Myc, Aurora Kinase A, and mutant p53(R172H) co-operate in a mouse model of metastatic skin carcinoma.

Clinical observations, as well as data obtained from the analysis of genetically engineered mouse models, firmly established the gain-of-function (GOF) properties of certain p53 mutations. However, little is known about the underlying mechanisms. We have used two independent microarray platforms to perform a comprehensive and global analysis of tumors arising in a model of metastatic skin cancer progression, which compares the consequences of a GOF p53(R172H) mutant vs p53 deficiency. DNA profiling revealed a higher level of genomic instability in GOF vs loss-of-function (LOF) p53 squamous cell carcinomas (SCCs). Moreover, GOF p53 SCCs showed preferential amplification of Myc with a corresponding increase in its expression and deregulation of Aurora Kinase A. Fluorescent in situ hybridization confirmed amplification of Myc in primary GOF p53 SCCs and its retention in metastatic tumors. We also identified by RNA profiling distinct gene expression profiles in GOF p53 tumors, which included enriched integrin and Rho signaling, independent of tumor stage. Thus, the progression of GOF p53 papillomas to carcinoma was marked by the acquisition of epithelial-to-mesenchymal transition and metastatic signatures. In contrast, LOF p53 tumors showed enrichment of genes associated with cancer proliferation and chromosomal instability. Collectively, these observations suggest that genomic instability has a prominent role in the early stages of GOF p53 tumor progression (that is, papillomas), whereas it is implicated at a later stage in LOF p53 tumors (that is, SCCs). This model will allow us to identify specific targets in mutant p53 SCCs, which may lead to the development of new therapeutic agents for the treatment of metastatic SCCs.

The p53-Mdm2 network in progenitor cell expansion during mouse postnatal development.

Mdm2, an E3 ubiquitin ligase, negatively regulates the tumour suppressor p53. Loss of Mdm2 in mice results in p53-dependent apoptosis and embryonic lethality. This phenotype was rescued by the p53(515C) allele, which encodes an apoptosis-deficient p53R172P protein. However, these mice died within 2 weeks of birth, due to a severe impairment of progenitor cell expansion during postnatal haematopoiesis and cerebellar development, leading to p53-dependent cell cycle arrest. Loss of Mdm2 led to phosphorylation of the p53R172P protein, p53R172P stability and activation of the cell cycle inhibitor p21 in proliferating cells, but not in differentiated cells, in multiple tissue compartments. Proliferating cells of epithelial origin were not affected. The haematopoietic and neural defects were alleviated in mice lacking Mdm2 and containing one p53(515C) and one p53-null allele, but spermatogenesis was arrested. These findings establish a crucial role for the p53-Mdm2 network in regulating proliferation and progenitor expansion in many cell lineages and have important implications for the use of drugs that aim to disrupt the p53-Mdm2 interaction.

Genetic association and cellular function of MC1R variant alleles in human pigmentation.

We have examined MC1R variant allele frequencies in the general population of South East Queensland and in a collection of adolescent dizygotic and monozygotic twins and family members to define statistical associations with hair and skin color, freckling, and mole count. Results of these studies are consistent with a linear recessive allelic model with multiplicative penetrance in the inheritance of red hair. Four alleles, D84E, R151C, R160W, and D294H, are strongly associated with red hair and fair skin with multinomial regression analysis showing odds ratios of 63, 118, 50, and 94, respectively. An additional three low-penetrance alleles V60L, V92M, and R163Q have odds ratios 6, 5, and 2 relative to the wild-type allele. To address the cellular effects of MC1R variant alleles in signal transduction, we expressed these receptors in permanently transfected HEK293 cells. Measurement of receptor activity via induction of a cAMP-responsive luciferase reporter gene found that the R151C and R160W receptors were active in the presence of NDP-MSH ligand, but at much reduced levels compared with that seen with the wild-type receptor. The ability to stimulate phosphorylation of the cAMP response element binding protein (CREB) transcription factor was also apparent in all stimulated MC1R variant allele-expressing HEK293 cell extracts as assessed by immunoblotting. In contrast, human melanoma cell lines showed wide variation in the their ability to undergo cAMP-mediated CREB phosphorylation. Culture of human melanocytes of known MC1R genotype may provide the best experimental approach to examine the functional consequences for each MC1R variant allele. With this objective, we have established more than 300 melanocyte cell strains of defined MC1R genotype.

