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Nuclear factor-κB (NF-κB) is constitutively activated in diverse human malignancies. The MUC1 oncoprotein is overexpressed in human carcinomas and, like NF-κB, blocks cell death and induces transformation. The present studies demonstrate that MUC1 constitutively associates with NF-κB p65 in carcinoma cells. The MUC1 C-terminal subunit (MUC1-C) cytoplasmic domain binds directly to NF-κB p65 and, importantly, blocks the interaction between NF-κB p65 and its inhibitor IκBα. We show that NF-κB p65 and MUC1-C constitutively occupy the promoter of the Bcl-xL gene in carcinoma cells and that MUC1-C contributes to NF-κB-mediated transcriptional activation. Studies in non-malignant epithelial cells show that MUC1-C interacts with NF-κB in the response to TNFα stimulation. Moreover, TNFα induces the recruitment of NF-κB p65-MUC1-C complexes to NF-κB target genes, including the promoter of the MUC1 gene itself. We also show that a small peptide inhibitor of MUC1-C oligomerization blocks the interaction with NF-κB p65 in vitro and in cells. The MUC1-C inhibitor decreases MUC1-C and NF-κB p65 promoter occupancy and expression of NF-κB target genes. These findings indicate that MUC1-C is a direct activator of NF-κB p65 and that an inhibitor of MUC1 function is effective in blocking activation of the NF-κB pathway.
The NF-κB proteins (RelA/p65, RelB, c-Rel, NF-κB1/p50 and NF-κB2/p52) are ubiquitously expressed transcription factors. In the absence of stimulation, NF-κB proteins localize to the cytoplasm in complexes with IκBα and other members of the IκB family of inhibitor proteins (1). Phosphorylation of IκBα by the high molecular weight IκB kinase (IKKα, IKKβ, IKKγ) complex induces ubiquitination and degradation of IκBα and thereby release of NF-κB for nuclear translocation. In turn, activation of NF-κB target genes contributes to tumor development through regulation of inflammatory responses, cellular proliferation and survival (2). NF-κB p65, like other members of the family, contains an N-terminal Rel homology domain (RHD) that is responsible for dimerization and DNA binding. The RHD also functions as a binding site for ankyrin repeats in the IκBα protein, which blocks the NF-κB p65 nuclear localization signal (NLS). The NF-κB-IκBα complexes shuttle between the nucleus and cytoplasm (1). Activation of the canonical NF-κB pathway, for example in the cellular response to tumor necrosis α (TNFα), induces IKKβ-mediated phosphorylation of IκBα and its degradation, with a shift in the balance of NF-κB p65 to the nucleus. The nuclear NF-κB dimers engage κB consensus sequences, as well as degenerate variants, in promoter and enhancer regions (3, 4). Activation of NF-κB target genes is then further regulated by posttranslational modification of NF-κB p65 and its interaction with transcriptional coactivators (1). One of the many NF-κB target genes is IκBα, the activation of which results in de novo synthesis of IκBα and termination of the NF-κB transcriptional response.
Human mucin 1 (MUC1) is a heterodimeric glycoprotein that is aberrantly overexpressed by diverse carcinomas and certain hematologic malignancies. Overexpression of MUC1 confers anchorage-independent growth and tumorigenicity by mechanisms involving, at least in part, constitutive activation of the Wnt/β-catenin and NF-κB pathways (5-7). The MUC1 N-terminal subunit (MUC1-N), which contains variable numbers of extensively glycosylated tandem repeats, is dispensable for inducing a malignant phenotype (6). In this regard, the transmembrane MUC1 C-terminal subunit (MUC1-C) functions as a receptor (8) and contains a 72 amino acid cytoplasmic domain (MUC1-CD) that is sufficient for inducing transformation (6). The MUC1-C subunit is also targeted to the nucleus by a process dependent on its oligomerization (9). MUC1-CD functions as a substrate for phosphorylation by the epidermal growth factor receptor (10), c-Src (11), glycogen synthase kinase 3β (GSK3β) (12) and c-Abl (13). MUC1-CD also stabilizes the Wnt effector, β-catenin, through a direct interaction and thereby contributes to transformation (6). Other studies have demonstrated that MUC1-CD interacts directly with IKKβ and IKKγ, and contributes to activation of the IKK complex (7). Significantly, constitutive activation of NF-κB p65 in human carcinoma cells is downregulated by silencing MUC1, indicating that MUC1-CD has a functional role in regulation of the NF-κB p65 pathway (7). These findings have also suggested that MUC1-CD function could be targeted with small molecules to disrupt NF-κB signaling in carcinoma cells.
