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Keywords:

  • melanoma;
  • pigmentation;
  • progression;
  • skin;
  • p53;
  • apoptosis;
  • sunburn

Summary

  1. Top of page
  2. Summary
  3. Significance
  4. The p53 pathway members
  5. p53 – an ultraviolet (UV) target in normal melanocytes
  6. p53 mutation and expression in melanoma
  7. MDM2 expression in melanoma
  8. The p16INK4a/p14ARF locus and melanoma
  9. p53 apoptotic target genes –APAF-1 and PUMA
  10. Keratinocyte p53 is the UV transducer in the keratinocyte-melanocyte signaling cycle
  11. Acknowledgements
  12. References

p53 has a central role in skin pigmentation and may impact on melanoma at all stages, however, as it’s mutation frequency in melanoma is low, it’s role has been somewhat under-appreciated. During normal skin function, p53 in the keratinocyte is a transducer of the skin tanning signal and an essential component of what is effectively a keratinocyte-melanocyte signaling cycle that regulates skin pigmentation. It is clear that this cycle functions optimally in skin of dark pigmentation. When melanin biosynthesis is genetically disrupted in skin of white complexion, we propose that this cycle operates as a promoter of melanocyte proliferation. The cell autonomous function of p53 in melanocytes is not well described, however, the balance of the evidence suggests that p53 is an effective tumor suppressor and the myriad of mechanisms by which the p53 pathway may be dysregulated in tumors attests to it importance as a tumor suppressor. In this review, we outline the known mechanisms that impair p53 itself and its immediate regulators or target genes during melanomagenesis. Due to the importance of this pathway, it is clear that p53 disruptions may relate directly to a patient’s prognosis. This pathway will continue to be a focus of investigation, particularly with respect to targeted experimental chemotherapeutics.


Significance

  1. Top of page
  2. Summary
  3. Significance
  4. The p53 pathway members
  5. p53 – an ultraviolet (UV) target in normal melanocytes
  6. p53 mutation and expression in melanoma
  7. MDM2 expression in melanoma
  8. The p16INK4a/p14ARF locus and melanoma
  9. p53 apoptotic target genes –APAF-1 and PUMA
  10. Keratinocyte p53 is the UV transducer in the keratinocyte-melanocyte signaling cycle
  11. Acknowledgements
  12. References

Melanocytes find many ways to transform, as evidenced by the inherent molecular and phenotypic heterogeneity of melanoma. In keeping with this heterogeneity, the p53 pathway may be dysregulated in many ways during melanocyte transformation. This review outlines the known mechanisms for disrupting the p53 pathway during melanomagenesis and highlights the current questions and active areas of investigation in the field. Molecular status of a number of p53 pathway members are indicators of prognosis and these proteins are currently under study for potential chemotherapeutic targeting. The essential role p53 plays during skin pigmentation is also discussed and we outline how p53 may participate in an inherent sensitivity to UV induced melanocyte transformation in individuals with poor melanin synthesis capability.

The p53 pathway members

  1. Top of page
  2. Summary
  3. Significance
  4. The p53 pathway members
  5. p53 – an ultraviolet (UV) target in normal melanocytes
  6. p53 mutation and expression in melanoma
  7. MDM2 expression in melanoma
  8. The p16INK4a/p14ARF locus and melanoma
  9. p53 apoptotic target genes –APAF-1 and PUMA
  10. Keratinocyte p53 is the UV transducer in the keratinocyte-melanocyte signaling cycle
  11. Acknowledgements
  12. References

