RESUMO
Wnt/ß-catenin signaling plays an important role in melanocyte biology, especially in the early stages of melanocyte transformation and melanomagenesis. ß-catenin, encoded by the gene CTNNB1, is an intracellular signal transducer of Wnt signaling and activates transcription of genes important for cell proliferation and survival. Wnt/ß-catenin signaling is frequently activated in melanoma through oncogenic mutations of ß-catenin and elevated ß-catenin levels are positively correlated with melanoma aggressiveness. Molecular mechanisms that regulate ß-catenin expression in melanoma are not fully understood. MicroRNA-214 is known to function as a tumor suppressor by targeting ß-catenin in several types of cancer cells. Here, we investigated the regulation of ß-catenin by miR-214 and its role in melanoma. We show that ß-catenin mRNA levels are negatively correlated with miR-214 in melanoma. However, overexpression of miR-214 paradoxically increased ß-catenin protein levels and promoted malignant properties of melanoma cells including resistance to mitogen-activated protein kinase inhibitors (MAPKi). RNA-seq analysis revealed that melanoma cells predominantly express a ß-catenin mRNA isoform lacking miR-214 target site. Using matched miRNA and mRNA-seq and bioinformatics analysis, we identified novel miR-214 targets, ankyrin repeat domain 6 (ANKRD6) and C-terminal binding protein 1 (CTBP1), that are involved in negative regulation of Wnt signaling. Overexpression of miR-214 or knockdown of the novel miR-214 targets, ANKRD6 or CTBP1, increased melanoma cell proliferation, migration, and decreased sensitivity to MAPKi. Our data suggest that in melanoma cells ß-catenin is not regulated by miR-214 and the functions of miR-214 in melanoma are mediated partly by regulating proteins involved in attenuation of Wnt/ß-catenin signaling.
Assuntos
Melanoma/genética , MicroRNAs/genética , beta Catenina/genética , Oxirredutases do Álcool/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Proteínas do Citoesqueleto/genética , Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Melanoma/patologia , RNA Mensageiro/genética , Via de Sinalização Wnt/genéticaRESUMO
Both oncogenic and tumor suppressor roles have been assigned to Notch signaling in melanoma. In clinical trials, Notch inhibitors proved to be ineffective for melanoma treatment. Notch signaling has also been implicated in melanoma transdifferentiation, a prognostic feature in primary melanoma. In this study, we investigated the role of Notch signaling in melanoma tumor development and growth using the genetic model of mouse melanoma by crossing BRAFCA/+/Pten+/+/Tyr-CreER+ (B) and BRAFCA/+/Pten-/-/Tyr-CreER + (BP) mice with Notch1 or Notch2 floxed allele mice. The topical application of tamoxifen induced tumors in BP mice but not in B mice with or without the deletion of either Notch1 or Notch2. These data show that the loss of either Notch1 nor Notch2 can substitute the tumor suppressor function of Pten in BRAFV600E-induced melanomagenesis. However, in Pten-null background, the loss of either Notch1 or Notch2 appeared to accelerate BRAFV600E-induced tumor development, suggesting a tumor suppressor role for Notch1 and Notch2 in BRAFV600E/Pten-null driven melanomagenesis. Quantitative immunochemical analysis of a human cutaneous melanoma tissue microarray that consists of >100 primary tumors with complete clinical history showed a weak to moderate correlation between NOTCH protein levels and clinical and pathological parameters. Our data show that Notch signaling is involved during melanomagenesis and suggest that the identification of genes and signaling pathways downstream of Notch could help devise strategies for melanoma prevention.
RESUMO
The use of T-cell engagers (TCEs) to treat solid tumors is challenging, and several have been limited by narrow therapeutic windows due to substantial on-target, off-tumor toxicities due to the expression of low levels of target antigens on healthy tissues. Here, we describe TNB-928B, a fully human TCE that has a bivalent binding arm for folate receptor alpha (FRα) to selectively target FRα overexpressing tumor cells while avoiding the lysis of cells with low levels of FRα expression. The bivalent design of the FRα binding arm confers tumor selectivity due to low-affinity but high-avidity binding to high FRα antigen density cells. TNB-928B induces preferential effector T-cell activation, proliferation, and selective cytotoxic activity on high FRα expressing cells while sparing low FRα expressing cells. In addition, TNB-928B induces minimal cytokine release compared to a positive control TCE containing OKT3. Moreover, TNB-928B exhibits substantial ex vivo tumor cell lysis using endogenous T-cells and robust tumor clearance in vivo, promoting T-cell infiltration and antitumor activity in mouse models of ovarian cancer. TNB-928B exhibits pharmacokinetics similar to conventional antibodies, which are projected to enable favorable administration in humans. TNB-928B is a novel TCE with enhanced safety and specificity for the treatment of ovarian cancer.