The human melanocortin-1 receptor locus: analysis of transcription unit, locus polymorphism and haplotype evolution.

The complete sequence of the MC1R locus has been assembled, the coding region of the gene is intronless and placed within a 12 kb region flanked by the NULP1 and TUBB4 genes. The immediate promoter region has an E-box site with homology to the M-box consensus known to bind the microphthalmia transcription factor (MITF); however, promoter deletion analysis and transactivation studies have failed to show activation through this element by MITF. Polymorphism within the coding region, immediate 5' promoter region and a variable number tandem repeat (VNTR) minisatellite within the locus have been examined in a collection of Caucasian families and African individuals. Haplotype analysis shows linkage disequilibrium between the VNTR and MC1R coding region red hair variant alleles which can be used to estimate the age of these missense changes. Assuming a mean VNTR mutation rate of 1% and a star phylogeny, we estimate the Arg151Cys variant arose 7500 years before the present day, suggesting these variants may have arisen in the Caucasian population more recently than previously thought.

Human pigmentation genes: identification, structure and consequences of polymorphic variation.

The synthesis of the visible pigment melanin by the melanocyte cell is the basis of the human pigmentary system, those genes directing the formation, transport and distribution of the specialised melanosome organelle in which melanin accumulates can legitimately be called pigmentation genes. The genes involved in this process have been identified through comparative genomic studies of mouse coat colour mutations and by the molecular characterisation of human hypopigmentary genetic diseases such as OCA1 and OCA2. The melanocyte responds to the peptide hormones alpha-MSH or ACTH through the MC1R G-protein coupled receptor to stimulate melanin production through induced maturation or switching of melanin type. The pheomelanosome, containing the key enzyme of the pathway tyrosinase, produces light red/yellowish melanin, whereas the eumelanosome produces darker melanins via induction of additional TYRP1, TYRP2, SILV enzymes, and the P-protein. Intramelanosomal pH governed by the P-protein may act as a critical determinant of tyrosinase enzyme activity to control the initial step in melanin synthesis or TYRP complex formation to facilitate melanogenesis and melanosomal maturation. The search for genetic variation in these candidate human pigmentation genes in various human populations has revealed high levels of polymorphism in the MC1R locus, with over 30 variant alleles so far identified. Functional correlation of MC1R alleles with skin and hair colour provides evidence that this receptor molecule is a principle component underlying normal human pigment variation.

A polymorphism study of the human Agouti gene and its association with MC1R.

To determine whether the Agouti Signalling Protein (ASP) gene is associated with skin and hair coloration in humans, the complete coding region of ASP was screened for polymorphisms. Analysis of ASP in Caucasian, African-American, Spanish Basque, Hispanic, Apache and Australian Aboriginal populations revealed no amino acid substitutions. A single polymorphism in the 3' untranslated region occurred at a frequency of 0.2 in African-Americans. Variants of the Melanocortin 1 Receptor (MC1R) gene have been found to be associated with red hair and fair skin in humans. Red hair individuals are usually compound heterozygotes or homozygous for one of a number of MC1R polymorphisms associated with red hair. Some individuals however are heterozygous for only one of these polymorphisms and dizygotic twins can be concordant for MC1R variants but discordant for hair colour. A recent study has also identified rare redheads carrying no MC1R variants indicating that polymorphisms of the human MC1R gene are required but not sufficient for the red hair phenotype. To address the question of whether ASP also contributes to the red hair phenotype, individuals previously identified as having unexpected MC1R genotypes were screened for polymorphisms at the ASP locus. No polymorphisms were found in any of these individuals. Results indicate that the human ASP gene is unlikely to function in normal human pigmentation in the same way as MC1R.

MC1R genotype modifies risk of melanoma in families segregating CDKN2A mutations.

