RESUMO
BACKGROUND: Metastatic melanoma requires early detection, being treatment resistant. However, the earliest events of melanoma metastasis, and especially of dermal invasion, remain ill defined. RESULTS AND METHODS: Gene expression profiles of two clonal subpopulations, selected from the same human melanoma cell line, but differing in ability to cross the dermal-epidermal junction in skin reconstructs, were compared by oligonucleotide microarray. Of 26 496 cDNA probes, 461 were differentially expressed (>2-fold; P< 0.001), only 71 genes being upregulated in invasive cells. Among them, TSPAN8, a tetraspanin not yet described in melanoma, was upregulated at mRNA and protein levels in melanoma cells from the invasive clone, as assessed by RT-PCR, flow cytometry and western blot analysis. Interestingly, TSPAN8 was the only tetraspanin in which overexpression correlated with invasive phenotype. Flow cytometry of well-defined melanoma cell lines confirmed that TSPAN8 was exclusively expressed by invasive, but not non-invasive melanoma cells or normal melanocytes. Immunohistochemistry revealed that TSPAN8 was expressed by melanoma cells in primary melanomas and metastases, but not epidermal cells in healthy skin. The functional role of TSPAN8 was demonstrated by silencing endogenous TSPAN8 with siRNA, reducing invasive outgrowth from tumour spheroids within matrigel without affecting cell proliferation or survival. CONCLUSION: TSPAN8 expression may enable melanoma cells to cross the cutaneous basement membrane, leading to dermal invasion and progression to metastasis. TSPAN8 could be a promising target in early detection and treatment of melanoma.
Assuntos
Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica , Melanoma/metabolismo , Melanoma/patologia , Glicoproteínas de Membrana/metabolismo , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Antígenos de Neoplasias/genética , Apoptose , Biomarcadores Tumorais/metabolismo , Western Blotting , Movimento Celular , Proliferação de Células , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Metástase Linfática , Melanócitos/metabolismo , Melanoma/genética , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Cutâneas/genética , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Tetraspaninas , Células Tumorais CultivadasAssuntos
Cromossomos Humanos Par 13 , Conexinas/genética , Displasia Ectodérmica/genética , Mutação de Sentido Incorreto , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Arginina , Mapeamento Cromossômico , Conexina 30 , Feminino , Glicina , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Linhagem , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
This corrects the article DOI: 10.1038/onc.2016.219.
RESUMO
Melanoma is the deadliest form of skin cancer owing to its proclivity to metastasise, and recently developed therapies have not yielded the expected results, because almost all patients relapse. Therefore, understanding the molecular mechanisms that underlie early invasion by melanoma cells is crucial to improving patient survival. We have previously shown that, whereas the Tetraspanin 8 protein (Tspan8) is undetectable in normal skin and benign lesions, its expression arises with the progression of melanoma and is sufficient to increase cell invasiveness. Therefore, to identify Tspan8 transcriptional regulators that could explain the onset of Tspan8 expression, thereby conferring an invasive phenotype, we performed an innovative RNA interference-based screen, which, for the first time, identified several Tspan8 repressors and activators, such as GSK3ß, PTEN, IQGAP1, TPT1 and LCMR1. LCMR1 is a recently identified protein that is overexpressed in numerous carcinomas; its expression and role, however, had not previously been studied in melanoma. The present study identified Tspan8 as the first LCMR1 target that could explain its function in carcinogenesis. LCMR1 modulation was sufficient to positively regulate endogenous Tspan8 expression, with concomitant in vitro phenotypic changes such as loss of melanoma cell-matrix adherence and increase in invasion, and Tspan8 expression promoted tumourigenicity in vivo. Moreover, LCMR1 and Tspan8 overexpression were shown to correlate in melanoma lesions, and both proteins could be downregulated in vitro by vemurafenib. In conclusion, this study highlights the importance of Tspan8 and its regulators in the control of early melanoma invasion and suggests that they may be promising new therapeutic targets downstream of the RAF-MEK-ERK signalling pathway.
Assuntos
Complexo Mediador/genética , Melanoma/patologia , Neoplasias Cutâneas/patologia , Tetraspaninas/genética , Animais , Linhagem Celular Tumoral , Xenoenxertos , Humanos , Masculino , Complexo Mediador/metabolismo , Melanoma/genética , Melanoma/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Interferência de RNA , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Tetraspaninas/metabolismo , Transfecção , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
The X-linked lymphoproliferative syndrome (XLP) is an inherited immuno-deficiency to Epstein-Barr virus infection that has been mapped to chromosome Xq25. Molecular analysis of XLP patients from ten different families identified a small interstitial constitutional deletion in 1 patient (XLP-D). This deletion, initially defined by a single marker, DF83, known to map to interval Xq24-q26.1, is nested within a previously reported and much larger deletion in another XLP patient (XLP-739). A cosmid minilibrary was constructed from a single mega-YAC and used to establish a contig encompassing the whole XLP-D deletion and a portion of the XLP-739 deletion. Based on this contig, the size of the XLP-D deletion can be estimated at 130 kb. The identification of this minimal deletion, within which at least a portion of the XLP gene is likely to reside, should greatly facilitate efforts in isolating the gene.
