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1.
Mol Oncol ; 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38807304

RESUMO

Immunotherapy has achieved tremendous success in melanoma. However, only around 50% of advanced melanoma patients benefit from immunotherapy. Cyclin-dependent kinase inhibitor 2A (CDKN2A), encoding the two tumor-suppressor proteins p14ARF and p16INK4a, belongs to the most frequently inactivated gene loci in melanoma and leads to decreased T cell infiltration. While the role of p16INK4a has been extensively investigated, knowledge about p14ARF in melanoma is scarce. In this study, we elucidate the impact of reduced p14ARF expression on melanoma immunogenicity. Knockdown of p14ARF in melanoma cell lines diminished their recognition and killing by melanoma differentiation antigen (MDA)-specific T cells. Resistance was caused by a reduction of the peptide surface density of presented MDAs. Immunopeptidomic analyses revealed that antigen presentation via human leukocyte antigen class I (HLA-I) molecules was enhanced upon p14ARF downregulation in general, but absolute and relative expression of cognate peptides was decreased. However, this phenotype is associated with a favorable outcome for melanoma patients. Limiting Wnt5a signaling reverted this phenotype, suggesting an involvement of non-canonical Wnt signaling. Taken together, our data indicate a new mechanism limiting MDA-specific T cell responses by decreasing both absolute and relative MDA-peptide presentation in melanoma.

2.
Cancers (Basel) ; 16(9)2024 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-38730718

RESUMO

Elevated levels of peripheral blood and tumor tissue neutrophils are associated with poorer clinical response and therapy resistance in melanoma. The underlying mechanism and the role of neutrophils in targeted therapy is still not fully understood. Serum samples of patients with advanced melanoma were collected and neutrophil-associated serum markers were measured and correlated with response to targeted therapy. Blood neutrophils from healthy donors and patients with advanced melanoma were isolated, and their phenotypes, as well as their in vitro functions, were compared. In vitro functional tests were conducted through nonadherent cocultures with melanoma cells. Protection of melanoma cell lines by neutrophils was assessed under MAPK inhibition. Blood neutrophils from advanced melanoma patients exhibited lower CD16 expression compared to healthy donors. In vitro, both healthy-donor- and patient-derived neutrophils prevented melanoma cell apoptosis upon dual MAPK inhibition. The effect depended on cell-cell contact and melanoma cell susceptibility to treatment. Interference with protease activity of neutrophils prevented melanoma cell protection during treatment in cocultures. The negative correlation between neutrophils and melanoma outcomes seems to be linked to a protumoral function of neutrophils. In vitro, neutrophils exert a direct protective effect on melanoma cells during dual MAPK inhibition. This study further hints at a crucial role of neutrophil-related protease activity in protection.

