RESUMO
Mutational inactivation of EXT1 or EXT2 is the cause of hereditary multiple osteochondromas. These genes function in heparan sulphate proteoglycan (HSPG) biosynthesis in the Golgi apparatus. Loss of heterozygosity of the EXT1 locus at 8q24 is frequently found in solitary osteochondromas, whereas somatic mutations are rarely found. We investigated the expression of EXT1 and EXT2 (quantitative RT-PCR) and of different HSPGs (immunohistochemistry) in solitary and hereditary osteochondromas and in cases with malignant progression to secondary peripheral chondrosarcoma, in relation to possible mutations and promoter methylation. The mutation status of patients with multiple osteochondromas correlated with decreased EXT1 or EXT2 expression found in their resected tumours. We could not show somatic point mutations or promoter hypermethylation in 17 solitary tumours; however, EXT1 expression was decreased in 15 cases, whereas EXT2 was not. Intracellular accumulation of syndecan-2 and heparan sulphate-bearing isoforms of CD44 (CD44v3) was found in most tumours, which concentrated in the Golgi apparatus as shown by confocal microscopy. This contrasted with the extracellular expression found in normal growth plates. In conclusion, mutational inactivation of either EXT1 or EXT2 leads to loss of mRNA expression of the corresponding gene. We hypothesize that loss of EXT expression disrupts the function of the EXT1/2 complex in HSPG biosynthesis, resulting in the intracellular accumulation of HSPG core proteins that we found in these tumours.
Assuntos
Neoplasias Ósseas/química , Condrossarcoma/química , Proteoglicanas de Heparan Sulfato/análise , N-Acetilglucosaminiltransferases/administração & dosagem , N-Acetilglucosaminiltransferases/análise , Osteocondroma/química , Adolescente , Adulto , Idoso de 80 Anos ou mais , Neoplasias Ósseas/enzimologia , Neoplasias Ósseas/genética , Criança , Pré-Escolar , Condrossarcoma/enzimologia , Condrossarcoma/genética , Exostose Múltipla Hereditária/enzimologia , Exostose Múltipla Hereditária/genética , Exostose Múltipla Hereditária/metabolismo , Feminino , Complexo de Golgi , Humanos , Receptores de Hialuronatos/análise , Imuno-Histoquímica/métodos , Masculino , Metilação , Microscopia Confocal/métodos , Pessoa de Meia-Idade , Mutação/genética , Osteocondroma/enzimologia , Osteocondroma/genética , Reação em Cadeia da Polimerase/métodos , Sindecana-2/análiseRESUMO
Hedgehog (HH) signalling is important for specific developmental processes, and aberrant, increased activity has been described in various tumours. Disturbed HH signalling has also been implicated in the hereditary syndrome, Multiple Osteochondromas. Indian Hedgehog (IHH), together with parathyroid hormone-like hormone (PTHLH), participates in the organization of growth plates in long bones. PTHLH signalling is absent in osteochondromas, benign tumours arising adjacent to the growth plate, but is reactivated when these tumours undergo malignant transformation towards secondary peripheral chondrosarcoma. We describe a gradual decrease in the expression of Patched (PTCH) and glioma-associated oncogene homologue 1 (GLI1) (both transcribed upon IHH activity), and GLI2 with increasing malignancy, suggesting that IHH signalling is inactive and PTHLH signalling is IHH independent in secondary peripheral chondrosarcomas. cDNA expression profiling and immunohistochemical studies suggest that transforming growth factor-beta (TGF-beta)-mediated proliferative signalling is active in high-grade chondrosarcomas since TGF-beta downstream targets were upregulated in these tumours. This is accompanied by downregulation of energy metabolism-related genes and upregulation of the proto-oncogene jun B. Thus, the tight regulation of growth plate organization by IHH signalling is still seen in osteochondroma, but gradually lost during malignant transformation to secondary peripheral chondrosarcoma and subsequent progression. TGF-beta signalling is stimulated during secondary peripheral chondrosarcoma progression and could potentially regulate the retained activity of PTHLH.
Assuntos
Condrossarcoma/patologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Transdução de Sinais/fisiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Distribuição de Qui-Quadrado , Criança , Condrossarcoma/genética , Progressão da Doença , Feminino , Perfilação da Expressão Gênica , Lâmina de Crescimento/metabolismo , Proteínas Hedgehog , Humanos , Imuno-Histoquímica/métodos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Proteínas do Tecido Nervoso/genética , Análise de Sequência com Séries de Oligonucleotídeos , Osteossarcoma/genética , Osteossarcoma/patologia , Inibidor 1 de Ativador de Plasminogênio/genética , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-jun/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad2/genética , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , beta Catenina/genéticaRESUMO
Dysplasia epiphysealis hemimelica (DEH) and metachondromatosis (MC) are considered in the differential diagnosis of solitary and hereditary osteochondromas. Both are rare disorders with DEH demonstrating cartilaginous overgrowth of an epiphysis and MC exhibiting synchronous enchondromas and osteochondromas. Ten cases of DEH and two of MC were compared with osteochondromas at the histological and molecular level. Histologically, clumping of chondrocytes within a fibrillary chondroid matrix is characteristic of DEH, while osteochondromas and MC display the characteristic growth plate architecture. Using cDNA microarray analysis we demonstrate that DEH and MC cluster separately from osteochondromas and growth plates. The EXT genes, involved in the hereditary multiple osteochondromas syndrome, and downregulated in osteochondroma, were normally expressed in DEH and MC as shown by quantitative reverse transcriptase-polymerase chain reaction (qPCR). EXT is involved in heparan sulphate biosynthesis, important for Indian Hedgehog/ParaThyroid Hormone Like Hormone (IHH/PTHLH) growth plate signalling pathways. IHH/PTHLH signalling molecules were expressed in DEH and MC as shown by both qPCR and immunohistochemistry, suggesting that this pathway is active. This is in contrast to osteochondroma, in which PTHLH signalling is downregulated. Thus, lesions of DEH and MC are separate entities from osteochondroma as confirmed by their different cDNA and protein expression profiles. Downstream targets of EXT, which are downregulated in osteochondroma, are expressed in DEH and MC, suggesting that EXT signalling is not disturbed.