O receptor aril de hidrocarbonetos (AhR) em melanomas: associação com mutações BRAF, com a progressão da doença e identificação de ligantes / The aryl hydrocarbon receptor (AhR) in melanomas: association with BRAF mutations, with disease progression and ligand identification

O melanoma é um tipo de câncer de pele geneticamente diverso, que surge diante das transformações em melanócitos. A mutação BRAFV600E está presente em mais de 90% de todas as mutações em BRAF, sendo assim ocorre em cerca de 50% dos casos registrados. As mutações em NRAS, ocupam o segundo lugar entre as mutações mais prevalentes, cerca de 20% dos casos. Informações sobre as assinaturas genéticas, permitiram o desenvolvimento de terapia alvo dirigida. O Vemurafenib, inibidor da quinase BRAFV600E, apresentou inicialmente resultados bastante satisfatórios, contudo existe registro de casos de recidiva e resistência. O receptor aril de hidrocarbonetos é expresso em vários componentes da pele, e assim está relacionado a homeostase e fisiopatologia da pele. Diante disso, a avaliação da expressão do receptor em um painel de linhagens mutadas para NRAS e BRAF, e BRAF resistentes, mostrou-se maior do que a encontrada em melanócitos. Também encontramos maior expressão de mRNA de AhR em linhagens de melanoma derivadas de sítio primário e metastático, mutadas para BRAFV600E, quando comparadas ao melanócito. Agregado a isto, a análise in silico no TCGA (The Cancer Genome Atlas) mostrou que há 18% de alteração genética em AhR, sendo em maior parte a alta regulação de mRNA. Também, a análise do banco público GSE12391, mostrou aumento de mRNA de AhR na fase de crescimento vertical do melanoma. Assim, concluímos que há maior expressão de mRNA e sua importância nas fases de desenvolvimento do melanoma, tanto nos processos iniciais quanto em processos de migração, invasão e metástase. Ainda, encontramos maior mRNA do receptor em linhagens resistentes ao Vemurafenib. Este resultado sustenta a hipótese de que AhR pode ser considerado um marcador de resistência em melanomas. O AhR, inicialmente no citoplasma, quando ativado pode atuar como fator de transcrição regulando vários genes que apresentam sequências definidas, participando de respostas carcinogênicas. Compostos halogenados e moléculas endógenas derivadas das vias de metabolização do triptofano são agonistas do receptor. Anteriormente, nosso grupo mostrou que linhagens de melanoma incubadas com triptamina e DMT exibiram menor clonogenicidade. Diante de uma literatura escassa sobre o papel do DMT no melanoma e com base nestes resultados, nosso objetivo foi avaliar o papel de AhR nesta interface DMT-melanoma. Para isto, nosso objetivo foi construir linhagem editada geneticamente para AhR através da ferramenta CRISPR-Cas9. Vários foram os esforços, sem sucesso, utilizados nas tentativas de comprovar a manutenção de células editadas na cultura. Atrelamos a este resultado a possibilidade de haver duas subpopulações editadas geneticamente pós CRISPR-Cas9, onde uma destas manteve o padrão de crescimento semelhante às células wild type. Devido a este crescimento diferencial, não obtivemos congruências nos ensaios e postulamos a perda do possível nocaute. A partir disso, realizamos ensaios de interactoma para avaliar a interação de DMT-AhR. Nosso resultado sugere a interação de DMT ao receptor sigma 1, e não ao receptor aril de hidrocarbonetos. Desta forma, o interactoma sustenta a hipótese de que DMT não é um ligante de AhR. Para certificar este resultado análises de docking associados a ensaios biológicos, avaliando o papel do receptor, devem ser realizados para averiguar a afinidade e seletividade de DMT como ligante do receptor na linhagem de melanoma

