Glycoprotein nonmetastatic melanoma protein B impacts the malignant potential of bladder cancer cells through its hem-immunoreceptor tyrosine-based activation motif.

Bladder cancer is one of the most common cancers among men worldwide. Although multiple genomic mutations and epigenetic alterations have been identified, an efficacious molecularly targeted therapy has yet to be established. Therefore, a novel approach is anticipated. Glycoprotein nonmetastatic melanoma protein B (GPNMB) is a type I transmembrane glycoprotein that is highly expressed in various cancers. In this study, we evaluated bladder cancer patient samples and found that GPNMB protein abundance is associated with high-grade tumors, and both univariate and multivariate analyses showed that GPNMB is a prognostic factor. Furthermore, the prognosis of patients with high GPNMB levels was significantly poorer in those with nonmuscle invasive bladder cancer (NMIBC) than in those with muscle invasive bladder cancer (MIBC). We then demonstrated that knockdown of GPNMB in MIBC cell lines with high GPNMB inhibits cellular migration and invasion, whereas overexpression of GPNMB further enhances cellular migration and invasion in MIBC cell lines with originally low GPNMB. Therefore, we propose that GPNMB is one of multiple driver molecules in the acquisition of cellular migratory and invasive potential in bladder cancers. Moreover, we revealed that the tyrosine residue in the hemi-immunoreceptor tyrosine-based activation motif (hemITAM) is required for GPNMB-induced cellular motility.

The liver kinase B1 supports mammary epithelial morphogenesis by inhibiting critical factors that mediate epithelial-mesenchymal transition.

The liver kinase B1 (LKB1) controls cellular metabolism and cell polarity across species. We previously established a mechanism for negative regulation of transforming growth factor ß (TGFß) signaling by LKB1. The impact of this mechanism in the context of epithelial polarity and morphogenesis remains unknown. After demonstrating that human mammary tissue expresses robust LKB1 protein levels, whereas invasive breast cancer exhibits significantly reduced LKB1 levels, we focused on mammary morphogenesis studies in three dimensional (3D) acinar organoids. CRISPR/Cas9-introduced loss-of-function mutations of STK11 (LKB1) led to profound defects in the formation of 3D organoids, resulting in amorphous outgrowth and loss of rotation of young organoids embedded in matrigel. This defect was associated with an enhanced signaling by TGFß, including TGFß auto-induction and induction of transcription factors that mediate epithelial-mesenchymal transition (EMT). Protein marker analysis confirmed a more efficient EMT response to TGFß signaling in LKB1 knockout cells. Accordingly, chemical inhibition of the TGFß type I receptor kinase largely restored the morphogenetic defect of LKB1 knockout cells. Similarly, chemical inhibition of the bone morphogenetic protein pathway or the TANK-binding kinase 1, or genetic silencing of the EMT factor SNAI1, partially restored the LKB1 knockout defect. Thus, LKB1 sustains mammary epithelial morphogenesis by limiting pathways that promote EMT. The observed downregulation of LKB1 expression in breast cancer is therefore predicted to associate with enhanced EMT induced by SNAI1 and TGFß family members.

Promotion of squamous cell carcinoma tumorigenesis by oncogene-mediated THG-1/TSC22D4 phosphorylation.

Carcinoma cells possess high proliferative and invasive potentials and exhibit a resilience against stresses, metabolic disorder, and therapeutic efforts. These properties are mainly acquired by genetic alterations including driver gene mutations. However, the detailed molecular mechanisms have not been fully elucidated. Here, we provide a novel mechanism connecting oncogenic signaling and the tumorigenic properties by a transforming growth factor-ß1-stimulated clone 22 (TSC-22) family protein, THG-1 (also called as TSC22D4). THG-1 is localized at the basal layer of normal squamous epithelium and overexpressed in squamous cell carcinomas (SCCs). THG-1 knockdown suppressed SCC cell proliferation, invasiveness, and xenograft tumor formation. In contrast, THG-1 overexpression promoted the EGF-induced proliferation and stratified epithelium formation. Furthermore, THG-1 is phosphorylated by the receptor tyrosine kinase (RTK)-RAS-ERK pathway, which promoted the oncogene-mediated tumorigenesis. Moreover, THG-1 involves in the alternative splicing of CD44 variants, a regulator of invasiveness, stemness, and oxidative stress resistance under the RTK pathway. These findings highlight the pivotal roles of THG-1 as a novel effector of SCC tumorigenesis, and the detection of THG-1 phosphorylation by our established specific antibody could contribute to cancer diagnosis and therapy.

