RESUMEN
The one-carbon metabolism enzyme bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase 2 (MTHFD2) is among the most overexpressed proteins across tumors and is widely recognized as a promising anticancer target. While MTHFD2 is mainly described as a mitochondrial protein, a new nuclear function is emerging. Here, we observe that nuclear MTHFD2 protein levels and association with chromatin increase following ionizing radiation (IR) in an ataxia telangiectasia mutated (ATM)- and DNA-dependent protein kinase (DNA-PK)-dependent manner. Furthermore, repair of IR-induced DNA double-strand breaks (DSBs) is delayed upon MTHFD2 knockdown, suggesting a role for MTHFD2 in DSB repair. In support of this, we observe impaired recruitment of replication protein A (RPA), reduced resection, decreased IR-induced DNA repair protein RAD51 homolog 1 (RAD51) levels and impaired homologous recombination (HR) activity in MTHFD2-depleted cells following IR. In conclusion, we identify a key role for MTHFD2 in HR repair and describe an interdependency between MTHFD2 and HR proficiency that could potentially be exploited for cancer therapy.
RESUMEN
Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase-cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a 'folate trap'. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism.
Asunto(s)
Metilenotetrahidrofolato Deshidrogenasa (NADP) , Neoplasias , Metilenotetrahidrofolato Deshidrogenasa (NADP)/genética , Metilenotetrahidrofolato Deshidrogenasa (NADP)/metabolismo , Ácido Fólico/metabolismo , Formiatos , Purinas , TetrahidrofolatosRESUMEN
Mesenchymal stem/stromal cells (MSCs) are self-renewing and multipotent progenitors, which constitute the main cellular compartment of the bone marrow stroma. Because MSCs have an important role in the pathogenesis of multiple myeloma, it is essential to know if novel drugs target MSCs. Melflufen is a novel anticancer peptide-drug conjugate compound for patients with relapsed refractory multiple myeloma. Here, we studied the cytotoxicity of melflufen, melphalan and doxorubicin in healthy human bone marrow-derived MSCs (BMSCs) and how these drugs affect BMSC proliferation. We established co-cultures of BMSCs with MM.1S myeloma cells to see if BMSCs increase or decrease the cytotoxicity of melflufen, melphalan, bortezomib and doxorubicin. We evaluated how the drugs affect BMSC differentiation into adipocytes and osteoblasts and the BMSC-supported formation of vascular networks. Our results showed that BMSCs were more sensitive to melflufen than to melphalan. The cytotoxicity of melflufen in myeloma cells was not affected by the co-culture with BMSCs, as was the case for melphalan, bortezomib and doxorubicin. Adipogenesis, osteogenesis and BMSC-mediated angiogenesis were all affected by melflufen. Melphalan and doxorubicin affected BMSC differentiation in similar ways. The effects on adipogenesis and osteogenesis were not solely because of effects on proliferation, seen from the differential expression of differentiation markers normalized by cell number. Overall, our results indicate that melflufen has a significant impact on BMSCs, which could possibly affect therapy outcome.
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Células Madre Mesenquimatosas , Mieloma Múltiple , Médula Ósea/patología , Bortezomib/uso terapéutico , Doxorrubicina/farmacología , Doxorrubicina/uso terapéutico , Humanos , Melfalán/análogos & derivados , Melfalán/farmacología , Melfalán/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/patología , Fenilalanina/análogos & derivadosRESUMEN
Immunoglobulin light-chain (AL) amyloidosis is a rare disease caused by clonal plasma cell secretion of misfolded light chains that assemble as toxic amyloid fibrils, depositing in vital organs including the heart and kidneys, causing organ dysfunction. Plasma cell-directed therapeutics are expected to reduce production of toxic light chain by eliminating amyloidogenic cells in bone marrow, thereby diminishing amyloid fibril deposition and providing the potential for organ recovery. Melphalan flufenamide (melflufen) is a first-in-class peptide-drug conjugate that targets aminopeptidases and rapidly releases alkylating agents inside tumor cells. Melflufen is highly lipophilic, permitting rapid uptake by cells, where it is enzymatically hydrolyzed by aminopeptidases, resulting in intracellular accumulation of the alkylating agents, including melphalan. Previous data demonstrating sensitivity of myeloma cells to melflufen suggest that the drug might be useful in AL amyloidosis. We describe the effects of melflufen on amyloidogenic plasma cells in vitro and ex vivo, demonstrating enhanced cytotoxic effects in comparison to melphalan, as well as novel mechanisms of action through the unfolded protein response (UPR) pathway. These findings provide evidence that melflufen-mediated cytotoxicity extends to amyloidogenic plasma cells, and support the rationale for the evaluation of melflufen in patients with AL amyloidosis.