Melanoma is a genetically diverse type of skin cancer, which arises from changes in melanocytes. The BRAFV600E mutation is present in more than 90% of all BRAF mutations, so it occurs in about 50% of registered cases. Mutations in NRAS occupy the second place among the most prevalent mutations, about 20% of cases. Information on genetic signatures allowed the development of targeted therapy. vemurafenib, kinase inhibitor BRAFV600E, initially presented very satisfactory results, however there is a record of cases of relapse and resistance. The aryl hydrocarbon receptor is expressed in several components of the skin and is thus related to homeostasis and skin pathophysiology. Therefore, the evaluation of receptor expression in a panel of strains mutated to NRAS and BRAF, and resistant BRAF, proved to be greater than that found in melanocytes. We also found main expression of AhR mRNA in melanoma strains derived from primary and metastatic site, mutated to BRAFV600E, when compared to melanocyte. Added to this, the in silico analysis in TCGA (The Cancer Genome Atlas) showed that there is 18% of genetic alteration in AhR, being mostly the high regulation of mRNA. Also, an analysis by the public bank GSE12391, showed an increase in AhR mRNA in the vertical growth phase of melanoma. Thus, it is concluded that there is greater expression of mRNA and its importance in the stages of development of melanoma, both in recent processes and in the processes of migration, invasion and metastasis. In addition, we found higher receptor mRNA in strains resistant to vemurafenib. This result supports the hypothesis that AhR can be considered a marker of resistance in melanomas. AhR, initially in the cytoplasm, when activated can act as a transcription factor regulating several genes that have defined sequences, participating in carcinogenic responses. Along with this, we show that along the tumor progression, there is an increase in AhR in the radial growth phase of melanoma. Halogenated compounds and endogenous molecules derived from the tryptophan metabolism pathways are receptor agonists. Previously, our group showed that melanoma strains incubated with tryptamine and DMT exhibited less clonogenicity. In view of a scarce literature on the role of DMT in melanoma and based on these results, our objective was to evaluate the role of AhR in this DMT-melanoma interface. For this, our goal was to build genetically edited strain for AhR using the CRISPR-Cas9 tool. Several efforts were unsuccessful in attempts to prove the maintenance of cells edited in the culture. We linked to this result the possibility of having two subpopulations genetically edited after CRISPR-Cas9, where one of them maintained the growth pattern like wild type cells. Due to this differential growth, we did not obtain congruence in the tests and postulated the loss of the possible knockout. From that, we performed interactome assays to evaluate the DMT-AhR interaction. Our result suggests the interaction of DMT with the sigma 1 receptor, and not the aryl hydrocarbon receptor. Thus, the interactome supports the hypothesis that DMT is not an AhR ligand. To certify this result, docking analyses associated with biological assays, evaluating the role of the receptor, should be performed to ascertain the affinity and selectivity of DMT as a ligand of the receptor in the melanoma lineage

CD34 defines melanocyte stem cell subpopulations with distinct regenerative properties.

Melanocyte stem cells (McSCs) are the undifferentiated melanocytic cells of the mammalian hair follicle (HF) responsible for recurrent generation of a large number of differentiated melanocytes during each HF cycle. HF McSCs reside in both the CD34+ bulge/lower permanent portion (LPP) and the CD34- secondary hair germ (SHG) regions of the HF during telogen. Using Dct-H2BGFP mice, we separate bulge/LPP and SHG McSCs using FACS with GFP and anti-CD34 to show that these two subsets of McSCs are functionally distinct. Genome-wide expression profiling results support the distinct nature of these populations, with CD34- McSCs exhibiting higher expression of melanocyte differentiation genes and with CD34+ McSCs demonstrating a profile more consistent with a neural crest stem cell. In culture and in vivo, CD34- McSCs regenerate pigmentation more efficiently whereas CD34+ McSCs selectively exhibit the ability to myelinate neurons. CD34+ McSCs, and their counterparts in human skin, may be useful for myelinating neurons in vivo, leading to new therapeutic opportunities for demyelinating diseases and traumatic nerve injury.

Heterogeneous tumor subpopulations cooperate to drive invasion.

