Melanocyte Transformation

Models of melanoma have been difficult to develop due to the differences in anatomy and function of human versus mouse skin. Melanocytes in human skin are aligned on the basement membrane and are dispersed among the epidermal keratinocytes, whereas in mouse skin, melanocytes are absent from the epidermis and situated deep in hair follicles and dermis. When newborn mice are exposed to UVB, they can transform more readily because the melanocytes have not yet descended from the epidermis into the hair follicles.

Because of the major architectural and functional differences between mouse and human skin, our laboratory had started ~15 years ago to graft human skin to immunodeficient mice to better mimic the micro-environmental conditions in human skin. In subsequent studies, we exposed the human skin in a two-step carcinogenesis model to an initiator (DMBA) followed by an initiator/promoter (UVB) according to established protocols in mouse skin carcinogenesis. We could readily develop in the human skin squamous but only rarely melanocytic lesions and then only after 1 year of UVB exposure. When we switched to a growth factor as 'initiator', bFGF, we saw more frequent and more profound lentiginous lesions, i.e., the atypical melanocytes were lined along the basement membrane zone and rarely invaded the dermis.

The encouraging results prompted us to use three growth factors for over-expression in the skin at the same time, bFGF, SCF and ET-3. All are known mitogens for melanocytes. This combination led to a major breakthrough in melanoma model development because we now induced melanocytic lesions in over 90% of the treated skins within 3 weeks that were histologically diagnosable as melanomas after 4 weeks in 17 of the 50 specimens (34%) examined.

A limitation of the model is that once the growth factors were omitted, the lesions regressed suggesting that additional events are necessary for malignant transformation. This now seek to extend this model in order to induce malignant transformation so that melanocytes fulfill all six 'hallmarks of cancer'. This is possible because an additional technical advance has been made enabling us to graft artificial skin created in vitro (known as skin reconstructs, organotypic skin cultures, or skin equivalents) to mice.

In this model, the melanocytes align along the basement membrane as in natural human skin (this grafting technique is well known among wound healing experts. The organotypic cultures allow us to manipulate the expression/activity of specific genes prior to generation of the skin reconstructs. Specifically, we can increase expression of genes, down-regulate their expression, or introduce genes with specific mutations. Thus, we can theoretically 'rebuild' the epigenetic and genetic events leading to melanoma development and progression and thereby begin dissect and understanding the mechanisms of the disease process. The striking similarities between lesions developing in the experimental model and those occurring in patients make us confident that the model is suitable for studying early stages of melanomagenesis, which are difficult if not impossible to study using patients' material alone.

Melanoma-like lesions in human skin grafts induced by cutaneous expression of bFGF, ET-3, and SCF combined with irradiation with UVB. a,b: Human skin graft on a SCID mouse 2 weeks after beginning of treatment. A black lesion (a) has developed that histologically (b) shows hyperpigmentation, pigmented melanocytic nests (arrows) and single melanoma cells (arrowheads) that have left the basement membrane. c,d,e: Melanomas in human skin grafts in week 4 of treatment composed of big nests (arrows) of transformed melanocytic cells (c) that stain positive for S100 (d) and HMB45 (e). f,g: Colony formation in soft agar of melanocytic cells isolated from experimental lesions (f) and from established human melanoma cell lines (g). Scale bar 1 cm (a), 100 µm (b,c,d), 200 µm (e).

Yu, H., McDaid, R., Lee, J., Possik, P., Kumar, S.M., Elder, D.E., van Belle, P., Gimotty, P., Guerra, M., Hammond, R., Nathanson, K.L., Dalla Plama, M., Herlyn, M, Xu, X.: Role of BRAF mutation and p53 inactivation during transformation of a sub-population of primary human melanocytes. Am. J. Pathol. 174: 2367-2377, 2009. PMID19389934 (PMCID2684200)