Glycoprotein NMB promotes tumor formation and malignant progression of laryngeal squamous cell carcinoma.

Laryngeal squamous cell carcinoma (LSCC), although one of the most common head and neck cancers, has a static or slightly decreased survival rate because of difficulties in early diagnosis, lack of effective molecular targeting therapy, and severe dysfunction after radical surgical treatments. Therefore, a novel therapeutic target is crucial to increase treatment efficacy and survival rates in these patients. Glycoprotein NMB (GPNMB), whose role in LSCC remains elusive, is a type 1 transmembrane protein involved in malignant progression of various cancers, and its high expression is thought to be a poor prognostic factor. In this study, we showed that GPNMB expression levels in LSCC samples are significantly higher than those in normal tissues, and GPNMB expression is observed mostly in growth-arrested cancer cells. Furthermore, knockdown of GPNMB reduces monolayer cellular proliferation, cellular migration, and tumorigenic growth, while GPNMB protein displays an inverse relationship with Ki-67 levels. Therefore, we conclude that GPNMB may be an attractive target for future LSCC therapy.

Polyvinyl alcohol scaffolds and supplementation support 3D and sphere culturing of human cancer cell lines by reducing apoptosis and promoting cellular proliferation.

Three-dimensional (3D) culturing mimics the heterogeneous cellular conditions of the in vivo tumor microenvironment compared to 2D monolayer-cultured cells and 3D cultures of established cancer cell lines (sphere culture) or patient-derived cancer cells (organoid culture) are frequently used for cancer research or drug screening and evaluation. To establish more cost and time-efficient 3D culture methods for cancer cell lines, we supplemented sphere culture medium with polyvinyl alcohol (PVA) and found that 3D sphere cultures of breast and pancreatic cancer cell lines were significantly increased. Mechanistically, we found that PVA prevented cell death and promoted cellular proliferation while maintaining levels of stemness-related gene expression. Furthermore, we showed that polyvinyl formal resin (PVF) 3D scaffolds made by cross-linked PVA can function in serum-free, long-term 3D cultures to support maintenance of sphere- or tumor-like cell masses for diverse cancer cell types. Taken together, we demonstrate the effectiveness of PVA and PVF in human cancer cell line culture protocols.

Glycoprotein non-metastatic melanoma protein B functions with growth factor signaling to induce tumorigenesis through its serine phosphorylation.

Breast cancer is the most common cancer among women. Glycoprotein non-metastatic melanoma protein B (GPNMB), a type I transmembrane protein that is highly expressed in many cancers, including breast cancer, has been shown to be a prognostic factor. We previously reported that GPNMB overexpression confers tumorigenic potential, as evidenced by invasive tumor growth in vivo, sphere formation, and cellular migration and invasion to non-tumorigenic mammary epithelial cells. In this study, we focused on the serine (S) residue in the intracellular domain of GPNMB (S530 in human isoform b and S546 in mouse), which is predicted to be a phosphorylation site. To investigate the roles of this serine residue, we made an antibody specific for S530-phosphorylated human GPNMB and a point mutant in which S530 is replaced by an alanine (A) residue, GPNMB(SA). Established GPNMB(SA) overexpressing cells showed a significant reduction in sphere formation in vitro and tumor growth in vivo as a result of decreased stemness-related gene expression compared to that in GPNMB(WT)-expressing cells. In addition, GPNMB(SA) impaired GPNMB-mediated cellular migration. Furthermore, we found that tyrosine kinase receptor signaling triggered by epidermal growth factor or fibroblast growth factor 2 induces the serine phosphorylation of GPNMB through activation of downstream oncoproteins RAS and RAF.

PMEPA1/TMEPAI isoforms function via its PY and Smad-interaction motifs for tumorigenic activities of breast cancer cells.

PMEPA1 (prostate transmembrane protein, androgen-induced 1)/TMEPAI (transmembrane prostate androgen-induced protein) is highly expressed in diverse cancers, including breast, lung and prostate cancers. It consists of four isoforms with distinct extracellular regions (isoforms a-d). The expression and function of these isoforms are still poorly understood. Hence, we aimed to identify the preferentially expressed isoforms in breast cancer cells and analyze possible differences in tumorigenic functions. In this study, we used 5' Rapid Amplification of cDNA Ends (RACE) and Western blot analyses to identify the mRNA variants and protein isoforms of TMEPAI and found that TMEPAI isoform d as the major isoform expressed by TGF-ß stimulation in breast cancer cells. We then generated CRISPR/Cas9-mediated TMEPAI knockout (KO) breast cancer cell lines and used a lentiviral expression system to complement each isoform individually. Although there were no clear functional differences between isoforms, double PPxY (PY) motifs and a Smad-interaction motif (SIM) of TMEPAI were both essential for colony and sphere formation. Collectively, our results provide a novel insight into TMEPAI isoforms in breast cancer cells and showed that coordination between double PY motifs and a SIM of TMEPAI are essential for colony and sphere formation but not for monolayer cell proliferation.

