Campagne Cécile, Reyes-Gomez Edouard, Loiodice Sophia, Gadin Stéphanie, Ezagal Jacky, Bernex Florence, Abitbol Marie, Louise Anne, Beermann Friedrich, Panthier Jean-Jacques, Aubin-Houzelstein Geneviève, Egidy Giorgia
aINRA, UMR955 bUniversité Paris Est, Functional and Medical Genetics Unit, Alfort School of Veterinary Medicine (EnvA) cUniversité Paris-Est, Embryology, Histology and Pathological Anatomy Unit, EnvA, Maisons-Alfort dCytometry platform, Immunology Department, Institut Pasteur eInstitut Pasteur, Unit of Mouse Functional Genetics, CNRS URA 2578, Department of Developmental Biology, USC INRA, Paris, France fISREC, Swiss Federal Institute of Technology in Lausanne, Lausanne, Switzerland.
Melanoma Res. 2016 Feb;26(1):12-20. doi: 10.1097/CMR.0000000000000212.
The role of the Pax3 gene in embryonic development of pigment cells is well characterized. By contrast, the function of Pax3 in melanoma development is controversial. Indeed, data obtained from cultured cells suggest that PAX3 may contribute to melanomagenesis. PAX3 is found to be overexpressed in melanomas and also in nevi compared with normal skin samples. Pax3 homozygous loss of function is embryonic lethal. To assess the role of Pax3 in melanoma development in vivo, we analyzed Pax3 haploinsufficiency in a mouse model of melanoma predisposition. The Pax3(GFP/+) knock-in reporter system was combined with the Tyr::NRAS(Q61K); Cdkn2a(-/-) mouse melanoma model. Melanoma development was followed over 18 months. Histopathological, immunohistochemical, and molecular analyses of lesions at different stages of melanoma progression were carried out. Fluorescence-activated cell sorting on GFP of cells from primary or metastatic melanoma was followed by ex-vivo transformation tests and in-vivo passaging. We report here that Tyr::NRAS(Q61K); Cdkn2a(-/-); Pax3(GFP/+) mice developed metastasizing melanoma as their Tyr::NRAS(Q61K); Cdkn2a(-/-); littermates. Histopathology showed no differences between the two genotypes, although Pax3 mRNA and PAX3 protein levels in Pax3(GFP/+) lesions were reduced by half. The Pax3(GFP) allele proved to be a convenient marker to identify and directly sort heterogeneous populations of melanoma cells within the tumor bulk at each stage of melanoma progression. This new mouse model represents an accurate and reproducible means for identifying melanoma cells in vivo to study the mechanisms of melanoma development.
Pax3基因在色素细胞胚胎发育中的作用已得到充分表征。相比之下，Pax3在黑色素瘤发生发展中的功能存在争议。的确，从培养细胞获得的数据表明PAX3可能有助于黑色素瘤的发生。与正常皮肤样本相比，PAX3在黑色素瘤以及痣中均被发现过表达。Pax3纯合功能缺失在胚胎期是致死的。为了评估Pax3在体内黑色素瘤发生发展中的作用，我们在黑色素瘤易感性小鼠模型中分析了Pax3单倍剂量不足的情况。将Pax3(GFP/+)基因敲入报告系统与Tyr::NRAS(Q61K); Cdkn2a(-/-)小鼠黑色素瘤模型相结合。对黑色素瘤发展情况进行了18个月的跟踪观察。对黑色素瘤进展不同阶段的病变进行了组织病理学、免疫组织化学和分子分析。对来自原发性或转移性黑色素瘤细胞的GFP进行荧光激活细胞分选，随后进行体外转化试验和体内传代。我们在此报告，Tyr::NRAS(Q61K); Cdkn2a(-/-); Pax3(GFP/+)小鼠与它们的Tyr::NRAS(Q61K); Cdkn2a(-/-)同窝小鼠一样发生了转移性黑色素瘤。组织病理学显示两种基因型之间没有差异，尽管Pax3(GFP/+)病变中的Pax3 mRNA和PAX3蛋白水平降低了一半。Pax3(GFP)等位基因被证明是一种方便的标记，可用于在黑色素瘤进展的每个阶段识别并直接分选肿瘤块内异质性的黑色素瘤细胞群体。这个新的小鼠模型是在体内识别黑色素瘤细胞以研究黑色素瘤发生发展机制的一种准确且可重复的手段。