RESUMEN
Myeloma bone disease is a major complication in multiple myeloma affecting quality of life and survival. It is characterized by increased activity of osteoclasts, bone resorbing cells. Myeloma microenvironment promotes excessive osteoclastogenesis, a process of production of osteoclasts from their precursors, monocytes. The effects of two anti-myeloma drugs, melphalan flufenamide (melflufen) and melphalan, on the activity and proliferation of osteoclasts and their progenitors, monocytes, were assessed in this study. In line with previous research, differentiation of monocytes was associated with increased expression of genes encoding DNA damage repair proteins. Hence monocytes were more sensitive to DNA damage-causing alkylating agents than their differentiated progeny, osteoclasts. In addition, differentiated progeny of monocytes showed increased gene expression of immune checkpoint ligands which may potentially create an immunosuppressive microenvironment. Melflufen was ten-fold more active than melphalan in inhibiting proliferation of osteoclast progenitors. Furthermore, melflufen was also superior to melphalan in inhibition of osteoclastogenesis and bone resorption. These results demonstrate that melflufen may exert beneficial effects in patients with multiple myeloma such as reducing bone resorption and immunosuppressive milieu by inhibiting osteoclastogenesis.
RESUMEN
Introduction of the proteasome inhibitor bortezomib has dramatically improved clinical outcomes in multiple myeloma. However, most patients become refractory to bortezomib-based therapies. On the molecular level, development of resistance to bortezomib in myeloma cells is accompanied by complex metabolic changes resulting in increased protein folding capacity, and less dependency on the proteasome. In this study, we show that aminopeptidase B, encoded by the RNPEP gene, is upregulated in bortezomib-resistant myeloma cell lines, and in a murine in vivo model. Moreover, increased RNPEP expression is associated with shorter survival in multiple myeloma patients previously treated with bortezomib-containing regimens. Additionally, expression is increased in plasma cell precursors, a B-lymphoid compartment previously associated with myeloma stem cells. We hypothesized that increased aminopeptidase B expression in aggressive myeloma clones may be used therapeutically toward elimination of the cells via the use of a novel peptide-drug conjugate, melphalan flufenamide (melflufen). Melflufen, a substrate of aminopeptidase B, efficiently eliminates bortezomib-resistant myeloma cells in vitro and in vivo, and completely suppresses clonogenic myeloma growth in vitro at subphysiological concentrations. Thus, melflufen represents a novel treatment option that is able to eradicate drug-resistant myeloma clones characterized by elevated aminopeptidase B expression.
