaterials have an effect on wound healing, skin regeneration, and fibrosis reduction, we proposed that a microvascular-rich lipoconstruct with anisotropic topographical cues could be a clinically translatable vascularization approach. Murine lipofragments were formed with three polydimethylsiloxane molds (flat, 5 µm, and 50 µm parallel gratings) and implanted into the dorsal skinfold chamber of male C57BL/6 mice. Vascular ingrowth was observed through intravital microscopy over 21 days and further assessed by histology and protein identification. Our investigation revealed that topographical feature size influenced the commencement of neovascular ingrowth, with 5 µm gratings exhibiting early construct perfusion at 3 days post-operation, and 50 µm being delayed until day 5. We therefore postulate that surface structured lipoconstructs may serve as an easily obtained and produced construct suitable for providing soft tissue and ECs to tissue defects. STATEMENT OF SIGNIFICANCE: Skin graft failures due to inadequate or uneven perfusion frequently occur and can be even more complicated in deep, difficult to heal wounds, or bone coverage. In complex injuries, biomaterials are often used to cover bone structures with a standard split thickness graft; however, perfusion can take up to 3 weeks. Thus, any means to promote faster and uniform vascularization could significantly reduce healing time, as well as lower patient down-time. As pre-vascularized constructs have reported success in research, we created a cost-efficient, translatable method with no additional laboratory time as adipose tissue can be harvested and used immediately. We further used surface topography as an aspect to modulate construct perfusion, which has been reported for the first time here.


Assuntos
Tecido Adiposo/metabolismo , Neovascularização Fisiológica/fisiologia , Próteses e Implantes , Pele/irrigação sanguínea , Tecidos Suporte/química , Animais , Anisotropia , Colágeno/metabolismo , Dimetilpolisiloxanos/química , Epididimo/citologia , Fibrina/química , Masculino , Camundongos Endogâmicos C57BL , Microcirculação/fisiologia , Propriedades de Superfície , Engenharia Tecidual/métodos
14.
Pigment Cell Melanoma Res ; 33(2): 334-344, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31549767

RESUMO

NRAS mutations are the most common alterations among RAS isoforms in cutaneous melanoma, with patients harboring these aggressive tumors having a poor prognosis and low survival rate. The main line of treatment for these patients is MAPK pathway-targeted therapies, such as MEK inhibitors, but, unfortunately, the response to these inhibitors is variable due to tumor resistance. Identifying genetic modifiers involved in resistance toward MEK-targeted therapy may assist in the development of new therapeutic strategies, enhancing treatment response and patient survival. Our whole-genome CRISPR-Cas9 knockout screen identified the target Kelch domain-containing F-Box protein 42 (FBXO42) as a factor involved in NRAS-mutant melanoma-acquired resistance to the MEK1/2 inhibitor trametinib. We further show that FBXO42, an E3 ubiquitin ligase, is involved in the TAK1 signaling pathway, possibly prompting an increase in active P38. In addition, we demonstrate that combining trametinib with the TAK1 inhibitor, takinib, is a far more efficient treatment than trametinib alone in NRAS-mutant melanoma cells. Our findings thus show a new pathway involved in NRAS-mutant melanoma resistance and provide new opportunities for novel therapeutic options.


Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , GTP Fosfo-Hidrolases/genética , Genoma Humano , Melanoma/genética , Proteínas de Membrana/genética , Neoplasias Cutâneas/genética , Sequência de Bases , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Testes Genéticos , Humanos , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Melanoma/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Modelos Biológicos , Mutação/genética , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Piridonas/farmacologia , Piridonas/uso terapêutico , Pirimidinonas/farmacologia , Pirimidinonas/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico
15.
J Invest Dermatol ; 140(4): 878-890.e5, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31622599

RESUMO

Melanoma cells can switch between distinct gene expression profiles, resulting in proliferative or invasive phenotypes. Signaling pathways involved in this switch were analyzed by gene expression profiling of a cohort of 22 patient-derived melanoma cell lines. CDH1 negativity was identified as a surrogate marker for the invasive phenotype. CDH1 expression could be turned on and off by modulating activity of p38 or its downstream target MK2, suggesting that this pathway controls melanoma progression. Mechanistically, MK2 inhibition prevented melanoma-induced vascular barrier disruption, reduced the expression of PODXL and DEL-1, and prevented vascular dissemination in vivo. PODXL and DEL-1 expression in patients with melanoma were associated with poor survival and thus can be used as prognostic markers. Downstream targets of MK2 may thus serve as candidate therapeutics.


Assuntos
Regulação Neoplásica da Expressão Gênica , Melanoma/genética , Neoplasias Cutâneas/genética , Neoplasias Vasculares/prevenção & controle , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Humanos , Melanoma/metabolismo , Melanoma/patologia , Invasividade Neoplásica , Prognóstico , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células Tumorais Cultivadas , Neoplasias Vasculares/metabolismo , Neoplasias Vasculares/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/genética
16.
EMBO J ; 38(15): e95874, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31267558

