Altered TAB1:IKB Kinase Interaction Promotes Transforming Growth Factor B–Mediated Nuclear Factor-KB Activation During Breast Cancer Progression Jason R

Research Article

Altered TAB1:IKB Kinase Interaction Promotes Transforming Growth Factor B–Mediated Nuclear Factor-KB Activation during Breast Cancer Progression Jason R. Neil and William P. Schiemann

Department of Pharmacology, University of Colorado Health Sciences Center, Aurora, Colorado

Abstract their association with the co-mediator Smad, Smad4 (1, 3). Nuclear The conversion of transforming growth factor B (TGF-B) from translocation of these transcription factor complexes promotes a tumor suppressor to a tumor promoter occurs frequently their physical interaction with a variety of transcriptional acti- during mammary tumorigenesis, yet the molecular mecha- vators and repressors that ultimately regulate the expression of TGF-h–responsive genes in a cell- and promoter-specific manner nisms underlying this phenomenon remain undefined. We h show herein that TGF-B repressed nuclear factor-KB (NF-KB) (3). The complexity of TGF- signaling is increased through its activity in normal NMuMG cells, but activated this transcrip- activation of mitogen-activated protein kinases [MAPK; e.g., tion factor in their malignant counterparts, 4T1 cells, by in- extracellular signal–regulated kinase (ERK)-1/2, c-jun NH2-terminal ducing assembly of TGF-B–activated kinase 1 (TAK1)–binding kinase (JNK), and p38MAPK], which modulate the activity of protein 1 (TAB1):IKB kinase B (IKKB) complexes, which led to Smad2/3 or other downstream transcription factors (4–7). Phos- phorylation and activation of MAPKs are regulated by MAPK the stimulation of a TAK1:IKKB:p65 pathway. TAB1:IKKB complexes could only be detected in NMuMG cells following kinases (MAPKK/MKK), which in turn are phosphorylated and activated by upstream MAPK kinase kinases (MAPKKK/MKKK; ref. 8). their induction of epithelial-mesenchymal transition (EMT), h which, on TGF-B treatment, activated NF-KB. Expression of a TGF- –activated kinase 1 (TAK1) is a MAPKKK that is activated by receptors for the TGF-h, tumor necrosis factor a (TNF-a), and truncated TAB1 mutant [i.e., TAB1(411)] reduced basal and interleukin (IL)-1 (9–11). TAK1 activation is regulated via its asso- TGF-B–mediated NF-KB activation in NMuMG cells driven to ciation with the COOH-terminal binding domain of TAK1-binding undergo EMT by TGF-B and in 4T1 cells stimulated by TGF-B. protein 1 (TAB1) and via its association with TAB2 and TAB3, TAB1(411) expression also inhibited TGF-B–stimulated tumor which target TAK1 binding sites distinct from those bound by necrosis factor-A and cyclooxygenase-2 expression in 4T1 TAB1 (12–14). Activated TAK1 phosphorylates and activates cells. Additionally, the ability of human MCF10A-CA1a breast MKK3/MKK6 and MKK4, which then mediate stimulation of p38 cancer cells to undergo invasion in response to TGF-B abso- MAPK or JNK, respectively (6, 15). TAK1 also interacts with and lutely required the activities of TAK1 and NF-KB. Moreover, activates the InB kinase (IKK) complex (i.e., IKKa,IKKh, and IKKg/ small interfering RNA–mediated TAK1 deficiency restored the NEMO), which mediates nuclear factor nB (NF-nB) transcription cytostatic activity of TGF-B in MCF10A-CA1a cells. Finally, factor activation (16–18). On TAK1-mediated activation, the IKK expression of truncated TAB1(411) dramatically reduced the complex phosphorylates the InBa inhibitory protein, leading to its growth of 4T1 breast cancers in syngeneic BALB/c, as well degradation and subsequent activation of the p65 NF-nB tran- as in nude mice, suggesting a potentially important role of scription factor. Gene ablation studies in mice revealed that TAK1, NF-KB in regulating innate immunity by TGF-B. Collectively, but not its binding partner TAB1, is essential for TNF-a– and IL-1– our findings have defined a novel TAB1:TAK1:IKKB:NF-KB mediated activation of NF-nB (19). These studies also established a signaling axis that forms aberrantly in breast cancer cells and, critical role for TAB1 and TAK1 during embryonic development consequently, enables oncogenic signaling by TGF-B. [Cancer and in mediating inflammatory gene expression, whereas recent Res 2008;68(5):1462–70] evidence points to their involvement in mediating dysregulated signaling in cancer cells (20–27). Currently, relatively little is known Introduction about the roles of TAK1 and TAB1 in regulating the response of Transforming growth factor h (TGF-h) is a pleiotropic cytokine normal and malignant mammary epithelial cells (MEC) to TGF-h. that mediates a diverse array of physiologic activities in responsive This knowledge deficit is medically relevant because inappropriate cells and tissues through all stages of the metazoan life span (1, 2). and constitutive NF-nB activity has been linked to the development The biological actions of TGF-h are communicated across the and progression of human cancers (28) and to the conversion of plasma membrane through the combined actions of the TGF-h TGF-h from a suppressor to a promoter of mammary tumorigen- type I (ThR-I) and type II (ThR-II) Ser/Thr protein kinase receptor esis (29, 30). The aim of this study was to further our understanding complexes, which phosphorylate Smad2 and Smad3 and stimulate of the molecular events that contribute to dysregulated TGF-h and NF-nB signaling in developing and progressing breast cancers.

