Oncogene (2013), 1–7 & 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13 www.nature.com/onc
ORIGINAL ARTICLE
PPARg E3 ubiquitin ligase regulates MUC1-C oncoprotein stability Y Hou, J Gao, H Xu, Y Xu, Z Zhang, Q Xu and C Zhang MUC1-C oncoprotein is associated with colon, breast, ovarian, lung and pancreatic cancers. MUC1-C interacts with intracellular proteins to elicit signaling cascades that induce cell proliferation and tumor growth. Here we report that peroxisome proliferatoractivated receptor gamma (PPARg), an E3 ubiquitin ligase, is an inhibitor of MUC1-C-mediated cell proliferation. PPARg does so by binding to and inducing MUC1-C proteasome-dependent degradation that was independent of PPARg transcriptional activity. Lys134 residue was found to be critically important for PPARg-mediated MUC1-C degradation, as it terminated MUC1-C-mediated cell proliferation. These findings demonstrate PPARg induces MUC1-C ubiquitination and degradation that is critical to terminate MUC1-C signaling pathway-elicited cancer. Oncogene advance online publication, 2 December 2013; doi:10.1038/onc.2013.504 Keywords: PPARg; MUC1-C; ubiquitin; degradation; ubiquitination; cancer
INTRODUCTION MUC1 is expressed on the aptical surface of epithelial cells, such as the mammary gland, gastrointestinal, respiratory, urinary and reproductive tracts. Although MUC1 has an important role in protection and lubrication of epithelial surfaces, however, aberrant expression of MUC1 is often associated with colon, breast, ovarian, lung and pancreatic cancers.1 MUC1 N-terminal subunit is shed from the cell surface, leaving the MUC1 C-terminal subunit (MUC1-C, cytoplasmic tail) to elicit signaling cascades.2,3 ZAP-70 (zeta-chainassociated protein kinase 70), PKCd (protein kinase C d), GSK-3b (glycogen synthase kinase 3 beta), c-Src (c-Src tyrosine kinase) or Lck (lymphocyte-specific protein tyrosine kinase) can induce MUC1-C phosphorylation to trigger downstream pathway.3 As oncoprotein, MUC1-C interacts with IKK (the IkB kinase), NFkB/p65, Stat3 (Signal transducer and activator of transcription 3), p53 or BAX4–9 to activate downstream pathway associated with tumor growth. MUC1-C interacts with several cellular proteins to regulate tumorigenesis,1–9 but the intracellular inhibitor of MUC1-C is still unclear. Peroxisome proliferator-activated receptor gamma (PPARg) is a critical regulator of inflammation, adipocyte differentiation, glucose homeostasis and tumorigenesis,10,11 it has been implicated in the pathology of numerous diseases including cancer12,13 and can repress pro-inflammatory genes via transrepression and transcriptional squelching.14,15 Our previous report shows that PPARg E3 ubiquitin ligase induces NFkB/p65 degradation, leading to termination of NFkB activation.16 Other reports show that PPARg increases Muc1 gene expression in murine trophoblasts,17 but it has no effect on human MUC1 gene expression.18 However, it is unclear that the interaction of PPARg with MUC1-C, here we found that PPARg induced MUC1-C degradation, which terminated MUC1-C-elicited pathway. RESULTS PPARg interacts with MUC1-C To determine the interaction of PPARg with MUC1-C, the immunoprecipitation analysis was performed by using HT29 cell
lysates. Our results show that PPARg was directly bound to MUC1C (Figure 1a), which was further demonstrated by in vitro binding analysis (Figure 1b). The interaction of PPARg/MUC1-C complex was confirmed by Ni-NTA pull-down assay that showed PPARg was bound to MUC1-C (Figure 1c). Further analysis shows PPARg was bound to MUC1-C in the cytoplasm and nucleus (Figure 1d). To determine the specific region(s) of PPARg interacts with MUC1C, we generated mutants of PPARg. The results show that the 401– 505 fragment of PPARg was required for binding to MUC1-C (Figure 1e, Supplementary Figure 1).