The present studies demonstrate that MUC1-CD binds directly to NF-κB p65 and blocks the interaction between NF-κB p65 and IκBα. We show that the MUC1-C subunit associates with NF-κB p65 on the promoters of NF-κB target genes and promotes NF-κB-mediated transcription. The results also demonstrate that an inhibitor of MUC1-C oligomerization blocks the MUC1 interaction with NF-κB p65 and constitutive activation of the NF-κB pathway.
Human ZR-75-1 breast cancer and U-937 leukemia cells were grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. Human HeLa cervical and MCF-7 breast carcinoma cells were grown in Dulbecco’s modified Eagle’s medium with 10% FBS, antibiotics and L-glutamine. Human MCF-10A breast epithelial cells were grown in mammary epithelial cell growth medium (MEGM; Lonza, Walkersville, MD) and treated with 20 ng/ml TNFα (BD Biosciences, San Jose, CA). Transfection of the MCF-10A cells with siRNA pools (Dharmacon, Lafayette, CO) was performed in the presence of Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Cells were treated with 5 μM GO-201 and CP-1 synthesized by the MIT Biopolymer Laboratory (Cambridge, MA) (14).
Lysates from subconfluent cells were prepared as described (15). Soluble proteins were precipitated with anti-NF-κB p65 (Santa Cruz Biotechnology, Santa Cruz, CA). The immunoprecipitates and cell lysates were subjected to immunoblotting with anti-p65 (Santa Cruz Biotechnology), anti-p65(180-306) (clone 12H11; Millipore, Billerica, MA) anti-MUC1-C (Ab5; Lab Vision, Fremont, CA), anti-IκBα (Santa Cruz Biotechnology), anti-Bcl-xL (Santa Cruz Biotechnology) and anti-β-actin (Sigma, St. Louis, MO). Immune complexes were detected with horseradish peroxidase-conjugated secondary antibodies (GE Healthcare Biosciences, Piscataway, NJ) and enhanced chemiluminescence (GE Healthcare).
GST, GST-MUC1-CD, GST-MUC1-CD(1-45), GST-MUC1-CD(46-72) and GST-MUC1-CD(mSRM) were prepared as described (6, 7) and incubated with p65 and certain p65 deletion mutants. Purified GST-MUC1-CD was cleaved with thrombin to remove the GST moiety. GST-IκBα (Millipore, Billerica, MA) was incubated with p65(186-306) for 2 h at 25°C in the absence and presence of purified MUC1-CD. Adsorbates to glutathione-conjugated beads were analyzed by immunoblotting.
Cells were fixed and permeabilized as described (13). Incubation with anti-MUC1-C and anti-NF-κB p65 in blocking buffer was performed overnight at 4°C. The cells were blocked with 10% goat serum and stained with anti-MUC1-C, followed by FITC-conjugated secondary anti-hamster antibody. The cells were then incubated with anti-NF-κB p65 followed by Texas Red-conjugated anti-mouse Ig conjugate (Jackson Immuno-Research Laboratories, West Grove, PA). Nuclei were stained with 2 μM TO-PRO-3. Images were captured with a Zeiss LSM510 confocal microscope at 1024 × 1024 resolution.
