ABSTRACT
Cytoskeleton-associated protein 4 (CKAP4) is an endoplasmic reticulum protein that is also present in the cell surface membrane, where it acts as a receptor for Dickkopf1 (DKK1). In this study, we found that CKAP4 interacts with ß1 integrin and controls the recycling of α5ß1 integrin independently of DKK1. In S2-CP8 cells, knockdown of CKAP4 but not DKK1 enlarged the size of cell adhesion sites and enhanced cell adhesion to fibronectin, resulting in decreased cell migration. When CKAP4 was depleted, the levels of α5 but not ß1 integrin were increased in the cell surface membrane. A similar phenotype was observed in other cells expressing low levels of DKK1. In S2-CP8 cells, α5 integrin was trafficked with ß1 integrin and CKAP4 to the lysosome or recycled with ß1 integrin. In CKAP4-depleted cells, the internalization of α5ß1 integrin was unchanged, but its recycling was upregulated. Knockdown of sorting nexin 17 (SNX17), a mediator of integrin recycling, abrogated the increased α5 integrin levels caused by CKAP4 knockdown. CKAP4 bound to SNX17, and its knockdown enhanced the recruitment of α5ß1 integrin to SNX17. These results suggest that CKAP4 suppresses the recycling of α5ß1 integrin and coordinates cell adhesion sites and migration independently of DKK1.
Subject(s)
Integrin alpha5beta1/metabolism , Integrin beta1/metabolism , Membrane Proteins/metabolism , Cell Adhesion , Cell Line, Tumor , Cell Membrane/metabolism , Cell Movement , Gene Expression Regulation , HCT116 Cells , HeLa Cells , Humans , Intercellular Signaling Peptides and Proteins/metabolismABSTRACT
PURPOSE: The survival rate of pancreatic ductal adenocarcinoma (PDAC) is poor; thus, novel molecularly targeted therapy and companion diagnostics are required. We asked whether cytoskeleton-associated protein 4 (CKAP4), a novel Dickkopf1 (DKK1) receptor, is a candidate for PDAC diagnosis and therapy.Experimental Design: Whether CKAP4 can be secreted with small extracellular vesicles (SEV) from PDAC cells was examined. It was also investigated whether CKAP4 can be detected in sera from patients with PDAC by ELISA using newly generated anti-CKAP4 mAbs and whether anti-CKAP4 mAbs can show antitumor activity in vivo. RESULTS: CKAP4 was secreted with SEVs from PDAC cells, and the SEVs exhibited the characteristics of exosomes. The secretion of CKAP4-containing exosomes was mediated by DKK1-dependent endocytosis routes and required exosome biogenesis molecules. Two ELISAs capable of detecting tumor-secreted CKAP4 were developed. The serum CKAP4 levels were higher in patients with PDAC than healthy control individuals. CKAP4 was highly detected in the sera of pancreatic tumor-bearing xenografted mice and patients with PDAC, whereas CKAP4 was barely detectable in sera from normal mice and postoperative patients. Anti-CKAP4 mAbs with different epitopes demonstrated the inhibitory activities for the binding of DKK1 and CKAP4, AKT activity, and proliferation and migration of PDAC cells. Anti-CKAP4 mAbs also suppressed xenograft tumor formation in immunodeficient mice and extended the survival of mice receiving intraperitoneal or orthotopic injection of PDAC cells. CONCLUSIONS: CKAP4 secreted in exosomes may represent a biomarker for PDAC. Anti-CKAP4 mAbs can contribute to the development of novel diagnostic methods and therapeutics.
Subject(s)
Antineoplastic Agents/pharmacology , Biomarkers, Tumor/metabolism , Carcinoma, Pancreatic Ductal/diagnosis , Membrane Proteins/metabolism , Pancreatic Neoplasms/diagnosis , Adult , Aged , Aged, 80 and over , Animals , Antineoplastic Agents/therapeutic use , Biomarkers, Tumor/antagonists & inhibitors , Biomarkers, Tumor/blood , Carcinoma, Pancreatic Ductal/blood , Carcinoma, Pancreatic Ductal/mortality , Carcinoma, Pancreatic Ductal/therapy , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Exosomes , Female , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Male , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/blood , Mice , Mice, Knockout , Middle Aged , Molecular Targeted Therapy/methods , Pancreas/pathology , Pancreas/surgery , Pancreatectomy , Pancreatic Neoplasms/blood , Pancreatic Neoplasms/mortality , Pancreatic Neoplasms/therapy , Survival Analysis , Xenograft Model Antitumor AssaysABSTRACT
Dickkopf1 (DKK1) is a secretory protein that antagonizes oncogenic Wnt signaling by binding to the Wnt coreceptor low-density lipoprotein receptor-related protein 6 (LRP6). DKK1 may also regulate its own signaling to promote cancer cell proliferation, but the mechanism is not understood. Here, we identified cytoskeleton-associated protein 4 (CKAP4) as a DKK1 receptor and evaluated CKAP4-mediated DKK1 signaling in cancer cell proliferation. We determined that DKK1 binds CKAP4 and LRP6 with similar affinity but interacts with these 2 receptors with different cysteine-rich domains. DKK1 induced internalization of CKAP4 in a clathrin-dependent manner, further supporting CKAP4 as a receptor for DKK1. DKK1/CKAP4 signaling activated AKT by forming a complex between the proline-rich domain of CKAP4 and the Src homology 3 domain of PI3K, resulting in proliferation of normal cells and cancer cells. Expression of DKK1 and CKAP4 was frequent in tumor lesions of human pancreatic and lung cancers, and simultaneous expression of both proteins in patient tumors was negatively correlated with prognosis and relapse-free survival. An anti-CKAP4 antibody blocked the binding of DKK1 to CKAP4, suppressed AKT activity in a human cancer cell line, and attenuated xenograft tumor formation in immunodeficient mice. Together, our results suggest that CKAP4 is a potential therapeutic target for cancers that express both DKK1 and CKAP4.