3.
Cancers (Basel) ; 15(23)2023 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-38067230

RESUMO

INTRODUCTION: Patients with NRAS-mutant metastatic melanoma often have an aggressive disease requiring a fast-acting, effective therapy. The MEK inhibitor binimetinib shows an overall response rate of 15% in patients with NRAS-mutant melanoma, providing a backbone for combination strategies. Our previous studies demonstrated that in NRAS-mutant melanoma, the antitumor activity of the MEK inhibitor binimetinib was significantly potentiated by the BRAFV600E/K inhibitor encorafenib through the induction of ER stress, leading to melanoma cell death by apoptotic mechanisms. Encorafenib combined with binimetinib was well tolerated in a phase III trial showing potent antitumor activity in BRAF-mutant melanoma, making a rapid evaluation in NRAS-mutant melanoma imminently feasible. These data provide a mechanistic rationale for the evaluation of binimetinib combined with encorafenib in preclinical and clinical studies on NRAS-mutant metastatic melanoma. METHODS: The combination of BRAFi plus MEKi was tested in a monolayer culture of patient-derived cell lines and in corresponding patient-derived tissue slice cultures of NRAS-mutant melanoma. To investigate the treatment in vivo, NSG (NOD. Cg-PrkdcscidIl2rgtm1Wjl/SzJ) mice were subcutaneously injected with three different BRAF wild-type melanoma models harboring oncogenic NRAS mutations and treated orally with encorafenib (6 mg/kg body weight, daily) with or without binimetinib (8 mg/kg body weight, twice daily). In parallel, an individual healing attempt was carried out by treating one patient with an NRAS-mutated tumor. RESULTS: Encorafenib was able to enhance the inhibitory effect on cell growth of binimetinib only in the cell line SKMel147 in vitro. It failed to enhance the apoptotic effect found in two other NRAS-mutated cell lines. Encorafenib led to a hyperactivation of ERK which could be reduced with the combinational treatment. In two of the three patient-derived tissue slice culture models of NRAS-mutant melanomas, a slight tendency of a combinatorial effect was seen which was not significant. Encorafenib showed a slight induction of the ER stress genes ATF4, CHOP, and NUPR1. The combinational treatment was able to enhance this effect, but not significantly. In the mouse model, the combination therapy of encorafenib with binimetinib resulted in reduced tumor growth compared to the control and encorafenib groups; however, the best effect in terms of tumor growth inhibition was measured in the binimetinib therapy group. The therapy showed no effect in an individual healing attempt for a patient suffering from metastatic, therapy-refractory NRAS-mutated melanoma. CONCLUSION: In in vitro and ex vivo settings, the combination therapy was observed to elicit a response; however, it did not amplify the efficacy observed with binimetinib alone, whereas in a patient, the combinational treatment remained ineffective. The preclinical in vivo data showed no increased combinatorial effect. However, the in vivo effect of binimetinib as monotherapy was unexpectedly high in the tested regimen. Nevertheless, binimetinib proved to be advantageous in the treatment of melanoma in vivo and led to high rates of apoptosis in vitro; hence, it still seems to be a good base for combination with other substances in the treatment of patients with NRAS-mutant melanoma.

4.
J Exp Clin Cancer Res ; 42(1): 175, 2023 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-37464364

RESUMO

BACKGROUND: The mitogen-activated protein kinase (MAPK) signaling pathway is frequently hyperactivated in malignant melanoma and its inhibition has proved to be an efficient treatment option for cases harboring BRAFV600 mutations (BRAFMut). However, there is still a significant need for effective targeted therapies for patients with other melanoma subgroups characterized by constitutive MAPK activation, such as tumors with NRAS or NF-1 alterations (NRASMut, NF-1LOF), as well as for patients with MAPK pathway inhibitor-resistant BRAFMut melanomas, which commonly exhibit a reactivation of this pathway. p90 ribosomal S6 kinases (RSKs) represent central effectors of MAPK signaling, regulating cell cycle progression and survival. METHODS: RSK activity and the functional effects of its inhibition by specific small molecule inhibitors were investigated in established melanoma cell lines and patient-derived short-term cultures from different MAPK pathway-hyperactivated genomic subgroups (NRASMut, BRAFMut, NF-1LOF). Real-time qPCR, immunoblots and flow cytometric cell surface staining were used to explore the molecular changes following RSK inhibition. The effect on melanoma cell growth was evaluated by various two- and three-dimensional in vitro assays as well as with melanoma xenograft mouse models. Co-cultures with gp100- or Melan-A-specific cytotoxic T cells were used to assess immunogenicity of melanoma cells and associated T-cell responses. RESULTS: In line with elevated activity of the MAPK/RSK signaling axis, growth and survival of not only BRAFMut but also NRASMut and NF-1LOF melanoma cells were significantly impaired by RSK inhibitors. Intriguingly, RSK inhibition was particularly effective in three-dimensional growth settings with long-term chronic drug exposure and suppressed tumor cell growth of in vivo melanoma models. Additionally, our study revealed that RSK inhibition simultaneously promoted differentiation and immunogenicity of the tumor cells leading to enhanced T-cell activation and melanoma cell killing. CONCLUSIONS: Collectively, RSK inhibitors exhibited both multi-layered anti-tumor efficacy and broad applicability across different genomic melanoma subgroups. RSK inhibition may therefore represent a promising novel therapeutic strategy for malignant melanoma with hyperactivated MAPK signaling.