Mutations in the exons of the cyclin-dependent kinase inhibitor gene CDKN2A are melanoma-predisposition alleles which have high penetrance, although they have low population frequencies. In contrast, variants of the melanocortin-1 receptor gene, MC1R, confer much lower melanoma risk but are common in European populations. Fifteen Australian CDKN2A mutation-carrying melanoma pedigrees were assessed for MC1R genotype, to test for possible modifier effects on melanoma risk. A CDKN2A mutation in the presence of a homozygous consensus MC1R genotype had a raw penetrance of 50%, with a mean age at onset of 58.1 years. When an MC1R variant allele was also present, the raw penetrance of the CDKN2A mutation increased to 84%, with a mean age at onset of 37.8 years (P=.01). The presence of a CDKN2A mutation gave a hazard ratio of 13.35, and the hazard ratio of 3.72 for MC1R variant alleles was also significant. The impact of MC1R variants on risk of melanoma was mediated largely through the action of three common alleles, Arg151Cys, Arg160Trp, and Asp294His, that have previously been associated with red hair, fair skin, and skin sensitivity to ultraviolet light.

Melanocortin-1 receptor genotype is a risk factor for basal and squamous cell carcinoma.

MC1R gene variants have previously been associated with red hair and fair skin color, moreover skin ultraviolet sensitivity and a strong association with melanoma has been demonstrated for three variant alleles that are active in influencing pigmentation: Arg151Cys, Arg160Trp, and Asp294His. This study has confirmed these pigmentary associations with MC1R genotype in a collection of 220 individuals drawn from the Nambour community in Queensland, Australia, 111 of whom were at high risk and 109 at low risk of basal cell carcinoma and squamous cell carcinoma. Comparative allele frequencies for nine MC1R variants that have been reported in the Caucasian population were determined for these two groups, and an association between prevalence of basal cell carcinoma, squamous cell carcinoma, solar keratosis and the same three active MC1R variant alleles was demonstrated [odds ratio = 3.15 95% CI (1.7, 5.82)]. Three other commonly occurring variant alleles: Val60Leu, Val92Met, and Arg163Gln were identified as having a minimal impact on pigmentation phenotype as well as basal cell carcinoma and squamous cell carcinoma risk. A significant heterozygote effect was demonstrated where individuals carrying a single MC1R variant allele were more likely to have fair and sun sensitive skin as well as carriage of a solar lesion when compared with those individuals with a consensus MC1R genotype. After adjusting for the effects of pigmentation on the association between MC1R variant alleles and basal cell carcinoma and squamous cell carcinoma risk, the association persisted, confirming that presence of at least one variant allele remains informative in terms of predicting risk for developing a solar-induced skin lesion beyond that information wained through observation of pigmentation phenotype.

Melanocortin-1 receptor polymorphisms and risk of melanoma: is the association explained solely by pigmentation phenotype?

Risk of cutaneous malignant melanoma (CMM) is increased in sun-exposed whites, particularly those with a pale complexion. This study was designed to investigate the relationship of the melanocortin-1 receptor (MC1R) genotype to CMM risk, controlled for pigmentation phenotype. We report the occurrence of five common MC1R variants in an Australian population-based sample of 460 individuals with familial and sporadic CMM and 399 control individuals-and their relationship to such other risk factors as skin, hair, and eye color; freckling; and nevus count. There was a strong relationship between MC1R variants and hair color and skin type. Moreover, MC1R variants were found in 72% of the individuals with CMM, whereas only 56% of the control individuals carried at least one variant (P<.001), a finding independent of strength of family history of melanoma. Three active alleles (Arg151Cys, Arg160Trp, and Asp294His), previously associated with red hair, doubled CMM risk for each additional allele carried (odds ratio 2.0; 95% confidence interval 1. 6-2.6). No such independent association could be demonstrated with the Val60Leu and Asp84Glu variants. Among pale-skinned individuals alone, this association between CMM and MC1R variants was absent, but it persisted among those reporting a medium or olive/dark complexion. We conclude that the effect that MC1R variant alleles have on CMM is partly mediated via determination of pigmentation phenotype and that these alleles may also negate the protection normally afforded by darker skin coloring in some members of this white population.

Human pigmentation genetics: the difference is only skin deep.

There is no doubt that visual impressions of body form and color are important in the interactions within and between human communities. Remarkably, it is the levels of just one chemically inert and stable visual pigment known as melanin that is responsible for producing all shades of humankind. Major human genes involved in its formation have been identified largely using a comparative genomics approach and through the molecular analysis of the pigmentary process that occurs within the melanocyte. Three classes of genes have been examined for their contribution to normal human color variation through the production of hypopigmented phenotypes or by genetic association with skin type and hair color. The MSH cell surface receptor and the melanosomal P-protein are the two most obvious candidate genes influencing variation in pigmentation phenotype, and may do so by regulating the levels and activities of the melanogenic enzymes tyrosinase, TRP-1 and TRP-2.