p53 is a transcription factor with a powerful tumor suppressor function. While most tumor suppressors are silenced in tumor cells by missense or frameshift mutations, p53 mutations are mainly missense mutations (80% of tumors) resulting in aberrant proteins with an increased half life and leading to the appearance of dominant negative and/or gain of function phenotypes that are associated with tumor progression and metastasis (Lozano, 2007). Melanoma is one of a number of tumor types where p53 is still wild type, indicating that other events are contributing to p53 inactivation. p53 is kept inactive and at low levels predominantly by Mdm2, the major E3 ubiquitin ligase that binds directly to p53 and ubiquitinates it for subsequent degradation (Iwakuma and Lozano, 2007). Various signals activate p53, such as DNA damage, oncogenic, genotoxic and oxidative stress, leading to the induction of pro-apoptotic genes (BAX, PUMA, APAF-1), cell cycle regulators (p21 and GADD45) or senescence genes depending on signal intensity and cell type (Lozano and Zambetti, 2005; Riley et al., 2008). In tumor cells, the selective pressure to delete or inactivate p53 is very high. This primarily occurs through mutations in p53, amplification/overexpression of its inhibitors like Mdm2, Mdm4 (Mdm2 family member) and loss or inactivation of upstream activators such as p14ARF (p19ARF in mice) and p16INK4a (Toledo and Wahl, 2006). Different combinations of these events aim at diminishing or abolishing wild type p53 levels and activity leading to defective apoptosis, uncontrolled proliferation, and cellular transformation. Thus p53 inactivation or deletion can cooperate with Mdm2 over-expression or deletion, and deletion of p16INK4a, or p19ARF to promote tumorigenesis (Sharpless et al., 2001; Terzian et al., 2008). As the p53 pathway is closely connected to multiple other critical pathways it is only expected that it has an impact on tumors carrying specific genetic or somatic defects such as in melanoma where B-RAF and NRAS mutations are predominant. We will discuss below what is known about p53 pathway alterations in melanoma, their clinical significance and how they may arise during melanoma progression.

p53 – an ultraviolet (UV) target in normal melanocytes

  1. Top of page
  2. Summary
  3. Significance
  4. The p53 pathway members
  5. p53 – an ultraviolet (UV) target in normal melanocytes
  6. p53 mutation and expression in melanoma
  7. MDM2 expression in melanoma
  8. The p16INK4a/p14ARF locus and melanoma
  9. p53 apoptotic target genes –APAF-1 and PUMA
  10. Keratinocyte p53 is the UV transducer in the keratinocyte-melanocyte signaling cycle
  11. Acknowledgements
  12. References

Melanocytes are hardy cells that are programmed to survive for the entire life of the organism in an environment that carries a high mutagenic stress, the same environment that stimulates apoptosis in neighboring keratinocytes. Furthermore, melanocytes are responsible for melanin production which itself is a toxic process that produces free radicals. In fact, melanin biosynthesis is restricted to membrane bound intracellular organelles known as melanosomes possibly to limit damage to the melanocyte. To facilitate survival under these conditions, melanocytes have developed an attenuated p53 dependent apoptotic response after UV exposure. Melanocytes are known to express high levels of BCL2, an important inhibitor of the p53 target gene, BAX (Nishimura et al., 2005). Bcl2-null mice develop almost complete greying by 3 weeks of age due to melanocyte death. Melanocytes also employ other mechanisms to ensure survival after UV exposure, one of which is to upregulate the GADD45a gene that inhibits apoptosis (Fayolle et al., 2008). GADD45a is a p53 responsive gene, however, in melanocytes and melanoma cells, GADD45a expression is upregulated in a p53 independent fashion. Intrinsic apoptosis resistance through these and other mechanisms is thought to account for the inherent chemotherapeutic resistance of melanoma (Soengas and Lowe, 2003).

As UV irradiation of melanocytes clearly induces p53 stabilization and activation, UV responsive genes in the melanocyte are also potential p53 target genes. Nevertheless, while Bax and p21 may be readily activated in melanocytes after UV exposure (Marrot et al., 2005), a number of reports suggest that specific pigmentation genes may be upregulated by p53 after UV (Khlgatian et al., 2002; Nylander et al., 2000). A complete picture of p53 responsive gene activation without the broad spectrum effects of UV has to our knowledge not yet been reported. One study has reported microarray gene expression profiling on UVB irradiated normal melanocytes in culture and observed a clear p53 expression signature (Yang et al., 2006). Studies in normal melanocytes and in melanoma cells have shown that tyrosinase, the rate limiting enzyme in melanogenesis, is upregulated by p53 (Khlgatian et al., 2002). However, no p53 binding site has been identified in tyrosinase regulatory sequences, so the interaction may be indirect. The tyrosinase related protein TRP1 is also involved in melanogenesis and has a verified p53 binding site in its immediate regulatory sequences (Nylander et al., 2000). While other pigmentation genes demonstrate increased expression after UV, there have not yet been reports of cell autonomous p53 regulation.

