Substrate-biased activity-based probes identify the urokinase-plasminogen axis as a master regulator of metastatic signaling by orphan membrane receptor CDCP1

Abstract

CDCP1 is an oncogenic orphan transmembrane receptor and a promising target for detection and treatment of cancer. Extracellular proteolysis of CDCP1 by poorly defined mechanisms induces pro-metastatic signaling. We describe a novel approach for rapid identification of proteases responsible for key proteolytic events exploiting a substrate-biased activity-based probe (sbABP) that incorporates a substrate cleavage motif grafted onto a peptidyl-diphenyl-phosphonate warhead for specific target protease capture, isolation and identification. Using a CDCP1-biased probe we identify urokinase (uPA) as the master regulator of CDCP1 proteolysis, both by direct cleavage and via activation of CDCP1-cleaving plasmin. We show that co-expression of uPA and CDCP1 is strongly predictive of poor disease outcome across multiple cancers and demonstrate that uPA-mediated CDCP1 proteolysis promotes metastasis in disease-relevant preclinical in vivo models. These results highlight CDCP1 cleavage as a potential target to disrupt cancer and establish sbABP technology as a new approach to identify disease-relevant proteases.

Preclinical Evaluation of a Fluorescent Probe Targeting Receptor CDCP1 for Identification of Ovarian Cancer

“…CDCP1 is a 135 kDa cell surface protein that can undergo proteolytic cleavage in vitro and in vivo by serine proteases including matriptase 34 and proteases of the plasminogen activation system. 32,33 After proteolysis, CDCP1 is present as a 70 kDa membrane spanning the carboxyl-terminal fragment (CTF) and a 65 kDa amino-terminal fragment (ATF) that binds to the plasma membrane via CDCP1-CTF or full-length CDCP1 or is shed from the cell surface and can be detected in conditioned cell culture media and colorectal cancer patient blood. 34,35 Because 10D7 binds to CDCP1-ATF, shedding of this fragment from the cell surface would prevent the antibody binding to CDCP1 to deliver a fluorescent payload such as ICG.…”

“…A previously described tissue microarray (TMA) with duplicate 0.6 mm cores from 134 endometrioid and 35 mucinous ovarian cancer cases was obtained from the Cheryl Brown Ovarian Cancer Outcomes Unit, University of British Columbia, Vancouver, Canada. 26,27,33 Analysis of human samples in TMAs was approved by the Mater Health Services Human Research Ethics Committee, and all procedures were performed in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Immunohistochemistry for CDCP1 expression was performed using antibody #4115 as previously described on a Ventana Benchmark Ultra Automated Staining System (Ventana Medical Systems, Tucson, AZ) with staining assessed and scored by an anatomical pathologist.…”

Detection and Isolation of Circulating Tumor Cells from Breast Cancer Patients Using CUB Domain-Containing Protein 1

“…Urokinase (uPA) cleaves CDCP1, and uPA can be downregulated under hypoxia, providing an explanation for the accumulation of full-length CDCP1 under hypoxia. 44,45 Because hypoxia promotes the formation of an aggressive tumor phenotype, including CTCs, 46 the isolation of hypoxia-exposed CTCs by MACS is simplified by using the entire extracellular domain of CDCP1 for cell catching.…”