RESUMO
BACKGROUND: Malignant melanoma has an increasing incidence rate and the metastatic disease is notoriously resistant to standard chemotherapy. Loss of cell cycle checkpoints is frequently found in many cancer types and makes the cells reliant on compensatory mechanisms to control progression. This feature may be exploited in therapy, and kinases involved in checkpoint regulation, such as Wee1 and Chk1/2, have thus become attractive therapeutic targets. METHODS: In the present study we combined a Wee1 inhibitor (MK1775) with Chk1/2 inhibitor (AZD7762) in malignant melanoma cell lines grown in vitro (2D and 3D cultures) and in xenografts models. RESULTS: Our in vitro studies showed that combined inhibition of Wee1 and Chk1/2 synergistically decreased viability and increased apoptosis (cleavage of caspase 3 and PARP), which may be explained by accumulation of DNA-damage (increased expression of γ-H2A.X)--and premature mitosis of S-phase cells. Compared to either inhibitor used as single agents, combined treatment reduced spheroid growth and led to greater tumour growth inhibition in melanoma xenografts. CONCLUSIONS: These data provide a rationale for further evaluation of the combination of Wee1 and Chk1/2 inhibitors in malignant melanoma.
Assuntos
Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/genética , Sinergismo Farmacológico , Melanoma/tratamento farmacológico , Proteínas Nucleares/genética , Proteínas Tirosina Quinases/genética , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase do Ponto de Checagem 2/antagonistas & inibidores , Humanos , Melanoma/genética , Melanoma/patologia , Camundongos , Proteínas Nucleares/antagonistas & inibidores , Inibidores de Proteínas Quinases/administração & dosagem , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Pirimidinonas , Neoplasias Cutâneas , Tiofenos/administração & dosagem , Ureia/administração & dosagem , Ureia/análogos & derivados , Ensaios Antitumorais Modelo de Xenoenxerto , Melanoma Maligno CutâneoRESUMO
The novel synthetic retinoid, CD437, shows potent anti-tumor activity in a range of different cancer cell lines and now serves as a prototype for development of new retinoid related molecules (RRMs). The purpose of this study was to examine the effect and cellular targets of CD437 in the human metastatic melanoma cell lines FEMX-1 and WM239. We showed that treatment with CD437 led to cell cycle arrest and induced apoptosis through both the extrinsic- and intrinsic pathways (caspase 8, -9 and PARP cleavage) in both cell lines. Interestingly, apoptosis was induced independently of DNA-fragmentation in FEMX-1 cells, and appeared partially caspase-independent in the WM239 cells. Additionally, up-regulation of CHOP mRNA and cathepsin D protein expression, following retinoid treatment, suggests involvement of the endoplasmatic reticulum (ER) and lysosomes, respectively. Combination of suboptimal concentrations of CD437 and lexatumumab, a TRAIL death receptor-2 agonist, resulted in synergistic reduction of viable cells, along with increased PARP cleavage. These results indicate that CD437 has a strong anti-neoplastic effect alone and in combination with lexatumumab in melanoma cell lines.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Melanoma/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Retinoides/farmacologia , Neoplasias Cutâneas/metabolismo , Caspase 8/metabolismo , Caspase 9/metabolismo , Catepsina D/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Poli(ADP-Ribose) Polimerases/metabolismo , RNA Mensageiro/biossíntese , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/biossíntese , Fator de Transcrição CHOP/biossíntese , Regulação para Cima , Receptor fas/biossínteseRESUMO
BACKGROUND/AIMS: Breast cancer metastasis suppressor 1 (BRMS1) blocks metastasis in melanoma xenografts; however, its usefulness as a biomarker in human melanomas has not been widely studied. The goal was to measure BRMS1 expression in benign nevi, primary and metastatic melanomas and evaluate its impact on disease progression and prognosis. METHODS: Paraffin-embedded tissue from 155 primary melanomas, 69 metastases and 15 nevi was examined for BRMS1 expression using immunohistochemistry. siRNA mediated BRMS1 down-regulation was used to study impact on invasion and migration in melanoma cell lines. RESULTS: A significantly higher percentage of nevi (87%), compared to primary melanomas (20%) and metastases (48%), expressed BRMS1 in the nucelus (p < 0.0001). Strong nuclear staining intensity was observed in 67% of nevi, and in 9% and 24% of the primary and metastatic melanomas, respectively (p < 0.0001). Comparable cytoplasmic expression was observed (nevi; 87%, primaries; 86%, metastases; 72%). However, a decline in cytoplasmic staining intensity was observed in metastases compared to nevi and primary tumors (26%, 47%, and 58%, respectively, p < 0.0001). Score index (percentage immunopositive celles multiplied with staining intensity) revealed that high cytoplasmic score index (≥ 4) was associated with thinner tumors (p = 0.04), lack of ulceration (p = 0.02) and increased disease-free survival (p = 0.036). When intensity and percentage BRMS1 positive cells were analyzed separately, intensity remained associated with tumor thickness (p = 0.024) and ulceration (p = 0.004) but was inversely associated with expression of proliferation markers (cyclin D3 (p = 0.008), cyclin A (p = 0.007), and p21Waf1/Cip1 (p = 0.009)). Cytoplasmic score index was inversely associated