ADAM12 transmembrane and secreted isoforms promote breast tumor growth: a distinct role for ADAM12-S protein in tumor metastasis

Abstract

Increased levels of ADAM12 have been reported in a variety of human cancers. We have previously reported that urinary ADAM12 is predictive of disease status in breast cancer patients and that ADAM12 protein levels in urine increase with progression of disease. On the basis of these findings, the goal of this study was to elucidate the contribution of ADAM12 in breast tumor growth and progression. Overexpression of both the ADAM12-L (transmembrane) and ADAM12-S (secreted) isoforms in human breast tumor cells resulted in a significantly higher rate of tumor take and increased tumor size. Cells expressing the enzymatically inactive form of the secreted isoform, ADAM12-S, had tumor take rates and tumor volumes similar to those of wild-type cells, suggesting that the tumor-promoting activity of ADAM12-S was a function of its proteolytic activity. Of the two isoforms, only the secreted isoform, ADAM12-S, enhanced the ability of tumor cells to migrate and invade in vitro and resulted in a higher incidence of local and distant metastasis in vivo. This stimulatory effect of ADAM12-S on migration and invasion was dependent on its catalytic activity. Expression of both ADAM12 isoforms was found to be significantly elevated in human malignant breast tissue. Taken together, our results suggest that ADAM12 overexpression results in increased tumor take, tumor size, and metastasis in vivo. These findings suggest that ADAM12 may represent a potential therapeutic target in breast cancer.

FIGURE 1.

ADAM12 expression is elevated in malignant human breast tissue. IHC analyses for ADAM12 in a human tissue array. Low or no ADAM12 was detected in normal breast tissue, whereas significantly higher ADAM12 protein was detected in carcinoma in situ (CIS) and invasive carcinoma (IDC) tissues (A). B, the frequency of positive ADAM12 expression was significantly higher in malignant tissue as compared with normal breast tissue. C, average ADAM12 staining intensity (range 0–3, where 0 = no staining and 3 = maximum staining intensity) was also significantly higher in malignant tissue as compared with normal breast tissue. These results are expressed as the mean ± S.D. Student's t test was performed. *, p = 0.001, **, p ≤ 0.0001. D, a majority (80%) of the matched sets of primary tumor and metastatic LN analyzed had positive expression of ADAM12 for both tissues. E and F, transcript levels of both ADAM12 isoforms are significantly higher in malignant breast tumor tissue. Relative ADAM12-L and ADAM12-S transcript levels were normalized to β-actin and calibrated to the mean mRNA level (arbitrary value of 1) in normal tissue (gray bars). The fold increase in gene expression relative to the mean value for each disease sample (ADAM12-S, *, p < 0.05; ADAM12-L, **, p < 0.005) is indicated (E). ADAM12-L is preferentially expressed in Stage I and II breast cancer, whereas both forms are expressed in the later stages of disease (F).

FIGURE 2.

ADAM12 expression is elevated in aggressive breast cancer cells. A, real-time RT-PCR analysis indicated significantly higher expression of ADAM12-L in highly aggressive ER-negative cells as compared with ER-positive, less invasive breast cancer cells. *, p = 0.005; **, p ≤ 0.0001. B–G, stable expression of ADAM12 isoforms in MCF-7 cells. Individual ADAM12-l-expressing clones are indicated as LC1, LC2, LC3, and LC4. B, real-time RT-PCR analysis of ADAM12-L in WT MCF-7 and representative ADAM12-L-overexpressing clones. *, p = 0.001; **, p ≤ 0.0001. Cell-surface ADAM12-L expression was confirmed via FACS analysis. C, a representative ADAM12-L clone, LC2, had considerably higher cell surface staining as compared with the WT MCF-7. D, immunoblot analysis of cell lysates indicating increased expression of ADAM12-L in transfectants as compared with WT MCF-7 cells. Three distinct bands representing ADAM12-L were detected, including an ∼120-kDa latent form, an ∼90-kDa active form, and an ∼68-kDa truncated form, respectively. E, ADAM12-S stable clones displayed an ∼10- to 30-fold increase in mRNA expression as compared with WT MCF-7 cells. *, p ≤ 0.01; **, p ≤ 0.0001. Immunoblot of serum-free CM demonstrated expression of latent and the active species of ADAM12-S (F) and ADAM12-S^catmut (G) in the clones but not WT MCF-7 cells.

FIGURE 3.

The secreted isoform, ADAM12-S, promotes migration and invasion in breast cancer cells. Migration and Matrigel invasion analysis of ADAM12-S-expressing breast tumor cells. In vitro migration (A) and Matrigel invasion (B) analyses of representative ADAM12-S and ADAM12-S^catmut clones and WT MCF-7 cells. ADAM12-S-expressing cells displayed significantly (1.5- to 5-fold) higher migration and invasion rates as compared with WT MCF-7 or cells expressing enzymatically inactive ADAM12-S^catmut. C and D, the stimulation of migration and invasion was reversed when ADAM12-S expression was down-regulated using siRNA. Expression of the transmembrane form, ADAM12-L, did not stimulate migration or Matrigel invasion. E and F, migration rates of ADAM12-L clones were equal to or slightly (but not significantly) lower than WT MCF-7. G, ADAM12-S-expressing T47-D cells displayed enhanced migration rates compared with mock-transfected T47-D cells, cells expressing ADAM12-S^catmut, or ADAM12-L. *, p < 0.01. NS, not significant).

FIGURE 4.

ADAM12 expression promotes orthotopic tumor growth. A, mice injected with ADAM12-L- and ADAM12-S-expressing clones formed larger tumors (center panels) than WT MCF-7 cells (left panel) or ADAM12-S^catmut (right panel). B and C, histograms and table indicate mean tumor volumes. B and C, ADAM12-S and ADAM12-L-expressing tumors displayed a significantly higher (≥ 75%) tumor take and tumor volumes (2- to 6-fold) as compared with the WT MCF-7 or ADAM12-S^catmut tumors. D, increased staining of ADAM12 in tumors expressing ADAM12-L and ADAM12-S, respectively, compared with WT MCF-7 tumors. Proliferation (Ki67-positive nuclei) for WT MCF-7and ADAM12-expressing cells remained the same (D), whereas apoptosis rates (TUNEL-positive nuclei) were significantly lower in the ADAM12-L and ADAM12-S clones, respectively (D and E). H&E staining of representative tumor sections show neatly demarcated boundaries for WT MCF-7 tumors (D), whereas tumor-invasive fronts in ADAM12-L-expressing tumors were slightly more uneven. ADAM12-S tumors had much wider and more irregular tumor invasive boundaries.

FIGURE 5.

ADAM12 expression promotes distant metastasis. A, H&E staining of lymph node sections indicated that metastatic cell clusters were present in LN from ADAM12-S (7 of 18, 40%, p = 0.047) and ADAM12-L-tumor bearing mice (3 of 14, 21%, p = 0.24), respectively, and completely absent in WT MCF-7 tumor-bearing animals. B and C, incidences of tumor cell lung colonization were significantly higher in ADAM12-S tumor-bearing mice (6 of 16, 38%, p < 0.05), whereas no colonies were detected for the animals bearing ADAM12-S^catmut (0%) or the WT MCF-7 tumors (0%) and 20% of animals bearing ADAM12-L tumors (3 of 15, p = 0.13), respectively. B, lung colonies of tumor cells were positive for ADAM12, ERα, and human cytokeratin immunostaining, confirming human and epithelial origin of these cells.