Assuntos
Melanoma , Proteínas Quinases S6 Ribossômicas 90-kDa , Humanos , Animais , Camundongos , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteínas Proto-Oncogênicas B-raf , Evasão da Resposta Imune , Linhagem Celular Tumoral , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Ciclo Celular , Melanoma Maligno Cutâneo
5.
Leukemia ; 37(1): 178-189, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36352190

RESUMO

Diffuse large B-cell lymphoma (DLBCL) is an aggressive disease that exhibits constitutive activation of phosphoinositide 3-kinase (PI3K) driven by chronic B-cell receptor signaling or PTEN deficiency. Since pan-PI3K inhibitors cause severe side effects, we investigated the anti-lymphoma efficacy of the specific PI3Kß/δ inhibitor AZD8186. We identified a subset of DLBCL models within activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL that were sensitive to AZD8186 treatment. On the molecular level, PI3Kß/δ inhibition decreased the pro-survival NF-κB and AP-1 activity or led to downregulation of the oncogenic transcription factor MYC. In AZD8186-resistant models, we detected a feedback activation of the PI3K/AKT/mTOR pathway following PI3Kß/δ inhibition, which limited AZD8186 efficacy. The combined treatment with AZD8186 and the mTOR inhibitor AZD2014 overcame resistance to PI3Kß/δ inhibition and completely prevented outgrowth of lymphoma cells in vivo in cell line- and patient-derived xenograft mouse models. Collectively, our study reveals that subsets of DLBCLs are addicted to PI3Kß/δ signaling and thus identifies a previously unappreciated role of the PI3Kß isoform in DLBCL survival. Furthermore, our data demonstrate that combined targeting of PI3Kß/δ and mTOR is effective in all major DLBCL subtypes supporting the evaluation of this strategy in a clinical trial setting.


Assuntos
Linfoma Difuso de Grandes Células B , Fosfatidilinositol 3-Quinases , Humanos , Animais , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais , Linfoma Difuso de Grandes Células B/patologia , Serina-Treonina Quinases TOR/metabolismo , Linhagem Celular Tumoral
6.
Cancers (Basel) ; 14(9)2022 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-35565423

RESUMO

BACKGROUND: Eosinophils appear to contribute to the efficacy of immunotherapy and their frequency was suggested as a predictive biomarker. Whether this observation could be transferred to patients treated with targeted therapy remains unknown. METHODS: Blood and serum samples of healthy controls and 216 patients with advanced melanoma were prospectively and retrospectively collected. Freshly isolated eosinophils were phenotypically characterized by flow cytometry and co-cultured in vitro with melanoma cells to assess cytotoxicity. Soluble serum markers and peripheral blood counts were used for correlative studies. RESULTS: Eosinophil-mediated cytotoxicity towards melanoma cells, as well as phenotypic characteristics, were similar when comparing healthy donors and patients. However, high relative pre-treatment eosinophil counts were significantly associated with response to MAPKi (p = 0.013). Eosinophil-mediated cytotoxicity towards melanoma cells is dose-dependent and requires proximity of eosinophils and their target in vitro. Treatment with targeted therapy in the presence of eosinophils results in an additive tumoricidal effect. Additionally, melanoma cells affected eosinophil phenotype upon co-culture. CONCLUSION: High pre-treatment eosinophil counts in advanced melanoma patients were associated with a significantly improved response to MAPKi. Functionally, eosinophils show potent cytotoxicity towards melanoma cells, which can be reinforced by MAPKi. Further studies are needed to unravel the molecular mechanisms of our observations.

7.
Cancer Biol Med ; 2021 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-34591417

RESUMO

OBJECTIVE: Cellular heterogeneity is regarded as a major factor affecting treatment response and resistance in malignant melanoma. Recent developments in single-cell sequencing technology have provided deeper insights into these mechanisms. METHODS: Here, we analyzed a BRAFV600E-mutant melanoma cell line by single-cell RNA-seq under various conditions: cells sensitive to BRAF inhibition with BRAF inhibitor vemurafenib and cells resistant to BRAF inhibition with vemurafenib alone or vemurafenib in combination with the MEK1/2 inhibitors cobimetinib or trametinib. Dimensionality reduction by t-distributed stochastic neighbor embedding and self-organizing maps identified distinct trajectories of resistance development clearly separating the 4 treatment conditions in cell and gene state space. RESULTS: Trajectories associated with resistance to single-agent treatment involved cell cycle, extracellular matrix, and de-differentiation programs. In contrast, shifts detected in double-resistant cells primarily affected translation and mitogen-activated protein kinase pathway reactivation, with a small subpopulation showing markers of pluripotency. These findings were validated in pseudotime analyses and RNA velocity measurements. CONCLUSIONS: The single-cell transcriptomic analyses reported here employed a spectrum of bioinformatics methods to identify mechanisms of melanoma resistance to single- and double-agent treatments. This study deepens our understanding of treatment-induced cellular reprogramming and plasticity in melanoma cells and identifies targets of potential relevance to the management of treatment resistance.

