Downregulation of the FTO m6A RNA demethylase promotes EMT-mediated progression of epithelial tumors and sensitivity to Wnt inhibitors.

Post-transcriptional modifications of RNA constitute an emerging regulatory layer of gene expression. The demethylase fat mass- and obesity-associated protein (FTO), an eraser of N6-methyladenosine (m6A), has been shown to play a role in cancer, but its contribution to tumor progression and the underlying mechanisms remain unclear. Here, we report widespread FTO downregulation in epithelial cancers associated with increased invasion, metastasis and worse clinical outcome. Both in vitro and in vivo, FTO silencing promotes cancer growth, cell motility and invasion. In human-derived tumor xenografts (PDXs), FTO pharmacological inhibition favors tumorigenesis. Mechanistically, we demonstrate that FTO depletion elicits an epithelial-to-mesenchymal transition (EMT) program through increased m6A and altered 3'-end processing of key mRNAs along the Wnt signaling cascade. Accordingly, FTO knockdown acts via EMT to sensitize mouse xenografts to Wnt inhibition. We thus identify FTO as a key regulator, across epithelial cancers, of Wnt-triggered EMT and tumor progression and reveal a therapeutically exploitable vulnerability of FTO-low tumors.

Acquired resistance to BRAFi reverses senescence-like phenotype in mutant BRAF melanoma.

Targeting MAPK pathway in mutant BRAF melanoma with the specific BRAF inhibitor vemurafenib showed robust initial responses in the majority of patients followed by relapses due to acquired resistance to the drug. In V600EBRAF melanoma cell lines, senescence-associated ß-galactosidase activity is often encountered in a constitutive manner or induced after MAPK inhibition. However, the link between the senescence-like phenotype and the resistance to BRAF inhibition is not fully understood yet. Our data validate a senescence-like phenotype (low cell proliferation, high cell volume, and high ß-Gal activity) in mutant BRAF cells. Vemurafenib increased ß-Gal activity in 4 out of 5 sensitive lines and in 2 out of 5 lines with intrinsic resistance to the drug. Interestingly, the 3 lines with acquired resistance to vemurafenib became depending on the drug for proliferation. In absence of drug, these lines showed a lower cell proliferation rate together with a substantial increase of ß-Gal activity both in vitro and in vivo. In all settings, the senescence-like phenotype was significantly associated with an inhibition of pRB and cyclin D1, explaining the inhibition of cell proliferation. In conclusion, ß-Gal activity is increased by V600EBRAF inhibition in the majority of sensitive and intrinsically resistant melanoma cells. Acquired resistance to vemurafenib is associated with a dependence to the drug for cell proliferation and tumor growth, and, in this case, drug removal stimulate ß-Gal activity suggesting that the senescence-like phenotype could contribute to the acquired resistance to BRAF inhibition.

Survey on skin-lightening practices and cosmetics in Kigali, Rwanda.

BACKGROUND: The use of skin-lightening (SL) cosmetics appears to be common throughout the world, especially among dark-skinned women from sub-Saharan Africa. OBJECTIVES: The aims of this study were to evaluate the extent of the practice of SL in Kigali, the capital city of Rwanda, the motivations behind this practice and the complexity of the adverse effects observed. METHODS: An inventory of products sold on the Kigali market and their contents were compared with the results of a survey investigating the products used by the local population in order to deduce the proportions of people who depigment. The prevalence and severity of SL side effects (dermatitis, skin cancers, etc.) were evaluated in collaboration with dermatologists and general practitioners through a specific questionnaire and interviews. The sociological profiles of adolescents and their motivations for practicing SL were studied using qualitative and descriptive approaches through semi-direct individual and collective interviews. RESULTS: A total of 27 creams were identified and classified according to labeled ingredients known to be depigmenting agents; 35% of the surveyed population were found to use products with skin-bleaching properties, but only 27% stated that they used the products specifically for these depigmenting properties. An inquiry into the motivations of adolescents indicated that they know about and practice SL but are restricted by family, religion, and Rwandese culture. Whenever side effects appear, consumers opt either to stop bleaching practices for a short period or to switch from their commercial topical product to another one with, presumably, a different composition. CONCLUSIONS: Albeit that many people acknowledge that there are possible side effects of using preparations commonly used in SL, the practice is generally continued. Although it is important to question the rationale behind the practice of SL, it is equally important to develop and propose safer products.

p53 Reactivation by PRIMA-1(Met) (APR-246) sensitises (V600E/K)BRAF melanoma to vemurafenib.