Promotion of cellular senescence by THG-1/TSC22D4 knockout through activation of JUNB.

Induction of cellular senescence in cancerous cells is an important strategy which is used in the treatment of cancer. However, cancer cells are capable of exhibiting resistance to cellular senescence through inactivation of tumor suppressors. Because of this, establishment of a route to cellular senescence induction in cancer cells is a crucial direction for developing future cancer therapies. In this study, we demonstrate the involvement of TSC-22 homologous gene-1 (THG-1, also called TSC22D4) in the suppression of cellular senescence. CRISPR/Cas9 gene editing was used to establish THG-1 knockout (KO) cells in a THG-1 positive esophageal tumor cell line. It was found that THG-1 KO cells exhibited delayed cell proliferation as well as cellular senescence. The elevated expression of the CDK inhibitor P21(CDKN1A) was also identified in senescent cells. Through the investigation of the upstream pathway for induction of P21(CDKN1A), the JUNB pathway was identified to play a critical role in P21(CDKN1A) transcription; in fact, the siRNA-mediated knockdown of JUNB reduced the abundance of P21(CDKN1A) mRNA and cellular senescence in THG-1 KO cells. These findings provide a novel insight into the induction of cellular senescence in THG-1 positive cancer cells.

THG-1 suppresses SALL4 degradation to induce stemness genes and tumorsphere formation through antagonizing NRBP1 in squamous cell carcinoma cells.

Knockdown of THG-1 in TE13 esophageal squamous cell carcinoma (ESCC) cells is known to suppress tumorsphere growth. THG-1 was identified as an NRBP1 binding protein, and NRBP1 was reported to downregulate an stemness-related transcriptional factor SALL4, so we decided to examine the possibility that tumorigenic function of THG-1 is achieved by the competition to the tumor-suppressive function of NRBP1. SALL4 was decreased in THG-1 deficient TE13 cells with reduced tumorsphere formation, while exogenous SALL4 expression in THG-1 deficient TE13 cells recovered expression of stemness genes (NANOG and OCT4) and partially, but significantly, recovered tumorsphere formation ability. Additionally, we found that NRBP1 induced ubiquitination of SALL4, and THG-1 interrupted the ubiquitination of SALL4 by antagonizing NRBP1 binding to SALL4. These results suggest that THG-1 promotes tumorsphere growth of ESCC cells by the stabilization of SALL4 protein and induction of the target stemness genes through competitive binding to NRBP1.

Role of the kringle-like domain in glycoprotein NMB for its tumorigenic potential.

Glycoprotein NMB (GPNMB) is highly expressed in many types of malignant tumors and thought to be a poor prognostic factor in those cancers, including breast cancer. Glycoprotein NMB is a type IA transmembrane protein that has a long extracellular domain (ECD) and a short intracellular domain (ICD). In general, the ECD of a protein is involved in protein-protein or protein-carbohydrate interactions, whereas the ICD is important for intracellular signaling. We previously reported that GPNMB contributes to the initiation and malignant progression of breast cancer through the hemi-immunoreceptor tyrosine-based activation motif (hemITAM) in its ICD. Furthermore, we showed that the tyrosine residue in hemITAM is involved in induction of the stem-like properties of breast cancer cells. However, the contribution of the ECD to its tumorigenic function has yet to be fully elucidated. In this study, we focused on the region, the so-called kringle-like domain (KLD), that is conserved among species, and made a deletion mutant, GPNMB(ΔKLD). Enhanced expression of WT GPNMB induced sphere and tumor formation in breast epithelial cells; in contrast, GPNMB(ΔKLD) lacked these activities without affecting its molecular properties, such as subcellular localization, Src-induced tyrosine phosphorylation at least in overexpression experiments, and homo-oligomerization. Additionally, GPNMB(ΔKLD) lost its cell migration promoting activity, even though it reduced E-cadherin expression. Although the interaction partner binding to KLD has not yet been identified, we found that the KLD of GPNMB plays an important role in its tumorigenic potential.

