Overexpression of ADAM12L in MCF10A breast epithelial cells induces a mesenchymal phenotype.

<p>MCF10A cells were infected with lentiviruses expressing either GFP-ADAM12L fusion protein (ADAM12L) or the control protein GFP (Control). After antibiotic selection, cells were further enriched using flow cytometry and cultured for 48 hours. (A) Bright field pictures were captured to show cell morphology. (B) Cells were fixed and immunostained for E-cadherin, vimentin or stained with rhodamine-conjugated phalloidin to monitor actin stress fibers cytoskeletal actin. (C) Real-time PCR analyses of epithelial (E-cadherin and β-catenin) and mesenchymal (N-cadherin, vimentin, Zeb1, fibronectin and Twist) markers. Results are expressed as fold changes in ADAM12L-overexpressing cells compared with control cells. (D) Western blot analyses of E-cadherin and vimentin in ADAM12L-overexpressing cells. (E and F) ADAM12-overexpressing MCF10A cells were transfected with 100 nM non-targeted siRNA (siControl) or ADAM12 siRNA (siADAM12). After 48 hours, E-cadherin expression was analyzed by Western blotting (E). The efficiency and specificity of RNA interference in cell extracts was confirmed by ADAM12 blots (F). (G) Western blot analyses of phosphorylated Ser465/Ser467P-Smad2, Ser423/Ser425P-Smad3, Ser473P-Akt, Thr202/Tyr204P-ERK, Thr180/Tyr182P-p38 and Thr183/Thr185P-JNK in ADAM12L-overexpressing cells and control cells, 48 hours post-seeding. Immunoblots for ADAM12L and HSC-70 are shown as control. Left, representative western blots. Right, densitometric analyses of protein amounts in cells. Results are expressed as mean±SD from 3 independent experiments (*, p<0.05; **, p<0.01).</p

List of oligonucleotide primer pairs used in real time RT-PCR.

<p>List of oligonucleotide primer pairs used in real time RT-PCR.</p

ADAM12 expression is induced in breast epithelial cell line treated with TGF-β.

<p>(A), MCF10A cells were stimulated with TGF-β (5ng/ml) for 24, 48 and 96 hours and control cells were incubated with the type I TGF-β receptor inhibitor, SB431542 (10 μM). Concanavalin A-immobilized magnetic nanoparticles were used for selective enrichment of glycoproteins including ADAM12. The levels of vimentin, E-cadherin and ADAM12 were determined by western blot analyses. Top, representative western blots. Bottom, densitometric analyses of protein amounts in cells. Results are expressed as mean±SD from 3 independent experiments (*, p<0.05). (B) Western blot analyses of protein extracts from MCF10A and K-RAS transformed MCF10A epithelial cells cultured for 48h. (C) Cells were stimulated with TGF-β (5ng/ml) for 24, 48 and 96 hours and levels of phosphorylated Ser465/Ser467P-Smad2, Ser473P-Ak and Thr180/Tyr182P-p38 were determined by western blot analysis. Immunoblots for ADAM12 and actin are shown as controls. Top, representative western blots. Bottom, densitometric analyses of protein amounts in cells. Results are expressed as mean±SD from 3 independent experiments (*, p<0.05).</p

ADAM12 is associated with mesenchymal-like phenotype of Basal-B cell lines.

<p>Based on gene expression data of breast cell lines (n = 51) from Neve et al.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139179#pone.0139179.ref036" target="_blank">36</a>] and Kao et al.[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139179#pone.0139179.ref037" target="_blank">37</a>], statistical analyses identified 635 overexpressed genes in Basal-B cell lines compared with Luminal and Basal-A cells (Detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139179#pone.0139179.s008" target="_blank">S2 Table</a>). (A) Text mining analysis of the 100 most statistically significant upregulated genes in Basal-B cell lines using CoPub tool. Nodes represent genes and edges represent co-citation in abstract. Black lines indicate ADAM12 connections. (B) Spearman correlation analyses of steady-state ADAM12L, ADAM12S, vimentin, E-cadherin and N-cadherin mRNA levels (Top). Comparative distribution of mRNA levels of ADAM12, vimentin, E-cadherin, N-cadherin and TGF-β between Lum/Basal-A and Basal B cell lines (Bottom). (***,p<0.001; **,p<0.01; *, p<0.005).</p

Expression of ADAM12 in human breast cancer biopsies (n = 79).