RESUMEN
Multiple myeloma (MM) is characterized by extensive immunoglobulin production leading to an excessive load on protein homeostasis in tumor cells. Aminopeptidases contribute to proteolysis by catalyzing the hydrolysis of amino acids from proteins or peptides and function downstream of the ubiquitin-proteasome pathway. Notably, aminopeptidases can be utilized in the delivery of antibody and peptide-conjugated drugs, such as melflufen, currently in clinical trials. We analyzed the expression of 39 aminopeptidase genes in MM samples from 122 patients treated at Finnish cancer centers and 892 patients from the CoMMpass database. Based on ranked abundance, LAP3, ERAP2, METAP2, TTP2, and DPP7 were highly expressed in MM. ERAP2, XPNPEP1, DPP3, RNPEP, and CTSV were differentially expressed between relapsed/refractory and newly diagnosed MM samples (p < 0.05). Sensitivity to melflufen was detected ex vivo in 11/15 MM patient samples, and high sensitivity was observed, especially in relapsed/refractory samples. Survival analysis revealed that high expression of XPNPEP1, RNPEP, DPP3, and BLMH (p < 0.05) was associated with shorter overall survival. Hydrolysis analysis demonstrated that melflufen is a substrate for aminopeptidases LAP3, LTA4H, RNPEP, and ANPEP. The sensitivity of MM cell lines to melflufen was reduced by aminopeptidase inhibitors. These results indicate critical roles of aminopeptidases in disease progression and the activity of melflufen in MM.
RESUMEN
MX2 is an interferon inducible gene that is mostly known for its antiviral activity. We have previously demonstrated that MX2 is also associated with the tumorigenesis process in melanoma. However, it remains unknown which molecular mechanisms are regulated by MX2 in response to interferon signaling in this disease. Here, we report that MX2 is necessary for the establishment of an interferon-induced transcriptional profile partially through regulation of STAT1 phosphorylation and other interferon-related downstream factors, including proapoptotic tumor suppressor XAF1. MX2 and XAF1 expression tightly correlate in both cultured melanoma cell lines and in patient-derived primary and metastatic tumors, where they also are significantly related with survival. MX2 mediates IFN growth-inhibitory signals in both XAF1 dependent and independent ways and in a cell type and context-dependent manner. Higher MX2 expression renders melanoma cells more sensitive to targeted therapy drugs such as vemurafenib and trametinib; however, this effect is XAF1 independent. In summary, we uncovered a new mechanism in the complex regulation of interferon signaling in melanoma that can influence both survival and response to therapy.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Biomarcadores de Tumor/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Interferones/farmacología , Melanoma/tratamiento farmacológico , Terapia Molecular Dirigida , Proteínas de Resistencia a Mixovirus/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Antineoplásicos/farmacología , Apoptosis , Proteínas Reguladoras de la Apoptosis/genética , Biomarcadores de Tumor/genética , Proliferación Celular , Sinergismo Farmacológico , Humanos , Melanoma/metabolismo , Melanoma/patología , Proteínas de Resistencia a Mixovirus/genética , Fosforilación , Piridonas/farmacología , Pirimidinonas/farmacología , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Células Tumorales CultivadasAsunto(s)
Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Mieloma Múltiple , Proteínas de Neoplasias/biosíntesis , Oligopéptidos/uso terapéutico , Regulación hacia Arriba/efectos de los fármacos , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patologíaRESUMEN
BACKGROUND: Low survival rates in metastatic high-grade osteosarcoma (HGOS) have remained stagnant for the last three decades. This study aims to investigate the role of aminopeptidase N (ANPEP) in HGOS progression and its targeting with a novel lipophilic peptidase-enhanced cytotoxic compound melphalan flufenamide (melflufen) in HGOS. METHODS: Meta-analysis of publicly available gene expression datasets was performed to determine the impact of ANPEP gene expression on metastasis-free survival of HGOS patients. The efficacy of standard-of-care anti-neoplastic drugs and a lipophilic peptidase-enhanced cytotoxic conjugate melflufen was investigated in patient-derived HGOS ex vivo models and cell lines. The kinetics of apoptosis and necrosis induced by melflufen and doxorubicin were compared. Anti-neoplastic effects of melflufen were investigated in vivo. RESULTS: Elevated ANPEP expression in diagnostic biopsies of HGOS patients was found to significantly reduce metastasis-free survival. In drug sensitivity assays, melflufen has shown an anti-proliferative effect in HGOS ex vivo samples and cell lines, including those resistant to methotrexate, etoposide, doxorubicin, and PARP inhibitors. Further, HGOS cells treated with melflufen displayed a rapid induction of apoptosis and this sensitivity correlated with high expression of ANPEP. In combination treatments, melflufen demonstrated synergy with doxorubicin in killing HGOS cells. Finally, Melflufen displayed anti-tumor growth and anti-metastatic effects in vivo. CONCLUSION: This study may pave the way for use of melflufen as an adjuvant to doxorubicin in improving the therapeutic efficacy for the treatment of metastatic HGOS.