8.
Int J Radiat Oncol Biol Phys ; 111(4): 1072-1087, 2021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-34166770

RESUMO

Multifunctional Y-box binding protein-1 (YB-1) is highly expressed in different human solid tumors and is involved in various cellular processes. DNA damage is the major mechanism by which radiochemotherapy (RCT) induces cell death. On induction of DNA damage, a multicomponent signal transduction network, known as the DNA damage response, is activated to induce cell cycle arrest and initiate DNA repair, which protects cells against damage. YB-1 regulates nearly all cancer hallmarks described to date by participating in DNA damage response, gene transcription, mRNA splicing, translation, and tumor stemness. YB-1 lacks kinase activity, and p90 ribosomal S6 kinase and AKT are the key kinases within the RAS/mitogen-activated protein kinase and phosphoinositide 3-kinase pathways that directly activate YB-1. Thus, the molecular targeting of ribosomal S6 kinase and AKT is thought to be the most effective strategy for blocking the cellular function of YB-1 in human solid tumors. In this review, after describing the prosurvival effect of YB-1 with a focus on DNA damage repair and cancer cell stemness, clinical evidence will be provided indicating an inverse correlation between YB-1 expression and the treatment outcome of solid tumors after RCT. In the interest of being concise, YB-1 signaling cascades will be briefly discussed and the current literature on YB-1 posttranslational modifications will be summarized. Finally, the current status of targeting the YB-1 axis, especially in combination with RCT, will be highlighted.


Assuntos
Neoplasias , Proteínas de Transporte , Linhagem Celular Tumoral , Quimiorradioterapia , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proteína 1 de Ligação a Y-Box/genética , Proteína 1 de Ligação a Y-Box/metabolismo
9.
Cancers (Basel) ; 12(10)2020 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-33003386

RESUMO

The multifunctional protein Y-box binding protein-1 (YB-1) regulates all the so far described cancer hallmarks including cell proliferation and survival. The MAPK/ERK and PI3K/Akt pathways are also the major pathways involved in cell growth, proliferation, and survival, and are the frequently hyperactivated pathways in human cancers. A gain of function mutation in KRAS mainly leads to the constitutive activation of the MAPK pathway, while the activation of the PI3K/Akt pathway occurs either through the loss of PTEN or a gain of function mutation of the catalytic subunit alpha of PI3K (PIK3CA). In this study, we investigated the underlying signaling pathway involved in YB-1 phosphorylation at serine 102 (S102) in KRAS(G13D)-mutated triple-negative breast cancer (TNBC) MDA-MB-231 cells versus PIK3CA(H1047R)/PTEN(E307K) mutated TNBC MDA-MB-453 cells. Our data demonstrate that S102 phosphorylation of YB-1 in KRAS-mutated cells is mainly dependent on the MAPK/ERK pathway, while in PIK3CA/PTEN-mutated cells, YB-1 S102 phosphorylation is entirely dependent on the PI3K/Akt pathway. Independent of the individual dominant pathway regulating YB-1 phosphorylation, dual targeting of MEK and PI3K efficiently inhibited YB-1 phosphorylation and blocked cell proliferation. This represents functional crosstalk between the two pathways. Our data obtained from the experiments, applying pharmacological inhibitors and genetic approaches, shows that YB-1 is a key player in cell proliferation, clonogenic activity, and tumor growth of TNBC cells through the MAPK and PI3K pathways. Therefore, dual inhibition of these two pathways or single targeting of YB-1 may be an effective strategy to treat TNBC.

