Cassandre Yip

EGFR Activation and Signaling in Cancer Cells Are Enhanced by the Membrane-Bound Metalloprotease MT4-MMP

MT4-MMP (MMP-17) is a glycosylphosphatidyl inositol-anchored matrix metalloprotease expressed on the surface of cancer cells that promotes tumor growth and metastasis. In this report, we identify MT4-MMP as an important driver of cancer cell proliferation through CDK4 activation and retinoblastoma protein inactivation. We also determine a functional link between MT4-MMP and the growth factor receptor EGFR. Mechanistic experiments revealed direct association of MT4-MMP and its positive effects on EGFR phosphorylation in response to TGFα and EGF in cancer cells. Notably, the effects of MT4-MMP on proliferation and EGFR activation did not rely on metalloprotease activity. Clinically, MT4-MMP and EGFR expressions were correlated in human triple-negative breast cancer specimens. Altogether, our results identify MT4-MMP as a positive modifier of EGFR outside-in signaling that acts to cooperatively drive cancer cell proliferation.

Homologous Transcription Factors DUX4 and DUX4c Associate with Cytoplasmic Proteins during Muscle Differentiation

Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were recently shown to exit the nucleus via a novel mechanism of nuclear envelope budding. Following DUX4 or DUX4c overexpression in muscle cell cultures, we observed their association with similar nuclear buds. In conclusion, our study demonstrated unexpected interactions of DUX4/4c with cytoplasmic proteins playing major roles during muscle differentiation. Further investigations are on-going to evaluate whether these interactions play roles during muscle regeneration as previously suggested for DUX4c.

Dynamics of internalization and recycling of the prometastatic membrane type 4 matrix metalloproteinase (MT4‐MMP) in breast cancer cells

Membrane type 4 matrix metalloproteinase (MT4‐MMP) [matrix metalloproteinase (MMP) 17] is a GPI‐anchored membrane‐type MMP expressed on the cell surface of human breast cancer cells. In triple‐negative breast cancer cells, MT4‐MMP promotes primary tumour growth and lung metastases. Although the trafficking and internalization of the transmembrane membrane type 1 MMP have been extensively investigated, little is known about the regulatory mechanisms of the GPI‐anchored MT4‐MMP. Here, we investigated the fate and cellular trafficking of MT4‐MMP by analysing its homophilic complex interactions, internalization and recycling dynamics as compared with an inert form, MT4‐MMP‐E249A. Oligomeric and dimeric complexes were analysed by cotransfection of cells with FLAG‐tagged or Myc‐tagged MT4‐MMP in reducing and nonreducing immunoblotting and coimmunoprecipitation experiments. The trafficking of MT4‐MMP was studied with an antibody feeding assay and confocal microscopy analysis or cell surface protein biotinylation and western blot analysis. We demonstrate that MT4‐MMP forms homophilic complexes at the cell surface, and internalizes in early endosomes, and that some of the enzyme is either autodegraded or recycled to the cell surface. Our data indicate that MT4‐MMP is internalized by the clathrin‐independent carriers/GPI‐enriched early endosomal compartments pathway, a mechanism that differs from that responsible for the internalization of other membrane‐type MMP members. Although MT4‐MMP localizes with caveolin‐1, MT4‐MMP internalization was not affected by inhibitors of caveolin‐1 or clathrin endocytosis pathways, but was reduced by CDC42 or RhoA silencing with small interfering RNA. We provide a new mechanistic insight into the regulatory mechanisms of MT4‐MMP, which may have implications for the design of novel therapeutic strategies for metastatic breast cancer.

MT4-MMP and EGFR expression levels are key biomarkers for breast cancer patient response to chemotherapy and erlotinib.

Background:Triple-negative breast cancers (TNBC) are heterogeneous cancers with poor prognosis. We aimed to determine the clinical relevance of membrane type-4 matrix metalloproteinase (MT4-MMP), a membrane type matrix metalloproteinase that interacts with epidermal growth factor receptor (EGFR) overexpressed in >50% of TNBC.Methods:We conducted a retrospective immunohistochemical analysis on human TNBC samples (n=81) and validated our findings in in vitro and in vivo assays.Results:Membrane type-4 matrix metalloproteinase and EGFR are produced in 72.5% of TNBC samples, whereas those proteins are faintly produced by healthy tissues. Unexpectedly, tumour relapse after chemotherapy was reduced in samples highly positive for MT4-MMP. Mechanistically, this is ascribed to a higher sensitivity of MT4-MMP-producing cells to alkylating or intercalating chemotherapeutic agents, as assessed in vitro. In sharp contrast, MT4-MMP expression did not affect tumour cell sensitivity to paclitaxel that interferes with protease trafficking. Importantly, MT4-MMP expression sensitised cancer cells to erlotinib, a tyrosine kinase EGFR inhibitor. In a pre-clinical model, the growth of MT4-MMP overexpressing xenografts, but not of control ones, was reduced by epirubicin or erlotinib. The combination of suboptimal drug doses blocked drastically the growth of MT4-MMP-producing tumours.Conclusions:We demonstrate that MT4-MMP defines a sub-population of TNBC sensitive to a combination of DNA-targeting chemotherapeutic agents and anti-EGFR drugs.