Clonal selection and transcriptional reprogramming (e.g., epithelial-mesenchymal transition or phenotype switching) are the predominant theories thought to underlie tumor progression. However, a "division of labor" leading to cooperation among tumor-cell subpopulations could be an additional catalyst of progression. Using a zebrafish-melanoma xenograft model, we found that in a heterogeneous setting, inherently invasive cells, which possess protease activity and deposit extracellular matrix (ECM), co-invade with subpopulations of poorly invasive cells, a phenomenon we term "cooperative invasion". Whereas the poorly invasive cells benefit from heterogeneity, the invasive cells switch from protease-independent to an MT1-MMP-dependent mode of invasion. We did not observe changes in expression of the melanoma phenotype determinant MITF during cooperative invasion, thus ruling out the necessity for phenotype switching for invasion. Altogether, our data suggest that cooperation can drive melanoma progression without the need for clonal selection or phenotype switching and can account for the preservation of heterogeneity seen throughout tumor progression.

Melanoma conjuntival en niños / Conjuntival melanoma in children

Las lesiones melanocíticas de la conjuntiva abarcan un espectro que comprende desde nevus benignos y atípicos hasta el melanoma maligno. El melanoma conjuntival es un tumor poco común de personas adultas, con algunos casos reportados en niños. Las lesiones conjuntivales pigmentadas en niños son predominantemente nevus, y en raras ocasiones pueden sufrir transformación maligna. Se describirá el caso de una niña de 8 años con melanoma conjuntival, quien presentó antecedente de lesión pigmentada en la conjuntiva bulbar del ojo izquierdo desde los 3 años de edad, y la cual creció de forma progresiva.

Melanocytic lesions of the conjunctiva range from benign and atypical nevus to malignant melanoma. Conjuntival melanoma is an uncommon tumor among adults, and few cases have been reported in children. Pigmented conjuntival lesions in children are predominantly nevus, and on rare occasion can become malignant. A case is described of an 8-year old girl with conjuntival melanoma with antecedent of pigmented lesion in the bulbous conjunctiva of the left eye from age 3 which underwent progressive growth.

Two distinct types of mouse melanocyte: differential signaling requirement for the maintenance of non-cutaneous and dermal versus epidermal melanocytes.

Unlike the thoroughly investigated melanocyte population in the hair follicle of the epidermis, the growth and differentiation requirements of the melanocytes in the eye, harderian gland and inner ear - the so-called non-cutaneous melanocytes - remain unclear. In this study, we investigated the in vitro and in vivo effects of the factors that regulate melanocyte development on the stem cells or the precursors of these non-cutaneous melanocytes. In general, a reduction in KIT receptor tyrosine kinase signaling leads to disordered melanocyte development. However, melanocytes in the eye, ear and harderian gland were revealed to be less sensitive to KIT signaling than cutaneous melanocytes. Instead, melanocytes in the eye and harderian gland were stimulated more effectively by endothelin 3 (ET3) or hepatocyte growth factor (HGF) signals than by KIT signaling, and the precursors of these melanocytes expressed the lowest amount of KIT. The growth and differentiation of these non-cutaneous melanocytes were specifically inhibited by antagonists for ET3 and HGF. In transgenic mice induced to express ET3 or HGF in their skin and epithelial tissues from human cytokeratin 14 promoters, the survival and differentiation of non-cutaneous and dermal melanocytes, but not epidermal melanocytes, were enhanced, apparently irrespective of KIT signaling. These results provide a molecular basis for the clear discrimination between non-cutaneous or dermal melanocytes and epidermal melanocytes, a difference that might be important in the pathogenesis of melanocyte-related diseases and melanomas.

Photobiology of ocular melanocytes and melanoma.

There are two different types of ocular melanocytes and melanomas. Conjunctival melanocytes are located on the surface of the eye and are exposed to visible light and UV radiation. Recently, epidemiological studies demonstrated that sunlight plays a definite role in the occurrence of conjunctival melanoma, as it does in cutaneous melanoma. Uveal melanocytes consist of the iridal melanocytes, which are exposed to visible light and UV radiation; and the ciliary body melanocytes and choroidal melanocytes, which are not exposed to light radiation. Epidemiological studies demonstrated that sunlight may play a role in the occurrence of iridal melanoma, but may not be a major factor in the etiology of ciliary body and choroidal melanomas. Uveal melanocytes differ from epidermal melanocytes in that epidermal melanocytes respond to UV radiation and skin color becomes darker after exposure to sunlight; but uveal melanocytes do not respond to UV radiation and the iris color remains stable after exposure to sunlight. Recently, in vitro studies indicate that this phenomenon is determined both by cellular factors and environmental factors.