RESUMEN
Melphalan flufenamide (hereinafter referred to as "melflufen") is a peptide-conjugated drug currently in phase 3 trials for the treatment of relapsed or refractory multiple myeloma. Due to its lipophilic nature, it readily enters cells, where it is converted to the known alkylator melphalan leading to enrichment of hydrophilic alkylator payloads. Here, we have analysed in vitro and in vivo the efficacy of melflufen on normal and cancerous breast epithelial lines. D492 is a normal-derived nontumorigenic epithelial progenitor cell line whereas D492HER2 is a tumorigenic version of D492, overexpressing the HER2 oncogene. In addition we used triple negative breast cancer cell line MDA-MB231. The tumorigenic D492HER2 and MDA-MB231 cells were more sensitive than normal-derived D492 cells when treated with melflufen. Compared to the commonly used anti-cancer drug doxorubicin, melflufen was significantly more effective in reducing cell viability in vitro while it showed comparable effects in vivo. However, melflufen was more efficient in inhibiting metastasis of MDA-MB231 cells. Melflufen induced DNA damage was confirmed by the expression of the DNA damage proteins Æ´H2Ax and 53BP1. The effect of melflufen on D492HER2 was attenuated if cells were pretreated with the aminopeptidase inhibitor bestatin, which is consistent with previous reports demonstrating the importance of aminopeptidase CD13 in facilitating melflufen cleavage. Moreover, analysis of CD13high and CD13low subpopulations of D492HER2 cells and knockdown of CD13 showed that melflufen efficacy is mediated at least in part by CD13. Knockdown of LAP3 and DPP7 aminopeptidases led to similar efficacy reduction, suggesting that also other aminopeptidases may facilitate melflufen conversion. In summary, we have shown that melflufen is a highly efficient anti-neoplastic agent in breast cancer cell lines and its efficacy is facilitated by aminopeptidases.
Asunto(s)
Antineoplásicos Alquilantes/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Melfalán/análogos & derivados , Fenilalanina/análogos & derivados , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Antígenos CD13/genética , Antígenos CD13/metabolismo , Línea Celular Tumoral , Embrión de Pollo , Daño del ADN , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Histonas/metabolismo , Humanos , Leucil Aminopeptidasa/genética , Leucil Aminopeptidasa/metabolismo , Melfalán/farmacología , Fenilalanina/farmacología , Transducción de Señal , Proteína 1 de Unión al Supresor Tumoral P53/genética , Proteína 1 de Unión al Supresor Tumoral P53/metabolismoRESUMEN
MX2 protein is a dynamin-like GTPase2 that has recently been identified as an interferon-induced restriction factor of HIV-1 and other primate lentiviruses. A single nucleotide polymorphism (SNP), rs45430, in an intron of the MX2 gene, was previously reported as a novel melanoma susceptibility locus in genome-wide association studies. Functionally, however, it is still unclear whether and how MX2 contributes to melanoma susceptibility and tumorigenesis. Here, we show that MX2 is differentially expressed in melanoma tumors and cell lines, with most metastatic cell lines showing lower MX2 expression than primary melanoma cell lines and melanocytes. Furthermore, high expression of MX2 RNA in primary melanoma tumors is associated with better patient survival. Overexpression of MX2 reduces in vivo proliferation partially through inhibition of AKT activation, suggesting that it can act as a tumor suppressor in melanoma. However, we have also identified a subset of melanoma cell lines with high endogenous MX2 expression where downregulation of MX2 leads to reduced proliferation. In these cells, MX2 downregulation interfered with DNA replication and cell cycle processes. Collectively, our data for the first time show that MX2 is functionally involved in the regulation of melanoma proliferation but that its function is context-dependent.
