RESUMO
Tumour growth and invasiveness require extracellular matrix (ECM) degradation and are stimulated by the GALA pathway, which induces protein O-glycosylation in the endoplasmic reticulum (ER). ECM degradation requires metalloproteases, but whether other enzymes are required is unclear. Here, we show that GALA induces the glycosylation of the ER-resident calnexin (Cnx) in breast and liver cancer. Glycosylated Cnx and its partner ERp57 are trafficked to invadosomes, which are sites of ECM degradation. We find that disulfide bridges are abundant in connective and liver ECM. Cell surface Cnx-ERp57 complexes reduce these extracellular disulfide bonds and are essential for ECM degradation. In vivo, liver cancer cells but not hepatocytes display cell surface Cnx. Liver tumour growth and lung metastasis of breast and liver cancer cells are inhibited by anti-Cnx antibodies. These findings uncover a moonlighting function of Cnx-ERp57 at the cell surface that is essential for ECM breakdown and tumour development.
Assuntos
Neoplasias da Mama/enzimologia , Calnexina/metabolismo , Movimento Celular , Retículo Endoplasmático/enzimologia , Matriz Extracelular/metabolismo , Neoplasias Hepáticas/enzimologia , Neoplasias Pulmonares/enzimologia , Podossomos/enzimologia , Isomerases de Dissulfetos de Proteínas/metabolismo , Animais , Antineoplásicos Imunológicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Calnexina/antagonistas & inibidores , Linhagem Celular Tumoral , Retículo Endoplasmático/patologia , Matriz Extracelular/patologia , Feminino , Glicosilação , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Células NIH 3T3 , Invasividade Neoplásica , Podossomos/patologia , Transporte Proteico , Proteólise , Ensaios Antitumorais Modelo de Xenoenxerto , alfa-Galactosidase/metabolismoRESUMO
Within the family of NADPH oxidases, NOX4 is unique as it is predominantly localized in the endoplasmic reticulum, has constitutive activity, and generates hydrogen peroxide (H2O2). We hypothesize that these features are consequences of a so far unidentified NOX4-interacting protein. Two-dimensional blue native (BN) electrophorese combined with SDS-PAGE yielded NOX4 to reside in macromolecular complexes. Interacting proteins were screened by quantitative SILAC (stable isotope labeling of amino acids in cell culture) co-immunoprecipitation (Co-IP) in HEK293 cells stably overexpressing NOX4. By this technique, several interacting proteins were identified with calnexin showing the most robust interaction. Calnexin also resided in NOX4-containing complexes as demonstrated by complexome profiling from BN-PAGE. The calnexin NOX4 interaction could be confirmed by reverse Co-IP and proximity ligation assay, whereas NOX1, NOX2, or NOX5 did not interact with calnexin. Calnexin deficiency as studied in mouse embryonic fibroblasts from calnexin(-/-)mice or in response to calnexin shRNA reduced cellular NOX4 protein expression and reactive oxygen species formation. Our results suggest that endogenous NOX4 forms macromolecular complexes with calnexin, which are needed for the proper maturation, processing, and function of NOX4 in the endoplasmic reticulum.