Soluble chromatin was prepared as described (16) and precipitated with anti-p65, anti-MUC1-C or a control non-immune IgG. For Re-ChIP assays, complexes from the primary ChIP were eluted with 10 mM DTT, diluted in Re-ChIP buffer and reimmunoprecipitated with anti-p65. For PCR, 2 μl from a 50 μl DNA extraction was used with 25-35 cycles of amplification.
Cells were transfected with NF-κB-Luc (7) or pMUC1-Luc (17) and SV-40-Renilla-Luc (Promega, Madison, WI) in the presence of Lipofectamine. After 48 h, the cells were lysed in passive lysis buffer. Lysates were analyzed for firefly and Renilla luciferase activities using the dual luciferase assay kit (Promega).
To determine whether MUC1 interacts with NF-κB, anti-NF-κB p65 precipitates from ZR-75-1 breast cancer cells were immunoblotted with an antibody against the MUC1-C subunit cytoplasmic domain. The results demonstrate that MUC1-C coprecipitates with NF-κB p65 (Fig. 1A). Similar findings were obtained with lysates from MCF-7 breast cancer cells, which also overexpress endogenous MUC1 (Supplemental Fig. S1A). To determine whether the MUC1-N subunit is necessary for the association, studies were performed on U-937 cells that stably express exogenous MUC1-C and not MUC1-N (18). The coprecipitation of NF-κB p65 and MUC1-C in these cells demonstrated that MUC1-N is dispensable for the interaction (Supplemental Fig. S1B). Incubation of ZR-75-1 cell lysates with GST or a GST fusion protein containing the 72 amino acid MUC1-CD further demonstrated that MUC1-CD associates with NF-κB p65 (Supplemental Fig. S1C). To determine whether MUC1 binds directly to NF-κB, we incubated GST, GST-MUC1-CD, GST-MUC1-CD(1-45) or GST-MUC1-CD(46-72) with purified recombinant NF-κB p65 (Fig. 1B). Analysis of the adsorbates demonstrated that GST-MUC1-CD, and not GST, binds to NF-κB p65 (Fig. 1B). Incubation of MUC1-CD deletion mutants further demonstrated that this interaction is mediated by MUC1-CD(46-72), and not MUC1-CD(1-45) (Fig. 1B).
NF-κB p65 is a 551 amino acid protein that includes an N-terminal Rel homology domain (RHD) and a C-terminal transactivation domain (TAD) (Fig. 1C). Incubation of GST-MUC1-CD with purified NF-κB deletion mutants demonstrated binding to p65(1-306) and not p65(354-551) (Fig. 1C). To further define the MUC1-CD and NF-κB sequences responsible for the interaction, we incubated GST-p65(1-306) with MUC1-CD in the presence of peptides derived from MUC1-CD(46-72) (Fig. 1D). The results demonstrate that the GGSSLSY, and not the YNTPAVAATSANL, peptide blocked the interaction between MUC1-CD and p65(1-306) (Fig. 1D, left). Moreover, incubation of GST-MUC1-CD with mutation of the serine-rich motif (SRM; SAGNGGSSLS to AAGNGGAAAA) substantially decreased the interaction with p65(1-306) (Fig. 1D, right), further supporting dependence on the GGSSLSY sequence for the interaction.
The results further show that MUC1-CD binds to p65(1-180) (Supplemental Fig. S2A). As a control, there was no detectable interaction of GST-IκBα and p65(1-180) (Supplemental Fig. S2A). In that regard, IκBα binds to sequences just upstream to the NLS at amino acids 301-304 (19, 20). Notably, however, both MUC1-CD and IκBα formed complexes with p65(186-306) (Supplemental Fig. S2B). These findings indicate that, like IκBα, MUC1-CD binds directly to the NF-κ p65 RHD.
