RESUMO
Therapy of BRAF-mutant melanoma with selective inhibitors of BRAF (BRAFi) and MEK (MEKi) represents a major clinical advance but acquired resistance to therapy has emerged as a key obstacle. To date, no clinical approaches successfully resensitize to BRAF/MEK inhibition. Here, we develop a therapeutic strategy for melanoma using bromosporine, a bromodomain inhibitor. Bromosporine (bromo) monotherapy produced significant anti-tumor effects against established melanoma cell lines and patient-derived xenografts (PDXs). Combinatorial therapy involving bromosporine and cobimetinib (bromo/cobi) showed synergistic anti-tumor effects in multiple BRAFi-resistant PDX models. The bromo/cobi combination was superior in vivo to standard BRAFi/MEKi therapy in the treatment-naive BRAF-mutant setting and to MEKi alone in the setting of immunotherapy-resistant NRAS- and NF1-mutant melanoma. RNA sequencing of xenografts treated with bromo/cobi revealed profound down-regulation of genes critical to cell division and mitotic progression. Bromo/cobi treatment resulted in marked DNA damage and cell-cycle arrest, resulting in induction of apoptosis. These studies introduce bromodomain inhibition, alone or combined with agents targeting the mitogen activated protein kinase pathway, as a rational therapeutic approach for melanoma refractory to standard targeted or immunotherapeutic approaches.
Assuntos
Melanoma , Proteínas Proto-Oncogênicas B-raf , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Nucleares , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/metabolismo , Fatores de TranscriçãoRESUMO
BACKGROUND: While melanomas commonly harbor losses of 9p21, on which CDKN2A resides, the presence of additional tumor suppressor elements at this locus is incompletely characterized. Here we assess the expression levels and functional role of microRNA-876-3p (miR-876), whose gene also maps to 9p21. METHODS: Expression of miR-876 was assessed in human tissues and cell lines using quantitative miRNA reverse transcriptase polymerase chain reaction (qRT-PCR). MIR876 copy number was determined in The Cancer Genome Atlas (TCGA) melanoma cohort. The consequences of regulation of miR-876 expression were assessed on melanoma cell colony formation, migration, invasion, apoptosis, cell cycle progression, and drug sensitivity in culture, and on in vivo tumor growth in a xenograft model. Genome-wide transcriptomic changes induced by miR-876 overexpression were determined using RNA sequencing (RNA-Seq). RESULTS: miR-876 expression was significantly decreased in primary melanoma samples when compared with nevi, and in human melanoma cell lines when compared with human melanocytes. Analysis of the TCGA cohort revealed deletions in MIR876 in > 50% of melanomas. miR-876 overexpression resulted in decreased melanoma cell colony formation, migration, and invasion, which was accompanied by cell cycle arrest and increased apoptosis. Intra-tumoral injections of miR-876 significantly suppressed melanoma growth in vivo. RNA-Seq analysis of miR-876-treated tumors revealed downregulation of several growth-promoting genes, along with upregulation of tumor suppressor genes, which was confirmed by qRT-PCR analysis. Computational analyses identified MAPK1 (or ERK2) as a possible target of miR-876 action. Overexpression of miR-876 significantly suppressed luciferase expression driven by the MAPK1/ERK2 3' UTR, and resulted in decreased ERK protein expression in melanoma cells. MAPK1/ERK2 cDNA overexpression rescued the effects of miR-876 on melanoma colony formation. miR-876 overexpression sensitized melanoma cells to treatment with the BRAF inhibitor vemurafenib. CONCLUSIONS: These studies identify miR-876 as a distinct tumor suppressor on 9p21 that is inactivated in melanoma and suggest miR-876 loss as an additional mechanism to activate ERK and the mitogen activated protein kinase (MAPK) pathway in melanoma. In addition, they suggest the therapeutic potential of combining miR-876 overexpression with BRAF inhibition as a rational therapeutic strategy for melanoma.
