Papel cooperativo de c-MYC e E6/E7 de duas variantes moleculares do Papilomavírus Humano tipo 16 na proliferação e transformação in vitro de queratinócitos humanos primários

As funções das oncoproteínas E6 e E7 do Papilomavírus Humano tipo 16 (HPV-16) na progressão de células epiteliais imortalizadas para tumores invasivos não são totalmente compreendidas. Aqui, estabelecemos uma nova ligação entre E6 e E7 de duas variantes moleculares de HPV-16 (AA e E-350G) e c-MYC, em relação à cooperação na promoção da transformação maligna de queratinócitos humano primários de prepúcio de recém-nascido (PHK). Nosso objetivo foi estudar os efeitos sinérgicos de E6/E7 e c-MYC na proliferação e no potencial de transformação in vitro de PHKs. Avaliou-se a proliferação celular através da expressão proteica do Antígeno Nuclear de Células Proliferantes (PCNA). Também avaliamos a capacidade de transformação, in vitro, dos PHKs através de dois ensaios complementares. Observamos que E-350G-c-MYC PHKs exibiram um aumento discreto na expressão de PCNA e formaram significativamente mais colônias tanto nos ensaios de soft-ágar quanto nos ensaios em placas de cultura de baixa adesão. No geral, concluímos que a variante E-350G co-transfectada com c-MYC pode promover a transformação celular maligna com eficiência maior do que a variante AA-c-MYC. As propriedades oncogênicas exibidas pela variante E-350G permitem entender em maior detalhe os mecanismos que podem levar à neoplasia cervical humana, dada a maior frequência de sua ocorrência na progressão de lesões precursoras de alto grau para carcinomas invasivos.The roles of E6 and E7 oncoproteins of Human Papillomavirus type 16 (HPV-16) in the progression of immortalized epithelial cells to invasive tumors are not fully understood. Here, we establish a novel link between E6 and E7 of two molecular variants of HPV-16 (AA and E-350G), and c-MYC, regarding the cooperation in promoting malignant transformation of primary human foreskin keratinocytes (PHK). We aimed to study the synergistic effects of E6/E7 and c-MYC upon proliferation, and the in vitro transformation potential of PHK. We evaluated cellular proliferation through the expression of the Proliferating Cell Nuclear Antigen (PCNA) protein and colony formation abilities using soft agar and low attachment plates. We observed that E-350G-c-MYC PHKs exhibited discrete higher PCNA levels and formed significantly more colonies in both soft-agar and when growth in low-adhesion culture plates. Overall, we concluded that the E-350G variant co-transfected with c-MYC might promote malignant cellular transformation with a better efficiency than the AA-c-MYC counterpart. The enhanced oncogenic properties exhibited by the E-350G-c-MYC variant offer insights into mechanisms that may operate in human cervical neoplasia, given the higher frequency of its occurrence in the progression of high-grade precursor lesions to invasive carcinomas

Hypoxia, acidification and oxidative stress in cells cultured at large distances from an oxygen source

Hypoxia is a condition frequently encountered by cells in tissues, whether as a normal feature of their microenvironment or subsequent to deregulated growth. Hypoxia can lead to acidifcation and increased oxidative stress, with profound consequences for cell physiology and tumorigenesis. Therefore, the interplay between hypoxia and oxidative stress is an important aspect for understanding the efects of hypoxic microenvironments on cells. We have used a previously developed variant of the method of coverslip-induced hypoxia to study the process of acidifcation in a hypoxic microenvironment and to simultaneously visualize intracellular levels of hypoxia and oxidative stress. We observed high accumulation of CO2 in hypoxic conditions, which we show is the main contributor to acidifcation in our model. Also, increased levels of oxidative stress were observed in moderately hypoxic cells close to the oxygen source, where the mitochondrial membrane potential was preserved. Conversely, cells at large distances from the oxygen source showed higher levels of hypoxia, milder oxidative stress and reduced mitochondrial membrane potential. Our results contribute to characterize the interplay between reduced oxygen levels, acidifcation and oxidative stress in a simple in vitro setting, which can be used to model cell responses to an altered environment, such as the early tumor microenvironment

Estudio de los efectos de un microambiente hipóxico en queratinocitos humanos in vitro y su correlato con alteraciones del microambiente en la patología de liquen plano oral