The conserved RHD is responsible for DNA binding, dimerization and association with the IκB inhibitory proteins (21, 22). To determine whether binding of MUC1 to the RHD region affects the association with IκBα, we first studied ZR-75-1 cells that are stably silenced for MUC1 with a MUC1siRNA (Supplemental Fig. S3). Silencing of MUC1 was associated with increased binding of NF-κB p65 and IκBα (Fig. 2A). In addition, stable expression of exogenous MUC1 in HeLa cells (7) decreased the interaction between NF-κB p65 and IκBα (Fig. 2B). Stable expression of MUC1-CD in 3Y1 cells (6) was also sufficient to block binding of NF-κB p65 and IκBα (Fig. 2C), confirming that the MUC1-C cytoplasmic domain, and not other regions of this subunit, is responsible for the interaction. To determine whether MUC1 directly affects binding of NF-κB p65 and IκBα, we performed competition studies in which binding of IκBα to p65(186-306) was assessed in the presence of MUC1-CD. As expected, binding of IκBα to p65(186-306) was detectable in the absence of MUC1-CD (Fig. 2D). Significantly, however, the addition of increasing amounts of MUC1-CD was associated with a progressive decrease in the interaction IκBα and p65(186-306) (Fig. 2D). These findings indicate that NF-κB p65 forms mutually exclusive complexes with IκBα and MUC1-CD.
Confocal analysis of ZR-75-1 cells showed nuclear colocalization of MUC1-C and NF-κB p65 (Fig. 3A). In addition and consistent with MUC1-CD competing for binding to NF-κB p65, silencing MUC1 in the ZR-75-1 cells was associated with localization of nuclear NF-κB p65 to the cytoplasm (Fig. 3A). Previous studies demonstrated that MUC1 contributes to the upregulation of Bcl-xL expression (7). To determine if MUC1-C affects the NF-κB p65 transcription complex, we performed ChIP assays with anti-p65. Immunoprecipitation of the NF-κB responsive element (RE) in the promoter of the Bcl-xL gene (GGGACTGCCC; -366 to -356) (23) was analyzed by semiquantitative PCR. In ZR-75-1 cells, occupancy of the Bcl-xL promoter by NF-κB p65 was decreased by silencing MUC1 (Fig. 3B). As a control, there was no detectable signal in immunoprecipitates performed with non-immune IgG (Fig. 3B). There was also no detectable NF-κB p65 occupancy of a control region (CR; -1001 to -760) of the Bcl-xL promoter upstream to the NF-κB-RE (Fig. 3B). Analysis of HeLa cells further demonstrated that expression of exogenous MUC1 is associated with increased NF-κB p65 occupancy of the Bcl-xL promoter (Fig. 3C). To determine whether MUC1-C is present in the NF-κB transcription complex, ChIP assays were performed with anti-MUC1-C. Using chromatin from ZR-75-1 cells, MUC1-C occupancy was detectable on the NF-κB-RE and not on the control region (Fig. 3D, left). In Re-ChIP assays, the anti-MUC1-C complexes were released, reimmunoprecipitated with anti-p65 and then analyzed by PCR. Anti-p65 precipitated the NF-κB-RE region after release from anti-MUC1-C (Fig. 3D, right), indicating that MUC1-C is constitutively present in the Bcl-xL promoter region occupied by the NF-κB transcription complex.
The non-malignant MCF-10A breast epithelial cells (24, 25) express endogenous MUC1, but at levels lower than that found in breast carcinoma cells (7). We found, however, that stimulation of the MCF-10A cells with TNFα is associated with a substantial upregulation of MUC1 expression (Fig. 4A). In contrast to breast cancer cells, the MCF-10A cells exhibited little if any constitutive interaction between NF-κB p65 and MUC1-C (Fig. 4B). In turn, stimulation of the MCF-10A cells with TNFα induced the interaction between NF-κB p65 and MUC1-C (Fig. 4B). NF-κB engages consensus and degenerate κB binding sequences (5′-GGGRNWYYCC-3′, where R is a purine, N is any base, W is an adenine or thymine and Y is a pyrimidine). The MUC1 promoter contains such a potential sequence for NF-κB binding (5′-GGAAAGTCC-3′; -589 to -580) (26) (Fig. 4C). ChIP analysis of TNFα-stimulated, but not unstimulated, MCF-10A cells demonstrated MUC1-C occupancy of the MUC1 promoter NF-κB binding motif (Fig. 4C). Re-ChIP analysis further demonstrated that NF-κB p65 and MUC1-C occupy the same region of the MUC1 promoter (Fig. 4D). These findings indicate that, in contrast to breast cancer cells, the interaction between NF-κB p65 and MUC1—C and their occupancy of the NF-κB binding motif in the MUC1 promoter is inducible in MCF-10A cells.