Note: Supplementary data for this article are available at Cancer Research Online Materials and Methods (http://cancerres.aacrjournals.org/). Requests for reprints: William P. Schiemann, Department of Pharmacology, Materials. Recombinant human TGF-h1 and mouse TNF-a were University of Colorado Health Sciences Center, RC1 South Tower, Room L18-6110, obtained from R&D Systems. The constructs used in this work were (a) 12801 East 17th Avenue, P.O. Box 6511, Aurora, CO 80045. Phone: 303-724-1541; kinase-dead TAK1 [pcDNA3-HA-TAK1(K63M)], provided by Dr. Gary L. Fax: 303-724-3663; E-mail: [email protected]. I2008American Association for Cancer Research. Johnson (University of North Carolina, Chapel Hill, NC); (b) human TAB1a doi:10.1158/0008-5472.CAN-07-3094 cDNA, provided by Dr. Jiahuai Han (The Scripps Research Institute, La Jolla,

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CA); (c) mammalian InBa-null (pMSCV-InBa-null-GFP) and NF-nB pro- anti-Flag M2 (1:1,000; Sigma); (b) anti–h-actin (1:5,000; Sigma); (c) anti– moter–driven luciferase reporter, provided by Dr. John M. Routes (Medical COX-2 (1:2,000; Cayman Chemical Company); (d) anti–histone H1 (1:200); College of Wisconsin, Milwaukee, WI); (d) glutathione S-transferase (GST)- (e) anti-IKKh (1:500; Cell Signaling); (f ) anti–phospho-p65 (1:1,000); (g) InBa, provided by Dr. Robert Scheinman (University of Colorado Health anti-TAB1 (1:500; Cell Signaling and Novus Biologicals); (h) anti-TAK1 Sciences Center, Denver, CO); (e) human TNF-a promoter–driven luciferase (1:500; Cell Signaling); and (i) anti–X-linked inhibitor of apoptosis (xIAP; reporter (containing bases À639 to À162 from start site), provided by Dr. 1:500; Cell Signaling). The resulting immunocomplexes were visualized by Harvey F. Lodish (Whitehead Institute, Cambridge, MA); and (f ) cyclo- enhanced chemiluminescence. Differences in protein loading were moni- oxygenase 2 (COX-2) promoter–driven luciferase reporter, provided by Dr. tored by reprobing stripped membranes with anti–h-actin antibodies Bharat B. Aggarwal (University of Texas M. D. Anderson Cancer Center, (1:1,000; Rockland Immunology). Houston, TX). All additional supplies or reagents were routinely available. IKKB kinase assay. The ability of TGF-h to activate IKKh was Cell culture and retroviral infections. Normal murine NMuMG determined by an in vitro protein kinase assay that measured the extent mammary gland and malignant 4T1 breast cancer cells were obtained of immunoprecipitated IKKh to phosphorylate recombinant GST-InBa. from American Type Culture Collection, whereas human metastatic Briefly, NMuMG and 4T1 cells (350,000 per well) were stimulated with TGF- MCF10A-CA1a breast cancer cells were provided by Dr. Fred Miller h1 (5 ng/mL) for 0 to 3 h at 37jC as indicated, and were subsequently (Barbara Ann Karmanos Cancer Institute, Detroit, MI). Cell lines were harvested and solubilized on ice in Buffer H/1% Triton X-100. Clarified maintained and cultured in a constant atmosphere of 5% CO2 at 37jCas whole-cell extracts were prepared and incubated under continuous rotation previously described (31, 32). with anti-IKKh antibodies (2 AL/tube) for 4 h at 4jC. Immunocomplexes The following murine ecotropic retroviral constructs were used herein: were recovered by brief microcentrifugation and were subsequently washed (a) pMSCV-IRES-GFP or pMSCV-IRES-YFP (i.e., control vectors); (b) thrice in lysis buffer and twice in Buffer H. Phosphotransferase reactions pMSCV-DNThR-II-GFP (i.e., truncated ThR-II); (c) pMSCV-TAK1(K63M)- were done in a final volume of 40 AL, consisting of IKKh immunocomplexes YFP (i.e., kinase-dead TAK1); (d)pMSCV-InBa-null-GFP; and (e)pMSCV- and 2.5 AgofGST-InBa, and were initiated by addition of 10 ALof4Â assay TAB1(1–411)-YFP (i.e., truncated TAB1). Retroviral supernatants were buffer and allowed to proceed for 30 min at 30jC (35). The phosphorylation produced by EcoPack2 retroviral packaging cells (Clontech) and used to reactions were stopped by addition of 4Â sample buffer and boiled for infect NMuMG and 4T1 cells, and MCF10A-CA1a cells engineered to express 5 min before their fractionation through 10% SDS-PAGE gels. Phosphor- the ecotropic receptor as previously described (33). Forty-eight hours ylation of recombinant InBa by IKKh was detected by exposure of the dried postinfection, the infected cells were analyzed and isolated on a MoFlo cell gels to a phosphoscreen. Differences in protein loading were monitored by sorter (Cytomation) and were subsequently expanded to yield stable immunoblotting whole-cell extract aliquots (i.e., 10% of that subjected to polyclonal populations of control and transgene-expressing cells that were immunoprecipitation) with antibodies against IKKh and h-actin. z90% for expression of green fluorescent protein (GFP) or yellow TAB1:IKKB coimmunoprecipitation assay. Detergent-solubilized fluorescent protein (YFP). NMuMG and 4T1 whole-cell extracts (350,000 cells per tube) were pre- Luciferase reporter gene assays. Analysis of TGF-h–stimulated pared and incubated