mTOR pathway activation in cutaneous melanoma is associated with poorer prognosis characteristics

Dear Editor, The conserved serine ⁄ threonine kinase mTOR (the mammalian target of rapamycin), a downstream effector of the PI3K ⁄ AKT pathway, is a major regulator of survival, growth, proliferation and motility, in response to mitogens, energy and nutrient levels (reviewed in Guertin and Sabatini, 2007). mTOR is an attractive therapeutic target, and mTORC1 inhibitors, including the rapamycin analogues Deforolimus, Everolimus and Tensirolimus, alone or in combination with inhibitors of other pathways, are undergoing clinical trials for treating multiple cancers, including melanoma (reviewed in Dancey, 2006; Fecher et al., 2007). Alterations in molecules of the PI3K ⁄ AKT ⁄ mTOR pathway have been identified in cutaneous melanomas (reviewed in Hocker et al., 2008). In view of the possible involvement of the PI3K ⁄ AKT ⁄ mTOR pathway in melanoma, as well as its potential role as a therapeutic target, we aimed to clarify whether there is an association between the expression of the PI3K ⁄ AKT ⁄ mTOR pathway effectors, clinico-pathological and prognostic parameters, and BRAF and NRAS mutations in cutaneous melanomas. To evaluate mTOR pathway expression in cutaneous melanoma, tissue microarrays of 84 cutaneous melanomas cases (Supporting Information Table S1) were stained with antibodies against PI3K ⁄ AKT ⁄ mTOR and MAPK pathways effectors: PTEN, AKT, pAKT Ser473, pAKT Thr308, mTOR, pmTOR Ser2448, S6, pS6 Ser235 ⁄ 236, 4E-BP1, p4E-BP1 Thr37 ⁄ 46, ERK1 ⁄ 2 and pERK1 ⁄ 2 Thr202 ⁄ Tyr204 (methods detailed in Supporting Information Data S1 and Table S2). The results of examination of the stained sections were consistent with activation of the AKT ⁄ mTOR pathway in cutaneous melanoma, as we observed a high expression of its active forms in most of the cases studied (Figure 1). Similar conclusions were reported by Karbowniczek et al. (2008), who evaluated pS6 in a series of cutaneous melanomas. We found a significant positive association between pmTOR and the two phosphorylated forms of AKT – pAKT Ser473 (P < 0.01) and pAKT Thr308 (P = 0.03). pmTOR was also associated with the two downstream effectors of mTORC1 – pS6 (P = 0.01) and p4EBP1 (P < 0.01). A positive association was found between the staining level of pS6 and p4EBP1 (P = 0.01). Expression of pAKT Ser473 was positively associated with the two downstream effectors of mTORC1 – pS6 and p4EBP1 (P < 0.01). Moreover, we found that the two phosphorylated forms of AKT – pAKT Ser473 and pAKT Thr308 – were positively associated with pERK1 ⁄ 2 (P < 0.01 and P = 0.01, respectively). pmTOR expression was also positively associated with pERK1 ⁄ 2 (P = 0.04), along with its two effectors pS6 and p4EBP1 (P < 0.01 and P = 0.02, respectively). The positive correlations found between the expression of upstream and downstream effectors of the mTOR pathway suggest simultaneous activation of the signalling pathway in cutaneous melanoma. Also, the positive correlation observed between the expression of the phosphorylated forms of mTOR effectors and pERK1 ⁄ 2 suggests that the MAPK pathway is activated concurrently with the AKT ⁄ mTOR pathway in cutaneous melanoma, similar to the pattern seen by our group in ocular melanomas (Populo et al., 2010). The mean expression level of the studied markers was compared with the clinico-pathological parameters of the cases (Table 1). We found that expression of pAKT Ser473 was significantly elevated (P = 0.02) in tumours with more than 1 mm thick, and a borderline significant higher mean of pS6 was found in cases with epidermal ulceration (P = 0.05). A significantly higher mean expression of AKT (P = 0.04), pAKT Ser473 (P < 0.01) and pS6 (P = 0.02) was detected in cases with III ⁄ IV Clark level than in cases with I ⁄ II Clark level. pAKT Ser473, pmTOR and pS6 expression was also significantly elevated in tumours with a mitotic rate greater than 1 mitosis ⁄ 10 high-power fields (P = 0.01). The association found between the expression of several proteins of the mTOR pathway and a poorer prognosis suggests an association between mTOR pathway