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.

A switch in the expression of embryonic EMT-inducers drives the development of malignant melanoma.

Aberrant expression of embryonic epithelial-mesenchymal transition-inducing transcription factors (EMT-TFs) in epithelial cells triggers EMT, neoplastic transformation, stemness, and metastatic dissemination. We found that regulation and functions of EMT-TFs are different in malignant melanoma. SNAIL2 and ZEB2 transcription factors are expressed in normal melanocytes and behave as tumor-suppressor proteins by activating an MITF-dependent melanocyte differentiation program. In response to NRAS/BRAF activation, EMT-TF network undergoes a profound reorganization in favor of TWIST1 and ZEB1. This reversible switch cooperates with BRAF in promoting dedifferentiation and neoplastic transformation of melanocytes. We detected EMT-TF reprogramming in late-stage melanoma in association with enhanced phospho-ERK levels. This switch results in E-cadherin loss, enhanced invasion, and constitutes an independent factor of poor prognosis in melanoma patients.

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.

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.