with nuclear p-Akt (p = 0.013) and positively associated with cytoplasmic p-ERK1/2 expression (p = 0.033). Nuclear BRMS1 expression in ≥ 10% of primary melanoma cells was associated with thicker tumors (p = 0.016) and decreased relapse-free period (p = 0.043). Nuclear BRMS1 was associated with expression of fatty acid binding protein 7 (FABP7; p = 0.011), a marker of invasion in melanomas. In line with this, repression of BRMS1 expression reduced the ability of melanoma cells to migrate and invade in vitro. CONCLUSION: Our data suggest that BRMS1 is localized in cytoplasm and nucleus of melanocytic cells and that cellular localization determines its in vivo effect. We hypothesize that cytoplasmic BRMS1 restricts melanoma progression while nuclear BRMS1 possibly promotes melanoma cell invasion.Please see related article: http://www.biomedcentral.com/1741-7015/10/19.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Citoplasma/metabolismo , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Nevo/metabolismo , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Movimento Celular , Núcleo Celular/metabolismo , Progressão da Doença , Intervalo Livre de Doença , Feminino , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Melanoma/mortalidade , Melanoma/patologia , Invasividade Neoplásica/patologia , Nevo/mortalidade , Nevo/patologia , Prognóstico , Proteínas Repressoras , Células Tumorais CultivadasRESUMO
OBJECTIVE: To investigate the expression of MGST1 in primary tumors, solid metastases and metastatic effusions in advanced-stage serous ovarian carcinoma (OC) and analyze the association with clinicopathologic parameters, including chemotherapy resistance and survival. METHODS: MGST1 mRNA expression was investigated in 178 tumors (88 effusions, 38 primary carcinomas, 52 solid metastases) from 144 patients using real-time quantitative PCR (qRT-PCR). Forty-two of the 88 effusions were additionally analyzed for MGST1 protein expression by Western blotting. RESULTS: mRNA expression of MGST1 was higher in primary carcinomas and solid metastases compared to effusions (p=0.008 and p=0.012, respectively). In patient-matched samples, mRNA expression of MGST1 was higher in solid metastases compared to effusions (p=0.023), and a trend for higher MGST1 levels in solid metastases compared to primary tumors was observed (p=0.06). Biopsies from primary carcinomas obtained from patients with >200 ml ascites at diagnosis had higher mRNA expression of MGST1 compared to samples from patients with <200 ml ascites (p=0.037). MGST1 mRNA expression was not associated with age, histological grade, tumor stage, residual disease volume, response to chemotherapy, chemotherapy resistance or survival. Western blot analysis of patient-matched effusions showed high concordance between MGST1 protein and mRNA levels measured by qRT-PCR (p<0.001). CONCLUSIONS: The present study documents frequent MGST1 mRNA and protein expression in OC. The data suggest increased activity of oxidative response pathways, reflected by higher mRNA expression, in solid OC tumors compared to metastatic effusions. Additionally, a tumor microenvironment consisting of ascites may induce antioxidant activity.
Assuntos
Líquido Ascítico/enzimologia , Carcinoma/enzimologia , Glutationa Transferase/metabolismo , Neoplasias Ovarianas/enzimologia , Derrame Pleural Maligno/enzimologia , Ascite/enzimologia , Carcinoma/tratamento farmacológico , Carcinoma/patologia , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Estimativa de Kaplan-Meier , Metástase Neoplásica , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , RNA Mensageiro/metabolismo , Estatísticas não ParamétricasRESUMO
Although clinical management of melanoma has changed considerably in recent years, intrinsic treatment resistance remains a severe problem and strategies to design personal treatment regimens are highly warranted. We have applied a three-dimensional (3D) ex vivo drug efficacy assay, exposing disaggregated cells from 38 freshly harvested melanoma lymph node metastases and 21 patient derived xenografts (PDXs) to clinical relevant drugs for 7 days, and examined its potential to evaluate therapy response. A strong association between Vemurafenib response and BRAF mutation status was achieved (Pâ¯<â¯.0001), while enhanced viability was seen in some NRAS mutated tumors. BRAF and NRAS mutated tumors responded comparably to the MEK inhibitor Cobimetinib. Based on the ex vivo results, two tumors diagnosed as BRAF wild-type by routine pathology examinations had to be re-evaluated; one was subsequently found to have a complex V600E mutation, the other a double BRAF mutation (V600E/K601 N). No BRAF inhibitor resistance mechanisms were identified, but PIK3CA and NF1 mutations were identified in two highly responsive tumors. Concordance between ex vivo drug responses using tissue from PDXs and corresponding patient tumors demonstrate that PDX models represent an indefinite source of tumor material that may allow ex vivo evaluation of numerous drugs and combinations, as well as studies of underlying molecular mechanisms. In conclusion, we have established a rapid and low cost ex vivo drug efficacy assay applicable on tumor tissue from patient biopsies. The 3D/spheroid format, limiting the influence from normal adjacent cells and allowing assessment of drug sensitivity to numerous drugs in one week, confirms its potential as a supplement to guide clinical decision, in particular in identifying non-responding patients.