Assuntos
Cromossomos Humanos Par 9 , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Melanoma , MicroRNAs , Humanos , Melanoma/genética , Melanoma/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Linhagem Celular Tumoral , Animais , Cromossomos Humanos Par 9/genética , Movimento Celular/genética , Apoptose/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proliferação de Células/genética , Sequência de Bases , Invasividade NeoplásicaRESUMO
BACKGROUND: Technetium-99m-labeled Tilmanocept, a multivalent mannose, is readily internalized by the CD206 surface receptor on macrophages and dendritic cells which are abundantly present in lymph nodes. We want to examine the drainage patterns of Technetium-99m-labeled Tilmanocept to sentinel lymph nodes (SLNs) in melanoma patients following the 10% rule. METHODS: Multi-center retrospective review of patients with cutaneous melanoma undergoing SLN biopsy using Technetium-99m-labeled Tilmanocept between 2008 and 2014 was conducted. Statistical methods were used for data analyses. RESULTS: Of the 564 patients (mean age of 60.3 and 62% male) with preoperative lymphoscintigraphy showing at least one SLN, several primary tumor sites were included: 27% head/neck, 33% trunk, 21% upper extremity and 19% lower extremity. For the head/neck primary site, 36.5% of patients had multiple draining basins; for the trunk site, 36.4% of patients; for the upper extremity site, 13% of patients; and for the lower extremity, 27.4% of patients. A median of 3 (range 1-18) SLNs were identified and resected. Overall, 78% of patients had >1 SLN identified by Technetium-99m-labeled Tilmanocept. In a multivariate model, patients with >1 SLN were significantly associated with age, Breslow depth, tumor location and higher AJCC tumor stage. A total of 17.7% of patients (100/564) had a positive SLN identified. A total of 145 positive SLNs were identified out of 1,812 SLNs with a positive SLN rate of 8%. Positive SLN status was significantly associated with younger age, greater Breslow depth, mitosis rate, higher AJCC tumor stage, presence of ulceration and angiolymphatic invasion. CONCLUSIONS: Using the 10% rule, Technetium-99m-labeled Tilmanocept detects multiple SLNs in most melanoma patients.
Assuntos
Melanoma , Linfonodo Sentinela , Neoplasias Cutâneas , Humanos , Masculino , Pessoa de Meia-Idade , Feminino , Linfonodo Sentinela/diagnóstico por imagem , Linfonodo Sentinela/cirurgia , Linfonodo Sentinela/patologia , Linfocintigrafia/métodos , Melanoma/diagnóstico por imagem , Melanoma/cirurgia , Melanoma/patologia , Biópsia de Linfonodo Sentinela/métodos , Compostos Radiofarmacêuticos , Pentetato de Tecnécio Tc 99m , Tecnécio , Metástase Linfática/patologia , Neoplasias Cutâneas/diagnóstico por imagem , Neoplasias Cutâneas/cirurgia , Neoplasias Cutâneas/patologia , Linfonodos/diagnóstico por imagem , Linfonodos/cirurgia , Linfonodos/patologiaRESUMO
The invasive behavior of glioblastoma is essential to its aggressive potential. Here, we show that pleckstrin homology domain interacting protein (PHIP), acting through effects on the force transduction layer of the focal adhesion complex, drives glioblastoma motility and invasion. Immunofluorescence analysis localized PHIP to the leading edge of glioblastoma cells, together with several focal adhesion proteins: vinculin (VCL), talin 1 (TLN1), integrin beta 1 (ITGB1), as well as phosphorylated forms of paxillin (pPXN) and focal adhesion kinase (pFAK). Confocal microscopy specifically localized PHIP to the force transduction layer, together with TLN1 and VCL. Immunoprecipitation revealed a physical interaction between PHIP and VCL. Targeted suppression of PHIP resulted in significant down-regulation of these focal adhesion proteins, along with zyxin (ZYX), and produced profoundly disorganized stress fibers. Live-cell imaging of glioblastoma cells overexpressing a ZYX-GFP construct demonstrated a role for PHIP in regulating focal adhesion dynamics. PHIP silencing significantly suppressed the migratory and invasive capacity of glioblastoma cells, partially restored following TLN1 or ZYX cDNA overexpression. PHIP knockdown produced substantial suppression of tumor growth upon intracranial implantation, as well as significantly reduced microvessel density and secreted VEGF levels. PHIP copy number was elevated in the classical glioblastoma subtype and correlated with elevated EGFR levels. These results demonstrate PHIP's role in regulating the actin cytoskeleton, focal adhesion dynamics, and tumor cell motility, and identify PHIP as a key driver of glioblastoma migration and invasion.
