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. 2008 Mar 1;68(5):1462-70.
doi: 10.1158/0008-5472.CAN-07-3094.

Altered TAB1:I kappaB kinase interaction promotes transforming growth factor beta-mediated nuclear factor-kappaB activation during breast cancer progression

Jason R Neil  1 , William P Schiemann
Affiliations

Altered TAB1:I kappaB kinase interaction promotes transforming growth factor beta-mediated nuclear factor-kappaB activation during breast cancer progression

Jason R Neil et al. Cancer Res. .

Abstract

The conversion of transforming growth factor beta (TGF-beta) from a tumor suppressor to a tumor promoter occurs frequently during mammary tumorigenesis, yet the molecular mechanisms underlying this phenomenon remain undefined. We show herein that TGF-beta repressed nuclear factor-kappaB (NF-kappaB) activity in normal NMuMG cells, but activated this transcription factor in their malignant counterparts, 4T1 cells, by inducing assembly of TGF-beta-activated kinase 1 (TAK1)-binding protein 1 (TAB1):I kappaB kinase beta (IKK beta) complexes, which led to the stimulation of a TAK1:IKK beta:p65 pathway. TAB1:IKK beta complexes could only be detected in NMuMG cells following their induction of epithelial-mesenchymal transition (EMT), which, on TGF-beta treatment, activated NF-kappaB. Expression of a truncated TAB1 mutant [i.e., TAB1(411)] reduced basal and TGF-beta-mediated NF-kappaB activation in NMuMG cells driven to undergo EMT by TGF-beta and in 4T1 cells stimulated by TGF-beta. TAB1(411) expression also inhibited TGF-beta-stimulated tumor necrosis factor-alpha and cyclooxygenase-2 expression in 4T1 cells. Additionally, the ability of human MCF10A-CA1a breast cancer cells to undergo invasion in response to TGF-beta absolutely required the activities of TAK1 and NF-kappaB. Moreover, small interfering RNA-mediated TAK1 deficiency restored the cytostatic activity of TGF-beta in MCF10A-CA1a cells. Finally, expression of truncated TAB1(411) dramatically reduced the growth of 4T1 breast cancers in syngeneic BALB/c, as well as in nude mice, suggesting a potentially important role of NF-kappaB in regulating innate immunity by TGF-beta. Collectively, our findings have defined a novel TAB1:TAK1:IKK beta:NF-kappaB signaling axis that forms aberrantly in breast cancer cells and, consequently, enables oncogenic signaling by TGF-beta.