To determine whether MUC1 affects activation of NF-κB-mediated transcription, we silenced NF-κB p65 in control and TNFα-stimulated MCF-10A cells (Fig. 5A). Silencing NF-κB p65 attenuated TNFα-induced increases in MUC1-C expression (Fig. 5A), consistent with a potential role for NF-κB p65 in activating MUC1 gene transcription. As expected, silencing NF-κB p65 attenuated TNFα-induced activation of the NF-κB-Luc reporter (Fig. 5B, left). Significantly, TNFα-induced activation of the MUC1 promoter-Luc (pMUC1-Luc) was also attenuated by silencing NF-κB p65 (Fig. 5B, right). To assess the effects of MUC1-C, we silenced MUC1 expression in the MCF-10A cells with a MUC1siRNA (Fig. 5C). Consistent with the effects of MUC1 on NF-κB p65 occupancy of the NF-κB-RE, silencing MUC1 attenuated TNFα-induced activation of the NF-κB-Luc reporter (Fig. 5D, left). Moreover, silencing MUC1 attenuated activation of the pMUC1-Luc reporter (Fig. 5D, right). These findings indicate that MUC1 promotes NF-κB p65-mediated transcriptional activation of the MUC1 promoter.
To further define the role of MUC1 in NF-κB p65 function, we synthesized a peptide (GO-201) corresponding to MUC1-CD(1-15) which blocks oligomerization and thereby function of the MUC1-C cytoplasmic domain (9, 14). In addition, a control peptide, designated CP-1, was synthesized in which the CQC motif was mutated to AQA (Fig. 6A). A poly D-arginine transduction domain was included in the synthesis to facilitate entry of the peptides into cells (27) (Fig. 6A). GO-201 blocked the interaction between MUC1-CD and NF-κB p65 in vitro, indicating that MUC1-CD oligomerization is necessary for forming complexes with p65 (Fig. 6A, left). By contrast, CP-1 had little if any effect on this interaction (Fig. 6A, left). Treatment of MCF-10A cells with GO-201, but not CP-1, peptide also blocked the TNFα-induced interaction between MUC1-C and NF-κB p65 (Fig. 6A, right). ChIP analysis of the MUC1 promoter further showed that treatment with GO-201 decreased TNFα-induced MUC1-C and NF-κB p65 occupancy of the NF-κB binding motif (Fig. 6B). In concert with these results, treatment with GO-201 decreased TNFα-induced MUC1 expression (Fig. 6C). GO-201 also attenuated TNFα-induced Bcl-xL expression (Fig. 6C). These findings indicate that disruption of MUC1-C function with GO-201 attenuates (i) nuclear targeting of MUC1-C and (ii) NF-κB p65-mediated activation of MUC1 and Bcl-xL expression.
