SNPs at miR-155 binding sites of TYRP1 explain discrepancy between mRNA and protein and refine TYRP1 prognostic value in melanoma.

BACKGROUND: We previously demonstrated an inverse correlation between tyrosinase-related protein 1 (TYRP1) mRNA expression in melanoma metastases and patient survival. However, TYRP1 protein was not detected in half of tissues expressing mRNA and did not correlate with survival. Based on a study reporting that 3' untranslated region (UTR) of TYRP1 mRNA contains two miR-155-5p (named miR-155) binding sites exhibiting single-nucleotide polymorphisms (SNPs) that promote (matched miRNA-mRNA interaction) mRNA decay or not (mismatched), we aimed to investigate the role of miR-155 in the regulation of TYRP1 mRNA expression and protein translation accounting for these SNPs. METHODS: The effect of miR-155 on TYRP1 mRNA/protein expression was evaluated in two melanoma cell lines harbouring matched or mismatched miR-155-TYRP1 mRNA interaction after transfection with pre-miR-155. In parallel, 192 skin and lymph node melanoma metastases were examined for TYRP1 mRNA/protein, miR-155 and SNPs and correlated with patient survival. TYRP1 mRNA, SNPs at its 3'UTR and miR-155 were analysed by RT-qPCR, whereas TYRP1 protein was evaluated by western blot in cell lines and by immunohistochemistry in metastatic tissues. RESULTS: The miR-155 induced a dose-dependent TYRP1 mRNA decay and hampered its translation into protein in the line with the 'match' genotype. In melanoma metastases, TYRP1 mRNA inversely correlated with miR-155 expression but not with TYRP1 protein in the 'match' group, whereas it positively correlated with protein but not with miR-155 in the 'mismatch' group. Consequently, in the latter group, TYRP1 protein inversely correlated with survival. CONCLUSION: Polymorphisms in 3'UTR of TYRP1 mRNA can affect TYRP1 mRNA regulation by miR-155 and its subsequent translation into protein. These SNPs can render TYRP1 mRNA and protein expression nonsusceptible to miR-155 activity and disclose a prognostic value for TYRP1 protein in a subgroup of melanoma patients. These data support the interest in the prognostic value of melanogenic markers and propose TYRP1 to refine prognosis in patients with advanced disease.

Prognostic and predictive values of oncogenic BRAF, NRAS, c-KIT and MITF in cutaneous and mucous melanoma.

BACKGROUND: Mutations of BRAF, NRAS and c-KIT oncogenes are preferentially described in certain histological subtypes of melanoma and linked to specific histopathological features. BRAF-, MEK- and KIT-inhibitors led to improvement in overall survival of patients harbouring mutated metastatic melanoma. OBJECTIVES: To assess the prevalence and types of BRAF, NRAS, c-KIT and MITF mutations in cutaneous and mucous melanoma and to correlate mutation status with clinicopathological features and outcome. METHODS: Clinicopathological features and mutation status of 108 samples and of 98 consecutive patients were, respectively, assessed in one retrospective and one prospective study. Clinicopathological features were correlated with mutation status and the predictive value of these mutations was studied. RESULTS: This work identified significant correlations between BRAF mutations and melanoma occurring on non-chronic sun-damaged skin and superficial spreading melanoma (P < 0.05) on one hand, and between NRAS mutations and nodular melanoma (P < 0.05) on the other hand. Younger age (P < 0.05), microscopic (P < 0.05) and macroscopic (P < 0.05) lymphatic involvement at diagnosis of primary melanoma were significantly linked to BRAF mutations. A mutated status was a positive predictive factor of a response to BRAF inhibitors (OR = 3.44). Mutated melanoma showed a significantly (P = 0.038) higher objective response rate to cytotoxic chemotherapy (26.3%) than wild-type tumours (6.7%). CONCLUSION: Clinical and pathological characteristics of the primary melanoma differed between wild-type and BRAF- or NRAS-mutated tumours. Patients with BRAF-mutated tumours were younger at diagnosis of primary melanoma. Patients carrying mutations showed better responses better to specific kinase inhibitors and interestingly also to systemic cytotoxic chemotherapy.

