ABSTRACT
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.
Subject(s)
Breast Neoplasms/enzymology , Calnexin/metabolism , Cell Movement , Endoplasmic Reticulum/enzymology , Extracellular Matrix/metabolism , Liver Neoplasms/enzymology , Lung Neoplasms/enzymology , Podosomes/enzymology , Protein Disulfide-Isomerases/metabolism , Animals , Antineoplastic Agents, Immunological/pharmacology , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Calnexin/antagonists & inhibitors , Cell Line, Tumor , Endoplasmic Reticulum/pathology , Extracellular Matrix/pathology , Female , Glycosylation , Liver Neoplasms/drug therapy , Liver Neoplasms/pathology , Lung Neoplasms/secondary , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Nude , NIH 3T3 Cells , Neoplasm Invasiveness , Podosomes/pathology , Protein Transport , Proteolysis , Xenograft Model Antitumor Assays , alpha-Galactosidase/metabolismABSTRACT
Biologics such as peptides and antibodies are a well-established class of therapeutics. However, their intracellular delivery remains problematic. In particular, methods to efficiently inhibit intra-nuclear targets are lacking. We previously described that Pseudomonas Exotoxin A reaches the nucleoplasm via the endosomes-to-nucleus trafficking pathway. Here, we show that a non-toxic truncated form of PE can be coupled to peptides and efficiently reach the nucleoplasm. It can be used as a Peptide Nuclear Delivery Device (PNDD) to deliver polypeptidic cargos as large as Glutathione- S-transferase (GST) to the nucleus. PNDD1 is a fusion of PNDD to the c-myc inhibitor peptide H1. PNDD1 is able to inhibit c-Myc dependent transcription at nanomolar concentration. In contrast, H1 fused to various cell-penetrating peptides are active only in the micromolar range. PNDD1 attenuates cell proliferation and induces cell death in various tumor cell lines. In particular, several patient-derived Diffuse Large B-Cell Lymphomas cell lines die after exposure to PNDD1, while normal B-cells survive. Altogether, our data indicate that PNDD is a powerful tool to bring active cargo to the nucleus and PNDD1 could be the basis of a new therapy against lymphoma.
