Fig 1: TCGA analysis revealed co-expression of ADAM12 with KAT2A, TAK1 and TAK1-inducible genes signature in breast tumors. (A) ADAM12 expression in breast tumors and normal breast tissue from the TCGA database. The boxplots contain 50% of the values, with a notch at the median value, and a diamond at the average value. The whiskers depict the first and last quartiles, and outliers appear as black dots. (B) ADAM12, KAT2A, and TAK1 expression in normal breast tissues and a subset of breast cancer samples from the TCGA database, selected based on their low (first decile, green) or high (ninth decile, red) ADAM12 expression. (C) ADAM12, KAT2A, and TAK1 expression in normal breast tissues and breast cancer samples from the TCGA database stratified by subtype. (D) Definition of the TAK1 signature, using transcriptomic data for primary cells treated with TGF-β with or without the TAK1 inhibitor 5Z. The TAK1 signature contains the genes that are upregulated by TGF-β addition, but not upregulated in the TGF-β+5Z condition. (E) Hierarchical clustering with euclidean distance metric and Ward's linkage method of highest and lowest ADAM12 expressing tumors samples, and normal breast tissue samples. The unsupervised clustering of breast tumors according to the TAK1 signature almost perfectly segregates tumors according to ADAM12 expression. (F) TAK1 activation score (calculated from the expression of genes in the TAK1 signature) correlates positively with ADAM12 expression in 1104 breast tumors. The high-ADAM12 samples are shown in red and low-ADAM12 in green. The statistical analysis was performed with a two-way ANOVA test, followed by a Tukey HSD test (*** denotes P < 0.001). (G) TAK1-activation score negatively correlates with KAT2A expression in 1104 breast tumors. Statistics as in panel E.
Fig 2: Epistasis and interaction between KAT2A and TAK1. (A) RT-qPCR on the indicated genes in MRC5 cells transfected with siCtl (non-targeting siRNA), siKAT2A and/or siTAK1. ADAM12 is induced by the knockdown of KAT2A but this is abolished by the simultaneous knockdown of TAK1. Statistical analysis was performed with a two-way ANOVA followed by a Dunnett's test. (B) Western blot showing expression level of TAK1 and KAT2A after siRNA transfection. (C) Co-immunoprecipitation of endogenous TAK1 from MRC5 cells with or without stimulation by 5 ng/ml TGF-ß revealed interaction with KAT2A. TAB1, a known interactor of TAK1, served as a positive control.
Fig 3: TAK1 mediates ADAM12 induction in vitro and in vivo. (A) Results of the signaling screen: only TAK1 expression resulted in the activation of ADAM12. Yellow and red dots represent ADAM12 normalized Nanostring counts in IMR90 infected with the kinase, each dot representing one kinase. Black dots represent ADAM12 expression level in control cells (infected with empty vector). (B) RT-qPCR validation of ADAM12 reactivation by TAK1 in IMR90 cells. ADAM12 was activated by the wild-type form of TAK1 but not by the catalytic dead form of TAK1 (TAK1 CD). (C) Western blot analysis of ADAM12 protein levels following the overexpression of wild-type and catalytic dead TAK1 (TAK1CD). Ctl: cells infected with the empty vector. The white arrows represent the exogenous TAK1; TAK1CD is lower because it does not self-phosphorylate. Tubulin (TUB) is the loading control. For ADAM12, concanavalin A enrichment was performed. (D) Schematic representation of canonical and non-canonical TGF-ß pathways. TAK1 is a component of the non-canonical TGF-ß pathway. (E) RT-qPCR on the indicated genes in the presence or absence of the TAK1 kinase inhibitor (5Z)-7-Oxozeaenol (5Z). IMR90 were pre-treated with 0.3µM 5Z or DMSO for two hours, followed by stimulation with 5 ng/ml of TGF-ß for 6 h. ADAM12 induction by TGF-ß is abolished by 5Z. (F) Control western blot showing the phosphorylation of SMAD3, indicating the activation of canonical TGF-ß pathway even though TAK1 was inhibited by 5Z. (G) Testing the dependence of ADAM12 on TAK1 in vivo: reporter mice with the ADAM12 promoter (PADAM12) driving GFP expression were subjected to an injury (cardiotoxin injection into the tibialis muscle). Some of the mice were treated by intraperitoneal injection of TAK1 inhibitor (5Z) at 5 mg/kg before and after injury, while the controls were injected only with solvent (DMSO). Three days after the injury, mice were sacrificed and muscle tissue was dissected. (H) Immunofluorescence on injured muscle shows that 5Z treatment reduces GFP induction. The arrows indicate GFP-positive cells. Scale bar: 150 µm. (I) Quantification of panel H (10 fields counted in each condition). All the experiments were performed at least three time except for ADAM12 western blot and mice experiments. The statistical analysis was performed with one way ANOVA followed by Dunnett's test, except for panel I where a Mann–Whitney test was performed. In all figures, we used the following conventions: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.
