Distinct Functions of Natural ADAM-15 Cytoplasmic Domain Variants in Human Mammary Carcinoma

Levels of mRNA expression of ADAM15 isoforms in breast cancer tissues. Expression of ADAM-15A, ADAM-15B, ADAM-15C, and ADAM-15D isoforms was quantified by TaqMan real-time reverse transcription-PCR in 48 breast cancer tissues compared with 10 control normal mammary tissues. Points, mean; bars, SE. Significance was determined by t test.

Relationships between ADAM-15 isoform expression and survival in node-negative and node-positive breast cancer patients. Expression of ADAM-15A, ADAM-15B, and ADAM-15C mRNAs were quantified in a second independent cohort of 229 breast cancer patients, and correlated with clinical outcome. Patients were divided into high (>median) and low (<median) levels of the individual ADAM isoforms. Continuous line, low-level expression; dotted line, high-level expression. X axis, time after surgery in months; Y axis, relapse-free survival in percent (see Materials and Methods).

Characterization of ADAM-15 stably transfected MDA-MB-435 cells. A. Western blot analysis: Cell lysates were blotted with antibodies to the V5 epitope tag and to ADAM-15 as indicated. Representative clones of ADAM15A and ADAM15B that displayed equivalent levels of tagged protein were used for further studies. B. Cell size was determined by counting pixels in the images of 15 to 20 cells in fields chosen at random. C. Immunocytochemical analysis of FAK localization and morphology of the actin cytoskeleton. F-actin (top) was stained with rhodamine-phalloidin; FAK immunostaining (middle) was carried out as described in Materials and Methods. The merged image of FAK and actin staining is shown in the bottom panel. Scale bar, 20 μm.

Cell adhesion, migration, invasion, and metastasis of ADAM-15 stably transfected cells. A. Cell adhesion: 8 × 10⁴ MDA-MB-435 cells were seeded in triplicate in 96-well plates in serum-free medium and incubated for 1 h at 37°C, and then washed well with warm PBS, fixed, and stained with methylene blue; their absorbance was determined at 630 nm. Each clone was tested in triplicate (V, vector control; A, ADAM-15A; B, ADAM-15B). Columns, mean of three independent experiments; bars, SD. B. De-adhesion assay: Growing MDA-MB-435 cells were washed and incubated either in PBS or with EDTA-EGTA for 5 min as in Materials and Methods. The percentage of cells remaining attached were determined as for the adhesion assay (data are the SE values of two clones). C. De-adhesion assay with stably transfected MCF-7 cells: Cells were treated as in B either in PBS or with EDTA-EGTA for 5 min. The percentage of cells remaining attached were determined as for the adhesion assay (data are the SE values of two independent experiments with one clone of each type assayed in triplicate in each experiment.). D. Scratch wound assay: Confluent MDA-MB-435 cells in chamber slides were wounded by scratching using micropipette tips and were incubated with 2% FCS medium for 24 h. Cells migrating into the wound side were counted and data from three experiments were plotted (left); wound closure was also shown (right). Significance was determined by the t test (*P < 0.05, **P < 0.01). E. Matrigel invasion of MDA-MB-435 cells overexpressing ADAM-15 variants. Overexpression of variant A led to a significant induction (99%, P = 0.029) of Matrigel invasion, whereas there was no significant difference between the other groups (mean ± SE, V: 467.3 ± 55.4, n = 4; A: 931.3 ± 173.9, n = 4; B: 658.5 ± 96.7, n = 4). F. Experimental metastasis assay. Significant reduction of the number of X-gal–stained lung colonies by overexpression of ADAM-15A and ADAM-15B variants in MDA-MB-435 cells compared with the vector-transfected control group (V versus A, P = 0.022; V versus B, P = 0.004). Dots, result of one mouse; bar, mean (V: 276 ± 67.23 SE, n = 6; A: 17.33 ± 8.77, n = 6; B: 72.14 ± 19.27, n = 7).