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Orientador: Edgard Graner Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba Resumo: Recentes evidências indicam que as células tronco do câncer (CTC) tenham papel importante nas recorrências loco-regionais e metástases à distância de diversas neoplasias malignas, incluindo o carcinoma de células escamosas oral (CCEO). Uma caractéristica importante das CTC consiste na resistência a agentes quimioterápicos, que pode contribuir para a permanência de células residuais após o tratamento. A enzima ácido graxo sintase (FASN) tem sido descrita como uma oncoproteina metabólica, tendo sua expressão aumentada em neoplasias malignas originárias de diferentes tecidos. Sua expressão está também aumentada no CCEO e estudos in vivo mostraram que sua inibição com orlistat (ORL) diminui o volume de tumores ortotópicos de língua e reduz o número de metástases cervicais. O objetivo do presente trabalho foi avaliar a imunoexpressão de marcadores de superfície associados a CTC (CD44, CD326, CD133 e CD271) nas linhagens celulares derivadas de CCEO SCC-9 ZsG e LN-1A tratadas com paclitaxel (PTX), cisplatina (CIS) e com os inibidores de FASN ORL e TVB-3166, bem como isolar suas subpopulações por meio da imunoexpressão de CD44 e CD326. Para isto, as células foram tratadas com CIS, ORL e TVB-3166 em suas respectivas IC50 e com PTX a 1 µM e 50 µM. Os estudos de citometria de fluxo revelaram os fenótipos CD44+/CD326+ e CD44-/CD326+ em ambas as linhagens tratadas ou não com as drogas estudadas. PTX diminuiu a expressão de CD44+ e a população CD44+/CD326+ e aumentou a população CD44-/CD326+ nas duas linhagens celulares. CIS aumentou a expressão de CD44 e CD326 nas células SCC-9 ZsG e LN-1A. De modo geral, ORL e TVB-3166 não modificaram significativamente os fenótipos celulares, a não ser pela diminuição da expressão de CD44 na população com baixa expressão desta proteína e da expressão de CD326 na linhagem LN-1A. Para o isolamento de subpopulações, células SCC-9 ZsG e LN-1A foram marcadas com anticorpos contra CD44 e CD326 e, por meio de citometria de fluxo, subpopulações identificadas, separadas e avaliadas quanto a morfologia e expressão de proteínas de transição epitélio-mesenquimal (TEM), por meio de western blotting. Foram isolados seis fenótipos a partir da linhagem SCC-9 ZsG, dos quais destacam-se CD44Low/CD326+ e CD44-/CD326+, com características epiteliais e alta produção de e-caderina, e CD44+/CD326- e CD44+/CD326Low, com alta produção de slug, vimentina e n-caderina. A partir da linhagem LN-1A foram isolados 7 fenótipos, com destaque para CD44Low/CD326+ e CD44-/CD326+, de aspecto epitelial e produtores de e-caderina e CD44+/CD326- e CD44+/CD326Low, com alta expressão de slug, vimentina e n-caderina. Células CD44High/CD326-, com aspecto mesenquimal e alta produção de vimentina, n-caderina e slug, foram isoladas somente da linhagem LN-1A. Estes resultados mostram que PTX e CIS modificam o padrão de expressão de CD44 e CD326 nas células SCC-9 ZsG e LN-1A e que subpopulações com maior expressão de CD44 e ausência ou baixa expressão de CD326 apresentam características mesenquimais associadas a TEM, enquanto células com maior expressão a CD326 e com ausência ou baixa expressão de CD44 têm características epiteliais Abstract: Recent evidences indicate that cancer stem cells (CSC) have a central role in tumor recurrence and metastasis in several types of human malignancies, including oral squamous cell carcinoma (OSCC). An important feature of CSC is the resistance to chemotherapy, which may contribute the presence of residual cancer cells following the treatment. Fatty acid synthase (FASN) has been described as a metabolic oncoprotein overexpressed in malignancies derived from several tissues. Its expression is increased in OSCC and in vivo studies have shown that its inhibition with orlistat (ORL) decreases the volume of tongue orthotopic tumors as well as the number of cervical lymph node metastases. The aim of the present study is to evaluate the immunoexpression of CSC surface markers (CD44, CD326, CD133, and CD271) in the OSCC cell lines SCC-9 ZsG and LN-1A treated with paclitaxel (PTX), cisplatin (CIS), as well as the FASN inhibitors ORL and TVB-3166. Also, this