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International audienc

PKCθ-induced phosphorylations control the ability of Fra-1 to stimulate gene expression and cancer cell migration

International audienceThe AP-1 transcription factor Fra-1 is aberrantly expressed in a large number of cancers and plays crucial roles in cancer development and progression by stimulating the expression of genes involved in these processes. However, the control of Fra-1 transactivation ability is still unclear and here we hypothesized that PKCθ-induced phosphorylation could be necessary to obtain a fully active Fra-1 protein. Using MCF7 stable cells overexpressing equivalent levels of unphosphorylated Fra-1 or PKCθ-phosphorylated Fra-1, we showed that PKCθ-induced phosphorylation of Fra-1 was crucial for the stimulation of MMP1 and IL6 expression. Consistently, we found a significant positive correlation between PRKCQ (coding for PKCθ) and MMP1 mRNA expression levels in human breast cancer samples. PKCθ-induced phosphorylations, in part at T217 and T227 residues, strongly and specifically increased Fra-1 transcriptional activity through the stimulation of Fra-1 transactivation domain, without affecting JUN factors. More importantly, these phosphorylations were required for Fra-1-induced migration of breast cancer cells and phosphorylated Fra-1 expression was enriched at the invasion front of human breast tumors. Taken together, our findings indicate that PKCθ-induced phosphorylation could be important for the function of Fra-1 in cancer progression

PTPN13 induces cell junction stabilization and inhibits mammary tumor invasiveness

International audienceClinical data suggest that the protein tyrosine phosphatase PTPN13 exerts an anti-oncogenic effect. Its exact role in tumorigenesis remains, however, unclear due to its negative impact on FAS receptor-induced apoptosis. Methods: We crossed transgenic mice deleted for PTPN13 phosphatase activity with mice that overexpress human HER2 to assess the exact role of PTPN13 in tumor development and aggressiveness. To determine the molecular mechanism underlying the PTPN13 tumor suppressor activity we developed isogenic clones of the aggressive human breast cancer cell line MDA-MB-231 overexpressing either wild type or a catalytically-inactive mutant PTPN13 and subjected these to phosphoproteomic and gene ontology analyses. We investigated the PTPN13 consequences on cell aggressiveness using wound healing and Boyden chamber assays, on intercellular adhesion using videomicroscopy, cell aggregation assay and immunofluorescence. Results: The development, growth and invasiveness of breast tumors were strongly increased by deletion of the PTPN13 phosphatase activity in transgenic mice. We observed that PTPN13 phosphatase activity is required to inhibit cell motility and invasion in the MDA-MB-231 cell line overexpressing PTPN13. In vivo, the negative PTPN13 effect on tumor invasiveness was associated with a mesenchymal-to-epithelial transition phenotype in athymic mice xenografted with PTPN13-overexpressing MDA-MB-231 cells, as well as in HER2-overexpressing mice with wild type PTPN13, compared to HER2-overexpressing mice that lack PTPN13 phosphatase activity. Phosphoproteomic and gene ontology analyses indicated a role of PTPN13 in the regulation of intercellular junction-related proteins. Finally, protein localization studies in MDA-MB-231 cells and HER2-overexpressing mice tumors confirmed that PTPN13 stabilizes intercellular adhesion and promotes desmosome formation. Conclusions: These data provide the first evidence for the negative role of PTPN13 in breast tumor invasiveness and highlight its involvement in cell junction stabilization

Prognostic value of androgen receptor and FOXA1 co-expression in non-metastatic triple negative breast cancer and correlation with other biomarkers

International audienceBACKGROUND: In luminal androgen receptor (AR) tumours, FOXA1 may direct AR to sites occupied by ER in luminal tumours, thus stimulating proliferation.METHODS: AR and FOXA1 expression were evaluated by immunohistochemistry in 333 non-metastatic triple-negative breast cancers (TNBC). Positivity threshold was set at ≥ 1% staining. Lymphocytic infiltration, PD-L1expression, PIK3CA mutations, PTEN defects and BRCA1 promoter methylation were assessed.RESULTS: AR + /FOXA1 + tumours (42.4%) were more frequently: found in older patients, lobular, of lower nuclear grade, with more frequently PIK3CA mutations; exhibited less frequently BRCA1 promoter methylation, defects of PTEN and PD-L1 expression than others. Recurrence-free and overall survivals were significantly lower for AR + /FOXA1 + TNBC (median follow-up: 7.8 years).CONCLUSIONS: AR + /FOXA1 + expression defines a luminal-like TNBC subgroup affected with a worse outcome compared to other TNBC and a higher risk of late recurrences. This subgroup appears enriched in PIK3CA mutations, suggesting a role for PI3K inhibitors in this subgroup

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

International audienceRationale: 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

Immunotherapy of triple-negative breast cancer with cathepsin D-targeting antibodies

International audienceBackground: Triple-negative breast cancer (TNBC) treatment is currently restricted to chemotherapy. Hence, tumorspecific molecular targets and/or alternative therapeutic strategies for TNBC are urgently needed. Immunotherapy is emerging as an exciting treatment option for TNBC patients. The aspartic protease cathepsin D (cath-D), a marker of poor prognosis in breast cancer (BC), is overproduced and hypersecreted by human BC cells. This study explores whether cath-D is a tumor cell-associated extracellular biomarker and a potent target for antibody-based therapy in TNBC.Methods: Cath-D prognostic value and localization was evaluated by transcriptomics, proteomics andimmunohistochemistry in TNBC. First-in-class anti-cath-D human scFv fragments binding to both human and mousecath-D were generated using phage display and cloned in the human IgG1λformat (F1 and E2). Anti-cath-D antibodybiodistribution, antitumor efficacy and in vivo underlying mechanisms were investigated in TNBC MDA-MB-231 tumorxenografts in nude mice. Antitumor effect was further assessed in TNBC patient-derived xenografts (PDXs).Results: HighCTSDmRNA levels correlated with shorter recurrence-free survival in TNBC, and extracellular cath-D wasdetected in the tumor microenvironment, but not in matched normal breast stroma. Anti-cath-D F1 and E2 antibodiesaccumulated in TNBC MDA-MB-231 tumor xenografts, inhibited tumor growth and improved mice survival withoutapparent toxicity. The Fc function of F1, the best antibody candidate, was essential for maximal tumor inhibition in theMDA-MB-231 model. Mechanistically, F1 antitumor response was triggered through natural killer cell activation via IL-15upregulation, associated with granzyme B and perforin production, and the release of antitumor IFNγcytokine. The F1antibody also prevented the tumor recruitment of immunosuppressive tumor-associated macrophages M2 andmyeloid-derived suppressor cells, a specific effect associated with a less immunosuppressive tumor microenvironmenthighlighted by TGFβdecrease. Finally, the antibody F1 inhibited tumor growth of two TNBC PDXs, isolated frompatients resistant or not to neo-adjuvant chemotherapy.Conclusion: Cath-D is a tumor-specific extracellular target in TNBC suitable for antibody-based therapy.Immunomodulatory antibody-based strategy against cath-D is a promising immunotherapy to treat patients withTNB