Tyrosinase-related protein 1 mRNA expression in lymph node metastases predicts overall survival in high-risk melanoma patients.

BACKGROUND: Clinical outcome of high-risk melanoma patients is not reliably predicted from histopathological analyses of primary tumours and is often adjusted during disease progression. Our study aimed at extending our previous findings in skin metastases to evaluate the prognostic value of tyrosinase-related protein 1 (TYRP1) in lymph node metastases of stages III and IV melanoma patients. METHODS: TYRP1 mRNA expression in 104 lymph node metastases was quantified by real-time PCR and normalised to S100 calcium-binding protein B (S100B) mRNA expression to correct for tumour load. TYRP1/S100B ratios were calculated and median was used as cutoff value. TYRP1/S100B mRNA values were correlated to clinical follow-up and histopathological characteristics of the primary lesion. RESULTS: A high TYRP1/S100B mRNA ratio significantly correlated with reduced disease-free (DFS) and overall survival (OS; Cox regression analysis, P=0.005 and 0.01, respectively), increased Breslow thickness (Spearman's rho test, P<0.001) and the presence of ulceration (Mann-Whitney test, P=0.02) of the primaries. Moreover, high TYRP1/S100B was of better prognostic value (lower P-value) for OS than Breslow thickness and ulceration. Finally, it was well conserved during disease progression with respect to high/low TYRP1 groups. CONCLUSION: High TYRP1/S100B mRNA expression in lymph node metastases from melanoma patients is associated with unfavourable clinical outcome. Its evaluation in lymph node metastases may refine initial prognosis for metastatic patients, may define prognosis for those with unknown or non-evaluable primary lesions and may allow different management of the two groups of patients.

TYRP1 mRNA expression in melanoma metastases correlates with clinical outcome.

BACKGROUND: Clinical outcome of patients with high-risk melanoma cannot be reliably predicted on the basis of classical histopathological examination. Our study aimed to determine in melanoma metastases a gene expression profile associated with patient survival, and to identify and validate marker(s) of poor clinical outcome. METHODS: Skin and lymph node metastases from melanoma patients (training population) were used to identify candidate prognostic marker(s) based on DNA microarray analysis. Additional skin metastases (validation population) were used to assess the prognostic value of the first ranked gene by real-time PCR. RESULTS: We performed microarray analysis in the training population and generated a list of 278 probe sets associated with a shorter survival. We used the first ranked gene, tyrosinase-related protein 1 (TYRP1), further measured its expression in the validation population by real-time PCR and found it to be significantly correlated with distant metastasis-free survival (DMFS), overall survival (OS) and Breslow thickness. We also found that it was fairly well conserved in the course of the disease regardless of the delay to metastasis occurrence. Finally, although Tyrp1 protein (immunohistochemistry (IHC)) was only detected in about half of the samples, we showed that its expression also correlated with Breslow thickness. CONCLUSION: Our data indicate that TYRP1 mRNA expression level, at least in skin metastases, is a prognostic marker for melanoma, and is particularly useful when prognostic pathology parameters at the primary lesion are lacking. Its conserved expression further supports its use as a target for therapy.

A new mechanism of action for skin whitening agents: binding to the peroxisome proliferator-activated receptor.

Synopsis Octadecenedioic acid is known as a skin whitening agent but its activity is not mediated via a direct inhibition of tyrosinase. Based on the secondary properties of this molecule, such as its anti-inflammatory and anti-ageing effects, we postulated that octadecenedioic acid interacted with the peroxisome proliferator-activated receptor (PPAR) as this nuclear receptor also mediates these effects. Using reporter gene technology, we were indeed able to demonstrate binding of octadecenedioic acid to all three PPAR subtypes, in particular PPARgamma with an EC(50)-value of approx. 1 x 10(-6) m. Binding to PPARgamma of octadecenedioic acid or rosiglitazone, a known pharmaceutical PPARgamma agonist, led to reduced melanogenesis. Subsequently also tyrosinase mRNA (as measured by real-time polymerase chain reaction) and tyrosinase levels (as measured by Western blot) were reduced, suggesting the existence of a complete novel mechanism of skin whitening agents: binding to PPARgamma results in reduced tyrosinase mRNA expression which in turn results in less tyrosinase being formed. This in turn leads to reduced melanogenesis both in vitro and in vivo Because octadecenedioic acid binds not only to PPARgamma but also to PPARalpha and PPARdelta, other efficacies mediated via these receptors may also be expected.