Assuntos
Neoplasias Encefálicas/patologia , Adesões Focais/patologia , Glioblastoma/patologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neovascularização Patológica/patologia , Citoesqueleto de Actina/metabolismo , Animais , Encéfalo/patologia , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/genética , Adesão Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Estudos de Coortes , Progressão da Doença , Feminino , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioblastoma/irrigação sanguínea , Glioblastoma/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Microscopia Intravital , Camundongos , Microscopia Confocal , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neovascularização Patológica/genética , Imagem com Lapso de Tempo , Vinculina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is a devastating disease often detected at advanced stages when surgery cannot be performed. Conventional and targeted systemic therapies perform poorly, and therefore effective drugs are urgently needed. Different epigenetic modifications occur in CCA and contribute to malignancy. Targeting epigenetic mechanisms may thus open therapeutic opportunities. However, modifications such as DNA and histone methylation often coexist and cooperate in carcinogenesis. We tested the therapeutic efficacy and mechanism of action of a class of dual G9a histone-methyltransferase and DNA-methyltransferase 1 (DNMT1) inhibitors. APPROACH AND RESULTS: Expression of G9a, DNMT1, and their molecular adaptor, ubiquitin-like with PHD and RING finger domains-1 (UHRF1), was determined in human CCA. We evaluated the effect of individual and combined pharmacological inhibition of G9a and DNMT1 on CCA cell growth. Our lead G9a/DNMT1 inhibitor, CM272, was tested in human CCA cells, patient-derived tumoroids and xenograft, and a mouse model of cholangiocarcinogenesis with hepatocellular deletion of c-Jun-N-terminal-kinase (Jnk)-1/2 and diethyl-nitrosamine (DEN) plus CCl4 treatment (JnkΔhepa + DEN + CCl4 mice). We found an increased and correlative expression of G9a, DNMT1, and UHRF1 in CCAs. Cotreatment with independent pharmacological inhibitors G9a and DNMT1 synergistically inhibited CCA cell growth. CM272 markedly reduced CCA cell proliferation and synergized with Cisplatin and the ERBB-targeted inhibitor, Lapatinib. CM272 inhibited CCA tumoroids and xenograft growth and significantly antagonized CCA progression in JnkΔhepa + DEN + CCl4 mice without apparent toxicity. Mechanistically, CM272 reprogrammed the tumoral metabolic transcriptome and phenotype toward a differentiated and quiescent status. CONCLUSIONS: Dual targeting of G9a and DNMT1 with epigenetic small molecule inhibitors such as CM272 is a potential strategy to treat CCA and/or enhance the efficacy of other systemic therapies.
Assuntos
Neoplasias dos Ductos Biliares , Proliferação de Células/efeitos dos fármacos , Colangiocarcinoma , DNA (Citosina-5-)-Metiltransferase 1 , Inibidores Enzimáticos/farmacologia , Antígenos de Histocompatibilidade , Histona-Lisina N-Metiltransferase , Animais , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/fisiologia , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Antígenos de Histocompatibilidade/metabolismo , Código das Histonas/efeitos dos fármacos , Código das Histonas/fisiologia , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Camundongos , Resultado do Tratamento , Ubiquitina-Proteína Ligases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
The identification and targeting of key molecular drivers of melanoma and breast and lung cancer have substantially improved their therapy. However, subtypes of each of these three common, lethal solid tumors lack identified molecular drivers, and are thus not amenable to targeted therapies. Here we show that pleckstrin homology domain-interacting protein (PHIP) promotes the progression of these "driver-negative" tumors. Suppression of PHIP expression significantly inhibited both tumor cell proliferation and invasion, coordinately suppressing phosphorylated AKT, cyclin D1, and talin1 expression in all three tumor types. Furthermore, PHIP's targetable bromodomain is functional, as it specifically binds the histone modification H4K91ac. Analysis of TCGA profiling efforts revealed PHIP overexpression in triple-negative and basal-like breast cancer, as well as in the bronchioid subtype of nonsmall cell lung cancer. These results identify a role for PHIP in the progression of melanoma and breast and lung cancer subtypes lacking identified targeted therapies. The use of selective, anti-PHIP bromodomain inhibitors may thus yield a broad-based, molecularly targeted therapy against currently nontargetable tumors.
