GNAQ and BRAF mutations show differential activation of the mTOR pathway in human transformed cells

Somatic mutations in GNAQ gene were described as being the main oncogenic activation in uveal melanomas, whereas mutations in BRAF gene have been described as a key genetic alteration that contributes to skin melanoma development. We have previously reported differential activation of the MAPK and AKT/mTOR signalling pathways in uveal and skin melanomas harbouring, respectively, GNAQ and BRAF mutations. The aim of this work was to compare the functional effect of GNAQ and BRAF mutations in mTOR and MAPK pathway activation, cell proliferation and apoptosis. In this work, we performed transient transfection of HEK293 cells with BRAFWT, BRAFV 600E, GNAQWT, GNAQQ209P and GNAQQ209L vectors. We treated melanoma cell lines displaying different BRAF and GNAQ mutational status with the mTOR inhibitor RAD001 and with the MEK1/2 inhibitor U0126 and evaluated the effects in the growth of the cell lines and in mTOR and MAPK pathway effectors expression. At variance with the significant increase in the level of pmTOR Ser2448 and pS6 Ser235/236 proteins observed in cells transfected with BRAF vectors, no significant alteration in mTOR pathway effectors was observed in cells transfected with the three GNAQ expressing vectors. Also, GNAQ overexpression enhances Stat3 activation, which might mediate GNAQ oncogenic effects. None of the vectors led to significant differences in proliferation or apoptosis in the transfected cell lines. Cell lines harbouring a BRAF mutation were more sensitive to RAD001 treatment. U0126 leads to the reduction of MAPK and mTOR pathways activation in all cell lines tested. Our results indicate that GNAQ and BRAF activation drive distinct intracellular signalling pathways that may be useful for therapeutic decisions in human melanomas

Tendências do carcinoma espinocelular cutâneo no Hospital de Gaia (2004-20013)

Introduction: Cutaneous squamous cell carcinoma (cSCC) is the second most common skin cancer and its incidence has been rising. The objective of our study was to perform a descriptive and analytical analysis of the cutaneous squamous cell carcinoma excised in the Hospital Center Vila Nova de Gaia e Espinho (CHVNGE) over a period of 10 years and establish trends (incidence, survival and mortality).Material and Methods: Information was retrospectively gathered in the CHVNGE, from January 2004 to December 2013, using the regional cancer registry and the histopathological registry of the hospital. The aim of this study was to describe the characteristics and trends of cutaneous squamous cell carcinoma (incidence, association with actinic keratosis or basal cell carcinoma, survival and mortality rates).Results: 485 cutaneous squamous cell carcinoma were surgically removed in a total of 380 patients (56.1% men and 43.9% women). 361 patients presented invasive cutaneous squamous cell carcinoma and 124 in situ cutaneous squamous cell carcinoma. The Dermatology Department removed 70.4% of the cutaneous squamous cell carcinoma, followed by the Plastics (16.4%) and General Surgery Departments (4.7%). Cutaneous squamous cell carcinoma was more prevalent in the age-group ≥75-years in both sexes (p < 0.001).The mean age of invasive cutaneous squamous cell carcinoma was 76.7 years (±11.5 years), women being older than men (79.0 vs 74.0 years, p < 0.001). The face was the most common topographic location (42.1%), in both genders (p < 0.001). We observed a rising incidence in both genders, particularly in the last study period (16.2/100 000 person-year). The 5-year survival rate was 98.7%. The mean age of in situ cutaneous squamous cell carcinoma was lower than invasive disease (75.5 years ± 11.3). A previous basal cell carcinoma occurred in 20.6% and actinic keratosis were diagnosed more frequently in women (p = 0.040). The face was the most common location (30.8%). Incidence rates have risen, particularly in women and age-group ≥ 75-years.Conclusion: Our study reports a rapid increase of the incidence in an ageing Portuguese population and highlights the importance of improving the existing cancer registries in Portugal.Introdução: O carcinoma espinocelular cutâneo é o segundo cancro cutâneo mais comum e a sua incidência tem crescido. O objetivo do nosso estudo foi realizar uma análise descritiva e analítica dos carcinoma espinocelular cutâneo excisados no Centro Hospitalar Vila Nova de Gaia e Espinho (CHVNGE) num período de 10 anos e estabelecer tendências (incidência, sobrevida e mortalidade).Materiais e Métodos: A informação foi retrospetivamente recolhida nos Registos Oncológico e Histológico do CHVNGE entre o período de Janeiro de 2004 e Dezembro de 2013. O objetivo do nosso estudo foi descrever as características e tendências (incidência, associação a queratoses actínicas e carcinomas basocelulares, sobrevida e mortalidade) do carcinoma espinocelular cutâneo.Resultados: Foram removidas 485 lesões em 380 pacientes (56,1% homens e 43,9% mulheres). 361 pacientes apresentavam doença invasora e 124 doença in situ. O serviço de Dermatologia removeu a maioria das lesões (70,4%), seguido pelo serviço de Cirurgia Plástica (16,4%) e Cirurgia Geral (4,7%). A faixa etária ≥ 75 anos foi a mais atingida por carcinoma espinocelular cutâneo em ambos os sexos (p < 0,001). A média de idades dos pacientes com carcinoma espinocelular cutâneo invasor foi de 76,7anos (± 11,5), sendo mais elevada no sexo feminino (79,0 vs 74,0 anos, p < 0,001). A face foi a localização topográfica mais comum (42,1%) nos dois sexos (p = 0,002). Houve um aumento da taxa de incidência ajustada à idade em ambos os sexos, particularmente no último período do estudo (16,2/100 000 pessoas). A sobrevida aos 5 anos foi de 98,7%. A idade média do carcinoma espinocelular cutâneo in situ foi inferior à da doença invasora (75,5 anos ± 11,3). Dos doentes com carcinoma espinocelular cutâneo in situ, 20,6% tinham antecedentes de carcinoma basocelular e as mulheres apresentaram mais queratoses actínicas (p = 0,040). A face foi o local mais comum (30,8%). A taxa de incidência de carcinoma espinocelular cutâneo in situ aumentou, sendo maior nas mulheres e na faixa etária ≥75 anos.Conclusão: Este estudo demonstra um rápido aumento da incidência do carcinoma espinocelular cutâneo numa população portuguesa envelhecida e realça a necessidade de melhorar os registos oncológicos em Portugal