under continuous rotation with anti-TAB1 antibodies luciferase activity driven by the synthetic NF-nB, SBE, 3TP, TNF-a,or (2 mL/tube; Santa Cruz Biotechnology) for 6 h at 4jC. The resulting COX-2 promoters was done as previously described (34). NMuMG and 4T1 immunocomplexes were collected by microcentrifugation, washed, and MECs (25–30,000 per well) were cultured overnight onto 24-well plates and fractionated through 10% SDS-PAGE gels before their immobilization to were subsequently transfected the following morning by overnight exposure nitrocellulose membranes, which were subsequently probed with anti-IKKh to LT1-liposomes (Mirus) containing 300 ng/well of individual luciferase antibodies (1:500). Differences in protein loading were monitored by immu- reporter cDNA and 50 ng/well of CMV-h-gal cDNA, which was used to noblotting whole-cell extract aliquots with antibodies against h-actin as above. control for differences in transfection efficiency. The cells were washed TAK1 small interfering RNA knockdown. The creation of MCF10A- twice with PBS and stimulated overnight in serum-free medium with TGF- CA1a cells lacking TAK1 was accomplished with SMARTpool small h1 (0–5 ng/mL) or TNF-a (0–20 ng/mL) as indicated. Afterward, luciferase interfering RNA (siRNA; Dharmacon) according to the manufacturer’s and h-gal activities contained in detergent-solubilized cell extracts were recommendations and as previously described (31, 32). Briefly, MCF10A- determined. Data are the mean (F SE) luciferase activities of at least three CA1a cells (10,000 per well) were plated onto 96-well plates and cultured independent experiments normalized to untreated cells. overnight in antibiotic-free medium. The following morning, the cells were NF-KB biotinylated oligonucleotide capture assay. DNA binding transiently transfected overnight with DharmaFECT One reagent (Dharma- activity of NF-nB was monitored in quiescent 4T1 cells before and after con) supplemented with TAK1 siRNAs (50 nmol/L) and were subsequently their activation by TGF-h as indicated. Afterward, 4T1 cells were collected stimulated with TGF-h1 (5 ng/mL) for varying times at 37jC. On and fractionated into cytoplasmic and nuclear extracts with a Nuclear completion of agonist stimulation, the cells were harvested and prepared Extraction Kit according to the manufacturer’s instructions (Chemicon). for [3H]thymidine incorporation assays as described below. The extent of NF-nB binding activity was determined by incubating 600 Ag of nuclear siRNA-mediated TAK1 deficiency was monitored by immunoblotting whole- extract with 1 Ag of biotinylated double-stranded DNA oligonucleotides cell extracts with antibodies against TAK1. that contained a NF-nB consensus sequence site under continuous rotation Cell biological assays. The effect of manipulating TAB1, TAK1, or InBa at 4jC (forward probe, 5¶-GATCTAGGGACTTTCCGCTGGGGACTTTCCA- function on various TGF-h–stimulated activities in normal and malignant GTCGA; reverse probe, 5¶-TCGACTGGAAAGTCCCCAGCGGAAAGTCCCTA- MECs was determined as follows: (a) cell proliferation using 10,000 cells per GATC). The resulting NF-nB:oligonucleotide complexes were captured by well in a [3H]thymidine incorporation assay as previously described (33); (b) addition streptavidin-agarose beads (Pierce) and collected by microcen- cell invasion induced by 3% serum using 100,000 cells per well in a modified trifugation. Washed complexes were fractionated through 10% SDS-PAGE Boyden chamber coated with Matrigel matrices (diluted 1:25 in serum-free before their immobilization to nitrocellulose membranes, which were DMEM) as previously described (34); and (c) epithelial-mesenchymal subsequently probed with anti–phospho-p65 antibodies (1:1,000; Cell transition (EMT) induced by TGF-h1 (5 ng/mL) treatment as previously Signaling) and anti-p65 antibodies (1:500; Santa Cruz Biotechnology). described (31, 32). Differences in extract loading were monitored by immunoblotting 25 Agof Tumor growth study. Control (YFP) and truncated TAB1(411)-express- resolved nuclear extract aliquots with antibodies against histone H1 (1:200; ing 4T1 cells were resuspended in sterile PBS and injected (12,500 per Santa Cruz Biotechnology). mouse) orthotopically into the mammary fat pad of 6-week-old female Western blotting. Control and TGF-h–stimulated NMuMG and 4T1 cells BALB/c and nude mice (four mice per condition; The Jackson Laboratory). were lysed and solubilized in Buffer H/Triton X-100 (35) for 30 min on ice. Mice were monitored daily and primary tumors were measured with digital Clarified cell extracts were resolved on 10% SDS-PAGE gels, transferred calipers (Fisher Scientific) between days 10 and 24. Tumor volumes electrophoretically onto nitrocellulose membranes, and blocked in 5% milk were calculated using the following equation: tumor volume = (x2)(y)(0.5), before incubation with the following primary antibodies (dilutions): (a) where x is the tumor width and y is the tumor length. Twenty-four days www.aacrjournals.org 1463 Cancer Res 2008; 68: (5). March 1, 2008