Generation of non-standard macrocyclic peptides specifically binding TSC-22 homologous gene-1.

Transforming growth factor-ß 1 (TGFß1)-stimulated clone 22 (TSC22) family includes proteins containing a leucine zipper domain and a TSC-box that are highly conserved during evolution. Currently, limited data are available on the function of this protein family, especially of TSC-22 homologous gene-1 (THG-1)/TSC22 domain family member 4 (TSC22D4). Similar to other family members, THG-1 functions depending on its interaction with the partner proteins and it is suggested to mediate a broad range of biological processes. THG-1-specific binding molecules will be instrumental for elucidating its functions. Therefore, the Random non-standard Peptide Integrated Discovery (RaPID) system was modified using commercially available materials and used for selecting macrocyclic peptides (MCPs) that bind to THG-1. Several MCPs were identified to bind THG-1. Fluorescein- and biotin-tagged MCPs were synthesized and employed as THG-1 detection probes. Notably, a fluorescein-tagged MCP specifically detected THG-1-expressing cells. Biotin-tagged MCPs can be successfully used for Enzyme-Linked Protein Sorbent Assay (ELISA) like assay of THG-1 protein and affinity-precipitation of purified THG-1 and endogenous THG-1 in esophageal squamous cell carcinoma cell lysates. The modified RaPID system rapidly and successfully identified THG-1-binding MCPs in vitro and the synthesized THG-1 binding MCPs are useful alternatives acting for antibodies.

Stereodivergent Synthesis of Bispyrrolidinoindoline Alkaloidal Scaffolds and Generation of a Lead Candidate with Stereospecific Antiproliferative Activity.

The fungal secondary metabolites (+)-WIN 64821 and (-)-ditryptophenaline are biosynthesized through condensation of l-tryptophan and l-phenylalanine, followed by reductive dimerization with generation of stereochemical variations. Inspired by the stereodivergent biogenetic process, we designed and synthesized a collection of bispyrrolidinoindoline diketopiperazine alkaloids and their analogues with systematic diversification of the stereochemistry of the privileged structural motif of the fungal alkaloids. Not only the stereochemical modifications of (+)-WIN 64821 at the 3-/3'-, 11-/11'-, and 15-/15'-positions, but also ring cleavage of the diketopiperazine moieties, allowed the generation of a lead compound exhibiting potent growth inhibitory activity (IC50 =3.03 µm) toward human colon cancer cells. Structure-activity relationship studies revealed that all six stereogenic centers were essential for the pharmacophore. High cell densities dramatically intensified the cytotoxic activities of the lead compound.

Efficient DNA binding of NF-κB requires the chaperone-like function of NPM1.

NPM1/nucleophosmin is frequently overexpressed in various tumors, although the oncogenic role of NPM1 remains unclear. Here we revealed the link between NPM1 and nuclear factor-κB (NF-κB), a master regulator of inflammation. We found that NPM1 knockdown decreased NF-κB-mediated transcription of selected target genes by decreasing the recruitment of NF-κB p65 to the gene promoters. NPM1 is directly associated with the DNA binding domain of p65 to enhance its DNA binding activity without being a part of the DNA-NF-κB complex. This result suggests that NF-κB requires the chaperone-like function of NPM1 for DNA binding. Furthermore, we demonstrated that NPM1 was required for efficient inflammatory gene expression induced by tumor necrosis factor alpha (TNF-α) and lipopolysaccharide in fibroblasts and macrophages. The NF-κB-mediated invasion of breast cancer cells was significantly decreased by NPM1 knockdown. Our study suggests a novel mechanistic insight into the NF-κB-mediated transcription and an oncogenic role of NPM1 in both tumor cells and the tumor micro-environment through the regulation of NF-κB.

Regulation of c-MYC transcriptional activity by transforming growth factor-beta 1-stimulated clone 22.

c-MYC stimulates cell proliferation through the suppression of cyclin-dependent kinase (CDK) inhibitors including P15 (CDKN2B) and P21 (CDKN1A). It also activates E-box-mediated transcription of various target genes including telomerase reverse transcriptase (TERT) that is involved in cellular immortality and tumorigenesis. Transforming growth factor-beta 1 (TGF-ß1)-stimulated clone 22 (TSC-22/TSC22D1) encodes a highly conserved leucine zipper protein that is induced by various stimuli, including TGF-ß. TSC-22 inhibits cell growth in mammalian cells and in Xenopus embryos. However, underlying mechanisms of growth inhibition by TSC-22 remain unclear. Here, we show that TSC-22 physically interacts with c-MYC to inhibit the recruitment of c-MYC on the P15 (CDKN2B) and P21 (CDKN1A) promoters, effectively inhibiting c-MYC-mediated suppression of P15 (CDKN2B) and also P21 (CDKN1A) promoter activities. In contrast, TSC-22 enhances c-MYC-mediated activation of the TERT promoter. Additionally, the expression of TSC-22 in embryonic stem cells inhibits cell growth without affecting its pluripotency-related gene expression. These results indicate that TSC-22 differentially regulates c-MYC-mediated transcriptional activity to regulate cell proliferation.