Intrinsic and acquired resistance of metastatic melanoma to (V600E/K)BRAF and/or MEK inhibitors, which is often caused by activation of the PI3K/AKT survival pathway, represents a major clinical challenge. Given that p53 is capable of antagonising PI3K/AKT activation we hypothesised that pharmacological restoration of p53 activity may increase the sensitivity of BRAF-mutant melanoma to MAPK-targeted therapy and eventually delay and/or prevent acquisition of drug resistance. To test this possibility we exposed a panel of vemurafenib-sensitive and resistant (innate and acquired) (V600E/K)BRAF melanomas to a (V600E/K)BRAF inhibitor (vemurafenib) alone or in combination with a direct p53 activator (PRIMA-1(Met)/APR-246). Strikingly, PRIMA-1(Met) synergised with vemurafenib to induce apoptosis and suppress proliferation of (V600E/K)BRAF melanoma cells in vitro and to inhibit tumour growth in vivo. Importantly, this drug combination decreased the viability of both vemurafenib-sensitive and resistant melanoma cells irrespectively of the TP53 status. Notably, p53 reactivation was invariably accompanied by PI3K/AKT pathway inhibition, the activity of which was found as a dominant resistance mechanism to BRAF inhibition in our lines. From all various combinatorial modalities tested, targeting the MAPK and PI3K signalling pathways through p53 reactivation or not, the PRIMA-1(Met)/vemurafenib combination was the most cytotoxic. We conclude that PRIMA-1(Met) through its ability to directly reactivate p53 regardless of the mechanism causing its deactivation, and thereby dampen PI3K signalling, sensitises (V600E/K)BRAF-positive melanoma to BRAF inhibitors.

Melanoma cell attachment, invasion, and integrin expression is upregulated by tumor necrosis factor alpha and suppressed by alpha melanocyte stimulating hormone.

We have previously shown alpha-melanocyte stimulating hormone to protect melanocytes and melanoma cells from the proinflammatory actions of tumor necrosis factor-alpha. The aim of the study was to extend this work to look into the influence of tumor necrosis factor-alpha on melanoma cell attachment, invasion, and integrin expression and ask to what extent alpha-melanocyte stimulating hormone might protect cells from tumor necrosis factor-alpha stimulation of increased integrin expression. HBL human melanoma cells were studied under resting and stressed conditions using tumor necrosis factor-alpha as a proinflammatory cytokine. Functional information on the actions of tumor necrosis factor-alpha on melanoma cells was obtained by examining the strength of attachment of melanoma cells to substrates and the ability of melanoma cells to invade through fibronectin. alpha3, alpha4, and beta1 integrin expression was detected by Western immunoblotting and the ability of alpha-melanocyte stimulating hormone to oppose the actions of tumor necrosis factor-alpha was studied on HBL cell attachment, invasion, and integrin subunit expression. Our results show that tumor necrosis factor-alpha increases the number of melanoma cells attaching to collagen (types I and IV) and tissue culture polystyrene, increases ability to invade through fibronectin, and upregulates the expression of alpha3 (28%), alpha4 (90%), and beta1 (65%) integrin subunit expression. In contrast, alpha-melanocyte stimulating hormone reduced cell attachment, invasion, and integrin expression and opposed the stimulatory effects of tumor necrosis factor-alpha. In conclusion this study provides further evidence of alpha-melanocyte stimulating hormone acting to "protect" melanoma cells from proinflammatory cytokine action. Our data support a hypothesis that an inflammatory environment would promote melanoma invasion and that the anti-invasive actions of alpha-melanocyte stimulating hormone are consistent with its working in an anti-inflammatory capacity.