10.
Cancers (Basel) ; 12(8)2020 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-32824741

RESUMO

Secreted factors play an important role in intercellular communication. Therefore, they are not only indispensable for the regulation of various physiological processes but can also decisively advance the development and progression of tumours. In the context of inflammatory disease, Y-box binding protein 1 (YB-1) is actively secreted and the extracellular protein promotes cell proliferation and migration. In malignant melanoma, intracellular YB-1 expression increases during melanoma progression and represents an unfavourable prognostic marker. Here, we show active secretion of YB-1 from melanoma cells as opposed to benign cells of the skin. Intriguingly, YB-1 secretion correlates with the stage of melanoma progression and depends on a calcium- and ATP-dependent non-classical secretory pathway leading to the occurrence of YB-1 in the extracellular space as a free protein. Along with an elevated YB-1 secretion of melanoma cells in the metastatic growth phase, extracellular YB-1 exerts a stimulating effect on melanoma cell migration, invasion, and tumourigenicity. Collectively, these data suggest that secreted YB-1 plays a functional role in melanoma cell biology, stimulating metastasis, and may serve as a novel biomarker in malignant melanoma that reflects tumour aggressiveness.

11.
Cell Death Dis ; 11(7): 581, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32719412

RESUMO

Rad51 is an essential factor of the homologous recombination DNA repair pathway and therefore plays an important role in maintaining genomic stability. We show that RAD51 and other homologous recombination repair genes are overexpressed in metastatic melanoma cell lines and in melanoma patient samples, which correlates with reduced survival of melanoma patients. In addition, Rad51 expression in melanoma cells was regulated on a transcriptional level by the MAPK signaling pathway with Elk1 as the main downstream transcriptional effector. Most strikingly, melanoma cells which developed resistance towards MAPK inhibitors could be efficiently targeted by Rad51 inhibitors similar to their sensitive counterparts, leading to DNA damage, G2/M arrest and apoptosis. Furthermore, the treatment of MAPK inhibitor resistant cells with Rad51 inhibitors enhances the susceptibility of these cells for MAPK inhibitor treatment in vitro and in vivo. These data indicate that Rad51 plays a critical role in the survival of metastatic melanoma cells and is a promising target for the therapy of melanoma irrespective of its MAPK inhibitor resistance status.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Sistema de Sinalização das MAP Quinases , Melanoma/enzimologia , Melanoma/patologia , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/farmacologia , Rad51 Recombinase/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Modelos Biológicos , Rad51 Recombinase/antagonistas & inibidores , Proteínas Elk-1 do Domínio ets/genética , Proteínas Elk-1 do Domínio ets/metabolismo
12.
Am J Pathol ; 190(10): 2155-2164, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32679231

RESUMO

Aberrant DNA methylation is an epigenetic hallmark of melanoma, but the expression of DNA methyltransferase (Dnmt)-1 in melanocytic tumors is unknown. Dnmt1 expression was analyzed in primary melanocytes, melanoma cell lines, and 83 melanocytic tumors, and its associations with proliferation, mutational status, and response to B-Raf and mitogen-activated protein kinase kinase (MEK) inhibition were explored. Dnmt1 expression was increased incrementally from nevi [mean fluorescence intensity (MFI), 48.1; interquartile range, 41.7 to 59.6] to primary melanomas (MFI, 68.8; interquartile range, 58.4 to 77.0) and metastatic melanomas (MFI, 87.5; interquartile range, 77.1 to 114.5) (P < 0.001). Dnmt1 expression was correlated with Ki-67 expression (Spearman correlation, 0.483; P < 0.001) and was independent of BRAF mutation status (P = 0.55). In BRAF-mutant melanoma, Dnmt1 was down-regulated during response to B-Raf and MEK inhibition and was again up-regulated on drug resistance in vitro and in vivo. Degradation of Dnmt1 by the histone deacetylase inhibitor suberoylanilide hydroxamic acid was associated with decreased cell viability in B-Raf inhibitor-sensitive and -resistant cell lines. This study demonstrates that Dnmt1 expression is correlated with proliferation in melanocytic tumors, is increased with melanoma progression, and is associated with response to B-Raf and MEK inhibition. Given its strong expression in metastatic melanoma, Dnmt1 may be a promising target for combined epigenetic and immunotherapy.