Lumbar spinal meningeal melanocytoma of the l3 nerve root with paraspinal extension: a case report.

STUDY DESIGN: A case report of spinal meningeal melanocytoma with a dumbbell-shaped extension and its magnetic resonance imaging features is presented. OBJECTIVE: To present a rare spinal tumor with pathologic and radiologic features. SUMMARY OF BACKGROUND DATA: Meningeal melanocytomas are rare lesions usually found in the posterior fossa and upper cervical spine. The review of literature shows the variation in different studies. The characteristic magnetic resonance imaging features of meningeal melanocytoma have not yet been defined. METHODS: A 33-year-old woman presented with a 3-year history of backache and weakness of her left lower limb. Magnetic resonance imaging showed a large dumbbell tumor at L3-L4 with extension in the paraspinal region. Schwannoma was the first possibility suggested by the MRI features. Histopathology of the lesion showed a meningeal melanocytoma. RESULTS: The patient showed a significant recovery after surgery and a full course of radiotherapy. CONCLUSIONS: Radiologic presentation could be confusing in cases of spinal dumbbell-shaped tumors. Awareness of the lesion characteristics will facilitate diagnosis and treatment of this condition.

Studies on epidermis reconstructed with and without melanocytes: melanocytes prevent sunburn cell formation but not appearance of DNA damaged cells in fair-skinned caucasians.

To assess the photoprotective role of melanocytes in the epidermis, we studied the effects of ultraviolet B on an epidermis reconstructed with and without melanocytes. To address more specifically the role of melanin in fair-skinned individuals, experiments were done with cells obtained from human skin of low phototypes (II-III). To study the effect of constitutive melanin and possibly that of newly synthesized melanin precursors, a single dose of ultraviolet B (0.10 or 0.15 J per cm2, corresponding to a 4-5 minimal erythema dose in vivo) was administered to reconstructs and the effects were monitored over the first 24 h. When reconstructs with and without melanocytes were compared, no difference was found for DNA damage/repair assessed with antibodies to cyclobutane pyrimidine dimers and 6-4 photoproducts. More necrotic/apoptotic cells, however, were noted 24 h following ultraviolet B irradiation in reconstructs lacking melanocytes. Twenty-four hours following ultraviolet B irradiation the number of necrotic/apoptotic cells and the number of cyclobutane pyrimidine dimer positive cells was coarsely concentration-dependent. The number of cyclobutane pyrimidine dimer positive cells, however, was independent of the type of reconstruct used (with/without melanocytes). In conclusion, low phototype melanocytes seem to protect epidermal basal cells against ultraviolet B-induced apoptosis/necrosis and may preserve the overall integrity of the epidermis after ultraviolet B irradiation. On the contrary, such melanocytes do not seem to have a protective role against DNA damage and may not prevent cancer.

Indeterminate melanocytic proliferations of the conjunctiva.

OBJECTIVE: To test the hypothesis that a subset of conjunctival melanocytic proliferations exists that cannot be reproducibly classified as benign, malignant, or indeterminate. METHODS: Three groups of excisional biopsy specimens of conjunctival melanocytic proliferations were evaluated by a panel of 5 ophthalmic pathologists. These groups included lesions that we considered to represent benign (group 1 [n = 5]), malignant (group 2 [n = 5]), and indeterminate melanocytic proliferations (group 3 [n = 5]). The panel classified the same sections in all 3 groups in a randomized, masked fashion, first without and then with a clinical history of patient age, sex, and race. The kappa statistic was used to quantify the degree of agreement among observers. RESULTS: There was strong concordance among the panel members for both group 1 (benign [kappa = 0.76]) and group 2 (malignant [kappa = 0.70]) melanocytic proliferations. There was no concordance of the panel for group 3 (indeterminate) lesions (kappa = -0.045). The concordance for groups 1 and 2 and lack of concordance for group 3 lesions were independent of knowledge of clinical history of age, sex, and race. CONCLUSION: A subset of melanocytic proliferations of the conjunctiva exists that cannot be reproducibly classified by pathologists as benign, malignant, or indeterminate.

