Functional Analysis of a Breast Cancer-Associated Mutation in the Intracellular Domain of the Metalloprotease ADAM12

Abstract

A recently identified breast cancer-associated mutation in the metalloprotease ADAM12 alters a potential dileucine trafficking signal, which could affect protein processing and cellular localization. ADAM12 belongs to the group of A Disintegrin And Metalloproteases (ADAMs), which are typically membrane-associated proteins involved in ectodomain shedding, cell-adhesion, and signaling. ADAM12 as well as several members of the ADAM family are over-expressed in various cancers, correlating with disease stage. Three breast cancer-associated somatic mutations were previously identified in ADAM12, and two of these, one in the metalloprotease domain and another in the disintegrin domain, were investigated and found to result in protein misfolding, retention in the secretory pathway, and failure of zymogen maturation. The third mutation, p.L792F in the ADAM12 cytoplasmic tail, was not investigated, but is potentially significant given its location within a di-leucine motif, which is recognized as a potential cellular trafficking signal. The present study was motivated both by the potential relevance of this documented mutation to cancer, as well as for determining the role of the di-leucine motif in ADAM12 trafficking. Expression of ADAM12 p.L792F in mammalian cells demonstrated quantitatively similar expression levels and zymogen maturation as wild-type (WT) ADAM12, as well as comparable cellular localizations. A cell surface biotinylation assay demonstrated that cell surface levels of ADAM12 WT and ADAM12 p.L792F were similar and that internalization of the mutant occurred at the same rate and extent as for ADAM12 WT. Moreover, functional analysis revealed no differences in cell proliferation or ectodomain shedding of epidermal growth factor (EGF), a known ADAM12 substrate between WT and mutant ADAM12. These data suggest that the ADAM12 p.L792F mutation is unlikely to be a driver (cancer causing)-mutation in breast cancer.

An essential role of metalloprotease-disintegrin ADAM12 in triple-negative breast cancer

“…The third mutation, L792F, was found in TNBC, but this mutation does not alter the proteolytic activity of ADAM12L (ref. [47] and our unpublished observations). Thus, it appears that TNBCs express the active form of ADAM12L, which is consistent with its role in EGFR activation in TNBC described here.…”

Phenotypic Diversity of Breast Cancer-Related Mutations in Metalloproteinase-Disintegrin ADAM12

“…The current study, together with two other previous reports [16] , [17] , provides an insight into the structural/functional aspects of the currently known breast cancer-associated mutations in ADAM12. These mutations are scattered along the entire length of the protein, and they do not appear to cluster within a specific region in the three-dimensional model of ADAM12-L.…”

“…Trafficking, processing, and catalytic activities of human D301H or G479E mutants have not been examined before. The L792F mutation in the cytoplasmic tail of human ADAM12-L was reported to have no effect on ADAM12-L trafficking, processing, or catalytic activity [16] , [17] .…”

“…We have previously shown that the D299H and G477E mutations in mouse ADAM12 (which correspond to the D301H and G479E mutations in human ADAM12) are loss-of-function mutations that inhibit the intracellular trafficking and proteolytic activation of the nascent ADAM12 protein [16]. The L792F mutation in human ADAM12-L was reported not to affect protein processing, localization, or function [16], [17]. The other three mutations - T596A, R612Q, and G668A - have been identified more recently [14], [15], and their effects on the structure/function of ADAM12 are currently unknown.…”

New insight into the role of the ADAM protease family in breast carcinoma progression

“…Additionally, in functional analysis assays no differences were shown in cell proliferations or ectodomain sheddase of EGF (an ADAM12 substrate for mutant ADAM12 and WT). According to these data, ADAM12 p. L792F mutation is improbable to drive cancer causing mutations in BC [ 95 ]. As discussed above, ADAM12 is overexpressed in human breast carcinomas and is a chemoresistance prognosticator in estrogen receptor-negative cancers.…”