Asunto(s)
Ciclo Celular , Melanoma/patología , Proteínas de Resistencia a Mixovirus/metabolismo , Animales , Ciclo Celular/genética , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Melanoma/genética , Ratones Desnudos , Proteínas de Resistencia a Mixovirus/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de SeñalRESUMEN
CHK1 is an important regulator of the cell cycle and DNA damage response, and its altered expression has been identified in various tumors. Chk1 inhibitors are currently being evaluated as monotherapy and as potentiators of chemotherapy in clinical settings. However, to our knowledge, no previous study has investigated either the activation status or the therapeutic potential of CHK1 targeting in vulvar cancer. Therefore, we examined the expression status of activated CHK1 forms pCHK1Ser345 , pCHK1Ser317 , pCHK1Ser296 , and pCHK1Ser280 in 294 vulvar squamous cell carcinomas (VSCC) using immunohistochemistry and analyzed their relationships with various clinicopathological variables and clinical outcome. To aid translation of preclinical studies, we also assessed cell sensitivity to the Chk1 inhibition in two vulvar cancer cell lines. Compared to the levels of pCHK1Ser345 , pCHK1Ser317 , pCHK1Ser296 , and pCHK1Ser280 in normal vulvar squamous epithelium, high nuclear pCHK1Ser345 expression was found in 57% of vulvar carcinomas, whereas low nuclear pCHK1Ser317 , pCHK1Ser296 , and pCHK1Ser280 expressions were observed in 58%, 64%, and 40% of the cases, respectively. Low levels of pCHK1Ser317 and pCHK1Ser280 in the nucleus correlated significantly with advanced tumor behaviors and aggressive features. None of pCHK1Ser345 , pCHK1Ser317 , pCHK1Ser296 , and pCHK1Ser280 forms were identified as prognostic factors. In vitro inhibition of CHK1 by small molecular inhibitors or siRNA reduced viability by inducing DNA damage and apoptosis of vulvar cancer cell lines. In summary, we conclude that cellular functions regulated by CHK1 are phosphorylation/localization-dependent and deregulation of CHK1 function occurs in VSCC and might contribute to tumorigenesis. Targeting CHK1 might represent as a useful antitumor strategy for the subgroup of VSCC harboring p53 mutations.
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Carcinoma de Células Escamosas/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Inhibidores de Proteínas Quinasas/farmacología , Activación Transcripcional , Neoplasias de la Vulva/genética , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis , Biomarcadores de Tumor , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/terapia , Ciclo Celular/genética , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/antagonistas & inhibidores , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Daño del ADN , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Persona de Mediana Edad , Fosforilación , Pronóstico , Modelos de Riesgos Proporcionales , Inhibidores de Proteínas Quinasas/uso terapéutico , ARN Interferente Pequeño/genética , Estudios Retrospectivos , Neoplasias de la Vulva/tratamiento farmacológico , Neoplasias de la Vulva/metabolismo , Neoplasias de la Vulva/terapiaRESUMEN
Flavopiridol (FP) is a pan-cyclin dependent kinase inhibitor, which shows strong efficacy in inducing cancer cell apoptosis. Although FP is potent against most cancer cells in vitro, unfortunately it proved less efficacious in clinical trials in various aggressive cancers. To date, the molecular mechanisms of the FP resistance are mostly unknown. Here, we report that a small fraction human prostate cancer DU145 cells can survive long-term FP treatment and emerge as FP-resistant cells (DU145FP). These DU145FP cells show accumulated mitochondrial lesions with stronger glycolytic features, and they proliferate in slow-cycling and behave highly migratory with strong anti-apoptotic potential. In addition, the cells are less sensitive to cisplatin and docetaxel-induced apoptotic pressure, and over-express multiple stem cell associated biomarkers. Our studies collectively uncover for the first time that FP-resistant prostate cancer cells show metabolic remodeling, and the metabolic plasticity might be required for the FP resistance-associated cancer cell stemness up-regulation.