Abnormal melanosomes: ultrastructural markers of melanocytic atypia.

To identify possible ultrastructural markers of melanocytic atypia, a quantitative ultrastructural study was made of melanocytes found at the dermal-epidermal boundary of normal skin and in benign, premalignant, and malignant melanocytic lesions. There was a significant increase (p < 0.05) in the number of melanosomes per melanocyte in the premalignant and malignant lesions compared with the number observed in the benign lesions. There was a significantly higher number (p < 0.05) of abnormal melanosomes (with irregularities in the laminar matrix or with a granular or clumpy matrix) in the premalignant and malignant lesions, which suggests that the presence of a high percentage of abnormal melanosomes might act as a useful ultrastructural marker in the diagnosis of melanocytic atypia.

Antigen-presenting OKM5+ melanophages appear in human epidermis after ultraviolet radiation.

Ultraviolet radiation of murine skin in vivo or epidermal cells (EC) in vitro dramatically inhibits the antigen-presenting capacity of EC in vitro and results in the inhibition of immune responses to antigen challenge. In humans, UV exposure in vivo markedly inhibits alloantigen presentation by EC in the EC-lymphocyte reaction (ELR) when EC are harvested immediately after the administration of 4 times the minimal erythema dose (4 MED), whereas EC harvested 72 h after 4 MED (UV-EC) exhibit enhanced allostimulatory capacity in the ELR. This enhanced ELR reactivity is due to the appearance, in the epidermis, of bone marrow-derived OKT6- DR+ cells which are distinct from Langerhans cells (LC) in their lack of surface OKT6 and in their ultrastructural morphology. This report focuses on the phenotype and function of T6- Dr+ UV-EC and on their relationship to known human antigen presenting cell (APC) subsets. Approximately 60% of T6- Dr+ UV-EC bore the monocyte marker defined by monoclonal antibody OKM5, but lacked determinants recognized by OKM1, Leu M1, Leu M3, Leu M4, Leu M5, and Mac1. All T6- Dr+ UV-EC bore the class II MHC antigen HLA-DQ (DC/DS), which is associated with a specialized subset of antigen-presenting monocytes capable of stimulation in the autologous mixed leukocyte reaction (AMLR). Panning of OKM5+ UV-EC resulted in a population of cells which was markedly enriched in melanophages and which exhibited potent alloantigen-presenting capacity in the ELR. Since OKM5+ T6- Dr+ UV-EC were similar to the specialized APC minor subset of OKM1- OKM5+ blood monocytes both in phenotype and in apparent phagocytic function, we examined other APC functions of UV-EC to assess the extent of this analogy. Relative to control EC (containing only LC as APC), UV-EC (containing functionally inactivated LC but many T6- Dr+ APC) induced significantly greater degrees of T-cell proliferation in the presence of either tetanus toxoid antigen or the mitogen concanavalin A. UV-EC, as well as panning-purified OKM5+ UV-EC, were also able to induce autologous T-cell proliferation in the absence of added antigen (autologous ELR), in contrast to control EC which were poor stimulators of an autologous ELR. Thus, although human EC 72 h after UV exposure are numerically and functionally depleted of LC, at least 2 additional subsets of T6- Dr+ APC appear in the epidermis.(ABSTRACT TRUNCATED AT 400 WORDS)

[Intraepidermal melanocytic proliferation. A practical guide to the classification of ambiguous melanotic skin lesions and of possible precursors of malignant melanomas]. / Les prolifeérations mélanocytaires intra-épidermiques. Guide pratique de classification des lésions mélaniques cutanées ambigués et de quelques précurseurs possibles des mélanomes malins.

A group of senior Pathologists engaged in review work on international randomized trials for the W.H.O and/or the E.O.R.T.C., propose a new simplified classification of melanocytic lesions with an intra-epidermal component, applicable in routine. This classification attempts to introduce standards to permit morphological identification of a large group of intra-epidermal melanocytic proliferations with three classes of atypia (slight, mild, severe) and group of malignant melanomas especially those without dermal invasion. The new definitions and objective criteria (at cytological and architectural level) of diagnosis are given with examples of equivalence between some established entities and this new universal terminology.