The laboratory uses biological approaches to understand cancer development and progression. The main model of investigations is melanoma. The melanoma related investigations span a wide area that includes gene regulation in normal cells, tissue morphogenesis, carcinogenesis, autocrine and paracrine regulation of growth, and cell-cell and cell-matrix interactions during angiogenesis and metastasis.
The unifying theme of our studies is on melanoma progression (Fig.), manifested as clinicopathologically defined lesions: step 0) melanocytes; step 1) common acquired and congenital nevi with structurally normal melanocytes; step 2) dysplastic nevi with structural and architectural atypia; step 3) RGP, non-tumorigenic primary melanomas without metastatic competence; step 4) VGP, tumorigenic primary melanomas with competence for metastasis; and step 5) metastatic melanoma (1). A recent refinement by Clark of this classification of nevi and primary melanomas (4) divides lesions into three classes: class I represents "precursor" nevi; class II lesions are "intermediates," confined to the epidermis or with microinvasion into the dermis and represented by in situ and invasive RGP melanomas; class III are VGP tumorigenic melanomas (2). Cell lines derived from each of these steps provide valuable tools for the study of melanomagenesis.
Figure 1: Melanoma development and progression
The model, developed by Drs. Clark, Elder, and Guerry, implies that melanoma commonly develops and progresses in a sequence of steps from nevic lesions which can be histologically identified in approximately 35% of cases. Thus, melanoma may also develop directly from normal (and precursor cells). The roles of melanocyte stem cells, melanoblasts (immature melanocytes) in melanogenesis remain poorly defined. Cells from lesions show persistence but non-tumorigenic lesions tend to disappear through apoptotic or differentiation pathways which are also poorly defined.
These lesions are clinically and histologically characterized, but the underlying biological events leading to melanoma development and progression toward an increasingly aggressive phenotype are poorly understood. Our laboratory uses biological, immunological, and molecular techniques to dissect each step and to determine the biological importance of individual genes, with the long-term goal of developing new approaches to cancer therapy.
The study of tumor progression has been and remains an important focus for our program. The concept of tumor progression in the melanocytic cell system is widely accepted today and we and others are expected to further contribute to our understanding of the molecular mechanisms of progression of cells from benign neoplasms to tumorigenic melanomas. As we are continuing this work, we are also beginning to consider the potential mechanisms for transformation in the large group of melanomas (representing more than 50% of all sporadic melanomas) that do not appear to arise from precursor nevi. We are proposing that melanocyte precursors are readily transformed, potentially not transiting through classical precursor stages. The search for such precursor cells is ongoing (see chapter on stem cells). Thus, melanomas may develop from precursor populations. In addition, the tumor microenvironment is playing an increasingly important role in our understanding of progression. The role of fibro blasts, endothelial cells and inflammatory cells is discussed elsewhere.
Figure 2: Human skin reconstructs and growth of human melanoma.
A: Normal skin reconstruct with well differentiated epidermis and dermis (H&E, 200x). B: RGP-like growing melanoma cells stained for S100 in red (200x) C: RGP/VGP-like growing melanoma cells stained for S100 in red (200x) D: Metastatic melanoma cells invading the reconstructed dermis. Stain for collagen IV in red demonstrates positivity around the tumor cells and along the basement membrane at the epidermal-dermal junction (200x).
Figure 3: Melanoma progression and the skin environment.
Our understanding of melanoma development and progression is changing towards a more complex model, one in which first genetic alterations are already observed in nevi. Mature, fully differentiated melanocytes are likely less prone to transforming than melanocyte precursor or stem cells, but neither has yet been identified in human skin. Tumor infiltrating fibroblasts (Fibr.), endothelial (Endo.) cells, and inflammatory and immune (Inflam.) cells play significant roles in tumor progression of primary (prim.) or metastatic (met.) lesions and can have either stimulatory or inhibitory activity. Normal melanocytes, nevus cells, or primary melanoma cells can undergo differentiation and death but we know little about the mechanisms.
Fukunaga-Kalabis, M., Martinez, G., Telson, S.M., Liu, Z.-J., Balint, K., Juhasz, I., Elder, D.E., Perbal, B., Herlyn, M.: Downregulation of CCN3 expression as a potential mechanism for melanoma progression. Oncogene 27(18): 2552-2560, 2007.
Fukunaga-Kalabis, M., Martinez, G., Nguyen, T-T.K, Kim, D., Santiago-Walker, A., Roesch, A., Herlyn, M.: Tenascin promotes melanoma progression by maintaining the ABCB5-positive side population. Oncogene 29(46): 6115-24, 2010.
Activated Notch signaling promotes melanoma progression
Aberrant Notch signaling has a major role in melanoma progression. Activated Notch signaling not only enhances primary melanoma cell proliferation in vitro and anchorage-dependent growth in soft-agar, but also accelerates xenografted primary melanoma growth in vivo and promotes metastasis formation in an experimental metastasis model, in which the malignant cells are injected intravenously. These data indicate that aberrantly regulated Notch signaling exerts an oncogenic effect on melanoma development and progression. However, it is unclear at present how Notch signaling is aberrantly activated during melanoma development.
Active form Notch 1 (NIC) enhances subcutaneous tumor growth in primary melanoma cells. WM 35 (RGP) melanoma cell line transfected with GFP or NIC were injected into SCID mice subcutaneously (3x106 /mouse) with 8 mice in each group. Tumors were harvested at week 7 (a); tumor weight is shown in (b). Each bar in b represents the mean ± s.e.
Balint, K., Xiao, M., Pinnix, C.C., Soma, A., Veres, I., Juhasz, I., Brown, E.J., Capobianco, A.J., Herlyn, M., Liu, Z.J.: Activation of Notch1 signaling is required for beta-catenin-mediated human primary melanoma progression. J Clin Invest 115:3166-3176, 2005. PMID16239965
Liu, Z.J., Xiao, M., Balint, K., Soma, A., Pinnix, C.C., Capobianco, A.J., Velazquez, O.C., Herlyn, M.: Inhibition of endothelial cell proliferation by Notch1 signaling is mediated by repressing MPAK and PI3K/Akt pathways and requires MAML1. FASEB J, 20: 1009-1011, 2006. PMID16571776
Liu, Z.-J., Xiao, M., Balint, K., Smalley, K.S.M., Brafford, P., Qiu, E., Pinnix, C.C., Li, X., Herlyn, M.: Notch1 signaling promotes primary melanoma progression by activating Mitogen-Activated Protein Kinase/Phosphatidylinositol 3-kKnase-Akt pathways and upregulating N-cadherin expression. Cancer Res 66: 4182-4190, 2006. PMID16618740
Pinnix, C. C., Lee, J.T., Liu, Z-J. McDaid, R., Balint, K., Beverly, L.J., Brafford, P.A., Xiao, M., Himes, B., Zabierowski, S.E., Yashiro-Ohtani, Y., Nathanson, K.L., Bengston, A., Pollock, P.M., Weeraratna, A.T., Nickoloff, B.J., Pear, W.E., Capobianco, A.J., Herlyn, M.: Active Notch1 protein confers a transformed phenotype to primary human melanocytes. Cancer Res. 69: 5312-5320, 2009. PMID19549918 (PMC2755513)