Few animal models exist for cutaneous malignant melanomas. The murine models require a combination of gain of function and loss of function mutations in a proto-oncogene and tumor suppressor gene, respectively . Severe sunburn in newborn mice may be needed to induce skin melanomas with high penetrance . By contrast, the MeLiM swine model exhibits spontaneously cutaneous melanomas with histopathological features similar to those of human melanomas . Here, we provide evidence that the hereditary disorder in MeLiM is useful for the identification of regulatory complexes involved in the development of melanomas in humans. Indeed, we found that the adaptor protein RACK1 in pig melanoma is overexpressed as in human melanoma. This is the first time that a prediction based on the MeLiM model finds confirmation in human melanoma. This suggests that similar mechanisms may be operating in the malignant transformation of melanocytes in pigs and man.
Global gene expression profiling on melanoma using various techniques including SAGE has been reported. The nature of the starting material determines the outcome of the comparisons. Noteworthy, the results seem complementary, and differential expression of RhoC , WNT5a , NOTCH2 , Ubc9  and genes associated with calcium signalling  among others, highlighted pathways involved in cell motility, tissue invasion and resistance to apoptosis. In our SAGE analysis, the RACK1 tag was more abundant in the library established from primary culture of metastatic melanoma cells than in the library from normal skin melanocytes. Consistently, RACK1 mRNA and protein were not detected in normal epidermal melanocytes, while they were both found at high levels in tumoral cells of cutaneous and metastatic melanomas. RACK1 was barely detected in melanocytes of normal skin in pigs and humans. This is opposed to cultured pig and human melanocytes, in which RACK1 mRNA and protein expression is readily detected [25, 26] (and our observations). One explanation to this discrepancy is the microenvironment itself : in culture, melanocytes are grown as a pure population of cells with close interaction , in a medium containing factors known to activate the RACK1 promoter . It is noteworthy that melanoma cell lines express even more RACK1 than cultured melanocytes . The latter observation suggests that RACK1 upregulation in MMC was maintained while deriving the corresponding melanoma cell lines. Hence, it is plausible that RACK1 upregulation in melanomas, once established, does not depend on environmental signals provided by stromal cells but is stably inherited from cell to cell.
This study shows that RACK1 mRNA and its corresponding protein are systematically overexpressed in tumoral cells of cutaneous and metastatic melanomas. This holds true for all pig and human samples examined to date, whether pigmented or not. Instead, we detected a conspicuously lower expression in nevi. GNB2L1 encodes RACK1, an adaptor protein that modulates signaling from PKC, Src, and β-integrin in various systems [29–31]. Evidence for a role of RACK1 in the pathogenesis of melanoma comes from the recent discovery of the capacity of RACK1 to increase survival of MeWo human melanoma cells following UV induced-apoptosis . Moreover, inhibition of RACK1 expression using siRNA was shown to reduce the tumorigenicity of MeWo in a xenograft tumor model . These authors further proposed a role for RACK1, specific to melanoma, involving a crosstalk between ERK and JNK signaling . In this model, RACK1 was shown to induce JNK activation by binding activated PKC, but the PKC isoforms involved were not identified.
PKC is a family of lipid-regulated serine/threonine kinase isozymes which differ in their activation and are differentially expressed in tissues and cell types . Upon activation, PKC translocates subcellularly by specific binding to anchoring proteins like RACK1 . PKC, the main target of TPA, has important roles in cell-cycle regulation, cell survival, malignant transformation and apoptosis . PKC in melanocytic lineage has been widely studied because TPA is essential for in vitro growth of normal melanocytes [35, 25] and because it was shown to affect the metastatic potential of these cells . Again, studies on cultured melanocytes and melanoma cell lines do not always reflect the actual situation in the whole organism. Hence, the contribution of PKC in melanoma is still controversial. WNT5A, identified as a robust marker of highly invasive human melanoma cells  was shown to mediate motility through activated classical PKCs . An additional effect of PKC on melanoma was recently underlined by a study of the aberrant expression of claudin-1 in melanoma . Conversely, PKCβ expression was reported lost in 90% of melanoma cell lines [39–41]. Recently, the expression of PKCα and δ isoforms, in contrast to PKCβ, was reported in skin and lung melanoma sections, but it was not specified whether the antibodies recognized the activated isoforms or not . Based on these observations, we analysed phospho-PKCαβ expression in human melanomas. The activated PKCαβ signal was stronger in malignant samples, but only partially colocalised with RACK1. RACK1 is known to bind activated PKCβ II  and to a lesser extent activated PKCα . Nuclear signal of phospho-PKCαβ could be ascribed mainly to PKCα  although nuclear PKCβ II has also been reported . Altogether, our results on the partial colocalisation of RACK1 and phospho-PKCαβ signal suggest that RACK1 binds more likely to activated PKCβ II than to PKCα in melanoma cells. The remaining RACK1 interacts probably with other PKC isoforms like PKCδ [41, 37] or other proteins.
Few studies have detected up-regulation of RACK1 in human cancer specimens [13, 46]. RACK1 mRNA was found to be strongly expressed in five non-small cell lung carcinomas, mainly in the endothelium of large vessels . RACK1 mRNA was also highly expressed in 11 cases of colorectal cancer, with a stronger expression in carcinoma cells than in the stroma . Recently, RACK1 upregulation was found to be part of the gene signature associated with shorter metastasis-free survival in breast cancer patients . RACK1 contains seven internal WD40 repeats which confer either stable or reversible binding capability to other proteins. Almost 60 proteins interacting with RACK1 have been described to date. RACK1 could affect cell transformation at multiple levels, increasing proliferation rate, migratory capacity, anchorage independent growth, or resistance to apoptosis .
Histopathological features of early stages of melanoma are still ill defined. Clearcut prognostic markers for melanoma which could be used to stratify patients for adjuvant treatments are lacking. Although this study is fairly descriptive, it provides a thorough analysis of RACK1 immunohistochemical detection in human melanoma samples at different stages. The number of samples in our study was limited and should be further increased, but the clear difference in the level and in the homogeneity of RACK1 expression between melanocytic cells in nevi and in cutaneous or metastatic melanoma suggests that RACK1 antigen could be used as a marker of malignancy in human melanocytic proliferation.