RESUMO
UNLABELLED: An emerging concept in melanoma biology is that of dynamic, adaptive phenotype switching, where cells switch from a highly proliferative, poorly invasive phenotype to a highly invasive, less proliferative one. This switch may hold significant implications not just for metastasis, but also for therapy resistance. We demonstrate that phenotype switching and subsequent resistance can be guided by changes in expression of receptors involved in the noncanonical Wnt5A signaling pathway, ROR1 and ROR2. ROR1 and ROR2 are inversely expressed in melanomas and negatively regulate each other. Furthermore, hypoxia initiates a shift of ROR1-positive melanomas to a more invasive, ROR2-positive phenotype. Notably, this receptor switch induces a 10-fold decrease in sensitivity to BRAF inhibitors. In patients with melanoma treated with the BRAF inhibitor vemurafenib, Wnt5A expression correlates with clinical response and therapy resistance. These data highlight the fact that mechanisms that guide metastatic progression may be linked to those that mediate therapy resistance. SIGNIFICANCE: These data show for the fi rst time that a single signaling pathway, the Wnt signaling pathway, can effectively guide the phenotypic plasticity of tumor cells, when primed to do so by a hypoxic microenvironment. Importantly, this increased Wnt5A signaling can give rise to a subpopulation of highly invasive cells that are intrinsically less sensitive to novel therapies for melanoma, and targeting the Wnt5A/ROR2 axis could improve the efficacy and duration of response for patients with melanoma on vemurafenib.
Assuntos
Antineoplásicos/uso terapêutico , Hipóxia Celular , Resistencia a Medicamentos Antineoplásicos , Indóis/uso terapêutico , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Sulfonamidas/uso terapêutico , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Melanoma/genética , Melanoma/secundário , Melanoma Experimental , Camundongos , Camundongos Nus , Metástase Neoplásica , Fenótipo , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Vemurafenib , Via de Sinalização WntRESUMO
We have previously shown that Wnt5A-mediated signaling can promote melanoma metastasis. It has been shown that Wnt signaling is antagonized by the protein Klotho, which has been implicated in aging. We show here that in melanoma cells, expressions of Wnt5A and Klotho are inversely correlated. In the presence of recombinant Klotho (rKlotho), we show that Wnt5A internalization and signaling is decreased in high Wnt5A-expressing cells. Moreover, in the presence of rKlotho, we observe an increase in Wnt5A remaining in the medium, coincident with an increase in sialidase activity, and decrease in syndecan expression. These effects can be inhibited using a sialidase inhibitor. In addition to its effects on Wnt5A internalization, we also demonstrate that Klotho decreases melanoma cell invasive potential by a second mechanism that involves the inhibition of calpain and a resultant decrease in filamin cleavage, which we demonstrate is critical for melanoma cell motility.
Assuntos
Movimento Celular , Proteínas Contráteis/metabolismo , Regulação Neoplásica da Expressão Gênica , Glucuronidase/deficiência , Melanoma/patologia , Proteínas dos Microfilamentos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Wnt/biossíntese , Proteínas Wnt/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Progressão da Doença , Filaminas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glucuronidase/genética , Glucuronidase/metabolismo , Proteoglicanas de Heparan Sulfato/metabolismo , Humanos , Proteínas Klotho , Melanoma/genética , Camundongos , Metástase Neoplásica , Transdução de Sinais/efeitos dos fármacos , Proteínas Wnt/genética , Proteína Wnt-5aRESUMO
Wnt signaling can be divided into three pathways, namely the canonical Wnt/beta-catenin pathway, and the non-canonical (or heretical) Wnt/Ca(2+) and planar cell polarity (PCP) pathways. Although the canonical Wnt/beta-catenin pathway is the best described in cancer, increasing data points to the importance of the heretical Wnt pathways in several aspects of tumor progression. The recent advances in understanding the players and mechanisms by which these Wnt pathways contribute to cancer progression have led to the identification of numerous molecules that are already, or could be considered, targets for cancer therapy.