p53 itself may be a melanoma risk gene. The hypomorphic Arg72Pro allele of p53 is associated with melanoma risk, particularly in individuals of dark complexion who do not carry MC1R variants (Stefanaki et al., 2007). While a clear picture is still emerging as to the functional consequences of p53 activation in melanocytes, the balance of these studies suggest that p53 plays a general anti-tumor role in melanocytes. Studies in transgenic mice expressing a melanocyte specific activated HRAS allele on a p53-null background provide strong evidence for p53 as an important melanoma tumor suppressor (Bardeesy et al., 2001).

p53 mutation and expression in melanoma

  1. Top of page
  2. Summary
  3. Significance
  4. The p53 pathway members
  5. p53 – an ultraviolet (UV) target in normal melanocytes
  6. p53 mutation and expression in melanoma
  7. MDM2 expression in melanoma
  8. The p16INK4a/p14ARF locus and melanoma
  9. p53 apoptotic target genes –APAF-1 and PUMA
  10. Keratinocyte p53 is the UV transducer in the keratinocyte-melanocyte signaling cycle
  11. Acknowledgements
  12. References

As p53 is the central member of a critical pathway for virtually all tumor types, it is important to know how alterations to this pathway occur during melanoma progression. A number of groups have reported mutational analysis of the p53 gene in melanoma. Low frequency (0–10%) of p53 mutation or loss of heterozygosity in melanoma was observed (Albino et al., 1994; Castresana et al., 1993; Hartmann et al., 1996; Lubbe et al., 1994; Montano et al., 1994; Papp et al., 1996; Sparrow et al., 1995b; Volkenandt et al., 1991). A high proportion of these mutations are UV signature mutations (Hocker and Tsao, 2007). These studies also identified frequent p53 protein expression in melanoma detected by immunohistochemistry, with both staining intensity and number of positive cells increasing with progression (Figure 1). In one report, 33% of nevi, 35% of primary melanomas and 70% of metastatic melanomas were positive for p53 (Sparrow et al., 1995b). Other studies described similar or lower frequencies of p53 protein expression in human melanoma (Albino et al., 1994; Essner et al., 1998; Poremba et al., 1995). In another report, high levels of p53 mRNA were observed suggesting that upregulation of p53 expression may be one mechanism to account for increased protein levels (Weiss et al., 1993, 1995a), however, due to the low frequency of this event, it might be assumed that protein stabilization is a much more important mechanism. As stable levels of wild type p53 is associated with better prognosis, patients with superficial spreading melanoma showed a longer relapse-free period and a better survival when p53 was detected (Florenes et al., 1994) and improved survival (Essner et al., 1998) with fewer relapses (Weiss et al., 1995b). However, two conflicting reports suggested that p53 overexpression may be associated with a worse prognosis (McGregor et al., 1993; Sparrow et al., 1995a).

image

Figure 1.  Schematic representation of protein expression changes for p53 pathway members during melanoma progression. Green triangles represent proteins such as p53 and MDM2 that accumulate in an increasing proportion of cases at each stage of progression. Red triangles represent proteins that lose expression during disease progression. Possible mechanisms to explain these expression changes are listed on each row. For example, MDM2, p21 and BAX are known targets of p53. Therefore it may be possible that elevated p53 levels also result in increases in expression of these target genes.

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Elevated p53 in melanoma may well have some functional implications since the p53 target gene, p21 tends to have a protein expression pattern that mirrors that observed for p53 where expression increases with progression (Sauroja et al., 2000; Sparrow et al., 1998; Trotter et al., 1997). BAX is a p53 pro-apoptotic target that also has a p53-like protein expression pattern during tumor progression, however, tumors with high levels of BAX are resistant to apoptosis, suggesting that other pro-apoptotic p53 targets over-ride the effects of BAX expression (Tang et al., 1998).

As another potential mechanism for attenuation of p53 activity in melanoma cells, small molecular weight isoforms of p53 are expressed during melanocyte transformation (Avery-Kiejda et al., 2008). The small p53 isoforms may play an inhibitory role for p53 transactivation, and the ratio of small isoforms to full-length protein may potentially regulate melanoma cell responses to apoptosis inducing drugs such as cisplatin. Recently, an ‘epithelial-like’ melanoma sub-type was identified that had significantly higher frequency of p53 inactivation (Shields et al., 2007). This melanoma subtype has wild type NRAS and BRAF together with low ERK activity and decreased levels of ERK regulated gene transcripts, suggesting that melanomagenesis is uniquely not dependent upon MAP Kinase pathway activation in these cases.