La hipoxia es un factor fundamental en el proceso de génesis tumoral, así como en patologías precursoras de cáncer, como es el Liquen Plano Oral (LPO). Objetivo: Determinar si es posible establecer una correlación entre las alteraciones que sufren queratinocitos normales en un microambiente hipóxico in vitro y alteraciones que aparecen en los queratinocitos en el epitelio de la mucosa oral en el contexto de la patología LPO. Métodos: Se estudiaron los cambios morfológicos mediante microscopía de contraste de fases, y la detección de marcadores asociados a hipoxia de queratinocitos humanos (HaCaT), como modelo celular oral, en un microambiente hipóxico generado por la variante del método “Hipoxia inducida por cubreobjetos”. Resultados: Mediante microscopía confocal se observó la presencia de los marcadores de hipoxia GLUT-1 y aductos de pimonidazol (Hipoxyprobe) en los cultivos celulares de HaCaT expuestos a un microambiente hipóxico. Además, se observó la presencia del marcador GLUT-1 mediante inmunohistoquímica en tejido epitelial humano derivado de biopsias de la patología LPO. Conclusiones: Se estableció una correlación entre las alteraciones detectadas en queratinocitos humanos inducidos a un microambiente hipóxico in vitro y las alteraciones detectadas in vivo en tejido epitelial de la mucosa oral

Effect of genetic ancestry on leukocyte global DNA methylation in cancer patients

Background: the study of genetic variants alone is not enough to explain a complex disease like cancer. Alterations in DNA methylation patterns have been associated with different types of tumor. In order to detect markers of susceptibility for the development of cutaneous melanoma and breast cancer in the Uruguayan population, we integrated genetic and epigenetic information of patients and controls. Methods: we performed two case-control studies that included 49 individuals with sporadic cutaneous melanoma and 73 unaffected controls, and 179 women with sporadic breast cancer and 209 women controls. We determined the level of global leukocyte DNA methylation using relative quantification of 5mdC by HPLC, and we compared methylation levels between cases and controls with nonparametric statistical tests. Since the Uruguayan population is admixed and both melanoma and breast cancer have very high incidences in Uruguay compared to other populations, we examined whether individual ancestry influences global leucocyte DNA methylation status. We carried out a correlation analysis between the percentage of African, European and Native American individual ancestries, determined using 59 ancestry informative markers, and global DNA methylation in all participants. Results: we detected global DNA hypomethylation in leukocytes of melanoma and breast cancer patients compared with healthy controls (p < 0.001). Additionally, we found a negative correlation between African ancestry and global DNA methylation in cancer patients (p <0.005). Conclusions: these results support the potential use of global DNA methylation as a biomarker for cancer risk. In addition, our findings suggest that the ancestral genome structure generated by the admixture process influences DNA methylation patterns, and underscore the importance of considering genetic ancestry as a modifying factor in epigenetic association studies in admixed populations such as Latino ones

