Abstract
Six different somatic missense mutations in the human ADAM12 gene have been identified so far in breast cancer. Five of these mutations involve highly conserved residues in the extracellular domain of the transmembrane ADAM12-L protein. Two of these extracellular mutations, D301H and G479E, have been previously characterized in the context of mouse ADAM12. Three other mutations, T596A, R612Q, and G668A, have been reported more recently, and their effects on ADAM12-L protein structure/function are not known. Here, we show that ADAM12-L bearing the G668A mutation is largely retained in the endoplasmic reticulum in its nascent, full-length form, with an intact N-terminal pro-domain. The T596A and R612Q mutants are efficiently trafficked to the cell surface and proteolytically processed to remove their pro-domains. However, the T596A mutant shows decreased catalytic activity at the cell surface, while the R612Q mutant is fully active and comparable to the wild-type ADAM12-L. The D301H and G479E mutants, consistent with the corresponding D299H and G477E mutants of mouse ADAM12 described earlier, are not proteolytically processed and do not exhibit catalytic activity at the cell surface. Among all six breast cancer-associated mutations in ADAM12-L, mutations that preserve the activity - R612Q and L792F - occur in triple-negative breast cancers, while loss-of-function mutations - D301H, G479E, T596A, and G668A - are found in non-triple negative cancers. This apparent association between the catalytic activity of the mutants and the type of breast cancer supports a previously postulated role of an active ADAM12-L in the triple negative breast cancer disease.
Highlights
Disintegrin and metalloproteinase domain-containing protein ADAM12 is a member of the ADAM family of proteins that mediate cleavage of substrates at the cell surface and/or modulate intracellular signaling pathways [1,2]
The five breast cancer-associated mutations mapping to the extracellular portion of ADAM12-L are located in different domains and spread over a region spanning more than 300 amino acids (Figure 1A)
Staining of live cells with an antibody recognizing the extracellular domain of ADAM12-L and analysis by flow cytometry demonstrated that WT ADAM12-L and the T596A and R612Q mutants were readily detected at the cell surface, whereas the G668A mutant showed much weaker cell surface staining (Figure 2B)
Summary
Disintegrin and metalloproteinase domain-containing protein ADAM12 is a member of the ADAM family of proteins that mediate cleavage of substrates at the cell surface and/or modulate intracellular signaling pathways [1,2]. ADAM12 is highly upregulated in human breast tumors [3,4,5,6,7,8,9]. Among thirteen different ADAM genes that encode catalytically active proteases [1], ADAM12 is the most frequently somatically mutated gene in human breast cancers. As of September 2013, the COSMIC database (Catalogue of Somatic Mutations in Cancer, http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/) listed 6 confirmed somatic missense mutations in the ADAM12 gene per a total of 1104 unique breast carcinoma samples analyzed. The frequencies of breast cancer-associated missense mutations in other genes encoding catalytically active ADAMs were: 1/973 in ADAM9, 1/973 in ADAM10, 1/1147 in ADAM17, 1/1010 in ADAM19, 1/973 (plus one nonsense mutation) in ADAM20, and
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