MDM2 expression in melanoma

  1. Top of page
  2. Summary
  3. Significance
  4. The p53 pathway members
  5. p53 – an ultraviolet (UV) target in normal melanocytes
  6. p53 mutation and expression in melanoma
  7. MDM2 expression in melanoma
  8. The p16INK4a/p14ARF locus and melanoma
  9. p53 apoptotic target genes –APAF-1 and PUMA
  10. Keratinocyte p53 is the UV transducer in the keratinocyte-melanocyte signaling cycle
  11. Acknowledgements
  12. References

In melanoma progression, MDM2 was shown to be highly expressed in 6% of dysplastic nevi, 27% of melanoma in situ, and 56% of invasive primary and metastatic melanomas (Polsky et al., 2001). Amplification of the MDM2 locus was only observed in one melanoma, suggesting that its protein stability is somehow altered with tumor progression. In another study, approximately 3.8% of melanomas had DNA amplification of the CDK4/MDM2 region of 12q14 (Bastian et al., 2003). Nevertheless, amplification of MDM2 in melanoma is relatively infrequent while its overexpression is a more common event. In the follow-up studies, MDM2 protein expression was statistically associated with improved disease free survival (Berger et al., 2004; Polsky et al., 2002). Similar results have been observed for other tumor types where Mdm2 and p53 are both high (Valentin-Vega et al., 2007). The MDM2-p53 interaction has been targeted in many tumor types using rationally designed small molecule inhibitors of MDM2, however, few reports have appeared investigating this therapeutic strategy in melanoma, although early reports of molecules that interfere with MDM2 function showed some promising data on p53 activation in melanoma cell lines (Smalley et al., 2007). One possible explanation for MDM2 overexpression in melanoma is the kinase AKT3 which is commonly upregulated in melanoma, with recurrent amplification of a broad chromosomal region containing this gene (Stahl et al., 2004). AKT kinases are known to upregulate MDM2 (Moumen et al., 2007). Alternatively, MDM2 may be elevated because of elevated p53 as they are both involved in an autoregulatory loop where MDM2 is a transcriptional target of p53.

The p16INK4a/p14ARF locus and melanoma

  1. Top of page
  2. Summary
  3. Significance
  4. The p53 pathway members
  5. p53 – an ultraviolet (UV) target in normal melanocytes
  6. p53 mutation and expression in melanoma
  7. MDM2 expression in melanoma
  8. The p16INK4a/p14ARF locus and melanoma
  9. p53 apoptotic target genes –APAF-1 and PUMA
  10. Keratinocyte p53 is the UV transducer in the keratinocyte-melanocyte signaling cycle
  11. Acknowledgements
  12. References

The Ink4a/Arf locus contains the open reading frames of two proteins: p16INK4a and p14ARF. p16INK4a is a CDK4-associated protein that inhibits CDK4/6-mediated phosphorylation of the retinoblastoma tumor suppressor (pRb) (Serrano et al., 1993) resulting in cell cycle arrest. Somatic loss of p16INK4a is one of the most common events in human cancer [reviewed in (Ruas and Peters, 1998)].

p14ARF and p16INK4a share exons two and three while exon 1β encoding p14ARF occurs 13 kb upstream of exon 1α encoding p16INK4a. This alternative reading frame arrangement ensures that p14ARF and p16INK4a are not isoforms and share no amino acid homology and resulted in the designation ARF for the exon 1β derived transcript (Quelle et al., 1995). Genetic and biochemical studies have shown that p14ARF binds MDM2 and inhibits the ubiquitination of p53, resulting in p53 protein stabilization and activation (Kamijo et al., 1998; Pomerantz et al., 1998; Stott et al., 1998). p16INK4a can activate p53 independently of p14ARF (Sharpless et al., 2001; Terzian et al., 2008).