RESUMO

MAPK inhibitors (MAPKi) show outstanding clinical response rates in melanoma patients harbouring BRAF mutations, but resistance is common. The ability of melanoma cells to switch from melanocytic to mesenchymal phenotypes appears to be associated with therapeutic resistance. High-throughput, subcellular proteome analyses and RNAseq on two panels of primary melanoma cells that were either sensitive or resistant to MAPKi revealed that only 15 proteins were sufficient to distinguish between these phenotypes. The two proteins with the highest discriminatory power were PTRF and IGFBP7, which were both highly upregulated in the mesenchymal-resistant cells. Proteomic analysis of CRISPR/Cas-derived PTRF knockouts revealed targets involved in lysosomal activation, endocytosis, pH regulation, EMT, TGFß signalling and cell migration and adhesion, as well as a significantly reduced invasive index and ability to form spheres in 3D culture. Overexpression of PTRF led to MAPKi resistance, increased cell adhesion and sphere formation. In addition, immunohistochemistry of patient samples showed that PTRF expression levels were a significant biomarker of poor progression-free survival, and IGFBP7 levels in patient sera were shown to be higher after relapse.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Melanoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteômica/métodos , Proteínas de Ligação a RNA/metabolismo , Adulto , Idoso , Carbamatos/farmacologia , Adesão Celular , Linhagem Celular Tumoral , Progressão da Doença , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/sangue , Masculino , Melanoma/tratamento farmacológico , Melanoma/genética , Pessoa de Meia-Idade , Mapas de Interação de Proteínas , Análise de Sequência de RNA , Sulfonamidas/farmacologia , Análise de Sobrevida , Regulação para Cima , Vemurafenib/farmacologia
17.
Oncotarget ; 10(36): 3373-3384, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31164959

RESUMO

Background: Recent progress in the diagnosis and treatment of primary and metastatic cutaneous melanoma (CM) has led to a significant increase in the patients` expectancy of life. The development of additional primary tumors (APT) other than CM represents an important survival issue. Results: Of a total of 1764 CM patients, 80 (4.5%) patients developed APT. For tumors diagnosed after CM, there was a 2.7 fold excess risk for APT compared to the swiss german population. A significantly increased risk was noted for female breast (SIR, 2.46), male larynx (SIR, 76.92), male multiple myeloma (SIR, 11.2), male oesophagus (SIR, 10.8) and thyroid on males (SIR, 58.8) and females (SIR, 38.1). All thyroid cancer cases had a common papillary histological subtype and a high rate of BRAFV600E mutation. Melanoma was the primary cause of death in the vast majority of patients. Methods: We used the cancer registry from the Comprehensive Cancer Center Zurich (CCCZ) and retrospectively analyzed patients with CM and APT between 2008 and 2018. We calculated the risk of APT compared to the swiss german population using the standardized incidence ratio (SIR). Conclusions: Patients with CM have an increased risk for hematologic and solid APT. Long-term follow-up is indicated.

18.
Cancer Immunol Res ; 7(1): 77-85, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30425105

RESUMO

Many metastatic melanoma patients experience durable responses to anti-PD1 and/or anti-CTLA4; however, a significant proportion (over 50%) do not benefit from the therapies. In this study, we sought to assess pretreatment liquid biopsies for biomarkers that may correlate with response to checkpoint blockade. We measured the combinatorial diversity evenness of the T-cell receptor (TCR) repertoire (the DE50, with low values corresponding to more clonality and lack of TCR diversity) in pretreatment peripheral blood mononuclear cells from melanoma patients treated with anti-CTLA4 (n = 42) or anti-PD1 (n = 38) using a multi-N-plex PCR assay on genomic DNA (gDNA). A receiver operating characteristic curve determined the optimal threshold for a dichotomized analysis according to objective responses as defined by RECIST1.1. Correlations between treatment outcome, clinical variables, and DE50 were assessed in multivariate regression models and confirmed with Fisher exact tests. In samples obtained prior to treatment initiation, we showed that low DE50 values were predictive of a longer progression-free survival and good responses to PD-1 blockade, but, on the other hand, predicted a poor response to CTLA4 inhibition. Multivariate logistic regression models identified DE50 as the only independent predictive factor for response to anti-CTLA4 therapy (P = 0.03) and anti-PD1 therapy (P = 0.001). Fisher exact tests confirmed the association of low DE50 with response in the anti-CTLA4 (P = 0.041) and the anti-PD1 cohort (P = 0.0016). Thus, the evaluation of basal TCR repertoire diversity in peripheral blood, using a PCR-based method, could help predict responses to anti-PD1 and anti-CTLA4 therapies.


Assuntos
Imunoterapia , Melanoma/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Neoplasias Cutâneas/imunologia , Adulto , Idoso , Anticorpos Monoclonais Humanizados/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Antígeno CTLA-4/antagonistas & inibidores , Feminino , Humanos , Ipilimumab/uso terapêutico , Masculino , Melanoma/tratamento farmacológico , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Intervalo Livre de Progressão , Neoplasias Cutâneas/tratamento farmacológico
20.
Cancer Cell ; 34(1): 69-84.e14, 2018 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-30008323

RESUMO

Human melanomas frequently harbor amplifications of EZH2. However, the contribution of EZH2 to melanoma formation has remained elusive. Taking advantage of murine melanoma models, we show that EZH2 drives tumorigenesis from benign BrafV600E- or NrasQ61K-expressing melanocytes by silencing of genes relevant for the integrity of the primary cilium, a signaling organelle projecting from the surface of vertebrate cells. Consequently, gain of EZH2 promotes loss of primary cilia in benign melanocytic lesions. In contrast, blockade of EZH2 activity evokes ciliogenesis and cilia-dependent growth inhibition in malignant melanoma. Finally, we demonstrate that loss of cilia enhances pro-tumorigenic WNT/ß-catenin signaling, and is itself sufficient to drive metastatic melanoma in benign cells. Thus, primary cilia deconstruction is a key process in EZH2-driven melanomagenesis.


Assuntos
Movimento Celular , Proliferação de Células , Cílios/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Melanócitos/metabolismo , Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Cílios/genética , Cílios/patologia , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Feminino , GTP Fosfo-Hidrolases/genética , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Metástase Linfática , Masculino , Melanócitos/patologia , Melanoma/genética , Melanoma/secundário , Proteínas de Membrana/genética , Camundongos Nus , Camundongos Transgênicos , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismo
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