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2008 American Association for Cancer Research. Cancer Research postinoculation, the mice were sacrificed and their primary tumors were accumulation of phospho-p65/RelA in MCF10A-CA1a cells (Sup- excised and weighed. plementary Fig. S1A). Comparison of NF-nB–driven luciferase Animal studies were done two (i.e., BALB/c) or three (i.e., nude) times in activities in control (i.e., GFP) and truncated ThR-II–expressing accordance with the animal protocol procedures approved by the Institu- (i.e., dominant-negative ThR-II) MCF10A-CA1a cells revealed that tional Animal Care and Use Committee of University of Colorado. these cells are subjected to a significant amount of autocrine TGF-h signaling that contributes to NF-nB activation (Fig. 1B). Results Additionally, TGF-h stimulated the phosphotransferase activity of Mammary tumorigenesis alters TGF-B coupling to NF-KB IKKh against recombinant InBa in malignant 4T1 cells but not in activity. Activation of the TGF-h signaling system typically normal NMuMG cells (Fig. 1C). Likewise, TGF-h treatment of 4T1 represses NF-nB activity in normal epithelial cells, including those cells induced phosphorylation and binding of nuclear p65/RelA to of the breast (36). In using a NF-nB–driven luciferase reporter gene, a biotinylated oligonucleotide containing a NF-nB consensus we too find that TGF-h stimulation of normal NMuMG cells sequence site (Fig. 1D). We also monitored Smad2/3 activation significantly repressed (by 58%) their activation of NF-nB(Fig.1A). induced by TGF-h in normal and malignant MECs. These analyses When identical analyses were done in malignant, metastatic 4T1 showed that TGF-h stimulation of NMuMG, 4T1, or MCF10A-CA1a breast cancer cells, basal NF-nB–driven transcriptional activity was cells rapidly induced the phosphorylation of Smad2/3 as well as the significantly higher when compared with NMuMG cells (Fig. 1A). expression of SBE-luciferase (Supplementary Fig. S1B). Thus, these Moreover, administration of TGF-h to 4T1 cells was observed to findings indicate that mammary tumorigenesis does indeed con- induce significant NF-nB–driven transcriptional activity. Impor- vert TGF-h from an inhibitor to a stimulator of NF-nB activity. tantly, the relative difference in NF-nB activation regulated by TGF-B stimulation of NF-KB activity requires TAK1 and the TGF-h between normal and malignant MECs was f10-fold. The IKK complex. Activation of TAK1 by TGF-h has been linked to human MCF10A breast cancer system has been proposed to the stimulation of NF-nB activity in certain cancer models, in- represent a model of mammary tumorigenesis regulated by TGF-h cluding those of the prostate and liver (26, 27). Whether TAK1 (37). Our own analyses of these MCF10A derivatives also showed couples TGF-h to NF-nB activation in malignant MECs remains that increasing MEC malignancy did indeed convert TGF-h from unknown. We addressed this question by expressing in 4T1 cells a an inhibitor to a stimulator of NF-nB–driven luciferase activity kinase-dead TAK1(K63M), which functions in a dominant-negative (data not shown) and of NF-nB–mediated DNA binding activity manner to inhibit TAK1-mediated signaling (10), to monitor its (data not shown). TGF-h treatment readily induced the nuclear effects on TGF-h stimulation of p38MAPK and NF- nB activity.

Figure 1. Mammary tumorigenesis alters TGF-h coupling to NF-nB activity. A, TGF-h1 treatment of normal NMuMG cells significantly repressed the expression of luciferase activity driven by the NF-nB promoter. In starkcontrast, this same stimulation protocol resulted in the significant activation of NF- nB–driven luciferase activity in malignant metastatic 4T1 cells. Points, mean luciferase activities relative to unstimulated NMuMG cells (n = 3); bars, SE. *, P < 0.05, Student’s t test. B, control (i.e., GFP) and dominant-negative ThR-II–expressing MCF10A-CA1a cells were transiently transfected with NF-nB-luciferase and h-gal and were subsequently processed for determination of luciferase and h-gal activities contained in detergent-solubilized whole-cell extracts. Columns, mean luciferase activities relative to GFP-expressing cells (n = 3); bars, SE. *, P < 0.05, Student’s t test. C, quiescent NMuMG and 4T1 cells were stimulated with TGF-h1 (5 ng/mL) for 0 to 3 h as indicated, and IKKh was subsequently isolated by immunoprecipitation with anti-IKKh antibodies. IKKh phosphotransferase activity in the resulting immunocomplexes was monitored by in vitro GST-InBa protein kinase assay. Data from a representative experiment that was repeated thrice with similar results. D, quiescent 4T1 cells were stimulated with TGF-h1 (5 ng/mL) as above, and nuclear extracts were prepared and incubated with biotinylated NF-nB oligonucleotide probes. Afterward, p65/RelA:oligonucleotide complexes were captured with streptavidin-agarose beads and were subsequently visualized by immunoblotting with antibodies against phospho-p65/RelA or total p65/Rel A as indicated. Data from a representative experiment that was repeated twice with similar results.