Melanoma, ancestralidad y variantes MC1R en la población mestizada uruguaya

Malignant melanoma (MM) is the most dangerous type of skin cancer and the main cause of death produced by skin diseases. In Uruguay, the incidence rate is 3.8/100,000, one of the highest in Latin America. We analyzed the contribution of ancestry and MC1R as a candidate gene for sporadic melanoma in Uruguay. Our objective wasto investigate the possible associations between ancestry and the MC1R gene with sporadic melanoma in the Uruguayan population. To that end, one hundred patients with sporadic MM and 107 controls were recruited. Phenotypic factors and lifestyle were evaluated as risk factors. At the same time, we analyzed fiveancestry informative markers, the MC1R variants (R151, R160 and D294H) and five tag-SNPs. Phototype, atypical nevi, sunburns and recreational exposure were the main risk factors for MM in the Uruguayan population. We confirmed 16q as a candidate region for MM. R151C, and R160W showed an important association with risk of melanoma (OR= 3.85, P= 1 x 10-2; OR= 10.15, P= 7 x 10-3, respectively). Furthermore, three novel MC1R haplotypes from the promoter region were detected,and the two most common haplotypes for the coding region were different to the ones found in Europeans through HapMap. However, MC1R coding region haplotypes revealed a highly similar frequency to that of the Spanish population. Our results showed that the chromosomal 16q region confers susceptibility to MM risk in the Uruguayan population. In addition, the admixed genome structure of the MC1R region could be part of the explanation of melanoma etiology.El melanoma maligno (MM) es uno de los más peligrosos, y la principal causa de muerte producida por tumores de piel. En el Uruguay, la tasa de incidencia es de 3,8/100.000, una de las más altas de América Latina. En este trabajo analizamos la ancestría y el gen candidato MC1R entre los pacientes con MM del Uruguay. Nuestro objetivo fue investigar la posible asociación entre ancestría y el gen MC1R en pacientes con melanoma esporádico en la población uruguaya. Con tal finalidad, se reclutaron 100 pacientes con MM esporádico y 107 controles. Se evaluó el riesgo de factores fenotípicos y de estilo de vida. Además se analizaron cinco marcadores informativos de ancesdencia, variantes del gen MC1R (R151, R160 y D294H) y cinco tagSNPs. El fototipo, los nevos atípicos, quemaduras solares y la exposición recreativa fueron los principales factores de riesgo para MM en la población uruguaya. La región cromosómica 16q es candidata para MM, mientras que R151C y R160W mostraron una importante asociación con el riesgo para MM (OR= 3,85, P= 1 x 10-2; OR= 10,15, P= 7 x 10-3, respectivamente). Por otra parte, se detectaron tres nuevos haplotipos en la región promotora y los dos haplotipos más frecuentes en la región codificante son diferentes a los encontrados en la población europea. Sin embargo, los haplotipos de la región codificante presentan una frecuencia muy similar a las encontradas en la población española. Los resultados muestran que la región cromosómica 16q confiere susceptibilidad al riesgo de MM en la población uruguaya. Por otra parte, la estructura genómica mestizada de la región del MC1R podría explicar la etiología del melanoma

Human papillomavirus type 18 E5 oncoprotein cooperates with E6 and E7 in promoting cell viability and invasion and in modulating the cellular redox state

BACKGROUND High-risk human papillomaviruses (HR-HPVs) are the etiological agents of cervical cancer. Among them, types 16 and 18 are the most prevalent worldwide. The HPV genome encodes three oncoproteins (E5, E6, and E7) that possess a high transformation potential in culture cells when transduced simultaneously. In the present study, we analysed how these oncoproteins cooperate to boost key cancer cell features such as uncontrolled cell proliferation, invasion potential, and cellular redox state imbalance. Oxidative stress is known to contribute to the carcinogenic process, as reactive oxygen species (ROS) constitute a potentially harmful by-product of many cellular reactions, and an efficient clearance mechanism is therefore required. Cells infected with HR-HPVs can adapt to oxidative stress conditions by upregulating the formation of endogenous antioxidants such as catalase, glutathione (GSH), and peroxiredoxin (PRX). OBJECTIVES The primary aim of this work was to study how these oncoproteins cooperate to promote the development of certain cancer cell features such as uncontrolled cell proliferation, invasion potential, and oxidative stress that are known to aid in the carcinogenic process. METHODS To perform this study, we generated three different HaCaT cell lines using retroviral transduction that stably expressed combinations of HPV-18 oncogenes that included HaCaT E5-18, HaCaT E6/E7-18, and HaCaT E5/E6/E7-18. FINDINGS Our results revealed a statistically significant increment in cell viability as measured by MTT assay, cell proliferation, and invasion assays in the cell line containing the three viral oncogenes. Additionally, we observed that cells expressing HPV-18 E5/E6/E7 exhibited a decrease in catalase activity and a significant augmentation of GSH and PRX1 levels relative to those of E5, E6/E7, and HaCaT cells. MAIN CONCLUSIONS This study demonstrates for the first time that HPV-18 E5, E6, and E7 oncoproteins can cooperate to enhance malignant transformationANII: PD_NAC_2016_1_13332

Contributions of viral oncogenes of HPV‑18 and hypoxia to oxidative stress and genetic damage in human keratinocytes

