Chardes, T. | bioRxiv Science

bioRxiv2021

SPARC in cancer-associated fibroblasts is an independent poor prognostic factor in non-metastatic triple-negative breast cancer and exhibits pro-tumor activity

PurposeTriple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype and lacks specific targeted therapeutics. The current mechanistic evidence from cell-based studies suggests that the matricellular protein SPARC has a tumor-promoting role in TNBC; however, data on the clinical relevance of SPARC expression/secretion by tumor and stromal cells in TNBC are limited. Experimental DesignThis study analyzed the prognostic value of tumor and stromal cell SPARC expression in a large series of 148 patients with non-metastatic TNBC and long follow-up (median: 5.4 years). Fibrosis, tumor-associated macrophage (TAM) infiltration, tumor-infiltrating lymphocyte (TIL) density, PD-L1 and PD-1 expression were assessed. Tumor and stromal cell SPARC expression was studied by immunofluorescence, western blotting, and meta-analysis of published single-cell mRNA sequencing data. The biological role of fibroblast-secreted SPARC was analyzed using cell adhesion, wound healing, Transwell-based motility and invasion, and tumor spheroid assays. ResultsSPARC expression was detected in cancer cells (42.4%), cancer-associated fibroblasts (CAFs) (88.1%), TAMs (77.1%), endothelial cells (75.2%), and TILs (9.8%). Recurrence-free survival was significantly lower in patients with SPARC-expressing CAFs. SPARC expression in CAFs was an independent prognostic factor in multivariate analysis. Tumor and stromal cell SPARC expression was observed in TNBC cytosols, patient-derived xenografts, and cell lines. SPARC was expressed by different CAF subsets, including myofibroblasts and inflammatory CAFs. Fibroblast-secreted SPARC inhibited TNBC cell adhesion and stimulated their migration and invasion. ConclusionsSPARC expression in CAFs is an independent predictor of recurrence-free survival in TNBC. Patients with SPARC-expressing CAFs could be eligible for anti-SPARC-targeted therapy. Statement of translational relevanceHere, we identified a subgroup of patients with triple-negative breast cancer (TNBC) with worse prognosis and eligible for therapies that target extracellular matrix proteins in the tumor stroma. Specifically, we found that expression of the matricellular protein SPARC in cancer-associated fibroblasts (CAFs) is an independent prognostic marker of poor outcome in TNBC. Furthermore, we showed that in TNBC, SPARC is expressed by different CAF subpopulations, including myofibroblasts and inflammatory fibroblasts that are involved/associated with many tumor-related processes. We then found that SPARC secreted by fibroblasts has a pro-tumor-promoting role by inhibiting TNBC cell adhesion and stimulating their motility and invasiveness. Overall, our results support the need to consider SPARC expressed/secreted by CAFs as a novel therapeutic target in TNBC in the context of treatments to modulate the tumor stroma.

cancer biology↗

bioRxiv2020

Cathepsin D exacerbates SPARC-driven aggressiveness by limited proteolysis in triple-negative breast cancer

Extracellular matrix (ECM) remodeling by proteases results in the release of protein fragments that promote tumor progression and metastasis. The protease cathepsin D (cath-D), a marker of poor prognosis in triple-negative breast cancer (TNBC), is aberrantly secreted in the tumor microenvironment. Using degradomic analyses by TAILS, we discovered 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. Our study establishes a novel crosstalk between proteases and matricellular proteins in the tumor microenvironment through limited proteolysis of SPARC, revealing a novel targetable 9-kDa bioactive SPARC fragment for new TNBC treatments.

cancer biology↗

bioRxiv2019

Biasing HER4 Tyrosine Kinase Signaling with Antibodies: Induction of Cell Death by Antibody-Dependent HER4 Intracellular Domain Trafficking

HER4 isoforms have oncogenic or tumor suppressor functions depending on their susceptibility to proteolytic cleavage and HER4 Intracellular Domain (4ICD) translocation. Here, we report that the NRG1 tumor suppressor mechanism through the HER4 JMa/CYT1 isoform can be mimicked by the agonist anti-HER4 antibody C6. NRG1 induced cleavage of poly(ADP-ribose) polymerase (PARP) and sub-G1 DNA fragmentation, and also reduced the metabolic activity of HER3-negative/HER4-positive cervical (C-33A) and ovarian (COV318) cancer cells. This effect was confirmed in HER4 JMa/CYT1-, but not JMa/CYT2-transfected BT549 triple-negative breast cancer cells. NRG1 favored 4ICD cleavage and retention in mitochondria in JMa/CYT1-transfected BT549 cells, leading to Reactive Oxygen Species (ROS) production through mitochondrial depolarization. Similarly, the anti-HER4 antibody C6, which binds to a conformational epitope located on aa 575-592 and 605-620 of HER4 domain IV, induced 4ICD cleavage and retention in mitochondria, and mimicked NRG1-mediated effects on PARP cleavage, ROS production, and mitochondrial membrane depolarization in cancer cells. In vivo, C6 reduced growth of COV434 and HCC187 tumor cell xenografts in nude mice. Biasing 4ICD trafficking to mitochondria with anti-HER4 antibodies to mimic NRG1 suppressor functions could be an alternative anti-cancer strategy.

cancer biology↗