Inhibition of tumor necrosis factor-alpha stimulated NFkappaB/p65 in human keratinocytes by alpha-melanocyte stimulating hormone and adrenocorticotropic hormone peptides.

Alpha-melanocyte stimulating hormone (alpha-MSH) has pigmentary, anti-inflammatory, antipyretic, and general immunomodulatory roles. It can oppose several cytokines including tumor necrosis factor-alpha in a number of tissues, including skin. We have previously shown that alpha-MSH can inhibit tumor necrosis factor-alpha stimulated intercellular adhesion molecule 1 upregulation and nuclear factor kappaB (NFkappaB) transcription factor activation in melanocyte and melanoma cells. It is thought, however, that this MSH biology may also extend to other cells of the skin and in this study we extend our work to keratinocytes. We have investigated in detail the ability of three alpha-MSH peptides to inhibit tumor necrosis factor alpha stimulated NFkappaB activation in nonpigmentary HaCaT keratinocytes (alpha-MSH, L-Lys-L-Pro-L-Val, and L-Lys-L-Pro-D-Val) and two adrenocorticotropic hormone (ACTH) peptides (1-17 and 1-39), reported to be present in skin tissue. NFkappaB/p65 activation was analyzed by electrophoretic mobility shift assay and immunofluorescent microscopy. alpha-MSH, L-Lys-L-Pro-L-Val, and L-Lys-L-Pro-D-Val all significantly inhibited tumor necrosis factor alpha stimulated NFkappaB activation, whereas ACTH 1-17 and 1-39 did not, in the HaCaT keratinocytes. MSH peptides and ACTH 1-39 were effective, however, at inhibiting NFkappaB activation in normal human keratinocytes. Immunolabeling of inhibitor kappaBalpha of NFkappaB (IkappaBalpha) revealed an abnormal localization to the nucleus of HaCaT cells, which was unaffected by MSH/ACTH peptides. In contrast, normal human keratinocytes showed a normal IkappaBalpha distribution that responded to MSH/ACTH with nuclear translocation. Our data support previous work on the role of MSH/ACTH peptides as immunomodulatory/anti-inflammatory regulators, and extend this work to keratinocytes identifying a novel IkappaBalpha mechanism and extends findings to ACTH peptides, identifying an abnormal IkappaBalpha mechanism in the immortal HaCaT versus normal keratinocyte.

Prominent role of cyclic adenosine monophosphate signalling pathway in the sensitivity of (WT)BRAF/(WT)NRAS melanoma cells to vemurafenib.

Vemurafenib improves survival in patients with melanoma bearing the (V600E)BRAF mutation, but it did not show any benefit in clinical trials focusing on wild type tumours while it may well inhibit (WT)BRAF considering the dosage used and the bioavailability of the drug. As tumours may contain a mixture of mutant and wild type BRAF cells and this has been also put forward as a resistance mechanism, we aimed to evaluate the sensitivity/resistance of six, randomly selected, (WT)BRAF/(WT)NRAS lines to vemurafenib and found four sensitive. The sensitivity to the drug was accompanied by a potent inhibition of both phospho-ERK and phospho-AKT, and a significant induction of apoptosis while absent in lines with intrinsic or acquired resistance. Phospho-CRAF expression was low in all sensitive lines and high in resistant ones, and MEK inhibition can effectively potentiate the drug effect. A possible explanation for CRAF modulation is cyclic adenosine monophosphate (cAMP), a mediator of melanocortin receptor 1 (MC1R) signalling, since it can actually inhibit CRAF. Indeed, we measured cAMP and found that all four sensitive lines contained significantly higher constitutive cAMP levels than the resistant ones. Consequently, vemurafenib and cAMP stimulator combination resulted in a substantial synergistic effect in lines with both intrinsic and acquired resistance but only restricted to those where cAMP was effectively increased. The use of a cAMP agonist overcame such restriction. In conclusion, we report that (WT)BRAF/(WT)NRAS melanoma lines with low phospho-CRAF and high cAMP levels may be sensitive to vemurafenib and that CRAF inhibition through cAMP stimulation may overcome the resistance to the drug.

