MELANOMA DEVELOPMENT AND PROGRESSION IN TRANSGENIC MOUSE MODELS

Melanoma is the most rapidly increasing malignancy among young people in the United States. Once the disease has reached the metastatic stage, it is largely refractory to conventional treatments. Recent evidence supports the possibility that a more effective therapy might be based on eliciting an immune response by cytotoxic T lymphocytes against peptide products in the tumor cells that are related to the specialized proteins ordinarily generated in melanocytes, the cells in which the tumors originate.

Malignant tumors are in general characterized by increased cell proliferation at the expense of cell differentiation. Our program is based on the view that malignant cells do not completely lose their specialized identity, even in advanced stages. That is, they retain the ability to generate at least some versions of products specifically associated with the corresponding normally differentiated cells. In the case of melanomas, these variants would be encoded by some of the many "pigment genes" required for melanization. Our objectives are: to identify the variants, starting at the level of the mRNAs of these genes in normal skin; to learn if changes consistently occur in melanomas of our transgenic mouse models; and to immunize the mice with peptides predicted from novel or overexpressed mRNAs.

The transgenic SV40E inbred (C57BL/6 strain) mice produced in this laboratory develop malignant melanomas strikingly resembling human melanomas in their progression and metastasis. Unlike human melanomas, the tumors arise in genetically identical animals with many possibilities for single-gene substitution and experimental intervention. Inasmuch as the genes under study have human homologues, the mouse melanoma models may ultimately establish a basis for parallel approaches to immunotherapy in human melanoma.

The transgene in the germ line of our mouse models is transcriptionally activated by a sequence from the mouse tyrosinase gene, which is specifically expressed in cells of melanocytic lineages. The oncogenic component of the transgene acts as a tumor-initiating stimulus in those cells. In skin melanocytes, this results in a state of melanoma susceptibility. To obtain malignant tumors, a promoting stimulus must be added. We have previously reported that wound-healing factors active after skin grafting efficiently provide such a stimulus and this almost always results in a melanoma within the graft (Mintz and Silvers, Proc. Natl. Acad. Sci. USA 90:8817, 1993). A small piece of skin is taken from a transgenic donor of a line with relatively high transgene expression, and therefore high susceptibility, and is transferred to a transgenic host of a low-susceptibility line. The tumors progress in the grafted skin and metastasize into many organs of the hosts.

To determine if the occurrence of skin melanoma is influenced by the age or the anatomic source of the skin, skin was grafted from donors of different ages, or from different anatomic sites, to a standard (lateral trunk) site in adult recipients of the same transgenic strain (1). In 27 grafts of neonatal body skin, melanomas arose with a significantly shorter latency than in 37 grafts of older body skin. The difference may reflect not merely the larger number of extrafollicular melanocytes in a given area of neonatal skin, but their unusually high mitotic activity shortly after birth, and also the influence of other growing skin cells nearby. Each of these body-skin grafts usually developed a single tumor, situated near the graft edge. As maximal wound healing occurs at the edge of such full-thickness skin grafts, melanocytes near the edge would receive the highest exposure to growth factors and cytokines associated with wound healing. In contrast, grafts of snout skin yielded many melanomas, each originating from melanocytes within a vibrissa follicle rather than at the graft edge. The strong local tumorigenic stimulus may be attributable to intrafollicular growth factors normally involved in whisker growth. These experiments support the conclusion that agents in the immediate skin environment of the melanocyte, in addition to the state of the melanocyte itself, contribute to melanoma formation.

As previously described, we are analyzing primary mouse melanomas and their metastases by quantitative RTPCR in order to learn whether specific alternatively spliced transcripts of some of the pigment genes are overexpressed in the tumors relative to their levels in skin melanocytes (Le Fur et al., Proc. Natl. Acad. Sci. USA 94:5332, 1997). This was clearly shown to be the case for the tyrosinase gene. The transcript profiles of other pigment genes are under study and thus far some also appear to have relatively increased levels of specific alternative mRNAs in the tumors.

