A 9-kDa matricellular SPARC fragment released by cathepsin D exhibits pro-tumor activity in the triple-negative breast cancer microenvironment

Theranostics. 2021 Apr 15;11(13):6173-6192. doi: 10.7150/thno.58254. eCollection 2021.


Rationale: Alternative therapeutic strategies based on tumor-specific molecular targets are urgently needed for triple-negative breast cancer (TNBC). The protease cathepsin D (cath-D) is a marker of poor prognosis in TNBC and a tumor-specific extracellular target for antibody-based therapy. The identification of cath-D substrates is crucial for the mechanistic understanding of its role in the TNBC microenvironment and future therapeutic developments. Methods: The cath-D substrate repertoire was investigated by N-Terminal Amine Isotopic Labeling of Substrates (TAILS)-based degradome analysis in a co-culture assay of TNBC cells and breast fibroblasts. Substrates were validated by amino-terminal oriented mass spectrometry of substrates (ATOMS). Cath-D and SPARC expression in TNBC was examined using an online transcriptomic survival analysis, tissue micro-arrays, TNBC cell lines, patient-derived xenografts (PDX), human TNBC samples, and mammary tumors from MMTV-PyMT Ctsd-/- knock-out mice. The biological role of SPARC and its fragments in TNBC were studied using immunohistochemistry and immunofluorescence analysis, gene expression knockdown, co-culture assays, western blot analysis, RT-quantitative PCR, adhesion assays, Transwell motility, trans-endothelial migration and invasion assays. Results: TAILS analysis showed that the matricellular protein SPARC is a substrate of extracellular cath-D. In vitro, cath-D induced limited proteolysis of SPARC C-terminal extracellular Ca2+ binding domain at acidic pH, leading to the production of SPARC fragments (34-, 27-, 16-, 9-, and 6-kDa). Similarly, cath-D secreted by TNBC cells cleaved fibroblast- and cancer cell-derived SPARC at the tumor pericellular acidic pH. SPARC cleavage also occurred in TNBC tumors. Among these fragments, only the 9-kDa SPARC fragment inhibited TNBC cell adhesion and spreading on fibronectin, and stimulated their migration, endothelial transmigration, and invasion. Conclusions: Our study establishes a novel crosstalk between proteases and matricellular proteins in the tumor microenvironment through limited SPARC proteolysis, revealing a novel targetable 9-kDa bioactive SPARC fragment for new TNBC treatments. Our study will pave the way for the development of strategies for targeting bioactive fragments from matricellular proteins in TNBC.

Keywords: ECM; TNBC; bioactive fragment; matricellular protein; protease; tumor microenvironment.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Cathepsin D / deficiency
  • Cathepsin D / genetics
  • Cathepsin D / metabolism*
  • Cell Adhesion
  • Extracellular Matrix / metabolism*
  • Female
  • Fibroblasts
  • Gene Expression Regulation, Neoplastic
  • Humans
  • Hydrogen-Ion Concentration
  • Mammary Neoplasms, Experimental / enzymology
  • Mice
  • Mice, Knockout
  • Mice, Transgenic
  • Molecular Weight
  • Neoplasm Invasiveness
  • Neoplasm Proteins / genetics
  • Neoplasm Proteins / metabolism*
  • Osteonectin / genetics
  • Osteonectin / metabolism*
  • Peptide Fragments / metabolism
  • Peptide Fragments / pharmacology*
  • Protein Domains
  • Proteolysis
  • Substrate Specificity
  • Transendothelial and Transepithelial Migration
  • Triple Negative Breast Neoplasms / enzymology
  • Triple Negative Breast Neoplasms / pathology*
  • Tumor Microenvironment*


  • Neoplasm Proteins
  • Osteonectin
  • Peptide Fragments
  • SPARC protein, human
  • Cathepsin D
  • Ctsd protein, mouse