<p>ADAM12 expression is correlated with mesenchymal markers (A) and the presence of metastases (B). Total RNA was extracted from breast tissue samples and the steady-state ADAM12L and ADAM12S, N-cadherin, vimentin, TGF-β and E-cadherin mRNA levels were measured by real-time PCR. (A) Spearman correlation analyses of steady-state ADAM12L, ADAM12S, vimentin, E-cadherin, N-cadherin and TGF-β mRNA levels. (B) Comparative analysis of ADAM12L, ADAM12S and vimentin mRNA levels in breast samples from patients without (-) and with (+) metastases. (*, p<0.05; **,p<0.01 ***,p<0.001).</p

Overexpression of ADAM12L induces chemoresistance in MCF10A cells.

<p>(A) Cisplatin-induced apoptosis is analyzed by quantification of cell viability and caspase 3/7 activity. Dose effects are measured after 24h of treatment and kinetic effects are measured with a dose of 20μg/ml. (B) Fas-L induced apoptosis is analyzed by quantification of cell viability and caspase 3/7 activity. Dose effects are measured after 24h of treatment and kinetic effects are measured with a dose of 75 ng/ml. All results are expressed as the mean ±SD from four independent experiments (*, p<0.05; **, p<0.01). (C) Comparative analysis of cell viability between MCF10A cells overexpressing either ADAM12L, ADAM12L-E315Q, ADAM12L-Δcyto or ADAM12S compared with control cells after 24H of cisplatin treatment with a dose of 20μg/ml.</p

The cytoplasmic tail is required for ADAM12L-induced EMT.

<p>MCF10A cells were infected with lentiviruses expressing either the fusion proteins GFP-ADAM12L, the cytoplasmic domain deletion mutant (GFP-ADAM12L-Δcyto) or the control protein GFP (Control). After antibiotic selection, cells were further enriched using flow cytometry and cultured for 48 hours. (A) Cells were fixed and immunostained for E-cadherin (B) The levels of vimentin and E-cadherin were determined by western blot analyses. Left, representative western blots. Immunoblots for ADAM12 and HSC-70 are shown as control. Right, densitometric analysis of protein amounts. Results are expressed as mean±SD from 3 independent experiments (*, p<0.05).</p

Inhibition of TβRI or ERK-MAPK reverses ADAM12L-induced mesenchymal phenotype.

<p>MCF10A cells overexpressing GFP-ADAM12 were treated (+) or not (-) with (A) the selective inhibitor of TGFβRI, SB431542 (10μM), (B) a highly selective inhibitor of both MEK1 and MEK2, U0126 (10μM), and (C) the PI3K inhibitor, Wortmannin (10μM) for 72 hours. The expression of vimentin and E-cadherin was analyzed by western blot and quantified by densitometry. Data are expressed as mean±SD from four independent experiments (*, p<0.05; **, p<0.01).</p

ADAM12-dependent EMT does not require catalytic activity and is specific of ADAM12L.

<p>MCF10A cells were infected with lentiviruses expressing either the fusion proteins GFP-ADAM12L, GFP-ADAM12-E351Q, GFP-ADAM12S or the control protein GFP (Control). After antibiotic selection, cells were further enriched using flow cytometry and cultured for 48 hours. (A) Bright field pictures were captured to show cell morphology. Cells were fixed and immunostained for E-cadherin or stained with rhodamine-conjugated phalloidin to monitor actin stress fibers cytoskeletal actin. (B) The levels of vimentin and E-cadherin were determined by western blot analyses. Left, representative western blots. Immunoblots for ADAM12L, ADAM12S and HSC-70 are shown as control. Right, densitometric analysis of protein amounts. Results are expressed as mean±SD from 6 independent experiments (*, p<0.05; **, p<0.01).</p

Figure 6

<p>Putative function of the interaction between VHL and Aurora-A in the ccRCC.</p