RESUMO
Mutational activation of BRAF is the earliest and most common genetic alteration in human melanoma. To build a model of human melanoma, we generated mice with conditional melanocyte-specific expression of BRaf(V600E). Upon induction of BRaf(V600E) expression, mice developed benign melanocytic hyperplasias that failed to progress to melanoma over 15-20 months. By contrast, expression of BRaf(V600E) combined with Pten tumor suppressor gene silencing elicited development of melanoma with 100% penetrance, short latency and with metastases observed in lymph nodes and lungs. Melanoma was prevented by inhibitors of mTorc1 (rapamycin) or MEK1/2 (PD325901) but, upon cessation of drug administration, mice developed melanoma, indicating the presence of long-lived melanoma-initiating cells in this system. Notably, combined treatment with rapamycin and PD325901 led to shrinkage of established melanomas. These mice, engineered with a common genetic profile to human melanoma, provide a system to study melanoma's cardinal feature of metastasis and for preclinical evaluation of agents designed to prevent or treat metastatic disease.
Assuntos
Melanoma/genética , Melanoma/patologia , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas B-raf/genética , Alelos , Animais , Linhagem Celular Tumoral , Proliferação de Células , Alvo Mecanístico do Complexo 1 de Rapamicina , Melanoma/metabolismo , Camundongos , Camundongos Transgênicos , Complexos Multiproteicos , Metástase Neoplásica , PTEN Fosfo-Hidrolase/metabolismo , Proteínas , Proteínas Proto-Oncogênicas B-raf/metabolismo , Serina-Treonina Quinases TOR , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Malignant melanoma is a common and frequently lethal disease. Current therapeutic interventions have little effect on survival, emphasizing the need for a better understanding of the genetic, epigenetic, and phenotypic changes in melanoma formation and progression. We identified 17 genes that were not previously known to be silenced by methylation in melanoma using a microarray-based screen following treatment of melanoma cell lines with the DNA methylation inhibitor 5-Aza-2'-deoxycytidine. Eight of these genes have not been previously shown to undergo DNA methylation in any form of cancer. Three of the genes, QPCT, CYP1B1, and LXN, are densely methylated in >95% of uncultured melanoma tumor samples. Reexpression of either of two of the silenced genes, HOXB13 and SYK, resulted in reduced colony formation in vitro and diminished tumor formation in vivo, indicating that these genes function as tumor suppressors in melanoma.
Assuntos
Epigênese Genética/genética , Inativação Gênica , Melanoma/patologia , Proteínas Supressoras de Tumor/genética , Animais , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Células Cultivadas , Ilhas de CpG/genética , Metilação de DNA , Decitabina , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Predisposição Genética para Doença/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Melanoma/genética , Melanoma/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Nus , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Quinase Syk , Transfecção , Transplante Heterólogo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Conditional Cre-mediated recombination has emerged as a robust method of introducing somatic genetic alterations in an organ-specific manner in the mouse. Here, we generated and characterized mice harboring a 4-hydroxytamoxifen (OHT)-inducible Cre recombinase-estrogen receptor fusion transgene under the control of the melanocyte-specific tyrosinase promoter, designated Tyr::CreER(T2). Cre-mediated recombination was induced in melanocytes in a spatially and temporally controlled manner upon administration of OHT and was documented in embryonic melanoblasts, follicular bulb melanocytes, dermal dendritic melanocytes, epidermal melanocytes of tail skin, and in putative melanocyte stem cells located within the follicular bulge. Functional evidence suggestive of recombination in follicular melanocyte stem cells included the presence of Cre-mediated recombination in follicular bulb melanocytes 1 year after topical OHT administration, by which time several hair cycles have elapsed and the melanocytes residing in this location have undergone multiple rounds of apoptosis and replenishment. These Tyr:: CreER(T2) transgenic mice represent a useful resource for the evaluation of melanocyte developmental genetics, the characterization of melanocyte stem cell function and dynamics, and the construction of refined mouse models of malignant melanoma.