Assuntos
Neoplasias/metabolismo , Proteínas Wnt/antagonistas & inibidores , Antineoplásicos/farmacologia , Progressão da Doença , Humanos , Neoplasias/patologia , Proteínas Wnt/metabolismoRESUMO
Certain forms of hexavalent chromium [Cr(VI)] are human carcinogens. Our recent work has shown that a broad range protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SOV), abrogated both Cr(VI)-induced growth arrest and clonogenic lethality. Notably, SOV enhanced Cr(VI) mutation frequency, ostensibly through forced survival of genetically damaged cells. In the present study, co-treatment with this PTP inhibitor bypassed the Cr(VI)-induced G(1)/S checkpoint arrest in diploid human lung fibroblasts (HLF). Moreover, the PTP inhibitor abrogated the Cr(VI)-induced decrease in the expression of key effectors of the G(1)/S checkpoint [Cyclin D1, phospho Ser 807/811 Rb (pRB), p27]. Cr(VI)-induced G(1) arrest was associated with the cytoplasmic appearance of pRb and the nuclear localization of p27, both of which were reversed by the PTP inhibitor. The PTP inhibitor's reversal of G(1)/S checkpoint effector localization after Cr exposure was found to be Akt1-dependent, as this was abrogated by transfection with either akt1 siRNA or an Akt1-kinase dead plasmid. Furthermore, Akt1 activation alone was sufficient to induce G(1)/S checkpoint bypass and to prevent Cr(VI)-induced changes in pRb and p27 localization. In conclusion, this work establishes Akt1 activation to be both sufficient to bypass the Cr(VI)-induced G(1)/S checkpoint, as well as necessary for the observed PTP inhibitor effects on key mediators of the G(1)/S transition. The potential for Akt to bypass G(1)/S checkpoint arrest in the face of genotoxic damage could increase genomic instability, which is a hallmark of neoplastic progression.
Assuntos
Dano ao DNA , Fase G1 , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fase S , Linhagem Celular , Cromo/farmacologia , Ciclina D1/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Fase G1/efeitos dos fármacos , Humanos , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/metabolismo , Proteína do Retinoblastoma/metabolismo , Fase S/efeitos dos fármacos , Vanadatos/farmacologiaRESUMO
Cytoplasmic expression of claudin-1 in metastatic melanoma cells correlates to increased migration, and increased secretion of MMP-2 in a PKC dependent manner, whereas claudin-1 nuclear expression is found in benign nevi. Melanoma cells were transfected with a vector expressing CLDN-1 fused to a nuclear localization signal (NLS). Despite significant nuclear localization of claudin-1, there was still transport of claudin-1 to the cytoplasm. Phorbol ester treatment of cells transfected with NLS-claudin-1 resulted in an exclusion of claudin-1 from the nucleus, despite the NLS. To ascertain whether PKC or PKA were involved in this translocation, we mutated the putative phosphorylation sites within the protein. We found that mutating the PKC phosphorylation sites to mimic a non-phosphorylated state did not cause a shift of claudin-1 to the nucleus of the cells, but mutating the PKA sites did. Mutations of either site to mimic constitutive phosphorylation resulted in cytoplasmic claudin-1 expression. Stable claudin-1 transfectants containing non-phosphorylatable PKA sites exhibited decreased motility. These data imply that subcellular localization of claudin-1 can be controlled by phosphorylation, dicating effects on metastatic capacity.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Melanoma/patologia , Proteínas de Membrana/metabolismo , Proteína Quinase C/metabolismo , Transporte Biológico/genética , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Claudina-1 , Simulação por Computador , Citoplasma/genética , Citoplasma/metabolismo , Ativação Enzimática , Vetores Genéticos , Humanos , Imuno-Histoquímica , Metaloproteinase 2 da Matriz/metabolismo , Melanoma/metabolismo , Mutagênese Sítio-Dirigida , Invasividade Neoplásica , Metástase Neoplásica , Sinais de Localização Nuclear/genética , Sinais de Localização Nuclear/metabolismo , Fosforilação , Frações Subcelulares/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , TransfecçãoRESUMO