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Resistencia a Antineoplásicos , Flavonoides/uso terapéutico , Piperidinas/uso terapéutico , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Apoptosis/efectos de los fármacos , Biomarcadores de Tumor/metabolismo , División Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Cisplatino/farmacología , Docetaxel/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Flavonoides/farmacología , Fase G2/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Piperidinas/farmacología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Seudópodos/efectos de los fármacos , Seudópodos/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
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.
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Biomarcadores de Tumor/genética , MicroARNs/genética , Neoplasias Quísticas, Mucinosas y Serosas/genética , Neoplasias Ováricas/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , ADN (Citosina-5-)-Metiltransferasas/análisis , ADN Metiltransferasa 3A , Supervivencia sin Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica , Predisposición Genética a la Enfermedad , Humanos , Estimación de Kaplan-Meier , Persona de Mediana Edad , Análisis Multivariante , Clasificación del Tumor , Neoplasias Quísticas, Mucinosas y Serosas/química , Neoplasias Quísticas, Mucinosas y Serosas/mortalidad , Neoplasias Quísticas, Mucinosas y Serosas/secundario , Neoplasias Ováricas/química , Neoplasias Ováricas/mortalidad , Neoplasias Ováricas/patología , Fenotipo , Fosforilación , Modelos de Riesgos Proporcionales , Factores de Riesgo , Estatmina/análisis , Factores de Tiempo , Regulación hacia ArribaRESUMEN
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.
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Proteínas de Ciclo Celular/genética , Quinasa de Punto de Control 2/genética , Sinergismo Farmacológico , Melanoma/tratamiento farmacológico , Proteínas Nucleares/genética , Proteínas Tirosina Quinasas/genética , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quinasa de Punto de Control 2/antagonistas & inhibidores , Humanos , Melanoma/genética , Melanoma/patología , Ratones , Proteínas Nucleares/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/administración & dosificación , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirazoles/administración & dosificación , Pirimidinas/administración & dosificación , Pirimidinonas , Neoplasias Cutáneas , Tiofenos/administración & dosificación , Urea/administración & dosificación , Urea/análogos & derivados , Ensayos Antitumor por Modelo de Xenoinjerto , Melanoma Cutáneo MalignoRESUMEN
Cyclin B1-CDK1 complex plays an important role in the regulation of cell cycle. Activation of Cyclin B1 and CDK1 and the formation of the complex in G2/M are under multiple regulations involving many regulators such as isoforms of 14-3-3 and CDC25 and Wee1. Abnormal expression of Cyclin B1 and CDK1 has been detected in various tumors. However, to our knowledge no previous study has investigated Cyclin B1 and CDK1 in vulvar cancer. Therefore, we evaluated the statuses of CDK1Tyr15, pCDK1Thr161, Cyclin B1 (total) and pCyclin B1Ser126 in 297 cases of vulvar squamous cell carcinomas by immunohistochemistry. Statistical analyses were performed to explore their clinicopathological and prognostic values. In at least 25% of tumor cases high expression of CDK1Tyr15, pCDK1Thr161, Cyclin B1 (total) and pCyclin B1Ser126 was observed, compared to the low levels in normal vulvar squamous epithelium. Elevated levels of CDK1Tyr15, pCDK1Thr161, Cyclin B1 (total) and pCyclin B1Ser126 were correlated with advanced tumor behaviors and aggressive features. Although CDK1Tyr15, pCDK1Thr161, Cyclin B1 (total) and pCyclin B1Ser126 could not be identified as prognostic factors, combinations of (pCDK1Thr161 C+N + 14-3-3σN), (pCDK1Thr161 C+N + 14-3-3ηC), (pCDK1Thr161 C+N + Wee1C) and (pCDK1Thr161 C+N + 14-3-3σN + 14-3-3ηC + Wee1C) were correlated with disease-specific survival (p = 0.036, p = 0.029, p = 0.042 and p = 0.007, respectively) in univariate analysis. The independent prognostic significance of (pCDK1Thr161 C+N + 14-3-3σN + 14-3-3ηC + Wee1C) was confirmed by multivariate analysis. In conclusion, CDK1Tyr15, pCDK1Thr161, Cyclin B1 (total) and pCyclin B1Ser126 may be involved in progression of vulvar squamous cell carcinoma. The combination of pCDK1Thr161, 14-3-3σ, 14-3-3η and Wee1 was a statistically independent prognostic factor.
