Analysis of candidate genes through a proteomics-based approach in primary cell lines from malignant melanomas and their metastases

Objective: Proteomics provide a powerful approach for screening alterations in protein expression and post-translational modification which can be associated to particular human diseases. In this study, analysis of protein expression was focused on malignant melanoma (MM) in order to analyse candidate genes involved in tumour progression. Methods: Proteome of cultured melanocytes and cell lines from primary and metastatic lesions of one MM patient has been profiled using two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). Differentially expressed proteins have been confirmed by 2-DE and MS on additional four MM cell lines. Total RNA from the first subset of cell lines was used for quantitative RT-PCR of the candidate genes identified after proteomic analysis. Results: A very high similarity was observed in the 2-DE maps between two MM cell lines derived from primary and secondary lesions of the same patient. Mass spectrometry identified 37 proteins which were found more abundant in tumour cells in comparison to control melanocytes (as confirmed on additional cell lines), with a relatively high prevalence of stress proteins. Among them, eight candidate genes (PRDX2, HSP27, HSP60, HSPA8, HSP9B, STIP1, PDI, and P4HB) have been further characterised by also evaluating their mRNA expression levels through real time RT-PCR analysis. Overexpression of HSP27, HSP60 and HSPA8 as well as downregulation of PRDX2 was observed in cells from metastatic MM in comparison to those from primary melanoma. Conclusion: Although further investigations with higher numbers of paired normal and tumour samples are needed, our findings strongly suggest that disregulation of stress pathways may be involved in melanoma progression