Assuntos
Mama/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Melanoma/metabolismo , Domínios de Homologia à Plecstrina/fisiologia , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Ciclina D1/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
BACKGROUND: Mitotic rate is a strong, independent prognostic factor in patients with melanoma. However, incorporating it into the melanoma staging system has proved challenging. METHODS: The prognostic impact of mitotic rate was assessed in a melanoma cohort comprising 5050 patients from 2 geographically distinct populations. Computer-generated cut points for mitotic rate were constructed to determine its impact on melanoma-associated survival using Kaplan-Meier and multivariate regression analyses. The impact of mitotic rate also was assessed in randomly split training and validation sets. RESULTS: Mitotic rate had a nonlinear impact on survival, as evidenced by unequally spaced cut points. An index incorporating these cut points that was constructed from one population produced significantly more accurate predictions of survival in the other population than using the entire scale of mitotic rate. An index constructed from the combined cohort was found to be independently predictive of survival, with an impact comparable to that of ulceration. Optimal high-versus-low cut points for mitotic rate were generated separately for each T category (<2 mitoses/mm2 vs ≥2 mitoses/mm2 for T1 melanoma, <4 mitoses/mm2 vs ≥4 mitoses/mm2 for T2 melanoma, <6 mitoses/mm2 vs ≥6/mitoses/mm2 for T3 melanoma, and <7 mitoses/mm2 vs ≥7 mitoses/mm2 for T4 melanoma). Using Kaplan-Meier analysis, elevated mitotic rate was found to have an impact on survival comparable to that of ulceration within each T category. Application of the index for mitotic rate that was constructed from the training data set demonstrated an independent impact in the validation data set, with a significance similar to that of ulceration. CONCLUSIONS: The results of the current study demonstrated the comparable prognostic impact of mitotic rate and ulceration, providing support for its reincorporation into the T category.
Assuntos
Melanoma/genética , Índice Mitótico/métodos , Feminino , Humanos , Masculino , Melanoma/mortalidade , Estadiamento de Neoplasias , PrognósticoRESUMO
BACKGROUND: Patient-derived xenograft (PDX) mouse tumour models can predict response to therapy in patients. Predictions made from PDX cultures (PDXC) would allow for more rapid and comprehensive evaluation of potential treatment options for patients, including drug combinations. METHODS: We developed a PDX library of BRAF-mutant metastatic melanoma, and a high-throughput drug-screening (HTDS) platform utilising clinically relevant drug exposures. We then evaluated 34 antitumor agents across eight melanoma PDXCs, compared drug response to BRAF and MEK inhibitors alone or in combination with PDXC and the corresponding PDX, and investigated novel drug combinations targeting BRAF inhibitor-resistant melanoma. RESULTS: The concordance of cancer-driving mutations across patient, matched PDX and subsequent PDX generations increases as variant allele frequency (VAF) increases. There was a high correlation in the magnitude of response to BRAF and MEK inhibitors between PDXCs and corresponding PDXs. PDXCs and corresponding PDXs from metastatic melanoma patients that progressed on standard-of-care therapy demonstrated similar resistance patterns to BRAF and MEK inhibitor therapy. Importantly, HTDS identified novel drug combinations to target BRAF-resistant melanoma. CONCLUSIONS: The biological consistency observed between PDXCs and PDXs suggests that PDXCs may allow for a rapid and comprehensive identification of treatments for aggressive cancers, including combination therapies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Melanoma/tratamento farmacológico , Animais , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , MAP Quinase Quinase Quinases/antagonistas & inibidores , Melanoma/enzimologia , Melanoma/genética , Melanoma/patologia , Camundongos , Mutação , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Microphthalmia-associated transcription factor (MITF) plays a critical and complex role in melanocyte transformation. Although several downstream targets of MITF action have been identified, the precise mechanisms by which MITF promotes melanocytic tumor progression are incompletely understood. Recent studies identified an oncogenic role for the bromodomain plant homeodomain finger transcription factor (BPTF) gene in melanoma progression, in part through activation of BCL2, a canonical target of MITF signaling. Analysis of the BPTF promoter identified a putative MITF-binding site, suggesting that MITF may regulate BPTF expression. Overexpression of MITF resulted in up-regulation of BPTF in a panel of melanoma and melanocyte cell lines. shRNA-mediated down-regulation of MITF in melanoma cells was accompanied by down-regulation of BPTF and BPTF-regulated genes (including BCL2) and resulted in reduced proliferative capacity of melanoma cells. The suppression of cell growth mediated by MITF silencing was rescued by overexpression of BPTF cDNA. Binding of MITF to the BPTF promoter was demonstrated using ChIP analysis. MITF overexpression resulted in direct transcriptional activation of BPTF, as evidenced by increased luciferase activity driven by the BPTF promoter. These results indicate that BPTF transduces key prosurvival signals driven by MITF, further supporting its important role in promoting melanoma cell survival and progression.
