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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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BMP4/7-dependent expression of inhibitor of differentiation/DNA binding (Id) proteins 1 and 3 has been implicated in tumor progression and poor prognosis of malignant melanoma patients. Hyaluronic acid (HA), a pericellular matrix component, supports BMP7 signalling in murine chondrocytes through its receptor CD44. However, its role in regulating BMP signalling in melanoma is not clear. In this study we found that depletion of endogenously-produced HA by hyaluronidase treatment or by inhibition of HA synthesis by 4-methylumbelliferone (4-MU) resulted in reduced BMP4/7-dependent Id1/3 protein expression in mouse melanoma B16-F10 and Ret cells. Conversely, exogenous HA treatment increased BMP4/7-dependent Id1/3 protein expression. Knockdown of CD44 reduced BMP4/7-dependent Id1/3 protein expression, and attenuated the ability of exogenous HA to stimulate Id1 and Id3 expression in response to BMP. Co-IP experiments demonstrated that CD44 can physically associate with the BMP type II receptor (BMPR) ACVR2B. Importantly, we found that coordinate expression of Id1 or Id3 with HA synthases HAS2, HAS3, and CD44 is associated with reduced overall survival of cutaneous melanoma patients. Our results suggest that HA-CD44 interactions with BMPR promote BMP4/7-dependent Id1/3 protein expression in melanoma, contributing to reduced survival in melanoma patients.
Assuntos
Proteína Morfogenética Óssea 4/biossíntese , Proteína Morfogenética Óssea 7/biossíntese , Regulação Neoplásica da Expressão Gênica , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Proteína 1 Inibidora de Diferenciação/biossíntese , Proteínas Inibidoras de Diferenciação/biossíntese , Melanoma Experimental/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 7/genética , Humanos , Receptores de Hialuronatos/genética , Ácido Hialurônico/genética , Proteína 1 Inibidora de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/genética , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Proteínas de Neoplasias/genética , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologiaRESUMO
Given its importance in development and homeostasis, bone morphogenetic protein (BMP) signaling is tightly regulated at the extra- and intracellular level. The extracellular matrix (ECM) was initially thought to act as a passive mechanical barrier that sequesters BMPs. However, a new understanding about how the ECM plays an instructive role in regulating BMP signaling is emerging. In this mini-review, we discuss various ways in which the biochemical and physical properties of the ECM regulate BMP signaling.
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Proteínas Morfogenéticas Ósseas/metabolismo , Matriz Extracelular/metabolismo , Espaço Extracelular/metabolismo , Transdução de Sinais , Animais , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Homeostase , Humanos , Modelos BiológicosRESUMO
CRISPR/Cas9 has emerged as a powerful methodology for the targeted editing of genomic DNA sequences. Nevertheless, the intrinsic inefficiency of transfection methods required to use this technique with cultured cells requires the selection and isolation of successfully modified cells, which invariably subjects the cells to stress. Here we report a workflow that allows the isolation of genomically modified cells, even where loss of functional alleles constitutes a selective disadvantage owing to impaired ability to survive stress. Using targeted disruption of the Id1 and Id3 genes in murine B16-F10 and Ret melanoma cell lines as an example, we show that the method allows for the footprintless isolation of CRISPR/Cas9-modified aneuploid cancer cells. We also provide evidence that serial CRISPR/Cas9 modifications can occur, for example when initial homologous recombination events introduce cryptic PAM sequences, and demonstrate that multiple alleles can be successfully targeted in aneuploid cancer cells. By sequencing individual alleles we also found evidence for CRISPR/Cas9-induced transposable element insertion, albeit at a low frequency. This workflow should have broad application in the functional analysis of prosurvival gene function in cultured cells.
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Aneuploidia , Sistemas CRISPR-Cas , Proteína 1 Inibidora de Diferenciação/genética , Proteínas Inibidoras de Diferenciação/genética , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Reação em Cadeia da Polimerase/métodos , Fluxo de TrabalhoRESUMO
Measuring concentrations of the differentiation-promoting hormone retinoic acid (RA) in glioblastoma tissues would help to understand the reason why RA treatment has been inefficient in clinical trials involving brain tumor patients. Here, we apply a recently established extraction and measurement protocol to screen glioblastoma tissues for the levels of the RA precursor retinol and biologically active RA. Combining this approach with mRNA analyses of 26 tumors and 8 normal brains, we identify a multifaceted disturbance of RA synthesis in glioblastoma, involving multiple aldehyde dehydrogenase 1 family and retinol dehydrogenase enzymes. Through database studies and methylation analyses, we narrow down chromosomal deletions and aberrant promoter hypermethylation as potential mechanisms accounting for these alterations. Employing chromatin immunoprecipitation analyses and cell-culture studies, we further show that chromatin at RA target genes is poised to RA substitution, but most glioblastoma cell cultures are completely resistant to RA treatment. This paradoxical RA response is unrelated to alternative RA signaling through the fatty acid-binding protein 5/peroxisome proliferator-activated receptor delta axis. Our data suggest a multifaceted disturbance of RA synthesis in glioblastoma and contribute to reconsider current RA treatment strategies.