As the discovery of point mutations in p16INK4a in kindreds with familial melanoma, the p16INK4a/p19ARF locus at 9p21 has been intensively studied in melanoma. Germline p16INK4a/p19ARF mutations occur in around 20–40% of familial melanoma kindreds. Due to the sharing of exon 2, it has been estimated that as many as 40% of the mutations that affect p16INK4a also affect p14ARF (Hayward, 2000; Satyamoorthy and Herlyn, 2003). Gene defects affecting only p14ARF have been described in a few melanoma families, indicating that p14ARF is an independent melanoma susceptibility gene (Hewitt et al., 2002; Randerson-Moor et al., 2001; Rizos et al., 2001). LOH of the p16INK4a/p19ARF locus is present in approximately 50% of sporadic melanomas. This mutation frequency indicates that other mechanisms such as methylation are involved in silencing the remaining p16INK4a/p19ARF allele (Schwabe and Lubbert, 2007). Loss of protein expression of p16INK4a/p19ARF occurs in 50–70% of invasive melanomas with a high proliferative capacity (Dahl and Guldberg, 2007). Several reports have compared the differences in percentage of 9p LOH between various types of nevi, and have generally found lower ranges in banal compared with dysplastic nevi. Overall, the number of mutations in nevi is low. In a recent comparative study using p16Ink4a-null, p19Arf-null and p53-null mice, p19Arf was shown to have an important p53 independent antitumor role through regulation of melanocyte proliferation and replicative senescence (Ha et al., 2007).

p53 apoptotic target genes –APAF-1 and PUMA

  1. Top of page
  2. Summary
  3. Significance
  4. The p53 pathway members
  5. p53 – an ultraviolet (UV) target in normal melanocytes
  6. p53 mutation and expression in melanoma
  7. MDM2 expression in melanoma
  8. The p16INK4a/p14ARF locus and melanoma
  9. p53 apoptotic target genes –APAF-1 and PUMA
  10. Keratinocyte p53 is the UV transducer in the keratinocyte-melanocyte signaling cycle
  11. Acknowledgements
  12. References

Apoptosis protease-activating factor-1 (APAF-1) is a key regulator of the mitochondrial apoptosis pathway and a member of the multimeric apoptosome that also consists of procaspase-9 and cytochrome c. APAF-1 was initially identified through a microarray analysis of p53 responsive genes (Kannan et al., 2001) and the p53 transactivation site has been identified (Robles et al., 2001). As a key mediator of p53 dependent apoptosis, APAF-1 is downregulated at both the protein and mRNA level during melanoma progression and is another example of how melanoma cells evade apoptosis (Soengas et al., 2001). Normal melanocytes are positive for APAF-1 mRNA and protein, however, APAF-1 LOH was detected in 42% of metastatic melanoma samples, and methylation was demonstrated for the remaining allele. Other studies have demonstrated higher levels of APAF-1 protein staining in nevi than in melanomas and have observed a negative correlation between APAF-1 expression level and tumor thickness, and primary melanomas tend to stain more positive than metastatic melanomas (Campioni et al., 2005). Loss of APAF-1 is associated with melanoma progression and is considered an interesting target to try to restore function in melanoma therapy.

p53 upregulated modulator of apoptosis (PUMA) is a BH3-only mitochondrial protein belonging to the BCL2 family of apoptotic regulators. PUMA was independently discovered in 2001 by two groups each screening for p53-inducible target genes (Nakano and Vousden, 2001; Yu et al., 2001). When expressed, PUMA causes rapid apoptosis and growth inhibition, induces cytochrome c release, and activates procaspases 3 and 9. Using tissue microarrays containing dysplastic nevus tissue, primary and metastatic melanoma samples, immunostaining was used to show decreasing expression of PUMA during melanoma progression (Karst et al., 2005). These data were also used to compare PUMA expression with a 5-year survival data. It was clear that those patients with tumors that had the highest levels of PUMA expression had a significantly better survival. Forced expression of PUMA in melanoma cells using an adenoviral vector resulted in significant melanoma cell apoptosis. Thus during melanoma progression, PUMA is silenced by unknown mechanisms and clearly contributes to the patients response during the treatment process.

We have here summarized the major p53 pathway changes that are known to occur during melanoma progression. There is currently no explanation for why p53 levels are elevated with progression, nor is there an understanding as to the factors that determine whether p53 mutation will occur. We speculate that p16Ink4a deletion may be one mechanism to drive increases in p53 expression. p16INK4a/p14ARF deletion or loss of expression is an essential event in melanocyte transformation that is required to overcome melanocyte senescence induced by NRAS or BRAF activation. p53 is probably not frequently mutated in melanoma as since its apoptotic functions are inherently attenuated in melanocytes and p16INK4a/p14ARF are likely to encode a stronger anti-tumor activity. MDM2 may be itself elevated in expression as a result of high p53 levels, as is possible for p21 and BAX since all of these genes are transactivated by p53. It is possible also that MDM2 is up-regulated by AKT3 kinase. The other two p53 target genes that are well studied are PUMA and APAF-1. During melanoma progression these genes are down-regulated and loss of expression likely contributes to melanoma chemoresistance. A better understanding of the mechanisms of p53 pathway rearrangements in melanoma will be necessary to time the molecular steps that occur during transformation and to develop better chemotherapeutic strategies.

