As it is known, despite its location within the protective chromatin microenvironment, DNA is subject to repeated damage by exogenous agents, such as ultraviolet radiation (UV), which can impair replication and/or transcription and must be corrected to prevent mutations .
Chromatin dynamics tightly controls activation and function of genes, by modulating the access of regulatory factors to DNA, and highly influences cellular behavior of normal and neoplastic cells . A highly ordered coordination and organization of factors regulating the opening and closing of chromatin is crucial to ensure that the correct epigenetic code is maintained within the genome .
Genomic instability is a major hallmark of tumor progression. It contributes to the gain of the invasive-metastatic phenotype of neoplastic cells by favoring the accumulation of sequential genetic and epigenetic events. These latter include DNA methylation, histone modifications, small, non-coding RNAs, and factors involved in the regulation of chromatin architecture [34–36].
Chromatin dynamics control nucleosome assembly and higher order chromatin structure, and are responsible for the global nuclear organization and compartmentalization, tightly regulating gene expression and controlling protein-DNA interactions within the cell nucleus [37, 38]. In particular, it allows the recognition of DNA-damaged sequences and the segregation of the damaged chromosomes, potential cause of genomic instability, determining chromatin reshaping and warranting the correct progression of the cell cycle and the maintenance of genome integrity [39–41].
In melanomagenesis, the deregulation of cell proliferation results, at least in part, from the alteration of epigenetic control checkpoints. In particular, the inactivation of UV-specific pathways of DNA repair is a critical step.
In normal melanocytes, UV trigger histone acetylation and the immediate activation of the histone poly-(ADP-ribosyl)ation, a post-synthetic DNA epigenetic modification which causes the generation of intracellular signals, leading to the DNA repair or, in the case of excessive damage, to apoptosis [24, 41–43].
Recent data concerning the "chromatin ambient" of the nucleus indicate a highly qualified role for chromatin modifying proteins in the control of DNA replication and DNA-repair [27, 44, 45]. The CAF-1 molecular complex looks fundamental for the maintenance of the epigenetic information; in particular, the deletion of CAF-1 subunits in S. cerevisiae confers sensibility to UV radiation . CAF-1, to date, is the only known chromatin assembly factor able to drive nucleosome assembly onto newly synthesized DNA, and has a central role in ensuring chromatin replication in S phase through the interaction with the polymerase sliding clamp, PCNA [26, 30, 31, 46–48]. It constitutes the first example of a factor involved in chromatin dynamics useful to assess cell proliferation . The behavior of its p60 and p150 subunits shares a number of similarities with PCNA, which plays crucial roles in both DNA replication and repair. Experimental data indicate that particularly p60 is active in either the control of cell proliferation and DNA-repair. Silencing of the p60 subunit by RNAi leads to the accumulation of double-strand DNA breaks and to the induction of programmed cell death in proliferating but not quiescent human cells .
Recently, the involvement of CAF-1/p60 in neoplastic progression has been reported, and its expression has been proposed as a new tool to define the biological behavior of some types of human malignancies (breast, tongue, and prostate cancer) [30–32].
This opens up the possibility that its use could be extended to malignancies of different histogenesis.
Here we show results supporting a role of CAF-1 in predicting the aggressiveness of CM.
In our cases, the maximum level of over-expression of CAF-1/p60 was found in the vertical growth phase of CM characterized by a metastasizing behavior, besides the Breslow thickness, ulceration and/or high mitotic index. This suggests that CAF-1/p60 may shows a promising role as a relevant, adjunctive prognostic marker for CM. Our results are in-line with previous data concerning the alteration of the poly-(ADP-ribosil)ation process in highly aggressive CM . In particular, the occurrence of CAF-1/p60 hyperexpression in the same cases of CM previously found to bear an altered expression of PARP-1, indicates that the co-existence of the deregulation of two of the major epigenetic pathways responsible of DNA-repair and cell replication control, constitutes a hallmark predictive of metastasizing behavior in CM of photoexposed skin.
Investigations are being undertaken from our research group to determine whether the alteration of such mechanisms still retains with the same statistical significance among a multi-institutional greater series of cases. This perspective looks extremely attractive, because give us means to built up a model in which the evaluation of the CAF-1/p60 status of expression could get over the limitations of current prognostic evaluation, particularly for that concerning the "grey area" of CM with an intermediate thickness (Breslow >1 and <2 mm).
In addition, it would be worth investigating how the CAF-1 molecular pathway can be regulated, by modulating the expression of its subunits and their interactions with the other histone chaperones and ATP-dependent chromatin remodelers. It has to be remembered, to this regard, that in contrast to genetic alterations, epigenetic modifications, although heritable in cells, are progressive, quantitatively evaluable, potentially reversible, and may serve as potential targets for drug treatment [22, 50, 51]. Given the absence of effective therapy for metastatic melanoma, the CAF-1 pathway may provide a window of opportunity for novel post-surgical molecular therapies. Undoubtedly, future studies will provide many exciting advances towards fully understanding of the critical link between chromatin, DNA repair and cell proliferation control, and the biology of CM.
Considering its essential role for cell cycle progression, it might constitute a new therapeutic target with cytostatic and/or cytotoxic effects that deserve to be investigated in the future.
Basing on the results of the present study, however, we believe that CAF-1/p60 expression is an exciting chance to better predict the biological behavior of CM, besides the traditional prognostic parameters.