Infection with high-risk human papillomaviruses like HPV-16 and HPV-18 is highly associated with the evelopment of cervical and other cancers. Malignant transformation requires viral oncoproteins E5, E6 and E7, which promote cell proliferation and increase DNA damage. Oxidative stress and hypoxia are also key factors in cervical malignant transformation. Increased levels of reactive species of oxygen (ROS) and nitrogen (RNS) are found in the hypoxic tumor microenvironment, promoting genetic instability and invasiveness. In this work, we studied the combined effect of E5, E6 and E7 and hypoxia in increasing oxidative stress and promoting DNA damage and nuclear architecture alterations. HaCaT cells containing HPV-18 viral oncogenes (HaCaT E5/E6/E7-18) showed higher ROS levels in normoxia and higher levels of RNS in hypoxia compared to HaCaT parental cells, as well as higher genetic damage in hypoxia as measured by γH2AX and comet assays. In hypoxia, HaCaT E5/E6/E7-18 increased its nuclear dry mass and both cell types displayed marked heterogeneity in nuclear dry mass distribution and increased nuclear foci. Our results show contributions of both viral oncogenes and hypoxia to oxidative stress, DNA damage and altered nuclear architecture, exemplifying how an altered microenvironment combines with oncogenic transformation to promote tumor progression.PEDECIBA. ANII

Identificación y caracterización de regiones cromosómicas asociadas a melanoma esporádico en la Uruguay mediante el método de mapeo por mestizaje :MC1-R como posible gen candidato

Tribunal: Dra. Mónica Sans, Dra. Leda Roche, Dra. Adriana Mimbacas.Orientador: Dr. Bernardo Berton

Analysis of the impact of E6/E7 proteins of different molecular variants of HPV-16 upon MAPK signaling pathways

A infecção persistente por HPV-16 está fortemente associada ao risco de desenvolvimento de neoplasias do colo do útero, vagina, vulva, pênis, canal anal e orofaringe. O estudo detalhado da variabilidade nucleotídica intra-típica de HPV-16 resultou em importantes achados no que concerne à filogenia e evolução viral, e à história natural das infecções. Variantes Asiático-Americanas (AA) e E-350G de HPV-16 foram associadas com maior risco de persistência da infecção viral e desenvolvimento de câncer de colo de útero quando comparadas à variante Européia protótipo (E-P ou E-350T), embora esta ainda apresente alto risco quando comparada aos outros tipos virais. Mais recentemente, diferenças funcionais entre as proteínas E6/E7 das distintas variantes moleculares de HPV- 16 estão sendo descritas, a fim de explicar as diferenças nas associações epidemiológicas observadas. Dados do nosso grupo apontaram para a transcrição aumentada do gene MEK2 especificamente em queratinócitos humanos primários (PHKs) transduzidos com E6/E7 da variante E-350G. Pelo exposto, objetivou-se: (1) Analisar os níveis de ativação de proteínas efetoras das vias de transdução de sinal mediadas por MAPK e PI3K/AKT em queratinócitos imortalizados por E6/E7 de três variantes moleculares de HPV-16 (AA, E-P, E-350G); (2) Analisar os efeitos das proteínas E6/E7 dessas variantes sob as vias de MAPK quanto à indução de fatores de transcrição; (3) Analisar o potencial transformante de PHKs imortalizados pelas diferentes variantes, e em cooperação com a proteína celular c-MYC; (4) Analisar o potencial de migração e invasão em PHKs imortalizados pelas diferentes variantes de HPV-16, e em cooperação com a proteína celular c-MYC. Neste estudo observou-se que a variante AA de HPV-16 induziu a maior ativação das vias de sinalização estudadas (MAPK, e PI3K/AKT). Ademais, PHKs imortalizados por esta variante apresentaram maior capacidade de migração, de invasão através de uma matriz de colágeno, além de maior potencial transformante. Adicionalmente, as células imortalizadas pela variante AA apresentaram maior expressão da proteína mesenquimal vimentina e diminuição dos níveis da proteína epitelial E-caderina, sugerindo ativação parcial de Transição Epitélio Mesênquima (EMT) nestes queratinócitos. Ademais, quando o oncogene c-MYC foi co-transduzido nas diferentes linhagens infectadas por E6/E7 de HPV-16, foi observado que em PHKs imortalizados pela variante AA também houve maior ativação da via de MAPK-ERK, maior migração, e um potencial transformante semelhante, em relação às células co-transduzidas pela variante E-350G e c-MYC. Em conjunto, estes dados sugerem que a variante AA de HPV-16 possui vantagem seletiva sob as outras variantes em promover transformação celular, migração e invasão, e isto poderia explicar, ao menos em parte, a maior prevalência desta variante no câncer cervical. Os resultados gerados neste estudo são de extrema relevância para avaliar o impacto da variabilidade intra-típica de HPV-16 sobre o potencial oncogênico observado em estudos epidemiológicosPersistent infection with HPV-16 is strongly associated with risk of developing neoplasia in the uterine cervix, vagina, vulva, penis, anal canal and oropharynx. The detailed study of HPV-16 intra-typical nucleotide variability resulted in important findings regarding phylogeny and viral evolution, and the natural history of infections. Asian-American (AA) and E-350G variants of HPV-16 were associated with increased risk of persistent viral infection and development of cervical cancer compared to the European prototype (E-P or E-350T), although this variant still presents higher risk when compared to other viral types. More recently, functional differences between the E6/E7 proteins of distinct molecular variants of HPV-16 are being described, in order to explain the differences in the epidemiological associations observed. Data from our group pointed to increased transcription of the MEK2 gene specifically in primary human keratinocytes (PHKs) transducing E6/E7 of the E-350G variant. Consequently, the aims of this study were: 1) To examine the activation levels of effector proteins of the signal transduction pathways mediated by MAPK and PI3K/AKT in PHKs immortalized by E6/E7 of three different molecular variants of HPV-16 (AA, E-P, E-350G); (2) To analyze the effects of E6/E7 of different molecular variants of HPV-16 upon MAPK pathways concerning the induction of transcription factors; (3) To analyze the transforming potential of PHKs immortalized by different molecular variants of HPV-16, and in cooperation with the cellular protein c- MYC; (4) To analyze the potential of migration and invasion in PHKs immortalized by different molecular variants of HPV-16, and in cooperation with the cellular protein c- MYC. In this study we observed that the AA variant of HPV-16 induced higher activation of both signaling pathways studied (MAPK, and PI3K/AKT). Furthermore, this variant presented increased migration capacity, higher invasion through a collagen matrix, and greater transforming potential. Moreover, cells immortalized by the AA variant showed higher expression of the mesenchymal protein vimentin and a decrease of the epithelial protein E-cadherin, suggesting partial activation of Epithelial Mesenchymal Transition (EMT). In addition, when the c-MYC oncogene was co-transduced in the different cells lines infected with HPV-16 E6/E7, we observed that in PHKs immortalized by the AA variant there was also an enhanced activation of the MAPK-ERK pathway, a higher ability to migrate, and similar transformation potential in comparison with cells co-transduced with the E-350G variant and c-MYC. Taken together, this data suggest that the AA molecular variant of the HPV-16 has a selective advantage over the other variants to promote cell transformation, migration and invasion, and this could partly explain the higher prevalence of this variant in cervical cancer. The results generated in this study are very important to assess the impact of intra-typical variability of HPV-16 on the oncogenic potential observed in epidemiological studie