Our recent studies showed that maintenance of protein tyrosine phosphorylation by PTP inhibition enhanced cell growth, clonogenic survival, and mutagenesis after a single low-level Cr(VI) exposure, thereby suggesting that tyrosine phosphorylation-dependent signaling may govern inappropriate survival in human lung fibroblasts (HLFs). Our goal is to identify specific phospho-tyrosine regulator(s)/ downstream effectors involved in enhanced survival after Cr(VI) exposure and PTP inhibition. Phosphotyrosine profiling array showed that PTP inhibition following Cr(VI) exposure increased tyrosine phosphorylation of specific proteins, such as FGR and ABL, which are upstream regulators of both Erk and Akt pathways. To explore the roles of these pathways in the PTP-induced increase in clonogenic survival after Cr(VI) exposure, we examined the effect of combined Akt1 and Erk1/2 knockdown via siRNA technology. Akt1 and/or Erk1/2 silencing had no effect on the PTP inhibitor-induced increase in survival following Cr(VI) exposure, suggesting the presence of non-Akt/non-Erk-mediated survival signaling. Interestingly, geldanamycin, an HSP90 inhibitor and non-specific Raf inhibitor, abrogated the PTP inhibitor-mediated increase in survival following Cr(VI) exposure and abolished the expression/activity of c-Raf and activity of Mek. These findings prompted us to explore upstream regulators of Erk, i.e., Ras, c-Raf and Mek for their potential roles in clonogenic survival. GW5074, a specific c-Raf kinase inhibitor did not alter the effect of the PTP inhibitor but decreased Cr(VI)-mediated clonogenic lethality, potentially though Mek hyperactivation. A genetic approach with a c/a Mek1 mutant also showed that Mek activity was not directly associated with the PTP inhibitor effect. Finally, a genetic approach with d/n or c/a Ras and c-Raf mutants, showed that Ras and c-Raf activities play a substantive role in enhancing clonogenic survival by PTP inhibition following Cr(VI) insult. In conclusion, these studies highlight a novel pro-survival mechanism for clonogenic survival in the face of genotoxic stress in the presence of PTP inhibition via an Erk/Mek-independent and Ras/c-Raf-dependent regulation in normal human lung fibroblasts.
Assuntos
Cromo/toxicidade , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas ras/metabolismo , Benzoquinonas/farmacologia , Linhagem Celular , Sobrevivência Celular , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Indóis/farmacologia , Lactamas Macrocíclicas/farmacologia , MAP Quinase Quinase Quinases/metabolismo , Fenóis/farmacologia , Proteínas Tirosina Fosfatases/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
We have previously shown that Wnt5A and ROR2, an orphan tyrosine kinase receptor, interact to mediate melanoma cell motility. In other cell types, this can occur through the interaction of ROR2 with the cytoskeletal protein filamin A. Here, we found that filamin A protein levels correlated with Wnt5A levels in melanoma cells. Small interfering RNA (siRNA) knockdown of WNT5A decreased filamin A expression. Knockdown of filamin A also corresponded to a decrease in melanoma cell motility. In metastatic cells, filamin A expression was predominant in the cytoplasm, which western analysis indicated was due to the cleavage of filamin A in these cells. Treatment of nonmetastatic melanoma cells with recombinant Wnt5A increased filamin A cleavage, and this could be prevented by the knockdown of ROR2 expression. Further, BAPTA-AM chelation of intracellular calcium also inhibited filamin A cleavage, leading to the hypothesis that Wnt5A/ROR2 signaling could cleave filamin A through activation of calcium-activated proteases, such as calpains. Indeed, WNT5A knockdown decreased calpain 1 expression, and by inhibiting calpain 1 either pharmacologically or using siRNA, it decreased cell motility. Our results indicate that Wnt5A activates calpain-1, leading to the cleavage of filamin A, which results in a remodeling of the cytoskeleton and an increase in melanoma cell motility.