An ethnobotanical survey of medicinal plants used in Rwanda for voluntary depigmentation.

ETHNOPHARMACOLOGICAL RELEVANCE: Voluntary depigmentation, a very common practice in sub-Saharan Africa, often performed with pharmaceutical products diverted from their pharmacological use, may cause severe dermatological and systemic side effects. The present work aims at investigating whether and which herbs were used in Rwanda for similar purposes before the advent of the current depigmentation craze; this may give clues at herbal treatments possibly advantageous compared to current products. MATERIAL AND METHODS: Sixty-one traditional healers, mostly representatives of their associations, were surveyed by questionnaires for knowledge and practice of voluntary depigmentation. Recipes or plants used, plant parts, harvest area, preparation methods, dosage and route of administration were recorded. Most of the cited herbs were harvested with the help of traditional healers and identified by comparison with voucher specimens; herbal vouchers of the five most cited herbs were deposited in official herbaria. RESULTS: All surveyed traditional healers have knowledge of voluntary depigmentation; the population currently practicing do not recourse to their services but obtain bleaching products directly from the market. Traditional healers disclosed recipes prescribed or self-used (often by women) in their youth; others cited recipes are used to treat skin diseases with properties of "clarification", "black skin stain removal", in cases of hyperpigmentation, and/or "skin softening". Curiously, from the 28 recipes cited by traditional healers, all are mono-herbal preparations; most of the plants are mixed with butter for application to the skin. CONCLUSION: Compared to other pathophysiological conditions, there is currently a very limited use of herbal preparations for depigmentation. Five herbs had a citation percentage equal or above to 50%, Brillantaisia cicatricosa Lindau (Acanthaceae), Chenopodium ugandae (Aellen) Aellen (Chenopodiaceae), Dolichopentas longiflora Oliv. (Rubiaceae), Protea madiensis Oliv. (Proteaceae) and Sesamum angolense Welw. (Pedaliaceae); in vitro experiments indicated a modulation of melanogenesis by these plant extracts, confirming the information obtained from traditional healers.