Assuntos
Anticorpos Biespecíficos , Neoplasias Ovarianas , Animais , Anticorpos Biespecíficos/uso terapêutico , Carcinoma Epitelial do Ovário , Feminino , Receptor 1 de Folato/metabolismo , Receptor 1 de Folato/uso terapêutico , Humanos , Camundongos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Linfócitos TRESUMO
The therapeutic potential of targeting CD19 in B cell malignancies has garnered attention in the past decade, resulting in the introduction of novel immunotherapy agents. Encouraging clinical data have been reported for T cell-based targeting agents, such as anti-CD19/CD3 bispecific T-cell engager blinatumomab and chimeric antigen receptor (CAR)-T therapies, for acute lymphoblastic leukemia and B cell non-Hodgkin lymphoma (B-NHL). However, clinical use of both blinatumomab and CAR-T therapies has been limited due to unfavorable pharmacokinetics (PK), significant toxicity associated with cytokine release syndrome and neurotoxicity, and manufacturing challenges. We present here a fully human CD19xCD3 bispecific antibody (TNB-486) for the treatment of B-NHL that could address the limitations of the current approved treatments. In the presence of CD19+ target cells and T cells, TNB-486 induces tumor cell lysis with minimal cytokine release, when compared to a positive control. In vivo, TNB-486 clears CD19+ tumor cells in immunocompromised mice in the presence of human peripheral blood mononuclear cells in multiple models. Additionally, the PK of TNB-486 in mice or cynomolgus monkeys is similar to conventional antibodies. This new T cell engaging bispecific antibody targeting CD19 represents a novel therapeutic that induces potent T cell-mediated tumor-cell cytotoxicity uncoupled from high levels of cytokine release, making it an attractive candidate for B-NHL therapy.
Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Antineoplásicos Imunológicos/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Citocinas/metabolismo , Citotoxicidade Imunológica/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Linfoma não Hodgkin/tratamento farmacológico , Animais , Anticorpos Biespecíficos/farmacocinética , Anticorpos Monoclonais Humanizados/farmacocinética , Antígenos CD19/imunologia , Antineoplásicos Imunológicos/farmacocinética , Complexo CD3/antagonistas & inibidores , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Técnicas de Cocultura , Humanos , Células K562 , Linfoma não Hodgkin/imunologia , Linfoma não Hodgkin/metabolismo , Macaca fascicularis , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos NOD , Camundongos SCID , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The role of Notch signaling in melanoma drug resistance is not well understood. In this study, we show that although NOTCH proteins are upregulated in metastatic melanoma cell lines, Notch signaling inhibition had no effect on cell survival, growth, migration or the sensitivity of BRAFV600E-melanoma cells to MAPK inhibition (MAPKi). We found that NOTCH1 is downregulated in melanoma cell lines with intrinsic and acquired resistance to MAPKi. Forced expression of NICD1, the active form of Notch1, caused apoptosis of the NOTCHlo , MAPKi-resistant cells, but not the NOTCHhi , MAPKi-sensitive melanoma cell lines. Whole transcriptome-sequencing analyses of NICD1-transduced MAPKi-sensitive and MAPKi-resistant cells revealed differential regulation of endothelin 1 (EDN1) by NICD1, that is, downregulation in MAPKi-resistant cells and upregulation in MAPKi-sensitive cells. Knockdown of EDN1 partially mimicked the effect of NICD1 on the survival of MAPKi-resistant cells. We show that the opposite regulation of EDN1 by Notch signaling is mediated by the differential regulation of c-JUN by NICD1. Our data show that MAPKi-resistant melanoma cells acquire vulnerability to Notch signaling activation and suggest that Notch-c-JUN-EDN1 axis is a potential therapeutic target in MAPKi-resistant melanoma.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Melanoma/enzimologia , Melanoma/patologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Receptores Notch/metabolismo , Transdução de Sinais , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Endotelina-1/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Ligantes , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição HES-1/metabolismo , Transcriptoma/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Cyclic AMP (cAMP) is an important second messenger that regulates a wide range of physiologic processes. In mammalian cutaneous melanocytes, cAMP-mediated signaling pathways activated by G-protein-coupled receptors (GPCR), like melanocortin 1 receptor (MC1R), play critical roles in melanocyte homeostasis including cell survival, proliferation, and pigment synthesis. Impaired cAMP signaling is associated with increased risk of cutaneous melanoma. Although mutations in MAPK pathway components are the most frequent oncogenic drivers of melanoma, the role of cAMP in melanoma is not well understood. Here, using the Braf(V600E)/Pten-null mouse model of melanoma, topical application of an adenylate cyclase agonist, forskolin (a cAMP inducer), accelerated melanoma tumor development in vivo and stimulated the proliferation of mouse and human primary melanoma cells, but not human metastatic melanoma cells in vitro The differential response of primary and metastatic melanoma cells was also evident upon pharmacologic inhibition of the cAMP effector protein kinase A. Pharmacologic inhibition and siRNA-mediated knockdown of other cAMP signaling pathway components showed that EPAC-RAP1 axis, an alternative cAMP signaling pathway, mediates the switch in response of primary and metastatic melanoma cells to cAMP. Evaluation of pERK levels revealed that this phenotypic switch was not correlated with changes in MAPK pathway activity. Although cAMP elevation did not alter the sensitivity of metastatic melanoma cells to BRAF(V600E) and MEK inhibitors, the EPAC-RAP1 axis appears to contribute to resistance to MAPK pathway inhibition. These data reveal a MAPK pathway-independent switch in response to cAMP signaling during melanoma progression.Implications: The prosurvival mechanism involving the cAMP-EPAC-RAP1 signaling pathway suggest the potential for new targeted therapies in melanoma. Mol Cancer Res; 15(12); 1792-802. ©2017 AACR.