Assuntos
Antígenos Nucleares/metabolismo , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Melanócitos/citologia , Melanoma/patologia , Fator de Transcrição Associado à Microftalmia/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição/metabolismo , Antígenos Nucleares/genética , Apoptose , Sítios de Ligação , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , Imunofluorescência , Humanos , Luciferases/metabolismo , Melanócitos/metabolismo , Melanoma/genética , Melanoma/metabolismo , Fator de Transcrição Associado à Microftalmia/genética , Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
BACKGROUND: Melanoma brain metastasis is associated with an extremely poor prognosis, with a median overall survival of 4-5 months. Since 2011, the overall survival of patients with stage IV melanoma has been significantly improved with the advent of new targeted therapies and checkpoint inhibitors. We analyze the survival outcomes of patients diagnosed with brain metastasis after the introduction of these novel drugs. METHODS: We performed a retrospective analysis of our melanoma center database and identified 79 patients with brain metastasis between 2011 and 2015. RESULTS: The median time from primary melanoma diagnosis to brain metastasis was 3.2 years. The median overall survival duration from the time of initial brain metastasis was 12.8 months. Following a diagnosis of brain metastasis, 39 (49.4%), 28 (35.4%), and 24 (30.4%) patients were treated with anti-CTLA-4 antibody, anti-PD-1 antibody, or BRAF inhibitors (with or without a MEK inhibitor), with a median overall survival of 19.2 months, 37.9 months and 12.7 months, respectively. Factors associated with significantly reduced overall survival included male sex, cerebellar metastasis, higher number of brain lesions, and treatment with whole-brain radiation therapy. Factors associated with significantly longer overall survival included treatment with craniotomy, stereotactic radiosurgery, or with anti-PD-1 antibody after initial diagnosis of brain metastasis. CONCLUSIONS: These results show a significant improvement in the overall survival of patients with melanoma brain metastasis in the era of novel therapies. In addition, they suggest the activity of anti-PD-1 therapy specifically in the setting of brain metastasis.
Assuntos
Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/secundário , Melanoma/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/efeitos adversos , Antineoplásicos Imunológicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Antígeno CTLA-4/antagonistas & inibidores , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Estadiamento de Neoplasias , Prognóstico , Modelos de Riscos Proporcionais , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Estudos Retrospectivos , Resultado do Tratamento , Adulto JovemRESUMO
MicroRNAs (miRNAs) play a key role in cancer progression by coordinately repressing target genes involved in cell proliferation, migration, and invasion. miRNAs regulate gene expression by repressing translation or directing sequence-specific degradation of complementary mRNA. Here, we report that expression of miR-1280 is significantly suppressed in human melanoma specimens when compared with nevi, and in human melanoma cell lines when compared with cultured normal human melanocytes. The proto-oncogene Src was identified as a target of miR-1280 action. Levels of Src expression were significantly higher in melanoma samples and cell lines than in nevi and normal melanocytes. miR-1280 overexpression significantly suppressed the luciferase activity of reporter plasmids containing the full-length 3' untranslated region of Src. miR-1280-mediated suppression of Src led to substantial decreases in melanoma cell proliferation, cell cycle progression, invasion, as well as induced melanoma cell apoptosis. The effects of miR-1280 overexpression on melanoma cell proliferation and growth were reversed by Src overexpression. Intratumoral delivery of miR-1280 significantly suppressed melanoma cell growth in vivo. Our results demonstrate a novel role for miR-1280 as a tumor suppressor in melanoma, identify the Src signaling pathway as a target of miR-1280 action, and suggest a potential therapeutic role for miR-1280 in melanoma.