TMEPAI, a transmembrane TGF-beta-inducible protein, sequesters Smad proteins from active participation in TGF-beta signaling.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine of key importance for controlling embryogenesis and tissue homeostasis. How TGF-beta signals are attenuated and terminated is not well understood. Here, we show that TMEPAI, a direct target gene of TGF-beta signaling, antagonizes TGF-beta signaling by interfering with TGF-beta type I receptor (TbetaRI)-induced R-Smad phosphorylation. TMEPAI can directly interact with R-Smads via a Smad interaction motif. TMEPAI competes with Smad anchor for receptor activation for R-Smad binding, thereby sequestering R-Smads from TbetaRI kinase activation. In mammalian cells, ectopic expression of TMEPAI inhibited TGF-beta-dependent regulation of plasminogen activator inhibitor-1, JunB, cyclin-dependent kinase inhibitors, and c-myc expression, whereas specific knockdown of TMEPAI expression prolonged duration of TGF-beta-induced Smad2 and Smad3 phosphorylation and concomitantly potentiated cellular responsiveness to TGF-beta. Consistently, TMEPAI inhibits activin-mediated mesoderm formation in Xenopus embryos. Therefore, TMEPAI participates in a negative feedback loop to control the duration and intensity of TGF-beta/Smad signaling.

TIF1ß is phosphorylated at serine 473 in colorectal tumor cells through p38 mitogen-activated protein kinase as an oxidative defense mechanism.

TIF1ß is a pleiotropic regulator of a diverse range of cellular processes such as DNA repair or gene repression in stem cells. This functional switch depends on phosphorylation at serine residue 473 and multiple pathways exist to accomplish this. However, the effects of exogenous reactive oxygen species (ROS) generated by bacterial flora and dietary metabolites in the colonic lumen or chemotherapy on TIF1ß have not been determined. We report here that exposure of colorectal cancer (CRC) cell lines DLD-1 and HCT116 to hydrogen peroxide specifically induces TIF1ß Ser473 phosphorylation. Hydrogen peroxide also induces primarily p38 MAPK and some p42/44 MAPK phosphorylation. Chemical inhibition of p38 MAPK and p42/44 MAPK reduced phosphorylation of TIF1ß serine 473 and increased CRC cell death upon peroxide exposure. Taken together, this suggests that it is primarily peroxide-induced p38 MAPK that mediates Ser473 phosphorylation and activation of TIF1ß to enable more efficient DNA repair to assist in tumor cell survival against exogenous ROS.

Repression of Smad3 by Stat3 and c-Ski/SnoN induces gefitinib resistance in lung adenocarcinoma.

Cancer-associated inflammation develops resistance to the epidermal growth-factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in non-small cell lung cancers (NSCLCs) harboring oncogenic EGFR mutations. Stat3-mediated interleukin (IL)-6 signaling and Smad-mediated transforming growth factor-ß (TGF-ß) signaling pathways play crucial regulatory roles in cancer-associated inflammation. However, mechanisms how these pathways regulate sensitivity and resistance to EGFR-TKI in NSCLCs remain largely undetermined. Here we show that signal transducer and activator of transcription (Stat)3 represses Smad3 in synergy with the potent negative regulators of TGF-ß signaling, c-Ski and SnoN, whereby renders gefitinib-sensitive HCC827 cells resistant. We found that IL-6 signaling via phosphorylated Stat3 induced gefitinib resistance as repressing transcription of Smad3, whereas TGF-ß enhanced gefitinib sensitivity as activating transcription of Smad3 in HCC827 cells with gefitinib-sensitizing EGFR mutation. Promoter analyses showed that Stat3 synergized with c-Ski/SnoN to repress Smad2/3/4-induced transcription of the Smad3 gene. Smad3 was found to be an apoptosis inducer, which upregulated pro-apoptotic genes such as caspase-3 and downregulated anti-apoptotic genes such as Bcl-2. Our results suggest that derepression of Smad3 can be a therapeutic strategy to prevent gefitinib-resistance in NSCLCs with gefitinib-sensitizing EGFR mutation.