RESUMO
The objective of this study was to analyze the clinical role of 9 microRNAs (miRs) previously found to be overexpressed in ovarian carcinoma effusions compared with primary ovarian carcinomas. High-grade serous carcinoma effusions (n=148) were analyzed for expression of miR-29a, miR-31, miR-99b, miR-182, miR-210, miR-221, miR-222, miR-224, and miR-342 using quantitative polymerase chain reaction. Expression levels were analyzed for association with clinicopathological parameters and survival. miR-29a and miR-31 levels were further assessed for association with protein expression of their targets Stathmin and DNA methyltransferase-3A (DNMT3A) by immunohistochemistry and Western blotting, respectively. miRNA levels were unrelated to clinicopathological parameters. However, higher miR-29a levels were significantly related to longer overall survival in univariate (P=.007) and Cox multivariate survival analysis (P=.045). miR-29a levels were inversely related to those of its target DNMT3A (P=.048), and higher DNMT3A expression was significantly related to poor overall survival in univariate (P=.03) and Cox multivariate (P=.016) survival analysis. In contrast, miR-31 levels were directly related to cytoplasmic phospho-Stathmin expression (P=.029) and unrelated to Stathmin and nuclear phospho-Stathmin, and both Stathmin and phospho-Stathmin expressions were unrelated to survival. miR-29a and its target DNMT3A are novel candidate biomarkers of longer and shorter survival, respectively, in metastatic high-grade serous carcinoma.
Assuntos
Biomarcadores Tumorais/genética , MicroRNAs/genética , Neoplasias Císticas, Mucinosas e Serosas/genética , Neoplasias Ovarianas/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , DNA (Citosina-5-)-Metiltransferases/análise , DNA Metiltransferase 3A , Intervalo Livre de Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Humanos , Estimativa de Kaplan-Meier , Pessoa de Meia-Idade , Análise Multivariada , Gradação de Tumores , Neoplasias Císticas, Mucinosas e Serosas/química , Neoplasias Císticas, Mucinosas e Serosas/mortalidade , Neoplasias Císticas, Mucinosas e Serosas/secundário , Neoplasias Ovarianas/química , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Fenótipo , Fosforilação , Modelos de Riscos Proporcionais , Fatores de Risco , Estatmina/análise , Fatores de Tempo , Regulação para CimaRESUMO
Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an important oncogene contributing to cancer progression partially by regulating cMYC and AKT. We examined CIP2A expression in cutaneous melanomas, its association with clinicopathological parameters and mapped molecular mechanisms regulated by CIP2A in vitro. CIP2A expression was analyzed by immunohistochemistry in 17 nevi, 132 primary melanomas and 49 metastases. Effects of siRNA-mediated down-regulation on proliferation, apoptosis and signaling pathways were assessed in melanoma cell lines. In superficial spreading melanomas (SSM), high nuclear CIP2A expression was associated with poor overall survival (OS) (P = 0.0018). Surprisingly, high cytoplasmic expression was related to improved relapse-free (P = 0.031) and OS (P = 0.014) in nodular melanomas (NM). In vitro experiments revealed that CIP2A can regulate proliferation and/or apoptosis partially through the PI3K/AKT pathway but also independently. In summary, CIP2A could represent a potential therapeutic target in SSM. However, in NM cytoplasmic CIP2A is associated with improved prognosis indicating that CIP2A has distinct, complex functions dependent on the molecular context and histological subtype. As seen in other cancer types, CIP2A can influence cMYC and AKT, but our data also suggest that in melanoma it has additional targets which need to be identified.