Tyrosinase modulation by five Rwandese herbal medicines traditionally used for skin treatment.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional herbal medicines provide an interesting, largely unexplored source for the development of potential new drugs and skin-care cosmetics. Some herbal extracts are known to be inhibitors of melanin formation, sometimes more potent than the classical inhibitors, hydroquinone/arbutin or kojic acid, and are not associated with melanocytes cytotoxicity or mutagenicity. Such plants are used in traditional medicine in many countries, particularly in Africa, for skin lightening. AIM OF THE STUDY: To evaluate in vitro the ability of Rwandese medicinal plants, traditionally used for the treatment of skin (discoloration and attenuation of discolored spots), to modulate pigmentation and tyrosinase activity. MATERIALS AND METHODS: Based on an ethnopharmacological survey, five herbs [Brillantaisia cicatricosa Lindau (Acanthaceae), Chenopodium ugandae (Aellen) Aellen (Chenopodiaceae), Dolichopentas longiflora Oliv. (Rubiaceae), Protea madiensis Oliv. (Proteaceae) and Sesamum angolense Welw. (Pedaliaceae)] were selected. Twenty-seven extracts, obtained by treating the herbs with increasing polarities solvents, were investigated for their effects on cell viability (MTT test) and on pigmentation: inhibition of the enzyme tyrosinase (colorimetry of reaction products, measurement of enzyme activity, TLC-autography; studies on crude cellular extracts obtained from normal melanocytes and on a mushroom tyrosinase) and measurement of melanogenesis by human melanoma cells. RESULTS: None of the tested plant extracts were cytotoxic on tested human melanoma cell lines, except for Dolichopentas longiflora (IC50 of leaves n-hexane extract, 4µg/ml for MM028 and 4.5µg/ml for MM001; IC50 of roots ethyl acetate extract, 0.8µg/ml for MM028 and 3.9µg/ml for MM001). Almost all extracts inhibited melanogenesis in a melanoma whole cells overall pigmentation assay, a model reflecting the entire cycle of melanogenesis. All the Protea madiensis extracts quite strongly inhibited melanogenesis and, surprisingly, one of the Dolichopentas longiflora leaves extracts was found to increase melanogenesis. These results were confirmed by the modulation of pigmentation reactions by crude cellular extracts obtained from normal melanocytes; interestingly, one of the extracts (Dolichopentas longiflora ethyl acetate extract) is even more active (61% at 500µg/ml) than kojic acid (<3% at 142µg/ml and 68% at 1421µg/ml). In a mushroom tyrosinase inhibition assay, data obtained on some extracts fairly agree with pigmentation inhibition measured on melanocytes proteins as, for example, the methanol extract of Protea madiensis. While a few others extract display discording data, this probably reflects either differences between human and mushroom tyrosinase, interference with melanocytes enzymes at later steps than tyrosinase or the simultaneous presence of compounds with conflicting activities in a given extract. CONCLUSIONS: Ethnopharmacological data represent an efficient approach to discover active herbs. Some of the selected medicinal plants clearly show potent tyrosinase inhibitions while one extract significantly increases cell pigmentation; one extract contains potent growth melanocytes inhibitors.

Improving the spectrophotometric determination of the alkylating activity of anticancer agents: a new insight into the mechanism of the NBP method.

In this paper, the mechanism of the nitrobenzylpyridine (NBP) method to measure the alkylating activity of drugs originally described by Epstein et al. [J. Epstein, R.W. Rosenthal, R.J. Ess, Anal. Chem. 27 (1955) 1435-1439] and modified later by others was revisited using melphalan, m-sarcolysin, chlorambucil, cyclophosphamide and ifosfamide. Its direct application to determine the activity of these drugs in human serum and aqueous media is described and discussed. This method, based on the formation of a chromophore due to the reaction between the alkylating agent and NBP, was significantly improved by extracting as quickly as possible the reaction product(s) into chloroform before adding alkali to develop the color. This significantly limited the degradation by hydrolysis of the products and enhanced the yield of the end chromophore in the organic phase. The reaction time was optimized by monitoring each compound color development. The best reaction time for each compound was selected and a higher stability of the extracted color over at least 1h was obtained (compared to a couple of minutes in previous studies). Most interestingly, water evaporation due to heating had little or no effect on the linearity of standard curves evaluated in the micromolar concentration range. Both the sensitivity and reproducibility of the method were therefore significantly improved. There appears to be a direct correlation between compound hydrolysis and alkylation activity; the relative reactivity is different among the compounds owing to the rate of (i) production, (ii) the relative proportions and (iii) the hydrolysis of the intermediates. A general mechanism for the nucleophilic competitive substitution is proposed.

Evaluation of the prognostic significance of serum galectin-3 in American Joint Committee on Cancer stage III and stage IV melanoma patients.

Galectin-3 (Gal-3) is a member of the beta-galactoside-binding lectins family and has been implicated in angiogenesis, tumor invasion, and metastatic process in vitro and in vivo. As we showed recently that advanced melanoma patients presented high serum level of Gal-3, we investigated the association of this protein with the outcome of melanoma patients. Whether this protein could be a biomarker has not been assessed, and we compared the prognostic value of serum Gal-3 in multivariate analysis with lactate dehydrogenase, C-reactive protein and S100B. We conclude that Gal-3 could be of prognostic value in melanoma patients; more precisely, this protein has a strong independent prognostic signification with a cut-off value of 10 ng/ml. After these data, we believe that serum Gal-3 measurement can have an important role in the follow-up and management of advanced American Joint Commission on Cancer stage III and stage IV melanoma patients. Further studies will uncover whether Gal-3 will be able to open new therapeutic perspectives.