Keratinocyte p53 is the UV transducer in the keratinocyte-melanocyte signaling cycle

  1. Top of page
  2. Summary
  3. Significance
  4. The p53 pathway members
  5. p53 – an ultraviolet (UV) target in normal melanocytes
  6. p53 mutation and expression in melanoma
  7. MDM2 expression in melanoma
  8. The p16INK4a/p14ARF locus and melanoma
  9. p53 apoptotic target genes –APAF-1 and PUMA
  10. Keratinocyte p53 is the UV transducer in the keratinocyte-melanocyte signaling cycle
  11. Acknowledgements
  12. References

In addition to its role melanocytes, it is possible that p53 could influence melanoma risk through a non-cell autonomous signaling pathway. The observation that minimally pigmented European skin types are at an elevated melanoma and non-melanoma skin cancer risk compared with heavily pigmented African skin types (50 and 13 times higher in Whites than in Black or African-Americans, respectively) illustrates the importance of pigmentation in preventing all forms of skin cancer. The importance of the keratinocyte-melanocyte signaling cycle is illustrated during the skin sunburn response (Figure 2). After UV exposure of heavily pigmented skin, keratinocytes efficiently upregulate many basic melanocyte functions through secretion of paracrine factors resulting in increased transfer of photoprotective melanins into the surrounding keratinocytes (reviewed in Costin and Hearing, 2007). In contrast, lightly pigmented Caucasian skin has diminished pigment synthesis capability and diminished photoprotection. Therefore, p53 is highly induced in keratinocytes and we propose that this will lead to higher levels of expression of its target genes which include the paracrine factors that are responsible for upregulating melanogenesis and melanocyte proliferation. As the lightest Caucasian skin types are severely compromised in melanin production, we propose that the higher levels of melanocyte signaling factors will favor the melanocyte proliferative response at the same time the skin sustains high levels of UV DNA damage. Many of the paracrine signaling molecules released from keratinocytes after UV exposure are highly mitogenic for melanocytes and are capable of acting as melanocyte tumor promoters in studies using mouse xenografted human skin models (Berking et al., 2004). We therefore propose that this pathway may contribute to the well-known tumor promotion effect of UV irradiation. The idea that p53 may function as a tumor promoter in this unique skin environment is intriguing. Evidence in support of this proposal comes from a number of recent studies that place melanin in the keratinocyte as a UV screen that when present in dark skin strongly diminishes the level of p53 detectable after UV when compared with fair skin where p53 is highly stabilized (Yamaguchi et al., 2006). The observation that p53 in the keratinocyte is capable of binding to and transactivating the pro-opiomelanocortin (POMC) promoter, causing secretion of the subunit melanogenic peptides α-melanocyte stimulating hormone (α-MSH) and adrenocorticotrophic hormone (ACTH) identifies p53 as the transducer of the skin pigmentation signal (Cui et al., 2007).

image

Figure 2.  A role for p53 in the keratinocyte-melanocyte signaling cycle. (A) In dark skin, heavy melanin production limits the p53 dependent sunburn signal induced by UV light, and reduced levels of keratinocyte derived melanocyte growth and pigmentation factors are released from keratinocytes. As melanin synthesis is highly efficient in individuals with dark skin, low levels of signaling factors produce an optimal tanning response. (B) In individuals with fair skin color, melanin biogenesis is genetically compromised. Consequently sunburn results in strong induction of p53 and high production of melanocyte signaling factors. Since many of these signaling factors are mitogenic for melanocytes, this signaling cycle may account for some of the tumor promoting effects of UV irradiation.