Assuntos
Calpaína/metabolismo , Proteínas Contráteis/metabolismo , Melanoma/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Neoplasias Cutâneas/metabolismo , Proteínas Wnt/metabolismo , Calpaína/genética , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Quelantes/farmacologia , Citoesqueleto/fisiologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Filaminas , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/patologia , Melanoma/fisiopatologia , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/fisiopatologia , Proteínas Wnt/genética , Proteína Wnt-5aRESUMO
Although the consequences of genotoxic injury include cell cycle arrest and apoptosis, cell survival responses after genotoxic injury can produce intrinsic death-resistance and contribute to the development of a transformed phenotype. Protein tyrosine phosphatases (PTPs) are integral components of key survival pathways, and are responsible for their inactivation, while PTP inhibition is often associated with enhanced cell proliferation. Our aim was to elucidate signaling events that modulate cell survival after genotoxin exposure. Diploid human lung fibroblasts (HLF) were treated with Cr(VI) (as Na(2)CrO(4)), the soluble oxyanionic dissolution product of certain particulate chromates, which are well-documented human respiratory carcinogens. In vitro soluble Cr(VI) induces a wide spectrum of DNA damage, in both the presence and absence of a broad-range PTP inhibitor, sodium orthovanadate (SOV). Notably, SOV abrogated Cr(VI)-induced clonogenic lethality. The enhanced survival of Cr(VI)-exposed cells after SOV treatment was predominantly due to a bypass of cell cycle arrest, as there was no effect of the PTP inhibitor on Cr-induced apoptosis. Moreover, the SOV effect was not due to decreased Cr uptake as evidenced by unchanged Cr-DNA adduct burden. Additionally, the bypass of Cr-induced growth arrest by SOV was accompanied by a decrease in Cr(VI)-induced expression of cell cycle inhibiting genes, and an increase in Cr(VI)-induced expression of cell cycle promoting genes. Importantly, SOV resulted in an increase in forward mutations at the HPRT locus, supporting the hypothesis that PTP inhibition in the presence of certain types of DNA damage may lead to increased genomic instability, via bypass of cell cycle checkpoints.
Assuntos
Sobrevivência Celular/efeitos dos fármacos , Cromo/farmacologia , Inibidores Enzimáticos/farmacologia , Mutagênese/efeitos dos fármacos , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Linhagem Celular , Humanos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
There are currently no effective therapies for metastatic melanoma and targeted immunotherapy results in the remission of only a very small percentage of tumors. In this study, we show that the noncanonical Wnt ligand, Wnt5A, can increase melanoma metastasis in vivo while down-regulating the expression of tumor-associated antigens important in eliciting CTL responses (e.g., MART-1, GP100, and tyrosinase). Melanosomal antigen expression is governed by MITF, PAX3, and SOX10 and is inhibited upon signal transducers and activators of transcription 3 (STAT3) activation, via decreases in PAX3 and subsequently MITF expression. Increasing Wnt5A in Wnt5A-low cells activated STAT3, and STAT3 was decreased upon Wnt5A knockdown. Downstream targets such as PAX3, MITF, and MART-1 were also affected by Wnt5A treatment or knockdown. Staining of a melanoma tissue array also highlighted the inverse relationship between MART-1 and Wnt5A expression. PKC activation by phorbol ester mimicked Wnt5A effects, and Wnt5A treatment in the presence of STAT3 or PKC inhibitors did not lower MART-1 levels. CTL activation studies showed that increases in Wnt5A correspond to decreased CTL activation and vice versa, suggesting that targeting Wnt5A before immunotherapy may lead to the enhancement of current targeted immunotherapy for patients with metastatic melanoma.