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TAB1:IKK complexes form solely in breast cancer cells and mediate their activation of NF-KB by TGF-B. Our findings thus far show that mammary tumorigenesis converts TGF-h from a repressor to a promoter of NF-nB activation via a TAK1:IKKh– dependent mechanism (Figs. 1 and 2). Although currently unknown, altered expression of various components of the TAK1 signaling complex could underlie the initiation of this aberrant signaling phenomenon in malignant MECs. We tested this hypothesis by comparing the cellular levels of TAK1, TAB1, and xIAP in normal and malignant MECs. As shown in Fig. 3A (left), normal NMuMG and malignant 4T1 cells express similar quantities of TAK1, TAB1, or xIAP. Identical findings were observed when comparing the cellular levels of these proteins in derivatives of the human MCF10A cell system (data not shown). TAK1 binds and phosphorylates IKKh, leading to its activation (38). Thus, altered formation of TAK1:IKKh complexes could underlie the differential coupling to TGF-h to NF-nB in malignant MECs. We tested this hypothesis by monitoring the formation of TAK1:IKKh complexes in NMuMG and 4T1 cells and found that both cell lines housed similar quantities of TAK1:IKKh complexes (Fig. 3A, middle). Although mammary tumorigenesis had no effect on the expression of TAK1 signaling components, this pathologic process led to the formation of TAB1:IKKh complexes solely in malignant, metastatic 4T1 cells (Fig. 3A, right). These findings suggest that incorporation of TAB1 into TAK1:IKKh complexes couples TGF-h to stimulation of NF-nB. Along these lines, the interaction between TAB1 and IKKh was undetectable in normal MECs, but the formation of TAB1:IKKh complexes was readily apparent in these same cells following their stimulation with TGF-h to induce EMT (Fig. 3B), which manifested in their acquisition of a fibroblastoid morphology and reduced E-cadherin expression (Fig. 3B). Collectively, these findings suggest that the interaction of TAB1 with IKKh mediates TGF-h stimulation of NF-nB solely in malignant MECs or in normal MECs that have undergone an EMT in response to TGF-h. Figure 2. TGF-h stimulation of NF-nB activity requires TAK1 and the IKKh TAB1a is a modular adapter protein composed of three distinct activity. A, quiescent control (i.e., YFP) or TAK1(K63M)-expressing 4T1 cells were stimulated with TGF-h1 (5 ng/mL) for 0 to 3 h as indicated. Afterward, the functional domains: (a)anNH2-terminal PP2C-like domain; (b)a phosphorylation status of p38 MAPK was monitored by immunoblotting with central p38a MAPK binding domain; and (c) a COOH-terminal phospho-specific p38 MAPK antibodies. Differences in protein loading were TAK1 binding domain (ref. 39; Fig. 3C, top). TAB1 deficiency in mice monitored with anti-p38 antibodies. Images from a single experiment that was h repeated twice with similar results. B, transient TAK1(K63M) expression or alters some transcriptional responses regulated by TGF- (19), pharmacologic inhibition of IKKh (i.e., IKK inhibitor VI, 1 Amol/L) significantly and as such, we generated a truncated, dominant-negative TAB1 h n inhibited basal and TGF- 1–stimulated NF- B activity in 4T1 cells. Points, mean mutant [i.e., TAB1(411)] that lacked the binding domains for p38a luciferase activities relative to corresponding unstimulated controls (n =3);bars, SE. *, P < 0.05, Student’s t test. C, murine 4T1 cells were transiently transfected MAPK and TAK1 to assess its effects on NF-nB activity in normal with COX-2 luciferase, together with h-gal as above, and were subsequently and malignant MECs. As expected, transient TAB1(411) expression stimulated overnight with TGF-h1 (5 ng/mL) in the absence or presence of the n IKKh antagonist (1 Amol/L). Columns, mean luciferase activities relative to failed to effect basal NF- B activity in NMuMG cells, but signi- unstimulated controls (n = 2); bars, SE. *, P < 0.05, Student’s t test. ficantly reduced that observed in 4T1 cells to a level similar to that in normal NMuMG cells (Fig. 3C, bottom). This finding suggests As expected, TAK1(K63M) expression inhibited TGF-h–mediated that TAB1 indeed plays an important role in regulating NF-nB activation of p38MAPK in 4T1 cells (Fig. 2 A). Moreover, expression activity in malignant MECs. Along these lines, our finding that of TAK1(K63M) in 4T1 cells not only reduced their basal level of TGF-h stimulation of EMT in normal MECs promoted the forma- NF-nB activity but also uncoupled TGF-h from activation of NF-nB tion of TAB1:IKKh (Fig. 3B) implies that this event may be suffi- in these malignant MECs (Fig. 2B). Interestingly, pharmacologic cient in coupling TGF-h to NF-nB activation in post-EMT MECs. inhibition of IKKh activity (i.e., IKK-2 VI inhibitor administration) We tested this hypothesis by stably expressing TAB1(411) in wholly mimicked the uncoupling of TGF-h from NF-nB in 4T1 cells NMuMG cells and subsequently monitoring their ability to undergo (Fig. 2B), suggesting that IKKh lies downstream of TAK1 during EMT and activate NF-nB in response to TGF-h. Figure 3D shows TGF-h stimulation of NF-nB and its protumorigenic target genes. that following their induction of EMT by TGF-h administration, TGF-h treatment of 4T1 cells stimulated COX-2 promoter–driven NMuMG cells readily acquired the ability to activate NF-nB when luciferase activity, an effect that was blocked by treatment with the stimulated by TGF-h. Interestingly, although TAB1(411) expression IKK-2 VI inhibitor (Fig. 2C). Thus, the ability of TGF-h to stimulate had no effect on the ability of NMuMG cells to undergo EMT NF-nB and target gene expression in malignant MECs requires the stimulated by TGF-h (data not shown), its expression did uncouple activity of TAK1 and IKKh, which promote proinflammatory gene TGF-h from NF-nB activation in post-EMT NMuMG cells (Fig. 3D). expression in malignant MECs. Collectively, these findings indicate that incorporation of TAB1 into www.aacrjournals.org 1465 Cancer Res 2008; 68: (5). March 1, 2008