Frequency of TERT promoter mutations in human cancers

Reactivation of telomerase has been implicated in human tumorigenesis, but the underlying mechanisms remain poorly understood. Here we report the presence of recurrent somatic mutations in the TERT promoter in cancers of the central nervous system (43%), bladder (59%), thyroid (follicular cell-derived, 10%) and skin (melanoma, 29%). In thyroid cancers, the presence of TERT promoter mutations (when occurring together with BRAF mutations) is significantly associated with higher TERT mRNA expression, and in glioblastoma we find a trend for increased telomerase expression in cases harbouring TERT promoter mutations. Both in thyroid cancers and glioblastoma, TERT promoter mutations are significantly associated with older age of the patients. Our results show that TERT promoter mutations are relatively frequent in specific types of human cancers, where they lead to enhanced expression of telomerase.We thank to Mrs Mafalda Rocha for the excellent technical support in the sequencing work. This work was partially supported by the Portuguese Science and Technology Foundation (FCT) through BPD (SFRH/BPD/85249/2012 to H. P.), PhD (SFRH/BD/81940/2011 to J.V. and SFRH/BD/79135/2011 to A. A.) and BI grants, and the grant through the Program Ciencia 2008 (J.L.) and the project (PIC/IC/83037/2007). Further funding was obtained from the project 'Microenvironment, metabolism and cancer' partially supported by Programa Operacional Regional do Norte (ON.2-O Novo Norte), under the Quadro de Referencia Estrategico Nacional (QREN), and through the Fundo Europeu de Desenvolvimento Regional (FEDER). IPATIMUP is an associate laboratory of the Portuguese Ministry of Science, Technology and Higher Education and is partially supported by the FCT

Telomerase promoter mutations in cancer: an emerging molecular biomarker?

João Vinagre, Vasco Pinto and Ricardo Celestino contributed equally to the manuscript.Cell immortalization has been considered for a long time as a classic hallmark of cancer cells. Besides telomerase reactivation, such immortalization could be due to telomere maintenance through the “alternative mechanism of telomere lengthening” (ALT) but the mechanisms underlying both forms of reactivation remained elusive. Mutations in the coding region of telomerase gene are very rare in the cancer setting, despite being associated with some degenerative diseases. Recently, mutations in telomerase (TERT) gene promoter were found in sporadic and familial melanoma and subsequently in several cancer models, notably in gliomas, thyroid cancer and bladder cancer. The importance of these findings has been reinforced by the association of TERT mutations in some cancer types with tumour aggressiveness and patient survival. In the first part of this review, we summarize the data on the biology of telomeres and telomerase, available methodological approaches and non-neoplastic diseases associated with telomere dysfunction. In the second part, we review the information on telomerase expression and genetic alterations in the most relevant types of cancer (skin, thyroid, bladder and central nervous system) on record, and discuss the value of telomerase as a new biomarker with impact on the prognosis and survival of the patients and as a putative therapeutic target

The mTOR Signalling Pathway in Human Cancer

The conserved serine/threonine kinase mTOR (the mammalian target of rapamycin), a downstream effector of the PI3K/AKT pathway, forms two distinct multiprotein complexes: mTORC1 and mTORC2. mTORC1 is sensitive to rapamycin, activates S6K1 and 4EBP1, which are involved in mRNA translation. It is activated by diverse stimuli, such as growth factors, nutrients, energy and stress signals, and essential signalling pathways, such as PI3K, MAPK and AMPK, in order to control cell growth, proliferation and survival. mTORC2 is considered resistant to rapamycin and is generally insensitive to nutrients and energy signals. It activates PKC-α and AKT and regulates the actin cytoskeleton. Deregulation of multiple elements of the mTOR pathway (PI3K amplification/mutation, PTEN loss of function, AKT overexpression, and S6K1, 4EBP1 and eIF4E overexpression) has been reported in many types of cancers, particularly in melanoma, where alterations in major components of the mTOR pathway were reported to have significant effects on tumour progression. Therefore, mTOR is an appealing therapeutic target and mTOR inhibitors, including the rapamycin analogues deforolimus, everolimus and temsirolimus, are submitted to clinical trials for treating multiple cancers, alone or in combination with inhibitors of other pathways. Importantly, temsirolimus and everolimus were recently approved by the FDA for the treatment of renal cell carcinoma, PNET and giant cell astrocytoma. Small molecules that inhibit mTOR kinase activity and dual PI3K-mTOR inhibitors are also being developed. In this review, we aim to survey relevant research, the molecular mechanisms of signalling, including upstream activation and downstream effectors, and the role of mTOR in cancer, mainly in melanoma

