Alterations in gene expression associated with single-hit mutations of tumor suppressor genes in phenotypically normal cells appear to represent the earliest molecular changes during cancer development. Previous studies showed that the altered transcriptome and proteome signatures of phenotypically normal cells bearing these single inherited hits parallel many changes observed in the corresponding form of cancer (for review see [17
]). Thus, identification of these early alterations may help identify critical molecular targets in both the inherited and sporadic cancers.
In the present study we compared global gene expression profiles of normal SF from control individuals and phenotypically normal skin-derived fibroblasts from FM individuals with two different CDKN2A/p16 mutations. We examined differences in gene expression profile at baseline and following UV irradiation, leading to the following important findings.
First, the transcriptome signatures are altered by single hit CDK2NA/p16 mutations in phenotypically normal SF from FM families. The extent of altered gene expression is mutation-site specific, with R87P-p16-CDKN2A mutation being far more disruptive than V126D-p16 mutations. This is in agreement with previous studies showing that the R87P-p16 mutation appears to have a more severe phenotype in vitro. The R87P-p16 mutation is an inactivating mutation of CDK4/6 binding [15
], inhibits kinase activity [20
], results in the accumulation of G1 phase cells [20
], and has a predominantly nuclear localization [21
]. In contrast, results on the V126D-p16 mutation have been less clear, which may reflect its temperature-sensitive biological effects in vitro and ability to partially “retain” wild type CDK4/6 inhibition [15
Second, the differentially expressed genes at baseline in R87P-p16 mutation carriers exhibit changes in canonical signaling pathways associated with normal melanocyte development and melanomas. KIT and its downstream effector MITF are part of the canonical signaling pathway involved in melanocyte development and pigmentation. KIT signals via MAPK to activate MITF, which in turn is a potent transcriptional and lineage specific regulator of the three major melanocyte pigment enzymes: tyrosinase, TRP1/TyrpI and TRP2/Dct [22
A third important finding is that UV-irradiated SF from FM patients show deregulation of cell cycle and DNA damage response pathways that are diametrically opposite to the gene expression pattern seen following irradiation of fibroblasts from normal controls. For example, cell cycle related genes were downregulated in UV-irradiated fibroblasts from normal individuals, but these genes were upregulated in fibroblasts derived from FM individuals. Moreover, many of the changes in gene expression seen after UV-irradiation of fibroblasts derived from FM individuals are shared with a previously described Gene Expression Omnibus data set of genes deregulated in malignant melanomas, but not in benign nevi [14
]. For example, the protein tyrosine phosphatase PTPN13 has been proposed to function as a tumor suppressor; this is in line with its downregulation [24
]. In addition, AFF1 is coordinately downregulated in UV-irradiated fibroblasts and in melanoma, and is often dysregulated in cancer [25
A fourth important observation is that occurrence of the more “benign” V126D-p16 mutation or of the more “disruptive” R87P-p16 mutation within a given FM family is mutually exclusive, supporting the notion that personalized, cohort-based, intervention modalities, including clinical outcome should be adjusted accordingly. We posit that, direct or up/down-stream “pharmacologic normalization” of the R87P-p16 mutation, individually and together with BRAF intervention [26
], may enable more effective strategies to delay or even prevent FM, including the occurrence of melanoma in individuals who are otherwise at risk. In this regard, proclivity towards pancreatic cancer that is likely to be caused by CDKN2A mutations [6
], particularly among affected carriers of the R87P-p16 mutation, should be explored for early diagnosis/prognosis and intervention of pancreatic cancer.
Together, these data suggest that genomic profiling of phenotypically normal SF can help identify novel molecular targets for chemoprevention, including early specific biomarkers of melanoma risk among individuals who are heterozygous for CDKN2A mutations within FM families; these potential biomarkers would enable mechanism-based early detection of melanoma and personalized prevention strategies to target sporadic melanoma.