TAK1:IKKh complexes mediates activation of NF-nB by TGF-h in to alter the ability of TGF-h to activate Smad2/3–mediated gene MECs. Our results also dissociate NF-nB activity from the ability of expression (Supplementary Fig. S2C) and p38MAPK phosphory- TGF-h to stimulate EMT in normal MECs. lation (Fig. 4A). Finally, consistent with its lack of involvement in We also examined the effects of stable TAB1(411) expression on TNF-a signaling (19, 40), TAB1(411) expression failed to effect the ability of TGF-h to activate NF-nB in 4T1 cells. Stable TNF-a stimulation of NF-nB activity in 4T1 cells (Supplementary TAB1(411) expression in 4T1 cells resulted in a distorted, more Fig. S2D). Thus, these findings indicate that TAB1:IKKh complexes rounded cell morphology characteristic of altered cellular adhesion form and function specifically in mediating activation of NF-nB (Supplementary Fig. S2A). Similar to its full-length counterpart, and its downstream effectors in response to TGF-h treatment of truncated TAB1(411) interacted physically with ThR-I, indicating post-EMT normal and malignant MECs. that the molecular determinants that mediate TAB1 binding to TAK1 is essential for TGF-B stimulation of NF-KB and h T R-I are located NH2-terminal to its p38a MAPK binding domain invasion in metastatic breast cancer cells. Our findings thus far (Supplementary Fig. S2B). Expression of TAB1(411) in 4T1 cells have identified a novel TAB1:TAK1:IKKh signaling axis that prevented the ability of TGF-h to induce their (a) activation of selectively couples TGF-h to NF-nB in malignant MECs. We next IKKh (Fig. 4A); (b) expression of luciferase driven by NF-nB addressed the contribution of this signaling axis to breast cancer (Fig. 4B); (c) phosphorylation of p65/RelA (Fig. 4C); and (d) cell proliferation and invasion stimulated by TGF-h. Similar to expression of the proinflammatory genes, COX-2 (Fig. 4C) and 4T1 cells, the formation of TAB1:IKKh complexes was also readily TNF-a (Fig. 4D). The inhibitory effects of TAB1(411) expression detected in metastatic human MCF10A-CA1a cells (data not on TGF-h signaling were specific for the NF-nB pathway and failed shown), indicating that the formation of TAB1:IKKh complexes was

Figure 3. TAB1:IKKh complexes form solely in breast cancer cells or in normal MECs following their induction of EMT by TGF-h. A, detergent-solubilized whole-cell extracts prepared from NMuMG and 4T1 cells were immunoblotted with antibodies against TAK1, TAB1, xIAP, and h-actin as indicated (left)orwereimmunoprecipitated with antibodies against either TAK1 (middle) or TAB1 (right), followed by immunoblotting with antibodies against IKKh. Differences in protein loading were monitored by reprobing stripped membranes with h-actin antibodies. Images from representative experiments that were repeated thrice with identical results. B, NMuMG cells were incubated for 36 h in the absence or presence of TGF-h1(5ng/mL)toinduceEMT(left) and were subsequently subjected to TAB1 coimmunoprecipitation analysis. Induction of EMT by TGF-h1 was monitored by immunoblotting for diminished E-cadherin expression, whereas differences in protein loading were monitored by h-actin immunoblotting. Images from representative experiments that were repeated thrice 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). NMuMG and 4T1 cells were transiently transfected with NF-nB-luciferase and h-gal, together with either empty vector or TAB1(411) cDNA, as indicated. Afterward, luciferase and h-gal activities contained in detergent-solubilized whole-cell extracts were measured (bottom). Columns and points, mean luciferase activities relative to empty vector–expressing, unstimulated NMuMG cells (n =3);bars, SE. *, P < 0.05, Student’s t test. D, control (YFP) or TAB1(411)-expressing NMuMG cells were incubated overnight in the absence (pre)orpresence (post)ofTGF-h1 (5 ng/mL) to induce EMT. Afterward, the cells were transiently transfected with NF-nB-luciferase and h-gal, and subsequently were stimulated overnight with TGF-h1 as indicated prior to measuring the luciferase and h-gal activities contained in detergent-solubilized whole cell extracts. Points, mean (n = 3) luciferase activities relative to empty vector–expressing, unstimulated NMuMG cells that were not subjected to EMT; bars, SE. *, P < 0.05, Student’s t test.

