Alice Truong

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.

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.