NEW INSIGHTS ON ESTROGENS AND VITAMIN E DERIVATIVE TOCOTRIENOLS ON HUMAN MALIGNANT MELANOMA: TOWARDS NOVEL THERAPEUTIC INTERVENTIONS.

Malignant melanoma represents the deadliest form of skin cancer: despite surgical resection of cutaneous in situ melanoma warrants a good prognosis, metastatic melanoma is mostly an incurable disease, because of the fast developing of resistance to existing therapies. For this reason, the identification of novel molecular targets and more effective antitumor compounds might be helpful in the management of the pathology. Clinical observations indicate that estrogen receptor \u3b2 (ER\u3b2) is expressed in melanoma tissues and its expression decreases with tumor progression, suggesting its anticancer activity. Moreover, this receptor was found to mediate the antitumor effect of the vitamin E derivative \u3b4-tocotrienol on breast cancer cell lines. This project firstly focused on the characterization of the estrogenic system on human melanoma cell lines. ER\u3b2 is expressed in melanoma cell lines but one, and proliferation and transactivation studies indicated that specific ER\u3b2 ligands exert an antiproliferative activity inducing the classical, nuclear mechanism of action of steroid hormones receptors. This antitumor effect is due to a G1/S cell cycle blockade, evidenced by the induction of the cell cycle inhibitor p27 and the reduction of the expression of cyclin D1 and D3, rather than to the activation of apoptotic mechanisms; furthermore, the activation of the receptor can influence the DNA methylation pattern of melanoma cells, suggesting that it could also exert antitumor effects through epigenetic regulation. However, the antiproliferative activity is dependent on the specific Ras/Raf mutational status of the cells and/or the expression of specific ER\u3b2 isoforms. MTT assays indicated that \u3b4-tocotrienol induces a significant reduction in melanoma cell viability, independently of the specific mutational status of the cells, and the expression of ER\u3b2. Gene-reporter assays failed to associate the antitumor activity of \u3b4-tocotrienol and the activation of the estrogen receptor \u3b2 in melanoma cells. The molecular mechanisms underlying \u3b4-tocotrienol activity were then analysed, evidencing a cytotoxic/apoptotic effect, through inhibition of colony-formation and cleavage of caspase-3 and PARP. Such apoptotic effect was found to be related to the activation of the endoplasmic reticulum (ER) stress pathways, as demonstrated by the induction of ER stress markers PERK, IRE1\u3b1, ATF4 and CHOP and the cleavage of caspase-4. Furthermore, the apoptotic effect was partially reverted by the ER stress inhibitor salubrinal. However, the mitochondrial apoptotic way was also activated by \u3b4-tocotrienol, since cytochrome c release and Bax/Bcl-2 ratio increment were observed. In vivo studies were also performed, and the reduction of tumor mass and volume and the delay of tumor progression strongly support the effectiveness of this compound on melanoma. Finally, the population of cancer stem cells was characterized in the A375 melanoma cell line, through the ability to grow on suspended melanospheres, and the expression of the melanoma stem cell marker CD271 and the embryonic stem cell marker Oct4. The cytotoxic activity of \u3b4-tocotrienol was assessed on this population of cells, showing the reduction in the number of spheroids and in the expression of CD271. In conclusion, this doctoral project demonstrated two important points. 1- ER\u3b2 has a tumor suppressive role on melanoma cells, depending of the specific mutation found in the Ras/Raf pathway: these results enforce the usefulness of genetic profiling of melanoma samples in dictating the more effective therapeutic strategy. Future efforts should be done to assess the clinical application of ER\u3b2-based therapies. 2- The vitamin E derivative \u3b4-tocotrienol has a potent antitumor effect, independent of ER\u3b2 activation. The translation of these results to the clinics might be an important tool for the management of metastatic melanoma, at least as adjuvant therapy