Genotypic and gene expression studies in congenital melanocytic nevi: insight into initial steps of melanotumorigenesis.

Large congenital melanocytic nevi (CMNs) are said to have a higher propensity to malignant transformation compared with acquired nevi. Thus, they represent a good model for studying initial steps of melanotumorigenesis. We have performed genotypic (karyotype, fluorescence in situ hybridization, and mutational analyses) and differential expression studies on a large cohort of medium (n=3) and large (n=24) CMN. Chromosomal abnormalities were rare and single, a feature probably reflecting the benignity of these lesions. Mutational screening showed a high frequency of NRAS mutations in our series (19/27 cases, 70%), whereas BRAF mutations were less common (4/27 cases, 15%). Differential did not show significant alterations of cellular processes such as cell proliferation, cell migration/invasion, angiogenesis, apoptosis, and immune/inflammatory responses. However, significant downregulation of genes involved in pigmentation and upregulation of genes playing a role in DNA protection were observed. Lastly, our microarrays displayed upregulation of genes mediating chemoresistance in cancer. As alteration of pigmentation mechanisms can trigger oxidative damage, increased expression of genes involved in maintenance of DNA integrity might reflect the ability of nevocytic cells to self-protect against cellular stress. Furthermore, the observed alterations linked to chemoresistance might partially account for the well-known inefficacy of chemotherapy in malignant melanoma.

A novel transport and delivery mechanism underpins the effectiveness of prolyl-m-sarcolysyl-p-fluorophenylalanine (PSF) in a human melanoma xenograft nude-mouse model.

The alkylating peptide PSF shows very promising results in vitro on different cancer cells but its efficacy in animals has not been assessed. Here we evaluate the efficacy of PSF in human melanoma-bearing nude mice and examine the underlying mechanism. In melanoma-bearing nude mice, escalating doses of PSF showed dose-dependent responses and reached tumor regression with an optimal dose of 20 mg/kg for 1 month. A comparison of PSF with its free moiety m-sarcolysin and melphalan showed a highly significant advantage of PSF. Furthermore, dose fractionation yielded an even better control of tumor regrowth. In vitro studies unraveled an original delivery mechanism based on the rapid binding of PSF mainly due to red blood cells to form a pro-drug complex and the subsequent release of active metabolites by tumor-associated proteolytic enzymes. Blood kinetics showed one major metabolite partially released over time, while in the presence of melanoma cells three additional metabolites are generated. Interestingly, tumor-shed proteases also induce the production of these metabolites and varying combinations of enzyme inhibitors indicate the involvement of metallo- and other families of proteases in the delivery process. This particular transport and delivery of such an alkylating agent may have several benefits, mainly lowering the drug-free moiety in plasma and at the same time increasing its concentration in protease rich areas such as tumors.

Chromosomal translocations as a mechanism of BRAF activation in two cases of large congenital melanocytic nevi.

Genetic studies of melanocytic tumors have mainly demonstrated activation of oncogenes such as NRAS or BRAF through point mutations. In two cases of large congenital melanocytic nevi, we observed a chromosomal translocation involving the BRAF oncogene on chromosome 7q34, resulting in both cases in removal of the auto-inhibitory N-terminal regulatory domain (hence the Ras-guanosine triphosphate binding domain) of BRAF from its protein kinase domain. This is early evidence of BRAF activation through chromosomal translocation in melanocytic tumors. Because BRAF point mutations are rather rare in congenital melanocytic nevi and melanoma arising in non-sun-exposed area, the molecular mechanism of oncogenic activation as described here could be a recurrent molecular feature in these groups of melanocytic tumors.