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Carcinoma de Células Escamosas , Ciclina B1/biosíntesis , Quinasas Ciclina-Dependientes/biosíntesis , Regulación Neoplásica de la Expresión Génica , Proteínas de Neoplasias/biosíntesis , Neoplasias de la Vulva , Adulto , Anciano , Anciano de 80 o más Años , Proteína Quinasa CDC2 , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Ciclina B1/genética , Quinasas Ciclina-Dependientes/genética , Supervivencia sin Enfermedad , Femenino , Humanos , Persona de Mediana Edad , Proteínas de Neoplasias/genética , Fosforilación/genética , Tasa de Supervivencia , Neoplasias de la Vulva/genética , Neoplasias de la Vulva/metabolismo , Neoplasias de la Vulva/mortalidad , Neoplasias de la Vulva/patologíaRESUMEN
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.
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Autoantígenos/metabolismo , Melanoma/mortalidad , Proteínas de la Membrana/metabolismo , Nevo/mortalidad , Neoplasias Cutáneas/mortalidad , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis/fisiología , Proliferación Celular/fisiología , Estudios de Cohortes , Regulación hacia Abajo/fisiología , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Péptidos y Proteínas de Señalización Intracelular , Estimación de Kaplan-Meier , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Melanoma/metabolismo , Melanoma/patología , Persona de Mediana Edad , Nevo/metabolismo , Nevo/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Pronóstico , ARN Interferente Pequeño/metabolismo , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Células Tumorales CultivadasRESUMEN
Activating mutations in KIT have been identified in melanomas of acral and mucosal types and in those arising in chronically sun-damaged skin. Until now, KIT has been considered an oncogenic driver and a potential therapeutic target. However, data presented by Dhal et al. show that in cutaneous melanomas the KIT promoter is a target for hypermethylation, leading to its downregulation. Their observations suggest that signaling pathways downstream of KIT may have distinct and opposing roles in the pathogenesis of melanoma subtypes. This will have important implications for the use of KIT inhibitors in treating melanomas.
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Epigénesis Genética , Melanoma/genética , Proteínas Proto-Oncogénicas c-kit/genética , Neoplasias Cutáneas/genética , HumanosRESUMEN
We have previously shown that Wnt5A drives invasion in melanoma. We have also shown that Wnt5A promotes resistance to therapy designed to target the BRAF(V600E) mutation in melanoma. Here, we show that melanomas characterized by high levels of Wnt5A respond to therapeutic stress by increasing p21 and expressing classical markers of senescence, including positivity for senescence-associated ß-galactosidase (SA-ß-gal), senescence-associated heterochromatic foci (SAHF), H3K9Me chromatin marks, and PML bodies. We find that despite this, these cells retain their ability to migrate and invade. Further, despite the expression of classic markers of senescence such as SA-ß-gal and SAHF, these Wnt5A-high cells are able to colonize the lungs in in vivo tail vein colony-forming assays. This clearly underscores the fact that these markers do not indicate true senescence in these cells, but instead an adaptive stress response that allows the cells to evade therapy and invade. Notably, silencing Wnt5A reduces expression of these markers and decreases invasiveness. The combined data point to Wnt5A as a master regulator of an adaptive stress response in melanoma, which may contribute to therapy resistance.