Assuntos
Regulação Neoplásica da Expressão Gênica , Melanoma/genética , MicroRNAs/genética , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Neoplasias Cutâneas/genética , Regiões 3' não Traduzidas , Animais , Apoptose , Sequência de Bases , Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Camundongos Nus , MicroRNAs/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although melanomas with mutant v-Raf murine sarcoma viral oncogene homolog B1 (BRAF) can now be effectively targeted, there is no molecular target for most melanomas expressing wild-type BRAF. Here, we show that the activation of Pleckstrin homology domain-interacting protein (PHIP), promotes melanoma metastasis, can be used to classify a subset of primary melanomas, and is a prognostic biomarker for melanoma. Systemic, plasmid-based shRNA targeting of Phip inhibited the metastatic progression of melanoma, whereas stable suppression of Phip in melanoma cell lines suppressed metastatic potential and prolonged the survival of tumor-bearing mice. The human PHIP gene resides on 6q14.1, and although 6q loss has been observed in melanoma, the PHIP locus was preserved in melanoma cell lines and patient samples, and its overexpression was an independent adverse predictor of survival in melanoma patients. In addition, a high proportion of PHIP-overexpressing melanomas harbored increased PHIP copy number. PHIP-overexpressing melanomas include tumors with wild-type BRAF, neuroblastoma RAS viral (v-ras) oncogene homolog, and phosphatase and tensin homolog, demonstrating PHIP activation in triple-negative melanoma. These results describe previously unreported roles for PHIP in predicting and promoting melanoma metastasis, and in the molecular classification of melanoma.
Assuntos
Biomarcadores Tumorais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/secundário , Melanoma/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Sequência de Bases , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/genética , Melanoma/genética , Melanoma/secundário , Melanoma Experimental/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/genética , RNA Interferente Pequeno/genética , Transdução de SinaisRESUMO
According to the American Joint Commission on Cancer (AJCC) 8th edition guidelines, SLN biopsy is recommended for primary melanomas with a Breslow thickness of at least 1 mm. Additionally, the National Comprehensive Cancer Network (NCCN) recommends that a SLN biopsy may be considered for melanoma patients with T1b lesions, which are 0.8-1 mm thick or less than 0.8 mm thick with ulceration. It can also be considered for T1a lesions that are less than 0.8 mm thick but have other adverse features, such as a high mitotic rate, lymphovascular invasion, or a positive deep margin. To reduce the false negative rate of melanoma SLN biopsy, we have introduced the intraoperative use of Sentinella, a gamma camera, to enhance the identification rate of SLNs beyond that of the traditional gamma hand-held probe. At the Center for Melanoma Research and Treatment at the California Pacific Medical Center, a multidisciplinary approach has been established to treat melanoma patients when the diagnosis of primary melanoma is made with a referral to our melanoma center. This comprehensive approach at the melanoma tumor board, including the efforts of pathologists, radiologists, dermatologists, surgical, medical and radiation oncologists, results in a consensus to deliver personalized and high-quality care for our melanoma patients. This multidisciplinary program for the management of melanoma can be duplicated for other types of cancer. This article consists of current knowledge to document the published methods of identification of sentinel lymph nodes. In addition, we have included new data as developed in our melanoma center as newly published materials in this article to demonstrate the utility of these methods in melanoma sentinel lymph node surgery. Informed consent has been waived by our IRB regarding the acquisition of clinical data as presented in this study.
RESUMO
The evolution of primary melanoma to lymph node and distant metastasis is incompletely understood. We examined the genomic diversity in melanoma progression in matched primary melanomas and lymph node and distant metastases from 17 patients. FISH analysis revealed cancer cell fractions with monotonic copy number alterations, including PHIP gain and PTEN loss, in the metastatic cascade. By contrast, the cancer cell fraction with copy number alterations for BPTF and MITF was reduced in lymph node metastases but increased in distant metastases. Separately, the cancer cell fraction with NCOA3 copy number alteration was comparable between primary tumors and lymph node metastases yet increased in distant metastases. These results suggest enrichment of the phosphoinositide 3-kinase and MITF pathways in the transition through the metastatic cascade. By contrast, next-generation sequencing analysis did not identify a consistent pattern of changes in variant allele frequency while revealing several intriguing findings, including decreased variant allele frequency in distant metastases and distinct drivers in lymph node versus distant metastases. These results provide evidence that distant melanoma metastasis does not always emanate from lymph node metastasis. These results enhance our understanding of clonal patterns of melanoma metastasis, with possible implications for targeted therapy and metastasis competency.