CDK/CK1 inhibitors roscovitine and CR8 downregulate amplified MYCN in neuroblastoma cells.

To understand the mechanisms of action of (R)-roscovitine and (S)-CR8, two related pharmacological inhibitors of cyclin-dependent kinases (CDKs), we applied a variety of '-omics' techniques to the human neuroblastoma SH-SY5Y and IMR32 cell lines: (1) kinase interaction assays, (2) affinity competition on immobilized broad-spectrum kinase inhibitors, (3) affinity chromatography on immobilized (R)-roscovitine and (S)-CR8, (4) whole genome transcriptomics analysis and specific quantitative PCR studies, (5) global quantitative proteomics approach and western blot analysis of selected proteins. Altogether, the results show that the major direct targets of these two molecules belong to the CDKs (1,2,5,7,9,12), DYRKs, CLKs and CK1s families. By inhibiting CDK7, CDK9 and CDK12, these inhibitors transiently reduce RNA polymerase 2 activity, which results in downregulation of a large set of genes. Global transcriptomics and proteomics analysis converge to a central role of MYC transcription factors downregulation. Indeed, CDK inhibitors trigger rapid and massive downregulation of MYCN expression in MYCN-amplified neuroblastoma cells as well as in nude mice xenografted IMR32 cells. Inhibition of casein kinase 1 may also contribute to the antitumoral activity of (R)-roscovitine and (S)-CR8. This dual mechanism of action may be crucial in the use of these kinase inhibitors for the treatment of MYC-dependent cancers, in particular neuroblastoma where MYCN amplification is a strong predictor factor for high-risk disease.

The Usf-1 transcription factor is a novel target for the stress-responsive p38 kinase and mediates UV-induced Tyrosinase expression.

The stress-activated signalling cascade leading to phosphorylation of the p38 family of kinases plays a crucial role during development and in the cellular response to a wide variety of stress-inducing agents. Although alterations in gene expression characteristic of the stress response require the regulation of key transcription factors by the p38 family, few downstream targets for this signalling pathway have been identified. By examining the ability of pigment cells to respond to UV irradiation as part of the UV-induced tanning response, we show that while the microphthalmia-associated transcription factor Mitf regulates basal Tyrosinase expression, it is the ubiquitous basic helix-loop-helix-leucine zipper transcription factor Usf-1 that is required for the UV activation of the Tyrosinase promoter. Consistent with this we demonstrate that Usf-1 is phosphorylated and activated by the stress-responsive p38 kinase. The results suggest that activation of Usf-1 by p38 at a wide variety of viral and cellular promoters will provide a link between stimuli as diverse as UV irradiation, glucose, viral infection and pro-inflammatory cytokines, and the changes in gene expression associated with the stress response.

E-box activator of the C4 promoter is related to but distinct from the transcription factor upstream stimulating factor.

The murine C4 promoter contains a motif (E-C4) active in transcriptional activation whose structure complies with the E-box consensus sequence recognized by the helix-loop-helix transcription factors. This site is found also in human and rat C4 promoters and has the structure (-75) CACGTG (-70) characteristic of the class B subset of b-helix-loop-helix-zipper proteins. We have challenged the hypothesis that the protein factor responsible for the E-C4-mediated transcriptional activation is identical to one of the previously characterized nuclear factors. The molecular mass of the E-C4 factor is slightly bigger than that of Hela upstream stimulating factor (USF) (43/44 kDa). Moreover, the nucleotides immediately adjoining the E-C4 core sequence contribute to the distinctive fine specificity of the E-C4 factor. Optimized USF and MYC DNA-binding sites, which differ in the nucleotides bordering the hexanucleotide box displace the E-C4 factor in competition assays but with lesser efficiency than the E-C4 site itself. Finally, the E-C4 factor fails to exhibit the heat resistance characteristic of USF proteins. The results show that the E-C4 transcription factor has DNA binding properties overlapping those of other helix-loop-helix proteins but is structurally distinct from the factors so far described. Although C4 is not the first liver gene endowed with an E-box-mediated activation, it affords the first example where such activation takes place in the context of a TATA-less promoter, and is functionally linked to an initiator element-dependent transcription.

