Consideration of possible effects of vitamin D on established cancer, with reference to malignant melanoma.

Epidemiological studies indicate that Vitamin D has a beneficial, inhibitory effect on cancer development and subsequent progression, including melanoma (MM), and favourable MM outcome has been reported as directly related to vitamin D3 status, assessed by serum 25-hydroxyvitamin D3 (25[OH]D3 ) levels taken at diagnosis. It has been recommended that MM patients with deficient levels of 25(OH)D3 be given vitamin D3 . We examine possible beneficial or detrimental effects of treating established cancer with vitamin D3 . We consider the likely biological determinants of cancer outcome, the reported effects of vitamin D3 on these in both cancerous and non-cancerous settings, and how the effect of vitamin D3 might change depending on the integrity of tumour vitamin D receptor (VDR) signalling. We would argue that the effect of defective tumour VDR signalling could result in loss of suppression of growth, reduction of anti-tumour immunity, with potential antagonism of the elimination phase and enhancement of the escape phase of tumour immunoediting, possibly increased angiogenesis but continued suppression of inflammation. In animal models, having defective VDR signalling, vitamin D3 administration decreased survival and increased metastases. Comparable studies in man are lacking but in advanced disease, a likely marker of defective VDR signalling, studies have shown modest or no improvement in outcome with some evidence of worsening. Work is needed in assessing the integrity of tumour VDR signalling and the safety of vitamin D3 supplementation when defective.

Compromised vitamin D receptor signalling in malignant melanoma is associated with tumour progression and mitogen-activated protein kinase activity.

The aims of this study were to investigate, in cutaneous malignant melanoma (MM), the integrity of nuclear vitamin D receptor (VDR) signalling, as implied by VDR subcellular location; to investigate the relationship between VDR and tumour progression and the inhibitory effect on VDR by mitogen-activated protein kinase (MAPK) overactivity. Archived tissue from 34 benign melanocytic naevi, 149 MMs and 44 matched metastases were stained by immunohistochemistry for VDR and a subset of primary MMs were stained for phosphorylated-extracellular signal-regulated kinase as a marker of MAPK activity. MM cell lines were investigated to show the subcellular location of VDR and cell viability in response to ligand±MAPK inhibitor. Benign melanocytic naevi showed mainly a strong nuclear VDR staining in contrast to MM where decreased nuclear and emergent cytoplasmic VDRs were associated with malignant progression in terms of dermal invasion and metastasis. MMs that retained exclusive nuclear VDR at the tumour base did not metastasize, a potentially important prognostic indicator. Decreased nuclear VDR correlated with increased cytoplasmic staining, suggesting the failure of nuclear entry as a primary cause of defective VDR signalling in MM. The histological subset analysis and MM cell line studies confirmed the inhibitory effect of MAPK activity on VDR signalling, but the pattern of VDR subcellular localization suggested failure of VDR nuclear entry as a primary effect of MAPK activity rather than direct inhibition of VDR-regulated transcription. Furthermore, high MAPK activity in tumours expressing cytoplasmic VDR was associated with worsened prognosis.

The unfavorable effect of the A allele of the vitamin D receptor promoter polymorphism A-1012G has different mechanisms related to susceptibility and outcome of malignant melanoma.

The A allele of the A-1012G (rs4516035) vitamin D receptor (VDR) promoter polymorphism is associated with increased susceptibility and worsened outcome in malignant melanoma (MM). The A allele contains a GATA-3 binding site. There is a second polymorphism in the same promoter region, G-1520C (rs7139166), and there is potential for another GATA binding site in the G allele. Here, we tested the hypothesis that the G(-1520)A(-1012) haplotype might be a greater risk factor for MM than A-1012 alone. The A allele of A-1012G was preferentially linked to G of G-1520C and was more frequent in MM patients (p = 0.011) but G of G-1520C was not (p = 0.756). The CA haplotype was a very significant risk factor for MM (p = 0.0001) while the CG haplotype was protective (p = 0.014, combined model p = 0.00002). There was no effect of GA haplotype (p = 0.931), suggesting that that the difference in frequencies of the A allele between patients and controls was accounted for by the differences in frequencies of the CA haplotype. The A allele of A-1012G was more frequent in patients with metastasis (p = 0.054) than MM patients without metastasis, as was the G allele of G-1520C (p = 0.028). The GA haplotype was more frequent in patients with metastasis (p = 0.015), while frequencies of CA were similar. We suggest that the different roles of the A allele of A-1012G in susceptibility and metastasis risk may be a function of the availability of transcription factors in the differing cellular backgrounds related to susceptibility and progression of MM.

Evidence that oxidative stress is a risk factor for the development of squamous cell carcinoma in renal transplant patients.

