Basi molecolari per lo sviluppo di strategie chemiopreventive e terapeutiche innovative nel melanoma: recettore estrogenico beta, epigenetica e tocotrienoli

Although early detection and improved surgical techniques have led to an increase in melanoma patient survival, the prognosis of highly aggressive, metastatic melanoma is still very poor. For patients with surgically resected primary melanoma, the only effective adjuvant therapy is represented by interferon-gamma; however, the indication for this drug remains controversial, mainly due to the limited improvement of overall survival. For highly metastatic melanoma, both chemotherapy and immunotherapy did not show the expected improvement in overall survival. Overall, based on the increasing incidence, the poor prognosis and the disappointing results so far obtained with the available agents, the identification of novel molecular markers for the improvement of diagnosis and the development of more effective, and less toxic, therapeutical strategies is urgently needed.

Epidemiologic and experimental observations support the hypothesis that melanoma can be classified as an estrogen-responsive tumor and that locally expressed estrogen receptors (ERs) could mediate molecular responses associated with the neoplastic transformation of normal benign nevi.

Since, as a general concept, ERalpha behaves as a proliferative protein, while ERbeta is characteristically endowed with antiproliferative effects, it is believed that a perturbation in the ERalpha/ERbeta ratio expression might lead to cell transformation and tumor development in target tissues.

The proposed project is based on clinical observations from the Literature indicating that ERbeta, and not ERalpha, is the predominant estrogen receptor type in benign and malignant melanocytic lesions. More importantly, the level of expression of ERbeta markedly decreases during the progression of melanoma towards its metastatic phase, further supporting the tumor suppressor activity of this receptor also in this type of neoplasia. In preliminary experiments, Dr. Limonta's laboratory has demonstrated that human melanoma cells lines (A375 and BLM) express both ERs, indicating that these two cell lines might represent a useful tool to study the role of ERbeta and its regulation, by both epigenetic meachnisms and nutrients, in melanoma progression. Moreover, Dr. Comitato's Laboratory (the Collaborator in the present proposal) has consistently reported that tocotrienols (TTs) significantly reduce breast cancer cells proliferation through activation of ERbeta.

Based on this consistent experiemtnal evidence,. the present proposal will be aimed at giving insights on:

1) The expression and the role of ERbeta in melanoma cells. This task will be pursued by: 1.1) analyzing the expression of ERbeta in normal melanocytes as well as in a panel of human melanoma cell lines derived from tumors at different stages of progression; 1.2) investigating the effects of specific ERbeta activation on cell growth, cell viability, and apoptosis in human melanoma cells (A375 and BLM); 1.3) clarifying whether ERbeta activation might affect the metastatic behavior of human melanoma cells (A375, BLM).

2) The epigenetic regulation of ERbeta expression. Preliminary experiments will be performed to analyze the global DNA methylation and post-translational modifications of histones in normal melanocytes and in melanoma cell lines derived from tumors at different stages of progression (2.1 and 2.2). The relevance of epigenetics in melanoma growth will then be confirmed by investigating the effects of Aza (DNMT inhibitor) and TSA (HDAC inhibitor) on human melanoma cell (A375, BLM) growth (2.3). Finally, it will be clarified whether the ER promoter is hypermethylated in the two melanoma cell lines and whether its methylation status might be changed by Aza (2.4 and 2.5).

3) Tocotrienols and ERbeta cross-talk in melanoma. These experiments, performed using A375 and BLM melanoma cells, will be aimed at: 3.1) verifying the effects of either a TT-rich fraction from palm oli (PTFR) or purified TTs on melanoma cell growth and metastatic behavior; 3.2) clarifying whether TTs might exert their effects through modulation of ERbeta expression/activity (3.3) and/or ERbeta epigenetic status (3.4); based on the in vitro results, investigating the effects of tocotrienols on the growth of melanoma xenografts in nude mice fed orally with PTFR or purified TTs.

The short-term achievement of this proposal is a better understanding of the molecular pathways involved in melanoma progression (based on ERbeta, epigenetics and TTs). In the long term, these results might improve the prevention as well as the treatment modalities for melanoma.

These goals fit with the AICR objectives.