Complete sequence and polymorphism study of the human TYRP1 gene encoding tyrosinase-related protein 1.

The complete 24,667 nucleotide sequence spanning the human TYRP1 gene has been determined from the inserts of two overlapping lambda clones. A LINE-1 repeat element is immediately adjacent to and may demarcate the immediate 5' promoter region of the gene. A search for polymorphism within the seven TYRP1 coding exons has been performed by an RNase mismatch detection procedure. Analysis of the TYRP1 gene in 100 Caucasian individuals of varying hair color has found no amino acid sequence variation nor revealed any hemizygous mutant allele in the hypopigmented phenotype of two 9p- syndrome patients.

Rufous oculocutaneous albinism in southern African Blacks is caused by mutations in the TYRP1 gene.

Oculocutaneous albinism (OCA) is the most common autosomal recessive disorder among southern African Blacks. There are three forms that account for almost all OCA types in this region. Tyrosinase-positive OCA (OCA2), which is the most common, affects approximately 1/3,900 newborns and has a carrier frequency of approximately 1/33. It is caused by mutations in the P gene on chromosome 15. Brown OCA (BOCA) and rufous OCA (ROCA) account for the majority of the remaining phenotypes. The prevalence of BOCA is unknown, but for ROCA it is approximately 1/8,500. Linkage analysis performed on nine ROCA families showed that ROCA was linked to an intragenic marker at the TYRP1 locus (maximum LOD score = 3.80 at straight theta=.00). Mutation analysis of 19 unrelated ROCA individuals revealed a nonsense mutation at codon 166 (S166X) in 17 (45%) of 38 ROCA chromosomes, and a second mutation (368delA) was found in an additional 19 (50%) of 38 chromosomes; mutations were not identified in the remaining 2 ROCA chromosomes. In one family, two siblings with a phenotypically unclassified form of albinism were found to be compound heterozygotes for mutations (S166X/368delA) at the TYRP1 locus and were heterozygous for a common 2.7-kb deletion in the P gene. These findings have highlighted the influence of genetic background on phenotype, in which the genotype at one locus can be influenced by the genotype at a second locus, leading to a modified phenotype. ROCA, which in southern African Blacks is caused by mutations in the TYRP1 gene, therefore should be referred to as "OCA3," since this is the third locus that has been shown to cause an OCA phenotype in humans.

Characterization of melanocyte stimulating hormone receptor variant alleles in twins with red hair.

The association between MSHR coding region variation and hair colour in humans has been examined by genotyping 25 red haired and 62 non-red Caucasians, all of whom were 12 years of age and members of a twin pair study. Twelve amino acid substitutions were seen at 11 different sites, nine of these being newly described MSHR variants. The previously reported Val92Met allele shows no association with hair colour, but the three alleles Arg151Cys, Arg160Trp and Asp294His were associated with red hair and one Val60Leu variant was most frequent in fair/blonde and light brown hair colours. Variant MSHR genotypes are associated with lighter skin types and red hair (P < 0.001). However, comparison of the MSHR genotypes in dizygotic twin pairs discordant for red hair colour indicates that the MSHR gene cannot be solely responsible for the red hair phenotype, since five of 13 pairs tested had both haplotypes identical by state (with three of the five having both identical by descent). Rather, it is likely that additional modifier genes exist, making variance in the MSHR gene necessary but not always sufficient, for red hair production.

Chromosomal structure of the human TYRP1 and TYRP2 loci and comparison of the tyrosinase-related protein gene family.

The structures of the human tyrosinase-related protein genes TYRP1 and TYRP2 have been determined and compared with that of the tyrosinase gene (TYR). The TYRP1 protein is encoded in 7 exons spread over 24 kb of genomic DNA. Characterization of a 55-kb contig encompassing the human TYRP2 locus reveals that the protein coding region is divided into 8 exons. All three members of the TYRP gene family share a common C-terminal membrane spanning exon. Examination of the position of other intron junctions suggests that TYRP1 was derived from a TYR duplication and then was itself duplicated to give rise to the TYRP2 gene. The evidence also suggests that at least some of the introns within the TYR, TYRP1, and TYRP2 coding regions were gained after duplication and that intron slippage is unlikely to have occurred.