Proteomic Identification of Cysteine Cathepsin Substrates Shed from the Surface of Cancer Cells

Barbara Sobotič,Matej Vizovišek,Robert Vidmar,Petra Van Damme,Vasilena Gocheva,Johanna A Joyce,Kris Gevaert,Vito Turk,Boris Turk,Marko Fonović

doi:10.1074/mcp.m114.044628

Abstract

Extracellular cysteine cathepsins are known to drive cancer progression, but besides degradation of extracellular matrix proteins little is known about their physiological substrates and thus the molecular mechanisms they deploy. One of the major mechanisms used by other extracellular proteases to facilitate cancer progression is proteolytic release of the extracellular domains of transmembrane proteins or ectodomain shedding. Here we show using a mass spectrometry-based approach that cathepsins L and S act as sheddases and cleave extracellular domains of CAM adhesion proteins and transmembrane receptors from the surface of cancer cells. In cathepsin S-deficient mouse pancreatic cancers, processing of these cathepsin substrates is highly reduced, pointing to an essential role of cathepsins in extracellular shedding. In addition to influencing cell migration and invasion, shedding of surface proteins by extracellular cathepsins impacts intracellular signaling as demonstrated for regulation of Ras GTPase activity, thereby providing a putative mechanistic link between extracellular cathepsin activity and cancer progression. The MS data is available via ProteomeXchange with identifier PXD002192.

Highlights

From the ‡Department of Biochemistry, Molecular and Structural Biology, Jozef Stefan Institute, Jamova cesta 39, SI-1000 Ljubljana, Slovenia; §International Postgraduate School Jozef Stefan, Jamova 39, SI-1000 Ljubljana, Slovenia; ¶Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium; ʈDepartment of Medical Protein Research, VIB, B-9000 Ghent, Belgium; **Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065; ‡‡Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Jamova cesta 39, SI-1000 Ljubljana, Slovenia; §§Center of Excellence NIN, Ljubljana, Slovenia; ¶¶Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia
Secreted cathepsins were found to be involved in several processes that contribute to carcinogenesis, including extracellular matrix (ECM)1 degradation, activation of proteases such as urokinase-type plasminogen activator and matrix metalloproteinases (MMPs), and in E-cadherin cleavage [2]
Cathepsin Treatment Increases Migration and Invasion Rate of Cancer Cells—As there were a number of cell adhesion molecules (CAMs) molecules among the identified cathepsin targets, which have a major role in cell adhesion and establishment of cell– cell contacts [45], we investigated whether their shedding influenced cell migration and invasion capabilities

Summary

Introduction

From the ‡Department of Biochemistry, Molecular and Structural Biology, Jozef Stefan Institute, Jamova cesta 39, SI-1000 Ljubljana, Slovenia; §International Postgraduate School Jozef Stefan, Jamova 39, SI-1000 Ljubljana, Slovenia; ¶Department of Biochemistry, Ghent University, B-9000 Ghent, Belgium; ʈDepartment of Medical Protein Research, VIB, B-9000 Ghent, Belgium; **Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065; ‡‡Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins, Jamova cesta 39, SI-1000 Ljubljana, Slovenia; §§Center of Excellence NIN, Ljubljana, Slovenia; ¶¶Faculty of Chemistry and Chemical Technology, University of Ljubljana, Slovenia. Cells (30,000 cells per setup) were incubated in 300 ␮l PBS (Lonza) (pH 6.0, with 0.5 mM DTT (Fluka Biochemica)), with added human recombinant cathepsin L or S (0.05 ␮M) or inhibited cathepsin (0.05 ␮M cathepsin 1 h preincubated with 20 ␮M E-64 (Peptide Institute)) as a negative control.

Results

Conclusion