The present work demonstrates that the MUC1-C subunit associates with NF-κB p65 in cells and that the MUC1-C cytoplasmic domain binds directly to p65. More detailed binding studies showed that MUC1-CD(46-72) forms complexes with p65(1-306), but not p65(354-551), indicating that MUC1-CD interacts with the p65 RHD. This observation was confirmed with binding of MUC1-CD to p65(1-180) and p65(186-306). Studies using MUC1-CD(46-72)-derived peptides and a MUC1-CD(mSRM) mutant further identified the GGSSLSY sequence as responsible for conferring the interaction. Structural analysis of NF-κB and IκBα cocrystals has demonstrated that IκBα ankyrin repeats interact with amino acid residues just preceding the NLS that resides at the C-terminus of the NF-κB p65 RHD (19, 20). Binding of IκBα to this region of the NF-κB p65 RHD sterically masks the NLS (amino acids 287-300) and thereby targeting of NF-κB p65 to the nucleus. The finding that, like IκBα, MUC1-CD binds to p65(186-306) invoked the possibility that the MUC1-C subunit may interfere with the interaction between IκBα and NF-κB p65. Indeed, studies in cells with gain and loss of MUC1 expression indicated that MUC1 competes with IκBα for binding to NF-κB p65 and that MUC1-CD is sufficient for such competition. In concert with these results, silencing endogenous MUC1 in ZR-75-1 cells is associated with targeting of nuclear NF-κB p65 to the cytoplasm. Moreover, direct binding studies with purified proteins confirmed that MUC1-CD blocks the interaction between NF-κB p65 and IκBα. Whether MUC1-CD masks the NLS is not known at this time and, like studies with IκBα (19, 20), cocrystals of MUC1-CD and the NF-κB RHD may be necessary to address this issue. NF-κB p65 interacts with multiple proteins that affect DNA binding and transcription (28). However, to our knowledge, there are no reports of proteins that interact with the NF-κB p65 RHD and interfere with binding of IκBα. Thus, based on these findings, the overexpression of MUC1-C in human malignancies could subvert the cytoplasmic retention of NF-κB p65 by competitively blocking the NF-κB p65-IκBα interaction.
Nuclear NF-κB activates IκBα expression in a negative feed back loop that promotes the formation of new NF-κB-IκBα complexes and shuttling of NF-κB back to the cytoplasm (1). In this context, the association of MUC1-C with NF-κB p65 could attenuate downregulation of NF-κB signaling by blocking the interaction with IκBα. The present results provide support for a model in which binding of MUC1-C to NF-κB p65 results in targeting of NF-κB p65 to the promoters of NF-κB target genes (Fig. 6D). Stimulation of MCF-10A epithelial cells with TNFα was associated with binding of MUC1-C to NF-κB p65 and occupancy of these complexes on the NF-κB-RE in the Bcl-xL gene promoter. In ZR-75-1 cells, NF-κB p65 occupancy of the Bcl-xL NF-κB-RE was detectable constitutively and decreased by silencing MUC1. In concert with the findings obtained for the Bcl-xL NF-κB-RE, occupancy of the MUC1 NF-κB binding motif by NF-κB p65 and MUC1-C was constitutively detectable in ZR-75-1 breast cancer cells and inducible in MCF-10A epithelial cells. These findings and the demonstration that, like NF-κB p65, silencing of MUC1 attenuates activation of the NF-κB-Luc and pMUC1-Luc reporters indicate that MUC1-C is of importance to activation of the NF-κB p65 transcriptional function. Previous work has shown that downregulation of NF-κB signaling is delayed in the absence of IκBα (29, 30) and thus overexpression of MUC1 in human tumors could confer similar effects by inhibiting the NF-κB p65-IκBα interaction. Further studies will be needed to determine whether the MUC1-C-p65 complexes that occupy gene promoters are formed in the nucleus or whether these complexes are transported from the cytoplasm and, if so, by what import mechanism.