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Figure 4. Truncated TAB1(411) expression selectively blocks TGF-h–mediated NF-nB activation in 4T1 cells. A, quiescent control (i.e., YFP) and TAB1(411)- expressing 4T1 cells were stimulated with TGF-h1 (5 ng/mL) as indicated. Afterward, IKKh was isolated by immunoprecipitation and analyzed for GST-InBa 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 with antibodies against p38 MAPK. Data from a representative experiment that was repeated thrice with similar results. B, control (i.e., YFP) and TAB1(411)-expressing 4T1 cells were transiently transfected with NF-nB-luciferase and h-gal as above and were subsequently stimulated with increasing concentrations of TGF-h1 for 24 h. Points, mean luciferase activities relative to unstimulated control cells (n = 4); bars, SE. 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 h-actin immunoblotting. Images from a representative experiment that was repeated thrice with identical results. D, control (i.e., YFP) and TAB1(411)-expressing 4T1 cells were transiently transfected with TNF-a-luciferase and h-gal and were subsequently processed for determination of luciferase and h-gal activities contained in detergent-solubilized whole-cell extracts. Columns, mean luciferase activities relative to YFP-expressing cells (n = 3); bars, SE. not a phenomenon unique to murine 4T1 cells. Introduction of sion significantly reduced 4T1 tumor volume by f65% (Fig. 6A) kinase-dead TAK1(K63M) (Fig. 5A) or dominant-negative InBa and their resulting weights at the time of necropsy by f59% (Fig. 5B) into MCF10A-CA1a cells abrogated their invasion through (Fig. 6B). We also examined the effects of TAB1(411) expression synthetic basement membranes in response to TGF-h. Finally, on 4T1 tumor growth in nude mice. Contrary to our expectations, MCF10A-CA1a cells are refractory to the cytostatic activities of TGF- we again observed TAB1(411) expression to significantly reduce h (Fig. 5C; ref. 37). Surprisingly, TAK1 deficiency not only reduced the growth of 4T1 tumors in nude mice (Fig. 6C and D). Thus, the proliferative capacity of MCF10A-CA1a cells but also partially specific incorporation of TAB1 into TAK1:IKKh complexes in restored their cytostatic response to TGF-h (Fig. 5C). Collectively, malignant MECs plays a significant role in promoting TGF-h these findings indicate that activation of the TAB1:TAK1:IKKh stimulation of breast cancer development and progression, signaling axis is essential in mediating oncogenic signaling by presumably in part via NF-nB–mediated activation of the innate TGF-h, particularly its ability to inhibit TGF-h–mediated growth immune system and its ability to promote tumor progression (41). arrest and induce TGF-h–stimulated breast cancer cell invasion. TAB1(411) expression inhibits mammary tumor growth in mice. We tested the above hypothesis by orthotopically injecting Discussion the mammary fat pads of BALB/c mice with syngeneic 4T1 breast The conversion of TGF-h from a tumor suppressor to a tumor cancer cells that expressed control (YFP) or truncated TAB1(411) promoter plays a significant role in determining how developing (Figs. 3 and 4). Interestingly, stable TAB1(411) expression failed and progressing tumors interact with and respond to changes to alter the proliferation of 4T1 cells in vitro (data not shown); in their microenvironments. Intense research efforts over the last however, the growth of 4T1 tumors in BALB/c mice was signi- decade have identified a number of aberrant genetic and epigenetic ficantly decreased by their expression of TAB1(411) (Fig. 6). Indeed, events that potentially contribute to oncogenic signaling by TGF-h over the course of these 24-day experiments, TAB1(411) expres- in malignant MECs (1). Included in this list of oncogenic mediators www.aacrjournals.org 1467 Cancer Res 2008; 68: (5). March 1, 2008

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2008 American Association for Cancer Research. Cancer Research of TGF-h signaling is NF-nB, whose stimulation is normally for the ability of these cells to colonize the lung during the per- repressed by TGF-h, but which instead becomes activated by this formance of tail vein injection assays. Discordance between our cytokine during the course of carcinogenesis (26, 27, 42, 43). respective studies may reflect differences in the MECs studied or in Unfortunately, precisely how carcinogenesis converts the cellular the relative contribution of oncogenic Ras, whose activity clearly response to TGF-h and its coupling to NF-nB remains to be cooperates with the TGF-h (1) and NF-nB (47) signaling systems. determined definitively. This question is medically relevant because However, both studies show the overall importance of NF-nB in pro- NF-nB activation has been associated with tumor inflammation, as moting the growth and development of mammary tumors in mice. well as with elevated tumor angiogenesis, invasion, and resistance The ability of TAB1:IKKh complexes to link TGF-h to NF-nB to apoptotic stimuli (28, 41), and with TGF-h–mediated EMT (44). signaling in malignant MECs likely occurs through the interaction Thus, chemotherapeutic targeting of these molecular events may of TAB1 with ThR-I, a binding reaction dependent on structural h afford novel avenues to alleviate oncogenic signaling by TGF- in determinants located in the NH2 terminus of TAB1 (Supplementary patients with metastatic breast cancer. Fig. S2B). Once bound to ThR-I, COOH-terminal TAB1 sequences By comparing the NF-nB activity profiles regulated by TGF-h in coordinate the binding and activation of TAK1:IKKh and, con- normal and malignant MECs, we defined a novel TAB1:TAK1:IKKh sequently, the induction of NF-nB specifically by TGF-h, but not signaling axis that forms specifically in breast cancer cells and by TNF-a (Supplementary Fig. S2C). The specificity of TAB1 for mediates NF-nB activation by TGF-h. This cellular response con- TGF-h signaling is consistent with gene targeting experiments that trasted sharply with that observed in normal MECs, where TGF-h established the requirement for TAB2 and TAB3 in mediating typically inhibits NF-nB activation by inducing the expression of intracellular signaling by IL-1 and TNF-a (19, 40). Future studies the NF-nB inhibitor, InBa (36). Although p38MAPK and AKT have need to identify the molecular mechanisms whereby TAB1:IKKh been linked to NF-nB activation in cancer cells (45, 46), our results complexes only form in malignant MECs. indicate that the formation of TAB1:IKKh complexes represents A potentially important observation of our study concerns the a key molecular event that enables TGF-h to activate NF-nBin connection between the coupling of TGF-h to NF-nB activation malignant MECs (Figs. 3 and 4) and in normal MECs induced to and its consequential induction of proinflammatory cytokines, undergo EMT in response to TGF-h. It is interesting to note that whose production can promote tumor progression via activation of TAB1(411) expression, which inhibited the coupling of TGF-h the innate immune system (41). Indeed, whereas the link between to NF-nB activation (Figs. 3 and 4), had no effect on the ability of NF-nB and inflammation in mediating cancer progression has TGF-h to induce EMT in NMuMG cells, suggesting that NF-nB clearly been established (28), the molecular mechanisms that activation in NMuMG cells is dissociated from their ability to underlie this pathologic sequence of events have yet to be fully undergo EMT in response to TGF-h. This finding contradicts those elucidated. We observed TAB1(411) expression to prevent TGF-h of Huber et al. (44) who found NF-nB to be essential for the ability of stimulation of NF-nB and its production of the proinflammatory TGF-h to induce and stabilize EMT in EpRas-transformed MECs and genes COX-2 and TNF-a in 4T1 cells, and of COX-2, TNF-a, IL-6,

