Identification of ADAM12 as a Novel Basigin Sheddase

Abstract

The transmembrane glycoprotein basigin, a member of the immunoglobulin superfamily, stimulates matrix metalloproteinase (MMP)-mediated extracellular matrix (ECM) degradation and thereby drives cancer cell invasion. Basigin is proteolytically shed from the cell surface and high concentrations of soluble basigin in the blood dictates poor prognosis in cancer patients. A positive correlation between basigin and a disintegrin and metalloproteinase (ADAM)-12 in serum from prostate cancer patients has been reported. Yet, the functional relevance of this correlation is unknown. Here, we show that ADAM12 interacts with basigin and cleaves it in the juxtamembrane region. Specifically, overexpression of ADAM12 increases ectodomain shedding of an alkaline phosphatase-tagged basigin reporter protein from the cell surface. Moreover, CRISPR/Cas9-mediated knockout of ADAM12 in human HeLa carcinoma cells results in reduced shedding of the basigin reporter, which can be rescued by ADAM12 re-expression. We detected endogenous basigin fragments, corresponding to the expected size of the ADAM12-generated ectodomain, in conditioned media from ADAM12 expressing cancer cell-lines, as well as serum samples from a healthy pregnant donor and five bladder cancer patients, known to contain high ADAM12 levels. Supporting the cancer relevance of our findings, we identified several cancer-associated mutations in the basigin membrane proximal region. Subsequent in vitro expression showed that some of these mutants are more prone to ADAM12-mediated shedding and that the shed ectodomain can enhance gelatin degradation by cancer cells. In conclusion, we identified ADAM12 as a novel basigin sheddase with a potential implication in cancer.

Keywords: CD147; EMMPRIN; a disintegrin and metalloproteinase; ectodomain shedding.

Figure 1

A disintegrin and metalloproteinase (ADAM)-12 interacts with basigin (BSG) in human cells. (A) 293-VnR cells were transfected with A12-GFP or GFP alone, immunoprecipitated with a polyclonal anti-GFP antibody and analyzed by Western blot, using an antibody against the C-terminal part of BSG. Total cell lysates (TCL) were tested for A12-GFP (pro and mature forms) and endogenous BSG protein expression. Actin was used as the loading control. (B) 293-VnR cells were co-transfected with A12 and full-length BSG, immunoprecipitated with an antibody against BSG and analyzed by Western blot, using a rabbit polyclonal antibody against A12. Immunoprecipitation with control rabbit IgG served as a negative control. (C) 293-VnR cells were transfected with A12 or the catalytically inactive A12-E/A mutant, together with full-length BSG and tested for A12 expression by Western blot, using actin as the loading control. (D) Cells from C were tested for A12-BSG co-localization by the proximity ligation assay (PLA) (red signal), using antibodies against the extracellular parts of A12 and BSG. Reactions using either of the two antibodies alone served as negative controls. Scale bar = 8 µm. (E) Number of red foci were automatically counted in 50 cells from three independent experiments, using MetaMorph microscopy analysis software. The average number of foci per cells ± SEM is shown in the graph. * p < 0.05, Student’s t-test.

Figure 2

ADAM12 overexpression increases ectodomain shedding of a truncated BSG reporter substrate. (A) Amino acid sequence of the reporter substrate BSG-AP, consisting of the C-terminal part of BSG fused to alkaline phosphatase (AP). The truncated extracellular part of BSG is shown in red and the transmembrane domain is underlined. (B) Fold shedding in 293-VnR cells transfected with BSG-AP together with A12 or catalytically inactive A12-E/A, and treated with or without the metalloproteinase inhibitor Batimastat (BB94). Fold shedding is calculated as AP activity in the medium divided by the total AP activity in medium and cell lysate, and normalized to A12-E/A. (C) Fold shedding in 293-VnR cells transfected with BSG-AP and A12-E/A, A12, ADAM9 (A9) or ADAM17 (A17), calculated as in (C). (D) Western blot of total lysates from cells used in (C), showing comparable expression of the different ADAMs. (E) Fold shedding in 293-VnR cells transfected with BSG-AP alone (no A12) or with BSG-AP and A12 together in cells treated with control siRNA, siRNA against matrix metalloproteinase (MMP)-14, or the inhibitor BB94 as indicated. (F) Western blot of media and total cell lysates (TCL) from cells in (E), using actin as the loading control. For all graphs, values represent means ± SEM from three independent experiments. * p < 0.05, ANOVA.

Figure 3

CRISPR/Cas9-mediated ADAM12 knockout reduces BSG reporter shedding. (A) qPCR analysis of A12 mRNA in CRISPR/Cas9 generated A12 knockout (clones D7 and E7) and non-edited wildtype (clone D4) HeLa cells. (B) Immunofluorescence staining of A12 in CRISPR/Cas9 generated A12 knockout HeLa clones (KO) D7 and E7, and parental HeLa cells. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and scale bar = 12 μm. (C) Fold shedding of BSG-AP transfected parental HeLa cells, CRISPR/Cas9-generated A12 knockout (KO) HeLa clones D7 or E7 with or without A12 re-expressed, and the non-edited wildtype (WT) clone D4, as indicated. Values represent means ± SEM from three independent experiments. * p < 0.05, ** p < 0.005, ANOVA.

Figure 4

ADAM12 sheds endogenous BSG. (A) Schematic of the BSG structure, showing the two immunoglobulin domains (D1 and D2), the identified MMP14 and putative ADAM12 (A12) cleavage sites, and the transmembrane domain (TM). (B) Cytospin of wildtype or A12 expressing MCF7 cells were immuno-stained with the antibody PA5-29787, recognizing region aa. 70–206 of BSG. Scale bar = 8 µm. (C) Wildtype or A12 expressing MCF7 cells were surface biotinylated and incubated overnight in serum free media. Biotinylated cell surface proteins were pulled down with streptavidin–agarose from conditioned cell media or total cell lysates (TCL) and detected by Western blot, using antibodies against aa. 70–206 (PA5-29787) or the C-terminus of BSG. Actin was used as a loading control. (D) Western blot of BSG in serum from pregnant (pregn.) or non-pregnant (control) women. (E) Western blot of BSG in serum from five bladder cancer patients stained with antibodies recognizing either the C- or the N-terminus of BSG.

Figure 5

Cancer-associated BSG mutants are differentially shed by ADAM12. (A) Illustration of cancer-associated mutations (marked in grey) in the extracellular membrane proximal region (aa. 190–208) of BSG, identified in the Catalogue Of Somatic Mutations In Cancer (COSMIC) database (BSG, ENST00000333511; August 2017). (B) Fold shedding in 293-VnR cells transfected with A12 or the catalytically inactive A12-E/A mutant together with wildtype (BSG-AP), or various BSG-AP point mutations identified in (A). (C) Western blot of AP in total cell lysates (TCL) from cells in (B). Actin was used as loading control. (D) In situ gelatinase assay, using HT1080 cells grown on Oregon green-labeled gelatin and treated with conditioned media from untransfected 293-VnR cells (control) or 293-VnR cells transfected with ADAM12 together with a full-length BSG expression construct harboring the A207T mutation. Dark areas (without green fluorescence) represent gelatin degradation, scale bar = 10 µm. (E) Gelatin degradation was measured in µm² for 27 images, using MetaMorph software and the percentage degradation was calculated. For both graphs, values represent means ± SEM from three independent experiments. * p < 0.05, Student’s t-test or ANOVA.