To learn whether the proteins of these genes persist to different extents as the tumors progress, cutaneous melanomas and metastases were analyzed by Western immunoblotting with antisera specific for the carboxyl terminus of each of four melanocytic proteins (2). These were tyrosinase, tyrosinase-related protein-1 (TRP1), tyrosinase-related protein-2 (TRP2) and Pmel 17/silver. Of the 13 melanomas examined, there were 5 melanotic primary tumors, 5 amelanotic primary tumors, and 3 amelanotic metastases. The melanotic tumors expressed all of the markers to some extent. In contrast, the amelanotic tumors lacked detectable levels of one, two, or three of the proteins, except for an apparently amelanotic tumor sample in which all were expressed, but in which some melanotic cells were likely to have been present. Thus, despite some variability, there is clearly a downward trend in the presence of these proteins as the tumors become amelanotic-a pigmentary change associated with ongoing malignant progression. In the amelanotic tumors, tyrosinase was most often deficient while TRP2 was most often persistently expressed. These results indicate that tumors in the relatively early stages of malignancy might be more responsive than later-stage tumors to immunotherapy involving an ensemble of antigenic peptides of the tested gene products. Moreover, TRP2 peptides may be especially useful for therapeutic intervention at the later stages.

We can also promote malignant cutaneous melanomas without skin grafting, by exposing the "initiated" transgenic animals to ultraviolet radiation (3). Unlike the situation in other reports of UVR-induced melanoma in mice, chemical carcinogens are not required here and no other skin malignancies arise. In our experiments, line 12 hemizygotes, characterized by a very low level of transgene expression, were exposed neonatally for limited periods to UVR including 65% UVB (280-320 nm wavelength). In the most successful protocol, the mice received a total dose of 1.9 J/cm² UVR distributed over 5 days for a short period each day. After a latency ranging from 37-98 weeks, 26% of the treated animals developed invasive and metastatic melanoma. This new inbred mouse model introduces many novel possibilities for experimental analysis of the melanoma-promoting mechanisms of UVR, the effects of different UV wavelengths, and the presumed photoprotective role of melanin in the skin.

Antigenic peptides must become linked to MHC proteins and displayed on the surface of antigen-presenting cells in order to be recognized by T lymphocytes. The melanoma (target) cells must also display the peptide in association with MHC molecules in order to be lysed by the activated T cells. Antigens associated with MHC class I proteins are recognized by cytotoxic T lymphocytes in cell-based immunity. We are therefore analyzing examples of primary melanomas and their metastases to learn whether the cells continue to express class I molecules as they progress in malignancy. The D^b and K^bclass I proteins are characteristic of cells of the C57BL/6 mouse strain. The case in Figure 1 illustrates an analysis of tumor cells from a skin melanoma with distinct melanotic and amelanotic zones, of which the latter is more advanced. In this particular case, D^b expression declined as the tumor progressed; K^b expression (data not shown) also declined. The extent of heterogeneity among stages of a tumor, as well as among tumors, will be of interest.

We have also undertaken analyses of cell cycle changes in melanomas, to ascertain the extent to which changes in cell proliferation and/or cell death (by apoptosis or necrosis) contribute to tumor progression. The case in Figure 2 was based on determination of DNA content of each cell after staining with ethidium bromide.

PUBLICATIONS

1.   SILVERS, W.K., MINTZ, B. Differences in latency and inducibility of mouse skin melanomas depending on the age and anatomic site of the skin. Cancer Res. 58:630-632, 1998.

2.   ORLOW, S.J., SILVERS, W.K., ZHOU, B.-K., MINTZ, B. Comparative decreases in tyrosinase, TRP1, TRP2, and Pmel 17/silver antigenic proteins from melanotic to amelanotic stages of syngeneic mouse cutaneous melanomas and metastases. Cancer Res. 58:1521-1523, 1998.

3.   KELSALL, S.R., MINTZ, B. Metastatic cutaneous melanoma promoted by ultraviolet radiation in mice with transgene-initiated low melanoma susceptibility. Cancer Res. 58:4061-4065, 1998.

^a   S. Kelsall: Present address-University of New South Wales, Sydney, Australia

^b   S.J. Orlow, B.-K. Zhou: NYU Medical Center, New York, NY 10016

Illustrations or unpublished data in these reports should not be used without permission of the author.

Fox Chase Cancer Center

Scientific Report 1998