Renal transplant patients are at a greatly increased risk of skin malignancy, particularly squamous cell carcinoma (SCC), a tumor closely associated with UV exposure. There is also significant interindividual skin cancer risk among transplant patients, with evidence suggesting that this derives from variation in response to oxidative stress. Our aim was to assess urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), by liquid chromatography-tandem mass spectrometry, in renal transplant patients with and without SCC. The relationships between SCC and urinary 8-oxodG were analyzed by conditional logistic regression and those between 8-oxodG and other candidate variables by linear regression, correcting for the effect of SCC. In SCC patients, urinary 8-oxodG was significantly elevated (p=0.03), both pre- and post-tumor development, compared to non-SCC transplant patients. Secondary analyses indicated that 8-oxodG was related to current heavy smoking (p=0.02) and darker skin type (p=0.02), but not measures of previous chronic sun exposure or current age and gender. Although subject numbers were limited, immunosuppression with azathioprine was positively associated with 8-oxodG in all patients combined (p=0.02). These results demonstrate, for the first time, that a subpopulation of renal transplant patients is under greater oxidative burden, and it is this population that is particularly predisposed to skin cancer.

The development and characterization of an in vitro model of psoriasis.

In this study, the phenotype of psoriatic keratinocytes and fibroblasts in reconstructed skin models was compared to those constructed from normal cells. Characterization of this model by immunohistochemistry showed that classical markers of keratinocyte differentiation exhibited similar patterns of distribution in the psoriatic models to those derived from normal cells and generally reflected in vivo observations. Some crucial differences, however, were observed between normal and psoriatic models when pro-inflammatory gene expression and keratinocyte proliferation were investigated. Notably, the chemokine receptor CXCR2 was overexpressed in the psoriatic models, and, moreover, was localized to the granular layer of keratinocytes as seen in psoriasis in vivo. Pro-inflammatory genes (tumor necrosis factor alpha [TNF-alpha], interferon gamma [IFN-gamma], and interleukin 8 [IL-8]) were expressed at high levels in the psoriatic models, but were only minimally expressed in the normal models. Models derived from uninvolved psoriatic skin showed the same gene expression profile as those derived from involved skin along with an increased proliferation rate when compared to normal models. These results suggest that psoriatic individuals possess an inherent predisposition to develop the disease phenotype even in the absence of T cells. This study represents a comprehensive characterization of psoriatic human skin reconstructed in vitro, and demonstrates the potential of this model as a valuable tool in drug discovery.

Quantification of UVR-induced DNA damage: global- versus gene-specific levels of thymine dimers.

The induction and repair of DNA damage has been shown to occur heterogeneously throughout the mammalian genome. As a consequence, analysis of these parameters at a global genome level may not reflect important gene-level events. Few techniques have been established to explore quantitatively gene-specific DNA damage and repair. Most of these are polymerase chain reaction (PCR)-based assays and are relatively insensitive, relying on decreased PCR amplification arising from damage in template DNA. We have developed a quantitative assay that combines specific immunocapture of damaged DNA by an antiserum specific for thymine dimers (IgG479), with PCR amplification of a 149 bp fragment of the human H-ras proto-oncogene. Quantification of DNA damage was based upon proportionality between the amount of the PCR product and the initial amount of damage. Detection of thymine dimers was possible with nanogram amounts of genomic DNA and increased in a linear, dose-responsive manner. Using this assay, gene-level induction of thymine dimers was shown to be directly proportional to levels induced in the global genome of ultraviolet radiation (UVR)-exposed, extracted DNA as measured by gas chromatography-mass spectrometry (GC-MS). This result suggests that global damage assessments do indeed reflect gene-level events although we predict that this relationship may not be maintained when applied to a cellular system. These findings demonstrate the suitability of this approach to the detection of UVR-induced DNA damage at the level of individual genes.

Association between functional polymorphism in EGF gene and malignant melanoma.

BACKGROUND: Malignant melanoma, the most serious cutaneous malignancy, has attracted substantial attention because of its rapidly increasing incidence and the poor prognosis of some tumours. Little is known of the genetic factors that mediate susceptibility to, and outcome of, sporadic malignant melanoma. Because of its role in mitogenesis, which is especially relevant to wound healing, tumorigenesis, and proliferation of epidermal tissues, epidermal growth factor (EGF) is an attractive candidate in which to look for genetic polymorphisms. METHODS: We enrolled 135 white European patients with malignant melanoma and 99 healthy white European controls, and screened a selection of DNA samples for polymorphisms in the promoter and 5' untranslated region of the EGF gene by analysis. We then screened DNA samples from all participants for the identified polymorphism by restriction-fragment-length polymorphism (RFLP) analysis. In-vitro EGF production was measured in peripheral-blood mononuclear cells from 34 controls, and the results were compared with the individuals' EGF genotypes. FINDINGS: We identified a single nucleotide substitution (G to A) at position 61 of the EGF gene. Allele frequencies in the controls were 56% EGF 61*A and 44% EGF 61*G. Cells from individuals homozygous for the 61*A allele produced significantly less EGF than cells from 61*G homozygotes (p=0.0004) or heterozygous A/G individuals (p=0.001). Compared with the A/A genotype, G/G was significantly associated with Breslow thickness (p=0.045) and with risk of malignant melanoma (odds ratio 4.9 [95% CI 2.3-10.2], p<0.0001). INTERPRETATION: This study suggests that high EGF production might be important in the development of malignant melanoma.