Fig 4: Two screens to investigate the mechanisms controlling tissue-restricted gene reactivation converge on ADAM12. (A) Principle of the approach. Primary human cells were challenged by alterations in signaling pathways or chromatin regulators, and we determined whether these changes were sufficient to cause inappropriate expression of tissue-restricted genes. Red is used to represent repressive chromatin, and green permissive chromatin. (B) Design of the signaling screen: the primary cells were infected with 192 different genetically activated kinases, and gene expression assayed by Nanostring. (C) Results of the signaling screen: log2 fold change of expression for each of the 42 tissue-restricted genes following infection by the 192 activated kinases. Each infection is represented by a yellow dot. The negative controls are indicated as black dots. The only kinase/gene pair showing a Z-score >3.5 is the gene ADAM12 being highly activated by the kinase MAP3K7/TAK1 (red dot). (D) Design of the chromatin screen: the primary cells were transfected with 160 different siRNA pools, each targeting a specific chromatin regulator, then gene expression was assayed by Nanostring. (E) Results of the chromatin screen: log2 fold change of expression for each of the 42 tissue-restricted genes following transfection of the 160 siRNA pools. Each transfected sample is shown as a yellow dot, controls are shown as black dots. Only a few siRNA/gene pairs have a Z-score >2.5. These include the induction of ADAM12 by depletion of SIRT6 and KAT2A.
Fig 5: TAK1 is involved in ADAM12 expression in breast cancer cells. (A) SUM159PT breast cancer cells were treated with 0.3 µM or 1 µM 5Z for four days and the level of ADAM12 was assessed by RT-qPCR. Inhibition of TAK1 by 5Z reduced the level of ADAM12 transcripts. (B) Western blot analysis showing ADAM12 protein expression after 5Z treatment of SUM159PT cells. Concanavalin A enrichment was performed. (C) RT-qPCR showing the expression levels of TAK1 and ADAM12 in SUM159PT cells transfected with non-targeting control siRNA (siCtl) or siTAK1 for four days. Knockdown of TAK1 decreased the levels of ADAM12 transcripts. (D) Western blot showing ADAM12 expression after knockdown by siCtl or siTAK1 in SUM159PT cells. As in panel B. (E) Wound healing assay. SUM159PT cells were treated with 0.3 µM and 1 µM 5Z for 4 days, following treatment, a scratch was made in the dishes and it was imaged every 2 h using the Incucyte live cell system. The area of the wound at different time points was measured by ImageJ software, then a percentage of the area covered by time was determined and is plotted in the panel on the right. Inhibition by 5Z delays the wound healing process. The statistical analysis was performed employing a two way ANOVA test followed by a Dunnett's test. Bottom right panel: growth rate of the cells measured as their percentage of confluence after seeding in a non-scratched plate; 5Z does not affect the growth rate.
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