Figure 5. TAK1 is essential for TGF-h stimulation of NF-nB and invasion in metastatic breast cancer cells. MCF10A- CA1a cells were engineered to stably express either kinase-dead TAK1(K63M) (A) or dominant-negative InBa-null (B)and were subsequently induced to invade through synthetic basement membranes by serum (3%) in the presence or absence of TGF-h1 (5 ng/mL) as indicated. Columns, mean invasion relative to that induced by serum in GFP-expressing cells (n = 4); bars, SE. *, P < 0.05, Student’s t test. C, MCF10A-CA1a cells were rendered TAK1 deficient by siRNA transfection and were subsequently stimulated with TGF-h1 (5 ng/mL) for 48 h as indicated. Cellular DNA was radiolabeled with [3H]thymidine and quantified by scintillation counting. Columns, mean [3H]thymidine incorporation normalized to unstimulated control transfected cells (n = 3); bars, SE. *, P < 0.05, Student’s t test. TAK1 deficiency was monitored by immunoblotting whole-cell extracts with TAK1-specific antibodies. Differences in protein loading were monitored with anti–h-actin antibodies. Images from a single experiment that was repeated thrice with similar results.

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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 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 (three mice per condition per experiment); bars, SE. *, P < 0.05, Student’s t test.

and granulocyte macrophage colony-stimulating factor in post- mediating breast cancer cell invasion. In addition, TAK1 deficiency EMT NMuMG cells (data not shown). These findings are remi- not only inhibited breast cancer proliferation but also partially niscent of the gene expression profiles detected in response to restored the cytostatic function of TGF-h in malignant MECs either TAK1 inactivation (25) or TAB1 deficiency (20), and as such, resistant to its growth inhibitory activities. These findings, together further support the role of TAB1:TAK1:IKKh complexes in mediat- with those linking the TAB1:TAK1:IKKh signaling axis to inflamma- ing malignant MEC expression of proinflammatory genes when tory gene expression, suggest that measures capable of anta- stimulated by TGF-h. Once released into the tumor milieu, these gonizing this oncogenic pathway may prevent the development proinflammatory cytokines function in recruiting innate immune and progression of mammary tumors. Accordingly, overexpression of effectors, such as immature and tumor-associated macrophages, TAB1(411) in 4T1 cells significantly impaired their growth relative to neutrophils, and mast cells, which collectively induce the remo- parental 4T1 cells when implanted into the mammary fat pads of deling of tumor microenvironments to favor the growth, metasta- mice (Fig. 6). Thus, the selective antagonism of TAB1:TAK1:IKKh:NF- sis, and angiogenesis of developing and progressing mammary nB signaling activated by TGF-h holds the potential to one day tumors (28, 41). Our finding that TAB1(411) expression retains its improve the clinical course of patients with metastatic breast cancer. ability to suppress the growth of mammary tumors in nude mice (Fig. 6) suggests that abrogating TGF-h–mediated stimulation of NF-nB preferentially prevents the activation of the innate immune Acknowledgments system, and not that of the adaptive immune system. Future Received 8/10/2007; revised 12/5/2007; accepted 1/3/2008. studies need to further investigate the linkage between TGF-h and Grant support: NIH grants CA095519 and CA114039 (W.P. Schiemann). The costs of publication of this article were defrayed in part by the payment of page NF-nB in mediating activation of the innate immune system during charges. This article must therefore be hereby marked advertisement in accordance the development and progression of mammary tumors. with 18U.S.C. Section 1734 solely to indicate this fact. We thank the members of the Schiemann Laboratory for critical reading of the Finally, an important and somewhat surprising finding presented manuscript and the members of the Flow Cytometry at the University of Colorado herein was the essential requirement for TAK1 and NF-nBin Cancer Center for their technical expertise.

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Jason R. Neil and William P. Schiemann

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