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BACKGROUND: A minority of patients with T1 melanoma will have a positive sentinel lymph node (SLN) biopsy (SLNB) finding. Identifying who will develop metastatic disease is important in determining prognosis and treatment. OBJECTIVE: We sought to identify clinical and histologic features predictive of a positive SLNB result and determine its prognostic significance in patients with T1 melanoma. METHODS: Clinical and histologic parameters were evaluated in 484 patients with T1 melanoma for their ability to predict a positive SLNB result. The impact of various factors on SLN positivity was evaluated. SLN status was examined as a predictor of overall survival. RESULTS: In all, 34 patients had a positive SLNB finding. Four factors predicted a higher risk of SLN positivity: age 43 years or younger, Breslow depth 0.8 mm or greater, tumors on the lower extremity and trunk, and tumor-infiltrating lymphocyte level. By multivariate analysis, low tumor-infiltrating lymphocytes (P = .0015) and decreasing age (P = .0058) independently predicted SLN positivity. If 0 to 2 of these factors were present, the rate of a positive SLNB result was 3%; this increased to 15% with 3 factors present and to 30% if all 4 factors were present (P < .002). SLN-positive patients had significantly decreased survival (P = .003), and SLN status was the most powerful predictor of survival (P = .009). LIMITATIONS: Our data analysis includes patients from 1994 to 2007 and therefore information on mitotic rate, a recently defined T1b criterion, is not recorded for all patients. CONCLUSIONS: Combining clinical and histologic prognostic factors may help identify subgroups of T1 patients at higher risk of SLN positivity. SLN status has significant prognostic impact in patients with thin melanomas.
Assuntos
Melanoma/patologia , Biópsia de Linfonodo Sentinela , Neoplasias Cutâneas/patologia , Adulto , Feminino , Humanos , Masculino , PrognósticoRESUMO
Most patients who die from cancer succumb to treatment-refractory advanced metastatic progression. Although the early stages of tumor metastasis result in the formation of clinically silent micrometastatic foci, its later stages primarily reflect the progressive, organ-destructive growth of already advanced metastases. Early-stage metastasis is regulated by multiple factors within tumor cells as well as by the tumor microenvironment (TME). In contrast, the molecular determinants that control advanced metastatic progression remain essentially uncharacterized, precluding the development of therapies targeted against it. Here we show that the TME, functioning in part through platelet endothelial cell adhesion molecule 1 (PECAM-1), drives advanced metastatic progression and is essential for progression through its preterminal end stage. PECAM-1-KO and chimeric mice revealed that its metastasis-promoting effects are mediated specifically through vascular endothelial cell (VEC) PECAM-1. Anti-PECAM-1 mAb therapy suppresses both end-stage metastatic progression and tumor-induced cachexia in tumor-bearing mice. It reduces proliferation, but not angiogenesis or apoptosis, within advanced tumor metastases. Because its antimetastatic effects are mediated by binding to VEC rather than to tumor cells, anti-PECAM-1 mAb appears to act independently of tumor type. A modified 3D coculture assay showed that anti-PECAM-1 mAb inhibits the proliferation of PECAM-1-negative tumor cells by altering the concentrations of secreted factors. Our studies indicate that a complex interplay between elements of the TME and advanced tumor metastases directs end-stage metastatic progression. They also suggest that some therapeutic interventions may target late-stage metastases specifically. mAb-based targeting of PECAM-1 represents a TME-targeted therapeutic approach that suppresses the end stages of metastatic progression, until now a refractory clinical entity.