Estudio de los efectos de un microambiente hipóxico en queratinocitos humanos in vitro y su correlato con alteraciones del microambiente en la patología de liquen plano oral

La hipoxia es un factor fundamental en el proceso de génesis tumoral, así como en patologías precursoras de cáncer, como es el Liquen Plano Oral (LPO).  Objetivo: Determinar si es posible establecer una correlación entre las alteraciones que sufren queratinocitos normales en un microambiente hipóxico in vitro y alteraciones que aparecen en los queratinocitos en el epitelio de la mucosa oral en el contexto de la patología LPO.  Métodos: Se estudiaron los cambios morfológicos mediante microscopía de contraste de fases, y la detección de marcadores asociados a hipoxia de queratinocitos humanos (HaCaT), como modelo celular oral, en un microambiente hipóxico generado por la variante del método “Hipoxia inducida por cubreobjetos”.  Resultados: Mediante microscopía confocal se observó la presencia de los marcadores de hipoxia GLUT-1 y aductos de pimonidazol (Hipoxyprobe) en los cultivos celulares de HaCaT expuestos a un microambiente hipóxico. Además, se observó la presencia del marcador GLUT-1 mediante inmunohistoquímica en tejido epitelial humano derivado de biopsias de la patología LPO.  Conclusiones: Se estableció una correlación entre las alteraciones detectadas en queratinocitos humanos inducidos a un microambiente hipóxico in vitro y las alteraciones detectadas in vivo en tejido epitelial de la mucosa oral