Relevância da via de sinalização do mTOR na iniciação /progressão de tumores humanos

BiomedicinaDoctoral Programme in Biomedicin

Disruption of iron-sulphur cluster N2 from NADH: ubiquinone oxidoreductase by site-directed mutagenesis.

We have cloned and inactivated, by repeat-induced point mutations, the nuclear gene encoding the 19.3 kDa subunit of complex I (EC 1.6.5.3) from Neurospora crassa, the homologue of the bovine PSST polypeptide. Mitochondria from mutant nuo19.3 lack the peripheral arm of complex I while its membrane arm accumulates. Transformation with wild-type cDNA rescues this phenotype and assembly of complex I is restored. To interfere with assembly of a proposed bound iron-sulphur cluster, site-directed mutants were constructed by introducing cDNA with altered codons for two adjacent cysteines, Cys-101 and Cys-102. The mutant complexes were purified and their enzymic activities and EPR and UV/visible spectra were analysed. Either of the mutations abolishes assembly of iron-sulphur cluster N2, showing that this redox group is bound to the 19.3 kDa protein. We also observed an interference with the reduction of redox group X, suggesting that cluster N2 is the electron donor to this high-potential redox group

Melanocytic Tumour in a Black Sheep never exposed to Ultraviolet Radiation

A slow growing progressive head skin tumour in a 6-year-old “Serra da Estrela” (black variety) male sheep without previous history of ultraviolet radiation exposure or illness is described. The tumour consisted of a large (5.4cm) irregular mass subdivided into two polypoid-like regions and small alternating pigmented and non-pigmented areas in the surrounding thick epidermis. By light microscopy the tumour disclosed morphologic and immunohistochemistry features consistent with a melanocytoma type tumour, without signs of vascular and perineural invasion. The tumour cells contained Masson-Fontana positive pigment, expressed S100 protein and vimentin, and display a low proliferative (Ki67<1%) labelling index. No metastases were found in the autopsy study. Analyses of the homologous regions of the hotspot mutational exons of BRAF and NRAS (the most often mutated genes in human melanocytic tumours) did not reveal alterations, but silent polymorphic variations, and no variation was observed in GNAQ gene sequence, that is found mutated in human melanocytomas. In summary, we report the phenotypic and genotypic features of, as far as we are aware, the first case of a head skin melanocytoma type tumour with low-grade malignant potential in a black sheep non-exposed to environmental radiation

Clinico-pathological features of cutaneous melanomas.

<p>Clinico-pathological features of cutaneous melanomas.</p

Mitochondrial dynamics protein Drp1 is overexpressed in oncocytic thyroid tumors and regulates cancer cell migration.

Oncocytic cell tumors are characterized by the accumulation of morphologically abnormal mitochondria in their cells, suggesting a role for abnormal mitochondrial biogenesis in oncocytic cell transformation. Little is known about the reason for the dysmorphology of accumulated mitochondria. The proteins regulating the morphology of mitochondria, the "mitochondria-shaping" proteins, can modulate their size and number; however, nothing is known hitherto about a possible involvement of mitochondrial dynamics in oncocytic cell transformation in tumors. Our aim was to assess the status of the mitochondria morphology and its role in oncocytic cell transformation. We therefore evaluated the expression pattern of the main mitochondrial fusion and fission proteins in a series of thyroid cell tumor samples, as well as in thyroid tumor cell lines, with and without oncocytic cell features. The expression of mitochondrial fusion (Opa1, Mfn1 and Mfn2) and fission (Drp1 and Fis1) proteins were evaluated by immunohistochemistry (IHC) in a series of 88 human thyroid tumors. In vitro studies, for comparative purposes and to deepen the study, were performed using TPC1--a papillary thyroid carcinoma derived cell line--and XTC.UC1, an oncocytic follicular thyroid carcinoma-derived cell line. Both IHC and in vitro protein analyses showed an overall increase in the levels of "mitochondrial-shaping" proteins in oncocytic thyroid tumors. Furthermore, overexpression of the pro-fission protein Drp1 was found to be associated with malignant oncocytic thyroid tumors. Interestingly, genetic and pharmacological blockage of Drp1 activity was able to influence thyroid cancer cells' migration/invasion ability, a feature of tumor malignancy. In this study we show that unbalanced mitochondrial dynamics characterize the malignant features of thyroid oncocytic cell tumors, and participate in the acquisition of the migrating phenotype