Assuntos
Neoplasias Experimentais/secundário , Molécula-1 de Adesão Celular Endotelial a Plaquetas/fisiologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Apoptose , Transplante de Medula Óssea , Caquexia/terapia , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Células Endoteliais/fisiologia , Feminino , Humanos , Neoplasias Pulmonares/secundário , Neoplasias Pulmonares/terapia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Camundongos Transgênicos , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Neovascularização Patológica , Comunicação Parácrina , Fenótipo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologiaRESUMO
High-figure of merit (FoM) plasmonic microwave resonator is researched as a non-invasive on-body sensor to monitor the human body's blood glucose variation rate in adults for biomedical applications, e.g., diabetic patients. The resonance frequencies of the proposed sensor are measured to be around [Formula: see text] GHz and [Formula: see text] GHz over the frequency band of DC to 6GHz which are suitable for monitoring interstitial fluid (ISF) changing rate. The [Formula: see text] sensor is experimentally wrapped on the human body arm to monitor the blood glucose changing rate via amplitude and frequency variations of the sensor. Amplitude variation and frequency shift are measured to be around 7 dB and 30 MHz, respectively. The measured results demonstrate the high precision of the proposed approach to depict a valid diagram for glucose changing rate due to good impedance matching of the designed microwave sensor and human body. The sensor is shown to enhance the sensitivity by a factor of 5 compared to the conventional ones.
Assuntos
Automonitorização da Glicemia , Glicemia , Adulto , Humanos , Micro-Ondas , Impedância Elétrica , GlucoseRESUMO
The longitudinal monitoring of patient circulating tumor DNA (ctDNA) provides a powerful method for tracking the progression, remission, and recurrence of several types of cancer. Often, clinical and research approaches involve the manual review of individual liquid biopsy reports after sampling and genomic testing. Here, we describe a process developed to integrate techniques utilized in data science within a cancer research framework. Using data collection, an analysis that classifies genetic cancer mutations as pathogenic, and a patient matching methodology that identifies the same donor within all liquid biopsy reports, the manual work for research personnel is drastically reduced. Automated dashboards provide longitudinal views of patient data for research studies to investigate tumor progression and treatment efficacy via the identification of ctDNA variant allele frequencies over time.
RESUMO
The nanometer-scale spatial organization of immune receptors plays a role in cell activation and suppression. While the connection between this spatial organization and cell signaling events is emerging from cell culture experiments, how these results translate to more physiologically relevant settings like the tumor microenvironment remains poorly understood due to the challenges of high-resolution imaging in vivo. Here we perform super-resolution immunofluorescence microscopy of human melanoma tissue sections to examine the spatial organization of the immune checkpoint inhibitor programmed cell death 1 (PD-1). We show that PD-1 exhibits a variety of organizations ranging from nanometer-scale clusters to more uniform membrane labeling. Our results demonstrate the capability of super-resolution imaging to examine the spatial organization of immune checkpoint markers in the tumor microenvironment, suggesting a future direction for both clinical and immunology research.
RESUMO
Glioblastoma's (GBM) aggressive growth is driven by redundant activation of a myriad of signaling pathways and genomic alterations in tyrosine kinase receptors, such as epidermal growth factor receptor (EGFR), which is altered in over 50% of cases. Single agents targeting EGFR have not proven effective against GBM. In this study, we aimed to identify an effective anti-tumor regimen using pharmacogenomic testing of patient-derived GBM samples, in culture and in vivo. High-throughput pharmacological screens of ten EGFR-driven GBM samples identified the combination of erlotinib (EGFRi) and MLN0128 (a mammalian target of rapamycin inhibitor, or MTORi) as the most effective at inhibiting tumor cell viability. The anti-tumor activity of erlonitib+MLN0128 was synergistic and produced inhibition of the p-EGFR, mitogen-activated protein kinase (MAPK), and Phosphoinositide 3-kinase (PI3K) pathways in culture. Using an orthotopic murine model of GBM, we show that erlotinib+MLN0128 inhibited tumor growth in vivo and significantly prolonged the survival of tumor-bearing mice. Expression profiling of tumor tissues from treated mice revealed a unique gene signature induced by erlotinib+MLN0128, consisting of downregulation of immunosuppressive chemokines in the tumor microenvironment, including C-C motif chemokine ligand 2 (CCL2) and periostin. Lower periostin levels resulted in the inhibition of Iba1+ (tumor-promoting) macrophage infiltration of GBM xenografts. Taken together, our results demonstrate that pharmacological co-targeting of EGFR and MTOR using clinically available drugs represents an effective treatment paradigm for EGFR-driven GBMs, acting both by inhibiting tumor cell growth and modulating the immune tumor microenvironment.