The Study on Melanoma and Other Types of Skin Cancer

The incidence of skin cancer (SC) exceeds all other human cancers combined and its incidence is increasing at an alarming rate. One of the main reasons is the direct link between the risk of SC and increased UV radiation exposure is as a result of depletion of the ozone layer (Armstrong & Kricker, 1996; Gloster & Brodland, 1996). Skin cancer affects more than one million Americans every year and accounts for more than ten thousand deaths annually, which represents approximately 4% of all cancer deaths together (Stern, 2010). Ultraviolet radiation (UV), in particular UV-B, induces a plethora of effects ranging from erythema, sun burns, immune suppression, increased photo-aging and skin cancers including melanoma and non-melanoma skin cancers (NMSCs). Despite melanoma having less likely prevalence rate than NMSCs, it is ranked as the most lethal human skin cancer and its incidence has doubled in the last couple of decades (Hall, Miller, Rogers, & Bewerse, 1999; Wingo, Ries, Rosenberg, Miller, & Edwards, 1998).

Previous studies including our recent work indicated that Retinoblastoma (Rb) signaling pathway, orchestrated by the two tumor suppressor genes p53 and p16INK4a, is the key UV-target pathway disrupted in NMSCs and cutaneous malignant melanoma (CMM), respectively (Abd Elmageed et al., 2009; Davies et al., 2002; Kannan et al., 2003; Sharpless & DePinho, 1999). The p53/Rb and the p16/Rb sub-pathways play critical roles in regulating cell cycle and apoptotic programs in response to UV irradiation (Abd Elmageed et al., 2009; Ouhtit, Gorny, et al., 2000; Ouhtit, Muller, Gorny, & Ananthaswamy, 2000; Williams & Ouhtit, 2005). Intriguingly, a single UV insult to the skin tissue can selectively disrupt either the p53/Rb or p16/Rb pathway or both in keratinocytes (KCs) and melanocytes (MCs). In KCs, UV-induced DNA damage results in an elevation of p53 protein followed by the induction of its direct transcriptional target p21WAF1, a cyclin-dependent kinase (Cdk) inhibitor (Al-Mohanna, Manogaran, Al-Mukhalafi, K, & Aboussekhra, 2004; Decraene et al., 2005). p21 inactivates the Cdk-Cyclin complex by forming a Cdk2/A or E Cyclin/Proliferating Cell Nuclear Antigen PCNA/p21complex, which prevents both Rb phosphorylation and the release of E2F leading to G1 arrest to allow the repair of DNA damage (Maeda et al., 2005). If the damage is severe and left unrepaired, a second preventive mechanism, “apoptotic pathway” is induced to eliminate damaged KCs (Brash et al., 1996; Cotton & Spandau, 1997; Ouhtit, Muller, et al., 2000). UV-induced apoptosis is mediated by p53 leading to up-regulation of its transcriptional pro-apoptotic targets Bax and Fas, and down-regulation of its anti-apoptotic transcriptional target Bcl-2 (Hill et al., 1999; Ouhtit, Gorny, et al., 2000; Ouhtit, Muller, et al., 2000; Zhuang, Wang, & Sauder, 2000). Likely, MCs compared to KCs are generally believed to possess an intrinsic resistance to apoptosis. MCs are characterized by a broad expression of apoptotic inhibitors (Bowen et al., 2003), including high basal levels of the anti-apoptotic protein Bcl-2, which opposes apoptosis and increases the survival of MCs (Bivik, Andersson, & Rosdahl, 2005). However, an increase and a redistribution of Bax protein from different compartments within the cell have been found to initiate the apoptotic response to UVB in MCs (Bivik et al., 2005; Kim et al., 2000).

The p16/Rb pathway also plays a key role in the regulation of cell cycle in response to UVB induced DNA damage (Ahmed, Ueda, & Ichihashi, 1999; Chazal et al., 2002). p16INK4a is a cell cycle regulator that inhibits Cdk4/6 specifically and consequently results in cyclin D-dependent phosphorylation of Rb, leading to reduced E2F driven transcription of genes necessary for S phase entry (Shirodkar et al., 1992). In irradiated cells with low doses of UVB, p16INK4A is upregulated within 12–24 hours leading to cell cycle arrest at G1 (Chazal et al., 2002; Milligan, Gabrielli, Clark, Hayward, & Ellem, 1998; Pavey, Conroy, Russell, & Gabrielli, 1999) to allow DNA repair before resuming the cell cycle (Sarkar-Agrawal, Vergilis, Sharpless, DePinho, & Runger, 2004). Inactivation of p16INK4a via missense mutations, deletions, or methylation, has been reported in a large number of different human tumor types including CMM (Castellano et al., 1997; Nobori et al., 1994; Ruas & Peters, 1998; van der Velden et al., 2001), where the Rb protein is no longer maintained in its active form and cell replication goes unchecked.

Although human INK4a/ARF is the only bona fide genetic locus lost frequently in familial and occasionally in somatic CMM, the functional relationship between INK4a/ARF and UV radiation in the pathogenesis of CMM is still elusive. However, mouse melanoma models have served as an in vivo genetic platform where the roles and molecular targets of UV radiation can be examined. Recently, these models have provided genetic evidence in support of the epidemiological link between increased CMM risk and childhood sunburn and have facilitated identification of the components of the Rb pathway as the principal and rate-limiting target of UV's actions in CMM formation (Kannan et al., 2003; van Schanke et al., 2006).

The difference in the apoptotic responses to UVB between MCs and KCs might explain why INK4a/ARF mutations predispose to melanoma, but not to NMSC. This also provide a molecular explanation for the link between melanomagenesis and the regulation of cell cycle check points and their link with DNA repair in response to UV exposure. The high penetrance of melanoma in families carrying germline mutations in CDKN2A/p16INK4a suggests that melanocyte immortalization, via inactivation of the p16/Rb pathway, is a crucial step in early initiation of melanoma. As an extension of our previous work, our aim was to study the molecular changes in the expression/function of the different components of the Rb pathway associated with UVB-induced cell cycle and apoptotic programs using normal human melanocytes and melanoma cell lines inducible for wild and mutant p16INK4a.