EMILIN1 role in tumor growth after enzymatic degradation

EMILIN1 is a ECM glycoprotein involved in several cellular processes. In particular, EMILIN1 is involved in elastogenesis processes and in the maintenance of lymphatic vessels structures. EMILIN1 presents several regulatory properties exercised through its EMI domain that is located at N-terminal region and is able to regulate homeostasis of blood pressure, and its gC1q domain located at C-terminal region. In particular, gC1q domain is involved in the regulation of both cell adhesion and cell proliferation through the binding with α4β1 integrin. EMILIN1-/- mice present an increase of tumoral cell proliferation; this is due to the loss of the interaction between gC1q domain-α4β1 integrin, that determines the activation of the MAPK pathway, resulting in upregulation of cell proliferation. Studies previously published in our laboratory demonstrated that neutrophil elastase, released by neutrophils present in tumor microenvironment, is able to degrade EMILIN1 resulting in the loss of its regulatory functions. Other authors have proposed EMILIN1 a possible substrate of several matrix metalloproteinases: matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-9 (MMP-9) and matrix metalloproteinase 14 (MT1-MMP). We demonstrated that among these MMPs only MT1-MMP shows a weak proteolytic activity on EMILIN1. Moreover, we observed that MT1-MMP was not able to impair EMILIN1 functions. On the contrary we observe that the digestion of EMILIN1 with neutrophil elastase was able to impair EMILIN1 tumor suppressor role. At this step, we wanted to analyze the capability of neutrophil elastase to degrade the gC1q domain. We digested the gC1q domain with several proteases and we observed that among the tested proteases only neutrophil elastase was able to degrade the gC1q domain and to impair its functionality. Thus, we wanted to pinpoint the neutrophil elastase cleavage site on gC1q domain, in order to generate a mutant of gC1q domain resistant to neutrophil elastase cleavage. We consulted several peptidase database that contained predicted neutrophile elastase cleavage sites, on these basis we generated several gC1q mutants. Among these mutants that generated, we found that the mutant R914W, in which aminoacid arginine was substituted with aminoacid tryptophan, was resistant to neutrophil elastase cleavage. Functional adhesion and proliferation assays confirmed the capability of R914 mutant to maintain its properties after neutrophil elastase treatment