Assuntos
Proliferação de Células/efeitos dos fármacos , DNA/metabolismo , Melanoma/metabolismo , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Proto-Oncogênicas B-raf/metabolismo , Neoplasias Cutâneas/metabolismo , Linhagem Celular Tumoral , DNA/efeitos dos fármacos , Inibidores de Histona Desacetilases/uso terapêutico , Humanos , Melanócitos/efeitos dos fármacos , Melanócitos/metabolismo , Melanoma/genética , Melanoma/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/genética , Vorinostat/farmacologia , Melanoma Maligno Cutâneo
14.
Cell Death Dis ; 9(9): 930, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30206212

RESUMO

The efficacy of targeted MAPK signalling pathway inhibitors (MAPKi) in metastatic melanoma therapy is limited by the development of resistance mechanisms that results in disease relapse. This situation still requires treatment alternatives for melanoma patients with acquired resistance to targeted therapy. We found that melanoma cells, which developed resistance towards MAPKi show an enhanced susceptibility to platinum-based drugs, such as cisplatin and carboplatin. We found that this enhanced susceptibility inversely correlates with the expression level of the p53 family member TAp73. We show that the lower expression of the TAp73 isoform in MAPKi-resistant melanoma cells enhances accumulation of DNA double-strand breaks upon cisplatin and carboplatin treatment by reducing the efficiency of nucleotide excision repair. These data suggest that a subgroup of melanoma patients with acquired resistance to MAPKi treatment and low TAp73 expression can benefit from chemotherapy with platinum-based drugs as a second-line therapy.


Assuntos
Antineoplásicos/uso terapêutico , Carboplatina/uso terapêutico , Cisplatino/uso terapêutico , Melanoma/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteína Tumoral p73/biossíntese , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/fisiologia , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Melanoma/patologia , Paclitaxel/uso terapêutico , Estudos Retrospectivos , Proteína Supressora de Tumor p53/metabolismo
15.
Mol Cancer Res ; 16(7): 1149-1160, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29743296

RESUMO

Cutaneous melanoma represents one of the most aggressive human tumor entities possessing a high tendency to metastasize. Cancer cells frequently exploit a highly conserved developmental program, the epithelial-to-mesenchymal transition (EMT), to gain migratory and invasive properties promoting their metastatic spread. Cytoplasmic localization of the oncogenic transcription and translation factor Y-box binding protein 1 (YB-1) is a powerful inducer of EMT in breast carcinoma cells. Interestingly, EMT-like processes have also been observed in cutaneous melanoma despite its neural crest origin. Here, increased expression of YB-1 negatively affects patient survival in malignant melanoma and promotes melanoma cell tumorigenicity both in vitro and in vivo Intriguingly, this effect seems to be mainly mediated by cytoplasmic YB-1 that does not exhibit phosphorylation at serine-102 (S102). Moreover, S102 unphosphorylated YB-1 enhances the migratory and invasive potential of human melanoma cells in two-dimensional (2D) and three-dimensional (3D) culture systems and facilitates acquisition of a mesenchymal-like invasive phenotype in the chick embryo model. Collectively, these data demonstrate that the cytoplasmic activity of YB-1 stimulates tumorigenicity and metastatic potential of melanoma cells by promoting EMT-like properties.Implications: This study reveals for the first time that YB-1 efficiently drives tumorigenicity and invasiveness of melanoma cells in its S102 unphosphorylated cytoplasmic state and that YB-1 expression represents a negative prognostic factor in primary melanoma patients. Mol Cancer Res; 16(7); 1149-60. ©2018 AACR.


Assuntos
Carcinogênese/genética , Transição Epitelial-Mesenquimal/genética , Melanoma/genética , Neoplasias Cutâneas/genética , Proteína 1 de Ligação a Y-Box/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Embrião de Galinha , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/patologia , Camundongos , Invasividade Neoplásica/genética , Neoplasias Cutâneas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto , Melanoma Maligno Cutâneo
16.
Clin Cancer Res ; 23(20): 6203-6214, 2017 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-28724666