Recognition of the E-C4 element from the C4 complement gene promoter by the upstream stimulatory factor-1 transcription factor.

Activation of complement gene expression plays a major role in the response to antigenic challenge. The induction of complement synthesis occurs primarily in liver and in macrophages and is mediated, at least in part, by increased transcription of the complement genes. For example, transcription of the C4 complement gene, which plays a crucial role in the complement pathway, is induced in response to acute inflammation or tissue injury. Previous work has defined the elements present in the C4 complement gene promoter that are required for its expression. Particularly important is an E-box motif, E-C4, that is conserved between the mouse, human, and rat promoters and that directed up to 90% of transcription from the mouse C4 promoter. Here we have purified the E-C4-binding factor to homogeneity using a novel and rapid affinity purification procedure. Following N-terminal microsequencing and subsequent isolation of the corresponding cDNA, the factor binding the E-C4 element was identified as upstream stimulatory factor-1 (USF-1), a basic helix-loop-helix-leucine zipper transcription factor. We also show for the first time that in vivo USF-1 is a phosphoprotein, but that phosphorylation of USF-1 is severely reduced in cells in culture. Moreover, the phosphorylated form of USF-1 binds DNA preferentially, indicating that phosphorylation may enhance the ability of USF-1 to bind DNA. The implications of USF-1 phosphorylation for C4 complement gene expression and transcription regulation are discussed.

Promoter elements of the mouse complement C4 gene critical for transcription activation and start site location.

We have explored the template and factor requirements for transcription of the gene encoding the murine complement component C4, expressed predominantly but not exclusively in liver and mononuclear phagocytes. Competition experiments in transcription assays with liver nuclear extracts show that the regions upstream of the transcription initiation site are largely dispensable for obtaining basal levels of accurately initiated transcription. Activated transcription, however, depends on three upstream regulatory factors, two of which interact with target sites seemingly related to NF-1 (region -112/-87) and USF (region -85/-64), respectively. A third upstream regulatory factor has been detected by the surprising finding that double-stranded oligomers covering sequences proximal to the cap site (position -48 to -7) stimulate transcription from the C4 promoter specifically. Results of nucleotide deletions and site-directed mutations argue that the C4 initiator, that is, the most critical element for basal and accurate transcription of the gene, overlaps the cap site and extends into the transcribed sequences (-1 to +12). Immediately downstream of this region lies a last regulatory element (within the +5 to +43 boundaries) indispensable for high levels of transcription. These data assume wider interest because the C4 promoter does not contain TATA or CAAT boxes and does not feature any of the elements characteristic of the TATA-less genes so far reported.

Pax3 and regulation of the melanocyte-specific tyrosinase-related protein-1 promoter.

Previous work has established that the melanocyte-specific tyrosinase-related protein-1 (TRP-1) promoter is regulated positively by the microphthalmia-associated transcription factor Mitf, acting through the conserved M box and negatively by the T-box factor Tbx2, which can bind two "melanocyte-specific elements" termed the MSEu and MSEi. Both the MSEu and MSEi, which share a 6-base pair GTGTGA consensus, are also recognized by a previously unidentified melanocyte-specific factor, MSF. Here we show using a combination of DNA binding assays, proteolytic clipping, and anti-Pax3 antibodies that MSF is indistinguishable from Pax3, a paired homeodomain transcription factor implicated genetically in melanocyte development and the regulation of the Mitf promoter. Consistent with Pax3 being able to bind the TRP-1 promoter, Pax3 is expressed in melanocytes and melanomas, and TRP-1 promoter activity is up-regulated by Pax3. The results identify a novel role for Pax3 in the expression of TRP-1, and the potential role of Pax3 in the melanocyte lineage is discussed.