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Figures

Figure 1
Figure 1
Mammary tumorigenesis alters TGF-β coupling to NF-κB activity. (A) TGF-β1 treatment of normal NMuMG cells significantly repressed the expression of luciferase activity driven by the NF-κB promoter. In stark contrast, this same stimulation protocol resulted in the significant activation of NF-κB-driven luciferase activity in malignant metastatic 4T1 cells. Data are the mean (± SE; n=3) luciferase activities relative to unstimulated NMuMG cells. (*, P < 0.05; Student's T-Test). (B) Control (i.e., GFP) and dominant-negative TβR-II-expressing MCF10-CA1a cells were transiently transfected with NF-κB-luciferase and β-gal, and subsequently were processed for determination of luciferase and β-gal activities contained in detergent-solubilized whole cell extracts. Data are the mean (± SE; n=3) luciferase activities relative to GFP-expressing cells. (*, P < 0.05; Student's T-Test). (C) Quiescent NMuMG and 4T1 cells were stimulated with TGF-β1 (5 ng/ml) for 0-3 h as indicated, and IKKβ subsequently was isolated by immunoprecipitation with anti-IKKβ antibodies. IKKβ phosphotransferase activity in the resulting immunocomplexes was monitored using in vitro GST-IκBα protein kinase assay. Data are from a representative experiment that was repeated three times with similar results. (D) Quiescent 4T1 cells stimulated with TGF-β1 (5 ng/ml) as above, and nuclear extracts were prepared and incubated with biotinylated NF-κB oligonucleotide probes. Afterward, p65/RelA:oligonucleotide complexes were captured with streptavidin-agarose beads, and subsequently were visualized by immunoblotting with antibodies against phospho-p65/RelA or total p65/Rel A as indicated. Data are from a representative experiment that was repeated twice with similar results.
Figure 2
Figure 2
TGF-β stimulation of NF-κB activity requires TAK1 and the IKKβ activity. (A) Quiescent control (i.e., YFP) or TAK1(K63M)-expressing 4T1 cells were stimulated with TGF-β1 (5 ng/ml) for 0-3 h as indicated. Afterward, the phosphorylation status of p38 MAPK was monitored by immunoblotting with phospho-specific p38 MAPK antibodies. Differences in protein loading were monitored using anti-p38 antibodies. Images are from a single experiment that was repeated twice with similar results. (B) Transient TAK1(K63M) expression or pharmacological inhibition of IKKβ (i.e., IKK inhibitor VI, 1 μM) significantly inhibited basal and TGF-β1-stimulated NF-κB activity in 4T1 cells. Data are the mean (± SE; n=3) luciferase activities relative to corresponding unstimulated controls. (*, P < 0.05; Student's T-Test). (C) Murine 4T1 cells were transiently transfected with COX-2 luciferase, together with β-gal as above, and subsequently were stimulated overnight with TGF-β1 (5 ng/ml) in the absence or presence of the IKKβ antagonist (1 μM). Data are the mean (± SE; n=2) luciferase activities relative to unstimulated controls. (*, P < 0.05; Student's T-Test).
Figure 3
Figure 3
TAB1:IKKβ complexes form solely in breast cancer cells, or in normal MECs following their induction of EMT by TGF-β. (A) Detergent-solubilized whole cell extracts prepared from NMuMG and 4T1 cells were immunoblotted with antibodies against TAK1, TAB1, xIAP, and β-actin as indicated (left panel), or were immunoprecipitated with antibodies against either TAK1 (middle panel) or TAB1 (right panel), followed by immunoblotting with antibodies against IKKβ. Differences in protein loading were monitored by reprobing stripped membranes with β-actin antibodies. Images are from representative experiments that were repeated three times with identical results. (B) NMuMG cells were incubated for 36 hr in the absence or presence of TGF-β1 (5 ng/ml) to induce EMT (left panel), and subsequently were subjected to TAB1 co-immunoprecipitation analysis. Induction of EMT by TGF-β1 was monitored by immunoblotting for diminished E-cadherin expression, while differences in protein loading were monitored by β-actin immunoblotting. Images are from representative experiments that were repeated three times with identical results. (C) Schematic depicting the modular structure of full-length TAB1, as well as that of the truncated TAB1 mutant [i.e., TAB1(411)] that lacks the binding domains (BD) for p38 MAPK and TAK1 (top panel). NMuMG and 4T1 cells were transiently transfected with NF-κB-luciferase and β-gal, together with either empty vector or TAB1(411) cDNA as indicated. Afterward, luciferase and β-gal activities contained in detergent-solubilized whole cell extracts were measured (lower panel). Data are the mean (± SE; n=3) luciferase activities relative to empty vector-expressing, unstimulated NMuMG cells. (*, P < 0.05; Student's T-Test).
Figure 4
Figure 4
Truncated TAB1(411) expression selectively blocks TGF-β-mediated NF-κB activation in 4T1 cells. (A) Quiescent control (i.e., YFP) and TAB1(411)-expressing 4T1 cells were stimulated with TGF-β1 (5 ng/ml) as indicated. Afterward, IKKβ was isolated by immunoprecipitation and analyzed for GST-IκBα phosphotransferase activity. The phosphorylation status of p38 MAPK also was monitored by immunoblotting with phospho-specific p38 MAPK antibodies. Differences in protein loading were monitored using antibodies against p38 MAPK. Data are from a representative experiment that was repeated three times with similar results. (B) Control (i.e., YFP) and TAB1(411)-expressing 4T1 cells were transiently transfected with NF-κB-luciferase and β-gal as above, and subsequently were stimulated with increasing concentrations of TGF-β1 for 24 h. Data are the mean (± SE; n=4) luciferase activities relative to unstimulated control cells. (C) Detergent-solubilized whole cell extracts prepared from control (i.e., YFP) and TAB1(411)-expressing 4T1 cells were immunoblotted for phospho-p65/RelA, p65/RelA, or COX-2 as indicated. TAB1(411) expression was detected by anti-FLAG immunoprecipitation, followed by immunoblotting with anti-TAB1 antibodies as indicated. Differences in protein loading were monitored by β-actin immunoblotting. Images are from a representative experiment that was repeated three times with identical results. (D) Control (i.e., YFP) and TAB1(411)-expressing 4T1 cells were transiently transfected with TNF-α-luciferase and β-gal, and subsequently were processed for determination of luciferase and β-gal activities contained in detergent-solubilized whole cell extracts. Data are the mean (± SE; n=3) luciferase activities relative to YFP-expressing cells.
Figure 5
Figure 5
TAK1 is essential for TGF-β stimulation of NF-κB and invasion in metastatic breast cancer cells. MCF10A-CA1a cells were engineered to stably express either kinase-dead TAK1(K63M) (A) or dominant-negative IκBα null (B), and subsequently were induced to invade through synthetic basement membranes by serum (3%) in the presence or absence of TGF-β1 (5 ng/ml) as indicated. Data are the mean (± SE; n=4) invasion relative to that induced by serum in GFP-expressing cells. (*, **, P < 0.05; Student's T-Test). (C) MCF10ACA1a cells were rendered TAK1-deficient by siRNA transfection, and subsequently were stimulated with TGF-β1 (5 ng/ml) for 48 h as indicated. Cellular DNA was radiolabeled with [3H]thymidine and quantified by scintillation counting. Data are the mean (± SE; n=3) [3H]thymidine incorporation normalized to unstimulated control transfected cells. (*, **, P < 0.05; Student's T-test). TAK1-deficiency was monitored by immunoblotting whole cell extracts with the TAK1-specific antibodies. Differences in protein loading were monitored using anti-β-actin antibodies. Images are from a single experiment that was repeated thrice with similar results.
Figure 6
Figure 6
TAB1(411) expression inhibits mammary tumor growth in mice. Parental (i.e., YFP) or TAB1(411)-expressing 4T1 cells were injected orthotopically into the mammary fat pads of either syngeneic Balb/C (A and C) or nude mice (B and D). Tumor volumes were measured every other day beginning at day 10 and continued through day 24. Data are the mean (± SE) tumor volumes (A and C) or weights (B and D) at the time of necropsy observed in two (i.e., Balb/C mice) or three (i.e., nude mice) independent experiments (3 mice/condition/experiment). (*, P < 0.05; Student's T-Test).
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