C18 ORF1, a novel negative regulator of transforming growth factor-ß signaling.

Transforming growth factor (TGF)-ß signaling is deliberately regulated at multiple steps in its pathway from the extracellular microenvironment to the nucleus. However, how TGF-ß signaling is activated or attenuated is not fully understood. We recently identified transmembrane prostate androgen-induced RNA (TMEPAI), which is involved in a negative feedback loop of TGF-ß signaling. When we searched for a family molecule(s) for TMEPAI, we found C18ORF1, which, like TMEPAI, possesses two PY motifs and one Smad-interacting motif (SIM) domain. As expected, C18ORF1 could block TGF-ß signaling but not bone morphogenetic protein signaling. C18ORF1 bound to Smad2/3 via its SIM and competed with the Smad anchor for receptor activation for Smad2/3 binding to attenuate recruitment of Smad2/3 to the TGF-ß type I receptor (also termed activin receptor-like kinase 5 (ALK5)), in a similar fashion to TMEPAI. Knockdown of C18ORF1 prolonged duration of TGF-ß-induced Smad2 phosphorylation and concomitantly potentiated the expression of JunB, p21, and TMEPAI mRNAs induced by TGF-ß. Consistently, TGF-ß-induced cell migration was enhanced by the knockdown of C18ORF1. These results indicate that the inhibitory function of C18ORF1 on TGF-ß signaling is similar to that of TMEPAI. However, in contrast to TMEPAI, C18ORF1 was not induced upon TGF-ß signaling. Thus, we defined C18ORF1 as a surveillant of steady state TGF-ß signaling, whereas TMEPAI might help C18ORF1 to inhibit TGF-ß signaling in a coordinated manner when cells are stimulated with high levels of TGF-ß.

Cooperative induction of transmembrane prostate androgen induced protein TMEPAI/PMEPA1 by transforming growth factor-ß and epidermal growth factor signaling.

TMEPAI/PMEPA1 (transmembrane prostate androgen induced-RNA/prostate transmembrane protein, androgen induced 1) is a pro-tumorigenic factor induced by TGF-ß signaling and constitutive TMEPAI expression in lung cancer cells depends on activated autocrine TGF-ß signaling. Here we demonstrate a novel mechanism of TMEPAI transcriptional co-regulation by EGF signaling. Interestingly, we found that ELK-1, downstream of EGFR/Ras/MAPK pathway, modulates TMEPAI expression. ELK-1 binds to the first intron (+1037 to +1294) of the TMEPAI gene together with TGF-ß activated Smad3 and enhances the transcription of TMEPAI. Furthermore, TMEPAI gene activation by EGF and TGF-ß signaling was reduced by the MEK inhibitor U0126. Together, EGF signaling collaboratively regulates TGF-ß-induced TMEPAI expression.

Transforming growth factor-ß induces transcription factors MafK and Bach1 to suppress expression of the heme oxygenase-1 gene.

Transforming growth factor-ß (TGF-ß) has multiple functions in embryogenesis, adult homeostasis, tissue repair, and development of cancer. Here, we report that TGF-ß suppresses the transcriptional activation of the heme oxygenase-1 (HO-1) gene, which is implicated in protection against oxidative injury and lung carcinogenesis. HO-1 is a target of the oxidative stress-responsive transcription factor Nrf2. TGF-ß did not affect the stabilization or nuclear accumulation of Nrf2 after stimulation with electrophiles. Instead, TGF-ß induced expression of transcription factors MafK and Bach1. Enhanced expression of either MafK or Bach1 was enough to suppress the electrophile-inducible expression of HO-1 even in the presence of accumulated Nrf2 in the nucleus. Knockdown of MafK and Bach1 by siRNA abolished TGF-ß-dependent suppression of HO-1. Furthermore, chromatin immunoprecipitation assays revealed that Nrf2 substitutes for Bach1 at the antioxidant response elements (E1 and E2), which are responsible for the induction of HO-1 in response to oxidative stress. On the other hand, pretreatment with TGF-ß suppressed binding of Nrf2 to both E1 and E2 but marginally increased the binding of MafK to E2 together with Smads. As TGF-ß is activated after tissue injury and in the process of cancer development, these findings suggest a novel mechanism by which damaged tissue becomes vulnerable to oxidative stress and xenobiotics.