Assuntos
Autoantígenos/metabolismo , Melanoma/mortalidade , Proteínas de Membrana/metabolismo , Nevo/mortalidade , Neoplasias Cutâneas/mortalidade , Adulto , Idoso , Idoso de 80 Anos ou mais , Apoptose/fisiologia , Proliferação de Células/fisiologia , Estudos de Coortes , Regulação para Baixo/fisiologia , Feminino , Técnicas de Silenciamento de Genes , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Estimativa de Kaplan-Meier , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Melanoma/metabolismo , Melanoma/patologia , Pessoa de Meia-Idade , Nevo/metabolismo , Nevo/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Prognóstico , RNA Interferente Pequeno/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Células Tumorais CultivadasRESUMO
The multidrug resistance protein 1 (MRP1) protects cells from xenobiotics by extruding from the intracellular compartment glutathione (GSH)-S-conjugates, glucuronyl conjugates and sulfate conjugates and by the co-export of xenobiotic(s) and GSH. An ATP-dependent transport of aflatoxin B1 (AFB1) and its GSH conjugates by MRP1 has been previously demonstrated in vitro. In the present study, we have sought to investigate the in vivo role of MRP1 in AFB1 carcinogenicity, by comparing the incidence of tumors occurring in mrp1 (+/+) and mrp1 (-/-) mice 12 months after an 8 weeks exposure to AFB1. The carcinogen induced a similar number of lung and liver tumors in both strains. Most lung tumors were of the solid type and showed a moderate degree of differentiation in both mrp1 (+/+) and mrp1 (-/-) mice. These data provide direct evidence that in vivo MRP1 does not protect from AFB1 carcinogenicity. Due to the redundancy of transmembrane export pumps, other pump(s) may effectively vicariate for MRP1-mediated transport of AFB1 and its glutathione conjugates.
Assuntos
Aflatoxina B1/efeitos adversos , Carcinógenos/efeitos adversos , Proteínas de Ligação a DNA/genética , Genes MDR , Neoplasias Hepáticas/etiologia , Neoplasias Pulmonares/etiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos , Aflatoxina B1/toxicidade , Animais , Carcinógenos/toxicidade , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Knockout , Proteína 3 Homóloga a MutSRESUMO
Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) has been shown to induce apoptosis in malignant cells while leaving normal cells unharmed, making it a desirable anticancer target. In the present study, metastatic melanoma cell lines were treated with lexatumumab (Human Genome Sciences, Inc.) a high-affinity monoclonal antibody agonistic to TRAIL receptor 2 (DR5). Binding of the antibody to the receptor led to activation of the extrinsic apoptosis pathway in approximately 20% of the treated cells. However, by combining subtoxic concentrations of the protein translation inhibitor anisomycin with lexatumumab, we obtained synergistic effects on cell viability compared with single agent treatment. Even the low doses of anisomycin could inhibit protein synthesis in melanoma cells with up to 30%, which might result in the shift in the levels of the proteins involved in apoptosis. Co-treatment with anisomycin increased activation of caspases and cleavage of the anti-apoptotic protein Livin, leading to formation of truncated p30-Livin α and p28-Livin ß proteins with potential pro-apoptotic functions. Furthermore, ansiomcycin treatment decreased levels of antiapototic XIAP. In summary our results suggest that combinational treatment with anicomycin and lexatumumab represents a novel therapeutic strategy in the treatment of melanoma.
Assuntos
Anisomicina/farmacologia , Apoptose , Melanoma/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Melanoma/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/genéticaRESUMO
Notoriously resistant malignant melanoma is one of the most increasing forms of cancer worldwide; there is thus a precarious need for new treatment options. The Wee1 kinase is a major regulator of the G(2)/M checkpoint, and halts the cell cycle by adding a negative phosphorylation on CDK1 (Tyr15). Additionally, Wee1 has a function in safeguarding the genome integrity during DNA synthesis. To assess the role of Wee1 in development and progression of malignant melanoma we examined its expression in a panel of paraffin-embedded patient derived tissue of benign nevi and primary- and metastatic melanomas, as well as in agarose-embedded cultured melanocytes. We found that Wee1 expression increased in the direction of malignancy, and showed a strong, positive correlation with known biomarkers involved in cell cycle regulation: Cyclin A (p<0.0001), Ki67 (p<0.0001), Cyclin D3 (p = 0.001), p21(Cip1/WAF1) (p = 0.003), p53 (p = 0.025). Furthermore, high Wee1 expression was associated with thicker primary tumors (p = 0.001), ulceration (p = 0.005) and poor disease-free survival (p = 0.008). Transfections using siWee1 in metastatic melanoma cell lines; WM239(WTp53), WM45.1(MUTp53) and LOX(WTp53), further support our hypothesis of a tumor promoting role of Wee1 in melanomas. Whereas no effect was observed in LOX cells, transfection with siWee1 led to accumulation of cells in G(1)/S and S phase of the cell cycle in WM239 and WM45.1 cells, respectively. Both latter cell lines displayed DNA damage and induction of apoptosis, in the absence of Wee1, indicating that the effect of silencing Wee1 may not be solely dependent of the p53 status of the cells. Together these results reveal the importance of Wee1 as a prognostic biomarker in melanomas, and indicate a potential role for targeted therapy, alone or in combination with other agents.