RESUMO

Purpose: NRAS mutations in malignant melanoma are associated with aggressive disease requiring rapid antitumor intervention, but there is no approved targeted therapy for this subset of patients. In clinical trials, the MEK inhibitor (MEKi) binimetinib displayed modest antitumor activity, making combinations a requisite. In a previous study, the BRAF inhibitor (BRAFi) vemurafenib was shown to induce endoplasmic reticulum (ER) stress that together with inhibition of the RAF-MEK-ERK (MAPK) pathway amplified its proapoptotic activity in BRAF-mutant melanoma. The present study investigated whether this effect might extent to NRAS-mutant melanoma, in which MAPK activation would be expected.Experimental Design and Results: BRAFi increased pERK, but also significantly increased growth inhibition and apoptosis induced by the MEKi in monolayer, spheroids, organotypic, and patient-derived tissue slice cultures of NRAS-mutant melanoma. BRAFi such as encorafenib induced an ER stress response via the PERK pathway, as detected by phosphorylation of eIF2α and upregulation of the ER stress-related factors ATF4, CHOP, and NUPR1 and the proapoptotic protein PUMA. MEKi such as binimetinib induced the expression of the proapoptotic protein BIM and activation of the mitochondrial pathway of apoptosis, the latter of which was enhanced by combination with encorafenib. The increased apoptotic rates caused by the combination treatment were significantly reduced through siRNA knockdown of ATF4 and BIM, confirming its critical roles in this process.Conclusions: The data presented herein encourage further advanced in vivo and clinical studies to evaluate MEKi in combination with ER stress inducing BRAFi as a strategy to treat rapidly progressing NRAS-mutant melanoma. Clin Cancer Res; 23(20); 6203-14. ©2017 AACR.


Assuntos
Estresse do Retículo Endoplasmático/efeitos dos fármacos , GTP Fosfo-Hidrolases/genética , MAP Quinase Quinase Quinases/antagonistas & inibidores , Melanoma/genética , Melanoma/metabolismo , Proteínas de Membrana/genética , Mutação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Apoptose/efeitos dos fármacos , Proteína 11 Semelhante a Bcl-2/genética , Proteína 11 Semelhante a Bcl-2/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , MAP Quinase Quinase Quinases/metabolismo , Melanoma/patologia , Modelos Biológicos , Fosforilação , Proteínas Proto-Oncogênicas B-raf/metabolismo , Transdução de Sinais/efeitos dos fármacos
17.
Oncotarget ; 8(22): 35761-35775, 2017 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-28415756

RESUMO

The clinical availability of small molecule inhibitors specifically targeting mutated BRAF marked a significant breakthrough in melanoma therapy. Despite a dramatic anti-tumour activity and improved patient survival, rapidly emerging resistance, however, greatly limits the clinical benefit. The majority of the already described resistance mechanisms involve a reactivation of the MAPK signalling pathway. The p90 ribosomal S6 kinase (RSK), a downstream effector of the MAPK signalling cascade, has been reported to enhance survival of melanoma cells in response to chemotherapy. Here, we can show that RSK activity is significantly increased in human melanoma cells with acquired resistance to the BRAFV600E/K inhibitor vemurafenib. Interestingly, inhibition of RSK signalling markedly impairs the viability of vemurafenib resistant melanoma cells and is effective both in two-dimensional and in three-dimensional culture systems, especially in a chronic, long-term application. The effect of RSK inhibition can be partly replicated by downregulation of the well-known RSK target, Y-box binding protein 1 (YB-1). Intriguingly, RSK inhibition also retains its efficacy in melanoma cells with combined resistance to vemurafenib and the MEK inhibitor trametinib. These data suggest that active RSK signalling might be an attractive novel therapeutic target in melanoma with acquired resistance to MAPK pathway inhibitors.


Assuntos
Antineoplásicos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Indóis/farmacologia , Melanoma , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Vemurafenib , Proteína 1 de Ligação a Y-Box/metabolismo
18.
Exp Dermatol ; 26(7): 598-606, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28423208

