Estudo do envolvimento da metalo-peptidase PHEX em processos tumorais
Nenhuma Miniatura disponível
Data
2018-03-29
Autores
Neves, Raquel Leao [UNIFESP]
Orientadores
Barros, Nilana Meza Tenorio De [UNIFESP]
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
Mutações no gene PHEX (phosphateregulating
gene with homologies to
endopeptidase on the X chromosome) resultam em uma forma prevalente (1:
20.000) de raquitismo humano hereditário denominada XLH (XLinked
Hypophosphataemia). A XLH é caracterizada por deficiência na reabsorção de
fosfato, metabolismo da vitamina D e defeitos na mineralização óssea.
Utilizando diversas metodologias e o modelo murino da XLH, o Hyp mouse,
identificamos a OPN (osteopontina) como o primeiro substrato fisiológico da
PHEX. A OPN é uma fosfoglicoproteína multifuncional com atividades tecidoespecíficas
que, dentre suas funções, pode atuar no osso como um potente
inibidor da mineralização óssea, e em tumores, como promotora da progressão
tumoral. Neste contexto, ampliamos nossos estudos desta protease no
carcinoma espinocelular de cabeça e pescoço (HNSCC). No presente trabalho,
demonstramos pela primeira vez a elevada expressão de PHEX em células
tumorais não ósseas (SCC22B). Descrevemos também a modulação da OPN
tumoral (SCC22BOPN)
pela PHEX exógena, e caracterizamos a presença
temporal da proteína PHEX na membrana devido a ação de cisteínoproteases.
No intuito de progredir na compreensão do papel da PHEX nas células
SCC22B, foram realizados ensaios de colocar em português (woundhealing)
em dois diferentes modelos de células SCC22B construídos por nosso grupo,
SCC22B shRNAPHEX
(expressão do gene PHEX reduzida) e SCC22B
secPHEX (superexpressão da forma secretada da PHEX). As análises
realizadas indicaram que quando comparadas a SCC22B, a linhagem SCC22B
secPHEX apresentou redução na migração celular, enquanto que um
considerável aumento foi observado nas células SCC22B shRNAPHEX.
Recentemente, desenvolvemos também uma terceira linhagem que
superexpressa a PHEX com o domínio transmembrana íntegro (SCC22B
mbPHEX), e que está sendo caracterizada para realizar uma análise
comparativa com os outros dois modelos. Por fim, demonstramos a coexpressão
de PHEX e OPN nas células de leucemia Jurkat, Kasumi, KG1,
K562
e Lucena (modelos de tumores nãosólidos).
Os resultados obtidos
representam um avanço na caracterização funcional da PHEX, e demonstram que a sua expressão e/ou função não está restrita apenas a tecidos
mineralizados.
Mutations in PHEX gene result in a prevalent (1: 20,000) form of hereditary human rickets, the XLinked Hypophosphataemia (XLH). XLH is characterized by deficiency in phosphate reabsorption and vitamin D metabolism, and defects in bone mineralization. Using several methodologies and the murine XLH model, we identified the OPN (osteopontin) as the first PHEX physiological substrate. OPN is a multifunctional phosphoglycoprotein with tissuespecific activities that, among its functions, can act in bone as a potent inhibitor of bone mineralization, and in tumors, as a promoter of tumor progression. Considering these data, we extended our studies of this protease in head and neck squamous cell carcinoma (HNSCC). In the present work, we demonstrate for the first time the high expression of PHEX in nonbone tumor cells (SCC22B), and describe the modulation of tumor OPN (SCC22BOPN) by exogenous PHEX and characterize the temporal targeting of the PHEX protein to SCC22B cell membrane. We have found that cysteine proteases expressed in these cells can degrade/inactivate endogenous PHEX and the treatment of these cells with the E64 inhibitor results in increased of PHEX in membrane and the degradation of OPN tumor fragments (SCC22BOPN). To better understand the role of PHEX in SCC22B cells, proliferation and migration assays were performed using the wound healing method in two different SCC22B cell models constructed in our laboratory, SCC22B shRNAPHEX (reduced PHEX gene expression) and SCC22B secPHEX (overexpression of the secreted form of PHEX). The analyzes performed indicated that when compared to SCC22B, the SCC22B secPHEX cell lineage showed reduction in cell proliferation and migration, whereas a considerable increase was observed in SCC22B shRNAPHEX cells. In addition to these two models, recently we have also developed a third line that overexpresses PHEX with the integral transmembrane domain (SCC22B mbPHEX) and is being characterized by our group. Finally, using the Jurkat, Kasumi, KG1, K562 and Lucena cell lineages, we also demonstrated the expression of PHEX/PHEX in leukemia cells (nonsolid tumors). The results obtained represent an advance in the functional characterization of PHEX, and demonstrate that its expression and/or function are not restricted only to mineralized tissues.
Mutations in PHEX gene result in a prevalent (1: 20,000) form of hereditary human rickets, the XLinked Hypophosphataemia (XLH). XLH is characterized by deficiency in phosphate reabsorption and vitamin D metabolism, and defects in bone mineralization. Using several methodologies and the murine XLH model, we identified the OPN (osteopontin) as the first PHEX physiological substrate. OPN is a multifunctional phosphoglycoprotein with tissuespecific activities that, among its functions, can act in bone as a potent inhibitor of bone mineralization, and in tumors, as a promoter of tumor progression. Considering these data, we extended our studies of this protease in head and neck squamous cell carcinoma (HNSCC). In the present work, we demonstrate for the first time the high expression of PHEX in nonbone tumor cells (SCC22B), and describe the modulation of tumor OPN (SCC22BOPN) by exogenous PHEX and characterize the temporal targeting of the PHEX protein to SCC22B cell membrane. We have found that cysteine proteases expressed in these cells can degrade/inactivate endogenous PHEX and the treatment of these cells with the E64 inhibitor results in increased of PHEX in membrane and the degradation of OPN tumor fragments (SCC22BOPN). To better understand the role of PHEX in SCC22B cells, proliferation and migration assays were performed using the wound healing method in two different SCC22B cell models constructed in our laboratory, SCC22B shRNAPHEX (reduced PHEX gene expression) and SCC22B secPHEX (overexpression of the secreted form of PHEX). The analyzes performed indicated that when compared to SCC22B, the SCC22B secPHEX cell lineage showed reduction in cell proliferation and migration, whereas a considerable increase was observed in SCC22B shRNAPHEX cells. In addition to these two models, recently we have also developed a third line that overexpresses PHEX with the integral transmembrane domain (SCC22B mbPHEX) and is being characterized by our group. Finally, using the Jurkat, Kasumi, KG1, K562 and Lucena cell lineages, we also demonstrated the expression of PHEX/PHEX in leukemia cells (nonsolid tumors). The results obtained represent an advance in the functional characterization of PHEX, and demonstrate that its expression and/or function are not restricted only to mineralized tissues.
Descrição
Citação
NEVES, Raquel Leão. Estudo do envolvimento da metalo-peptidase PHEX em processos tumorais. 2018. Tese (Doutorado em Biologia Molecular) - Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, 2018.