NUTRITIONAL AND HORMONAL MODULATION OF HUMAN MELANOMA PROGRESSION

The tumoral microenvironment, also called “tumor reactive stroma”, is composed by several heterotypic cells like cancer-associated fibroblasts (CAFs), pro inflammatory cells, endothelial cells and perycites which stroma create a complex and continuative “cross-talk” made of direct cell-to-cell contacts and paracrine/exocrine signals (growth factors, cytokines, soluble factors) promoting tumor development through stimulation of cancer cells survival/proliferation, migration and metastatic ability. The mediators of chronic stress norepinephrine (NE) and epinephrine (E) exert stimulant effects on the invasion process of a number of neoplastic cells, and this effect seems to be due, at least in part, to the interaction with β-adrenoceptors (AR), which are expressed by various malignant tumours. In particular, the expression of β-AR has been studied in various human solid tumors, such as breast, colon, prostatic, ovary, nasopharyngeal and oral cancer, raising the possibility that such receptors could affect invasion and dissemination processes. My study aimed to evaluate the expression of β-AR and the influence of NE and E on the malignant behaviour of melanoma cell lines. Preliminary results assessed by immunohistochemistry on a series of human benign and malignant melanocytic lesions showed that both β1- and β2-AR were expressed in melanocytic tumors, according to neoplastic progression, being significantly more expressed in malignant than benign lesions, and, for β2-AR, significantly more present in atypical than common naevi. In vitro studies demonstrated that primary and metastatic cell lines expressed both β-AR types and were stimulated by NE and/or E to secrete higher amounts of IL-6, VEGF and IL-8, which contribute to melanoma progression, while NE enhanced motility through matrix metalloprotease-2. The pro-invasive effect of NE and E was inhibited by non-selective AR inhibitor propanolol, confirming β-AR involvement. In addition, VEGF increased the invasive spur induced by NE in A375 cell line, while IL-6 in association with NE could activate dermal fibroblasts toward a myofibroblastic phenotype, typical of tumour microenvironment. Taken together the results suggest that NE and E can dramatically affect the aggressiveness of melanoma cells and, since primary and metastatic melanomas exhibit strongly β-AR expression, could affect the course of the disease in melanoma patients too. These data support the hypothesis that the interaction of cathecolamines with β-AR could be a useful target in oncologic therapy in order to prevent melanoma metastatic progression. Beside and in synergy with signals produced by tumor micorenvironment, cancer cells are also able to completely modify their metabolic behaviour in order to sustain cancer progression. In particular, there is growing interest in defining the role of reactive stroma in the regulation of cancer cells metabolism. At this purpose, the “Warburg effect” is the best known tumor-specific alteration of common metabolic pathways, as cancer cells show increased glycolysis even in conditions of high oxygen tension (‘‘aerobic glycolysis’’), leading to high lactate production. In addition, cancer cells can use elevated amounts of glucose as a carbon source for anabolic reactions. The aim of the second part of my study is to focus on the ability of nutrients to regulate transcription factors involved in cancer cells metabolism. One of these factors is represented by hypoxia inducible factor-1α (HIF-1α). It is well established that, under hypoxic condition, HIF-1α activates target genes whose protein products mediate a switch from oxidative to glycolytic metabolism (ex. GLUT-1, LDH-A), while it is not well established yet the role of HIF-1α in regulating these complex pathways under normoxic condition. To this purpose, A375 primary melanoma cell line was cultured in normoxic condition in presence/absence of nutrients like glucose, pyruvate and lactate in order to understand how different nutrients can activate key pathways for cancer progression. Cytofluorimetric analysis demonstrate that A375 show strong dependance on glucose content of media in order to sustain survival, thereby according to a Warburg metabolic phenotype, and that nutrients alone are able to induce the expression of HIF-1α and of its target gene carbonic anidrase-IX (CA-IX), probably thorugh an increased reactive oxygen species (ROS) production. In order to further understand the role of nutrients-induced HIF-1α, we performed migration and invasion assays with Boyden chambers, respectively in absence or presence of Matrigel mimicking the extracellular matrix. Nutrients alone are also able to elicit a pro-invasive response of A375 and this effect is reverted after both treatment with Topotecan, a HIF-1α farmacological inhibitor, and HIF-1α gene silencing. Finally, data obtained so far allow us to hypothesize a novel important role of nutrients-induced HIF-1α normoxic stabilization in order to sustain cancer progression.