RESUMO

The BRAFV600E inhibitor vemurafenib achieves remarkable clinical responses in patients with BRAF-mutant melanoma, but its effects are limited by the onset of drug resistance. In the case of resistance, chemotherapy can still be applied as second line therapy. However, it yields low response rates and strategies are urgently needed to potentiate its effects. In a previous study, we showed that the inhibition of the PI3K-AKT-mTOR pathway significantly increases sensitivity of melanoma cells to chemotherapeutic drugs (J. Invest. Dermatol. 2009, 129, 1500). In this study, the combination of the mTOR inhibitor temsirolimus with the chemotherapeutic agent temozolomide significantly increases growth inhibition and apoptosis in melanoma cells compared to temsirolimus or temozolomide alone. The combination of temozolomide with temsirolimus is not only effective in established but also in newly isolated and vemurafenib-resistant metastatic melanoma cell lines. These effects are associated with the downregulation of the anti-apoptotic protein Mcl-1 and the upregulation of the Wnt antagonist Dickkopf homologue 1 (DKK1). Knock-down of DKK1 suppresses apoptosis induction by the combination of temsirolimus and temozolomide. These data suggest that the inhibition of the mTOR pathway increases sensitivity of melanoma cells towards temozolomide. Chemosensitisation is associated with enhanced expression of the Wnt antagonist DKK1.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Dacarbazina/análogos & derivados , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Melanoma/tratamento farmacológico , Sirolimo/análogos & derivados , Neoplasias Cutâneas/tratamento farmacológico , Antineoplásicos Alquilantes/administração & dosagem , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Dacarbazina/administração & dosagem , Dacarbazina/uso terapêutico , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Transferência de Genes , Humanos , Indóis/administração & dosagem , Lentivirus , Melanoma/metabolismo , Proteínas de Membrana/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais , Sirolimo/administração & dosagem , Sirolimo/uso terapêutico , Pele/metabolismo , Neoplasias Cutâneas/metabolismo , Sulfonamidas/administração & dosagem , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Temozolomida , Vemurafenib
19.
J Invest Dermatol ; 136(4): 788-797, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26767426

RESUMO

The protease activity of the paracaspase mucosa-associated lymphoid tissue lymphoma translocation gene 1 (MALT1) plays an important role in antigen receptor-mediated lymphocyte activation by controlling the activity of the transcription factor nuclear factor-κB and is thus essential for the expression of inflammatory target genes. MALT1 is not only present in cells of the hematopoietic lineage, but is ubiquitously expressed. Here we report that stimulation with zymosan or Staphylococcus aureus induced MALT1 protease activity in human primary keratinocytes. Inhibition of the Src family of kinases or novel protein kinase C isoforms as well as silencing of CARMA2 or BCL10 interfered with activation of MALT1 protease. Silencing or inhibition of MALT1 protease strongly decreased the expression of important inflammatory genes such as TNFα, IL-17C, CXCL8 and HBD-2. MALT1-inhibited cells were unable to mount an antimicrobial response upon zymosan stimulation or phorbolester/ionomycin treatment, demonstrating a central role of MALT1 protease activity in keratinocyte immunity and suggesting MALT1 as a potential target in inflammatory skin diseases.


Assuntos
Caspases/metabolismo , Inflamação/genética , Queratinócitos/citologia , Proteínas de Neoplasias/metabolismo , Zimosan/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Anti-Infecciosos/química , Proteína 10 de Linfoma CCL de Células B , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/metabolismo , Caspases/genética , Perfilação da Expressão Gênica , Inativação Gênica , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Humanos , Interleucina-17/genética , Interleucina-17/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Queratinócitos/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteína de Translocação 1 do Linfoma de Tecido Linfoide Associado à Mucosa , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Proteínas de Neoplasias/genética , Análise de Sequência com Séries de Oligonucleotídeos , Isoformas de Proteínas , Proteína Quinase C/metabolismo , Staphylococcus aureus , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , beta-Defensinas/genética , beta-Defensinas/metabolismo , Quinases da Família src/metabolismo
20.
Eur J Cell Biol ; 93(1-2): 61-70, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24461929

RESUMO

Y-box binding protein 1 (YB-1) is a multifunctional protein involved in various cellular processes including both transcriptional and translational regulation of target gene expression. Significantly increased YB-1 levels have been reported in a number of human malignancies and shown to be associated with poor prognosis and disease recurrence. Indeed, YB-1 can act as a versatile oncoprotein playing an important role in tumour cell proliferation and progression. Consequently, YB-1 not only proves to be a good prognostic tumour marker, but also may be a promising emerging molecular target for the development of new therapeutical strategies. In this review, we discuss both the role of YB-1 in cancer and specifically in malignant melanoma as well as possible translations into the clinics derived thereof.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias/diagnóstico , Neoplasias/metabolismo , Proteína 1 de Ligação a Y-Box/metabolismo , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais/genética , Núcleo Celular/metabolismo , Humanos , Melanoma/diagnóstico , Melanoma/metabolismo , Melanoma/patologia , Melanoma/terapia , Terapia de Alvo Molecular , Neoplasias/patologia , Neoplasias/terapia , Prognóstico , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/terapia , Proteína 1 de Ligação a Y-Box/genética
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