Assuntos
Transtornos Linfoproliferativos/genética , Mapeamento por Restrição , Cromossomo X , Adolescente , Linhagem Celular , Cromossomos Artificiais de Levedura , Clonagem Molecular , Cosmídeos , Deleção de Genes , Ligação Genética , Humanos , Masculino , SíndromeRESUMO
X-linked lymphoproliferative disease (XLP) is an inherited immunodeficiency characterised by selective susceptibility to Epstein-Barr virus and frequent association with malignant lymphomas chiefly located in the ileocecal region, liver, kidney and CNS. Taking advantage of a large bacterial clone contig, we obtained a genomic sequence of 197620 bp encompassing a deletion (XLP-D) of 116 kb in an XLP family, whose breakpoints were identified. The study of potential exons from this region in 40 unrelated XLP patients did not reveal any mutation. To define the critical region for XLP and investigate the role of the XLP-D deletion, detailed haplotypes in a region of approximately 20 cM were reconstructed in a total of 87 individuals from 7 families with recurrence of XLP. Two recombination events in a North American family and a new microdeletion (XLP-G) in an Italian family indicate that the XLP gene maps in the interval between DXS1001 and DXS8057, approximately 800 kb centromeric to the previously reported familial microdeletion XLP-D.
Assuntos
Ligação Genética , Transtornos Linfoproliferativos/genética , Cromossomo X , Sequência de Bases , Clonagem Molecular , Primers do DNA , Feminino , Deleção de Genes , Haplótipos , Humanos , Masculino , LinhagemRESUMO
The interfollicular epidermis is continuously renewed, thanks to a regulated balance between proliferation and differentiation. The ΔNp63 transcription factor has a key role in the control of this process. It has been shown that ΔNp63 directly regulates Runt-related transcription factor 1 (RUNX1) transcription factor expression in mouse keratinocytes. The present study showed for the first time that RUNX1 is expressed in normal human interfollicular epidermis and that its expression is tightly regulated during the transition from proliferation to differentiation. It demonstrated that ΔNp63 directly binds two different RUNX1 regulatory DNA sequences and modulates RUNX1 expression differentially in proliferative or differentiated human keratinocytes. It also showed that the regulation of RUNX1 expression by ΔNp63 is dependent on p53 and that this coregulation relies on differential binding and activation of RUNX1 regulatory sequences by ΔNp63 and p53. We also found that RUNX1 inhibits keratinocyte proliferation and activates directly the expression of KRT1, a critical actor in early keratinocyte differentiation. Finally, we described that RUNX1 expression, similar to ΔNp63 and p53, was strongly expressed and downregulated in basal cell carcinomas and squamous cell carcinomas respectively. Taken together, these data shed light on the importance of tight control of the functional interplay between ΔNp63 and p53 in regulating RUNX1 transcription factor expression for proper regulation of interfollicular epidermal homeostasis.
Assuntos
Diferenciação Celular , Proliferação de Células , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Queratinócitos/citologia , Proteínas de Membrana/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Células Cultivadas , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Regulação para Baixo , Células Epidérmicas , Epiderme/metabolismo , Humanos , Queratinócitos/metabolismoRESUMO
Ectodermal dysplasias (EDs) constitute a large and complex group of diseases characterized by various defects in hair, nails, teeth and sweat glands. Of the 170 EDs described so far, fewer than 30 have been explained at the molecular level with identification of the causative gene. This review proposes a new classification of EDs based on the function of the protein encoded by the mutated gene. The EDs are reviewed in light of the recent molecular and biochemical findings and an attempt is made to classify ED causative genes into four major functional subgroups: cell-cell communication and signalling; adhesion; transcription regulation; and development.
Assuntos
Displasia Ectodérmica/classificação , Adesão Celular/genética , Comunicação Celular/genética , Displasia Ectodérmica/genética , Doenças do Cabelo/classificação , Doenças do Cabelo/genética , Humanos , Doenças da Unha/classificação , Doenças da Unha/genética , Doenças das Glândulas Sudoríparas/classificação , Doenças das Glândulas Sudoríparas/genética , Anormalidades Dentárias/genética , Transcrição GênicaRESUMO
The HED (hidrotic ectodermal dysplasia) or Clouston syndrome gene (named ED2) has been mapped to the pericentromeric region of chromosome 13 (13q11) to a 2.4-cM interval flanked by markers D13S1828 and D13S1830. We have developed a BAC/PAC-based contig map of this region. This contig, comprising 23 clones and spanning 1.5 Mb, was established by mapping of 27 BAC/PAC end-derived STSs, 11 known polymorphic markers, 2 previously mapped genes, and 14 ESTs. The genomic clone overlaps were confirmed by restriction fragment fingerprint analysis. This contig provides the basis for genomic sequencing and gene identification in the ED2 critical region. Of the 14 ESTs mapped to the contig, 6 show homology to human genes and 8 appear to be novel. Expression patterns of the genes/ESTs were tested by Northern blot and RT-PCR. Full characterization of some of these genes, as well as the novel ESTs, will be useful in assessing their involvement in the HED/Clouston syndrome.