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In fact, UV exposure results in release of many paracrine factors that influence skin and melanocyte function. The keratinocyte derived melanocyte growth factors include c-kit ligand (KITLG), endothelin-1 (EDN1), hepatocyte growth factor (HGF), basic fibroblast growth factor (FGF2), α-MSH/ACTH, leukemia inhibitory factor (LIF) and colony stimulating factor 2 (CSF2) among others (reviewed in Costin and Hearing, 2007). These listed factors are responsible for stimulating melanogenesis and melanocyte proliferation. α-MSH/ACTH is thought to have a predominant melanogenic role while KITLG and EDN1 may stimulate melanogenesis and melanocyte proliferation and survival. FGF2, HGF and LIF are potent melanocyte mitogens while CSF2 may influence proliferation, melanogenesis. CSF2 and EDN1 are thought also to stimulate dendrite formation in melanocytes to facilitate pigment transfer to surrounding keratinocytes.

A literature review of skin cytokines that regulate melanocyte proliferation and melanogenesis reveals that many of these molecules are potentially transactivated by p53. Direct p53 binding and regulation of the LIF promoter has been shown in mouse uterus, where alterations in p53 regulated LIF levels influence implantation of blastocysts (Hu et al., 2007). In an interesting twist, the c-Met tyrosine kinase receptor has a functional p53 response element in its regulatory sequences (Seol et al., 1999), as has HGF, its keratinocyte expressed ligand (Metcalfe et al., 1997). These binding sites have not been verified for skin cells. It is possible that other ligand/receptor systems such as KITLG/c-KIT and α-MSH/MC1R are coordinately regulated by p53 in both keratinocytes and melanocytes. Based on p53 binding site analysis, most of the major paracrine factors that are released from keratinocytes after UV exposure are potentially regulated by p53 (Table 1; Wei et al., 2006). As a test, we performed a similar p53 binding site analysis on the CSF2 locus and located at least one potential p53 response element. The EDN1 family member EDN2 has a p53 response element in an intronic sequence and may be released from skin keratinocytes after UVC irradiation (Adur et al., 2007). No reports of a p53 binding site in the EDN1 promoter have yet appeared, however, functional p53 binding sites may be quite degenerate and to our knowledge, no one has performed this analysis. Taken together, these data suggest that keratinocyte p53 is a master regulator of skin cytokine signaling. Identification of the precise profile of p53 regulated signaling molecules that are released from keratinocytes, and fibroblasts, is a high priority.

Table 1.   UV induced keratinocyte derived melanogens and mitogens
Ligandp53 binding site locationReferences
KITLGFirst intronWei et al. (2006)
EDN1Not yet reported 
EDN2EDN2 – intronWei et al. (2006)
EDN3Not yet reported 
FGF25′ (27.5 kb)Wei et al. (2006)
HGF3′ (78.6 kb)Wei et al. (2006)
POMC (α-MSH/ACTH)5′ 300 bpCui et al. (2007)
LIFFirst intronHu et al. (2007)
GM-CSFNot yet reported 

The idea that these keratinocyte derived melanocyte proliferation factors may be important for melanoma comes from a variety of sources. First, a transgenic mouse model over-expressing HGF in keratinocytes is sensitive to melanoma development (Noonan et al., 2001). Second, adenovirus infections to sustain gene transfer into human skin xenografted onto the back of immuno-compromised mice shows that overexpression of FGF2 in combination with UV exposure is capable of inducing pigmented lesions in 100% of treatments with occasional melanoma (Berking et al., 2001). When adenoviruses expressing KITLG and the EDN1 family member, EDN3 were included in this protocol, melanomas developed in 89% of treatments after only 4 weeks (Berking et al., 2004). Third, melanoma progression is dependent on autocrine expression of these same growth factors (Giehl et al., 2007). It should be noted also that there is a body of literature stating that in the weeks after sunburn, normal adult melanocyte content increases in skin (Yamaguchi et al., 2008). These data suggest that the tanning response may include a melanocyte mitogenic component where additional melanocytes are recruited to the burned area. It is precisely this response that we propose is p53 regulated and accentuated in Caucasian skin with limited melanin content.

References

  1. Top of page
  2. Summary
  3. Significance
  4. The p53 pathway members
  5. p53 – an ultraviolet (UV) target in normal melanocytes
  6. p53 mutation and expression in melanoma
  7. MDM2 expression in melanoma
  8. The p16INK4a/p14ARF locus and melanoma
  9. p53 apoptotic target genes –APAF-1 and PUMA
  10. Keratinocyte p53 is the UV transducer in the keratinocyte-melanocyte signaling cycle
  11. Acknowledgements
  12. References