study aims to isolate subpopulations according to the expression of CD44 and CD326 from the same cell lines. Cells were treated CIS, ORL and TVB-3166 in their respective IC50 and with PTX at 1 µM and 50 µM. Flow cytometry experiments showed that CD44+/CD326- and CD44-/CD326+ populations are present in both cell lines treated or not with the studied drugs. PTX decreases CD44+ expression and the size of CD44+/CD326+ population as well as increases CD44-/CD326+ population in both cell lines. In contrast, CIS increases the expression of CD44 and CD326 in SCC-9 ZsG and LN-1A cells. ORL and TVB-3166 did not significantly modified cell phenotypes except by a decrease of CD44 expression in cells with low expression of this marker and of CD326 in LN-1A cells. Cell sorting was performed after labeling of SCC-9 ZsG and LN-1A cells with antibodies against CD44 and CD326 and each phenotype had the morphology and production of epithelial-mesenchymal transition (EMT) markers were evaluated. Six different phenotypes were isolated from SCC-9 ZsG cells, with emphasis to CD44Low/CD326+ and CD44-/CD326+, with epithelial aspect and high producers of e-cadherin, and CD44+/CD326- and CD44+/CD326Low with high expression of slug, vimentin and n-cadherin. From LN-1A cells seven phenotypes were isolated, with emphasis to CD44Low/CD326+ and CD44-/CD326+ with epithelial morphology and high e-cadherin production as well as CD44+/CD326- and CD44+/CD326Low with high expression of slug, vimentin and n-cadherin. A group of cells CD44High/CD326- with mesenchymal phenotype and high producers of vimentin, n-cadherin and slug was found in LN-1A cells only. Taken together our results show that PTX and CIS change CD44 e CD326 pattern of expression in SCC-9 ZsG and LN-1A cells and that subpopulations with high CD44 expression and absent or low CD326 expression have mesenchymal characteristics associated with EMT whereas subpopulations with high CD326 expression and absent or low CD44 expression have epithelial characteristics Doutorado Patologia Doutora em Estomatopatologia CNPQ 141536/2017-9

Agradecimentos: The study was supported by grants from the State of São Paulo Research Foundation, FAPESP, São Paulo, Brazil (#2009/54068-0). R.A.M. is supported by the National Postdoctoral Program of the Coordination of Training of Higher Education Graduate Foundation (PNPD/CAPES, Brasilia, Brazil), and R.D.C. is a research fellow at The National Council for Scientific and Technological Development, CNPq, Brasília, Brazil Abstract: Papillon-Lefèvre syndrome (PLS; MIM#245000) is a rare recessive autosomal disorder characterized by palmar and plantar hyperkeratosis, and aggressively progressing periodontitis leading to premature loss of deciduous and permanent teeth. PLS is caused by loss-of-function mutations in the CTSC gene, which encodes cathepsin C. PLS clinical expressivity is highly variable and no consistent genotype-phenotype correlation has been demonstrated yet. Here we report the clinical and genetic features of five PLS patients presenting a severe periodontal breakdown in primary and permanent dentition, hyperkeratosis over palms and soles, and recurrent sinusitis and/or tonsillitis. Mutation analysis revealed two novel homozygous recessive mutations (c.947T>C and c.1010G>C) and one previous described homozygous recessive mutation (c.901G>A), with parents carrying them in heterozygous, in three families (four patients). The fourth family presented with the CTSC c.628C>T mutation in heterozygous, which was inherited maternally. Patient carrying the CTSC c.628C>T mutation featured classical PLS phenotype, but no PLS clinical characteristics were found in his carrier mother. All mutations were found to affect directly (c.901G>A, c.947T>C, and c.1010G>C) or indirectly (c.628C>T, which induces a premature termination) the heavy chain of the cathepsin C, the region responsible for activation of the lysosomal protease. Together, these findings indicate that both homozygous and heterozygous mutations in the cathepsin C heavy chain domain may lead to classical PLS phenotype, suggesting roles for epistasis or gene-environment interactions on determination of PLS phenotypes FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPES Fechado