Assuntos
Cromossomos Humanos Par 13/genética , Displasia Ectodérmica/genética , Mapeamento Físico do Cromossomo , Mapeamento de Sequências Contíguas , Etiquetas de Sequências Expressas , Humanos , Repetições de Microssatélites , Sitios de Sequências RotuladasRESUMO
During routine paternity testing a mutation of a paternal allele at the HPRTB locus was observed. The opportunity was taken to analyse this mutation at a molecular level. The repeat sequence is flanked by an imperfect repeat sequence and this region could be involved in the mutation mechanism. For this reason, we also examined the structure of "intermediate" alleles. Sequencing confirmed the insertion of a perfect repeat motif and revealed a deletion of a dinucleotide some 50 nucleotides downstream from the repeat sequence for the intermediate alleles. It is likely that these intermediate alleles are rare biallelic deletion polymorphisms and are probably not involved in the mutation or variation mechanism of this locus.
Assuntos
Alelos , Hipoxantina Fosforribosiltransferase/genética , Mutação , Paternidade , Sequências de Repetição em Tandem/genética , Sequência de Bases , Feminino , Ligação Genética , Humanos , Masculino , Dados de Sequência Molecular , Cromossomo XRESUMO
Hidrotic ectodermal dysplasia (HED) or Clouston syndrome is a rare autosomal dominant disorder characterized by nail dystrophy, alopecia and palmoplantar hyperkeratosis, which maps to chromosome 13q11-q12.1. We confirmed linkage of HED to this region in a large French family. To define the critical region for HED, detailed haplotypes were constructed with new pericentromeric polymorphic markers. A recombination event in the family indicates that the HED locus maps centromeric to D13S1832. Our French family does not share a common haplotype with other pedigrees previously published (particularly French-Canadian), indicating that the mutations in these families are likely to be of different origin.
Assuntos
Cromossomos Humanos Par 13 , Displasia Ectodérmica/genética , Mutação , Feminino , Genótipo , Haplótipos , Humanos , Escore Lod , Masculino , Repetições de Microssatélites , LinhagemRESUMO
We have identified a novel human gene with strong homology to the mouse Pa2g4 cell cycle gene. This novel gene (called PA2G4) belongs to a gene family with members in several chromosome regions: 3q24-q25, 6q22, 9q21, 12q13, 18q12, 20p12 and Xq25. A composite cDNA of 1697 nucleotides was isolated. The sequence of this cDNA predicts a protein of 394 amino acids. The deduced amino acid sequence of this human protein shows very strong homology to the mouse protein p38-2G4. The cDNA analyzed probably corresponds to a functional copy found at 12q13.
Assuntos
Ciclo Celular/genética , Proteínas Nucleares/genética , Animais , Sequência de Bases , Northern Blotting , Bandeamento Cromossômico , Mapeamento Cromossômico , Cromossomos Humanos Par 12 , Cromossomos Humanos Par 20 , Cromossomos Humanos Par 3 , Cromossomos Humanos Par 6 , Cromossomos Humanos Par 9 , Clonagem Molecular , DNA Complementar/isolamento & purificação , Proteínas de Ligação a DNA , Feminino , Humanos , Intestinos/química , Leucócitos/química , Masculino , Camundongos , Dados de Sequência Molecular , Ovário/química , Próstata/química , Pseudogenes , RNA Mensageiro/análise , Proteínas de Ligação a RNA , Homologia de Sequência de Aminoácidos , Baço/química , Testículo/química , Cromossomo XRESUMO
X-linked lymphoproliferative syndrome is an inherited immunodeficiency for which the responsible gene is currently unknown. Several megabase-sized deleted regions mapping to Xq25 have been identified in XLP patients, and more recently a 130-kb deletion has been reported (Lamartine et al., 1996; Lanyi et al., 1996). To establish a physical map of this deleted region and to identify the XLP gene, two cosmid contigs were established (Lamartine et al., 1996). However, the physical map of this region is still uncompleted and controversial and three points remain unsolved: (1) the centromeric-telomeric orientation of the whole region, (2) the relative orientation of the two contigs, and (3) the size of the gap between the two contigs. To provide a definitive answer to these questions, high-resolution mapping by fluorescence in situ hybridization on combed DNA and molecular approaches were combined to establish the physical map of the XLP region over 600 kb. Our results identified a gap of 150 kb between the two contigs, established the relative orientation of one contig to the other, and determine the centromeric-telomeric orientation of the whole region. Our results show that the order of the marker over this region is: cen.1D10T7-DF83-DXS982.tel.