Previous work first demonstrated that the MUC1-CD contains a CQC motif that mediates the formation of cell surface heterodimeric complexes (31). The MUC1-C subunit also forms oligomers by a mechanism dependent on a CQC motif (9). MUC1-C oligomerization is necessary for its interaction with importin β and targeting to the nucleus (9). The GO-201 inhibitor, derived from the MUC1 cytoplasmic domain that includes the CQC motif, blocks oligomerization of MUC1-CD in vitro and of MUC1-C in cells (14). GO-201 also blocks nuclear localization of MUC1-C and induces death of human breast cancer cells (14). The present results show that GO-201 blocks the direct binding of MUC1-CD and NF-κB p65 in vitro, indicating that MUC1-CD oligomerization is, at least in part, necessary for the interaction. The TNFα-induced association of NF-κB p65 and MUC1-C in MCF-10A cells was also blocked by treatment with GO-201. The specificity of GO-201 is further supported by the lack of an effect of the CP-1 control on the interaction between MUC1-CD and NF-κB p65 in vitro and in cells. These findings and those demonstrating that the GGSSLSY motif mediates the interaction with p65 lend support to a potential model in which MUC1-CD oligomerization at the CQC sequence is associated with conformational changes in the cytoplasmic domain that are in turn necessary for direct binding of p65 at the downstream GGSSLSY region. Blocking the NF-κB p65-MUC1-C interaction with GO-201 was also associated with a decrease in occupancy of NF-κB p65 on the NF-κB binding motif in the MUC1 promoter and a decrease in MUC1 expression. GO-201 also decreased Bcl-xL expression. These findings thus provide support for the potential importance of the NF-κB p65-MUC1-C interaction in targeting of NF-κB p65 to the promoters of NF-κB target genes.
TNFα stimulation of TNF receptor 1 induces the formation of cell membrane complexes that lead to the activation of (i) NF-κB and survival or, alternatively, (ii) caspase-8 and apoptosis (32, 33). The overexpression of MUC1, as found in human breast carcinomas (34), blocks activation of caspase-8 and apoptosis in the response to TNFα and other death receptor ligands (18). In MCF-10A cells, MUC1-C interacts with caspase-8 and FADD as an induced response to death receptor stimulation and blocks recruitment of caspase-8 to the death receptor complex (18). Other work has demonstrated that MUC1-C associates with and activates the IKK complex (7) (Fig. 6D). MUC1-CD(1-45) interacts directly with IKKβ (7) and the present work demonstrates that the GGSSLSY region in MUC1-CD(46-72) binds to p65. Therefore, it is possible that MUC1-C may form complexes that include both IKKβ and p65. Previous work demonstrated that enforced expression of MUC1 in COS cells is associated with increases in NF-κB activation (35). Moreover, mutation of the 7 tyrosines in the MUC1 cytoplasmic domain attenuated NF-κB activity, indicating that MUC1-CD is responsible for this effect (35). As shown in the present work, TNFα-induced upregulation of MUC1-C expression in MCF-10A cells directly contributes to the activation of NF-κB p65. Thus, MUC1-C can activate the NF-κB pathway through direct interactions with both IKKs and p65, and thereby promote a survival response (Fig. 6D). In addition, the upregulation of MUC1-C protects against the induction of apoptosis by blocking caspase-8 activation. The present findings also indicate that through binding to NF-κB p65, MUC1-C can contribute to activation of the MUC1 gene in an auto-inductive loop and, as a result, prolong survival, albeit in a reversible manner. In this regard, MUC1 may play a physiologic role in transiently dictating cell fate in the inducible response to death receptor stimulation. Conversely, irreversible activation of MUC1 expression in carcinoma cells through a MUC1-C-NF-κB p65 regulatory loop could confer a phenotype that is stably resistant to cell death through persistent activation of NF-κB p65 and inhibition of caspase-8. Irreversible activation of a MUC1-C-NF-κB p65 loop and the upregulation of prosurvival NF-κB target genes could also contribute to the MUC1-induced block in the apoptotic response of human carcinoma cells to genotoxic, oxidative and hypoxic stress (15, 17, 36-38). Thus, a physiologic mechanism designed to protect epithelial cells during death receptor stimulation may have been exploited by human carcinomas for survival under adverse conditions.
This work was supported by Grants CA42802, CA97098 and CA100707 awarded by the National Cancer Institute. Drs. Raina and Kharbanda are employees and shareholders of Genus Oncology. Dr. Kufe is a founder of Genus Oncology and a consultant to the company.