PPARg induces MUC1-C proteasome-dependent degradation Figure 2a shows that MUC1-C protein undergoes degradation in the cancer cells (HT29, MCF-7 or LS 174T cells) with the expression of endogenous PPARg, because cells were treated with MG132 (proteasome inhibitor) resulting in inhibition of MUC1-C degradation. To further detect the relevance of PPARg with MUC1-C degradation, HT29 cells were transfected with PPARg shRNA (short hairpin RNA). The results show that silenced PPARg led to increased MUC1-C protein levels (Figure 2b). Similar results were observed that overexpression of PPARg decreased endogenous MUC1-C protein levels in HT29 cells (Figure 2c) and exogenous MUC1-C protein levels in HEK293T cells (Figure 2d). In contrast, cells were treated with MG132, resulting in inhibition of MUC1-C protein degradation (Figures 2e and f). Figure 2g shows that overexpression of PPARg had no effect on MUC1-C mRNA (messenger RNA) levels and PPARg-reduced cytoplasmic MUC1-C protein levels (Supplementary Figure 2), suggesting that PPARgreduced MUC1-C protein levels were not involved in its transcriptional activity. To determine the regulation of PPARg on MUC1-C protein stability under basal state, PPARg shRNA-silenced HT29 cells were treated with cycloheximide to inhibit protein synthesis and observed a rapid decrease in MUC1-C protein halflife (Figure 2h), which was consistent with pulse-chase analysis (Supplementary Figure 3a). In contrast, overexpression of PPARg significantly decreased the half-life of MUC1-C protein (Figure 2i),
Institute of Life Science, JiangSu University, Zhenjiang, China. Correspondence: Dr Y Hou, Institute of Life Science, JiangSu University, Zhenjiang, JiangSu Province 212013, China. E-mail: [email protected] Received 22 June 2013; revised 14 October 2013; accepted 18 October 2013
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Figure 1. PPARg binds to MUC1-C. (a) HT29 cell lysates were subjected to immunoprecipitation and western blot. (b) GST pull-down assay in which GST–PPARg bound to glutathione–agarose beads was incubated with recombinant MUC1-C. (c) HEK293T cells were transfected with his-MUC1-C, together with or without PPARg plasmids, as indicated for 36 h. Cell lysates were subjected to Ni-NTA pull-down and western blot. Cells were treated with MG132 (20 mM) for 6 h before cell lysis. (d) The cytoplasmic or nuclear extracts of HT29 were subjected to immunoprecipitation and western blot. (e) Left panel shows the constructs of PPARg or mutant. Right panel shows that HT29 cells were transfected with Flag-PPARg or mutant plasmids as indicated. Cell lysates were subjected to immunoprecipitation and western blot. Cells were treated with MG132 (20 mM) for 6 h before cell lysis.
which was further demonstrated by pulse-chase analysis (Supplementary Figure 3b). These findings suggest that PPARg induced MUC1-C proteasome-dependent degradation. PPARg induces MUC1-C ubiquitination HT29 cells treated with MG132 led to increased MUC1-C ubiquitination (Figure 3a) and inhibit MUC1-C degradation under basal state (Figure 2a). To detect the effect of PPARg on MUC1-C ubiquitination, HT29 cells were transfected with PPARg shRNA. The results show that silenced PPARg significantly decreased MUC1-C ubiquitination (Figure 3b). In contrast, overexpression of PPARg significantly induced MUC1-C ubiquitination (Figure 3c), which was further demonstrated by in vitro ubiquitination analysis (Figure 3d). As C139 is the activity site of PPARg E3 ubiquitin ligase,16 PPARg/C139A mutant did not induce MUC1-C ubiquitination (Figures 3d and e) and degradation (Supplementary Figure 4). To determine whether PPARg-induced MUC1-C degradation was independent of its transcriptional activity, the nuclear location signal (NLS; 184–189 amino acids) of PPARg was deleted.16 Our data show that PPARg-DNLS was still able to induce MUC1-C ubiquitination (Figure 3f) and degradation (Supplementary Figure 4). These results show that PPARg-induced MUC1-C degradation was independent of its transcriptional activity. Deleted 401–505 fragment of PPARg did not induce MUC1-C ubiquitination (Supplementary Figure 5a) and degradation (Supplementary Figure 5b), which was consistent with the 401–505 fragment of PPARg binding to MUC1-C (Figure 1e, Supplementary Figure 1). Oncogene (2013) 1 – 7
We have demonstrated that PPARg induced MUC1-C ubiquitination, although it is still unclear whether the polyubiquitin targets the lysine site of MUC1-C; to detect this, lysine residues of MUC1-C (Figure 4a) were replaced with arginine, and the mutant plasmids were co-transfected with PPARg into HEK293T cells. Western blot analysis showed that Lys134R of MUC1-C significantly inhibited PPARg-mediated MUC1-C ubiquitination (Figure 4b) and degradation (Figures 4c and d), suggesting that lys134 of MUC1-C was critical for PPARg-mediated MUC1-C ubiquitination and degradation. Liganded PPARg leads to increased MUC1-C degradation PPARg is activated by its ligands to enhance its E3 ligase activity;16 therefore, we next determine whether the activation of PPARg by its ligands would affect MUC-C protein levels. Figure 5a shows that HT29 cells treated with troglitazone (TROG) or pioglitazone (PIOG) led to decreased MUC1-C protein levels. To further analyze whether liganded PPARg-reduced MUC1-C protein levels were independent of its transcriptional activity, HEK293T cells were transfected with his-MUC1-C with PPARg or its mutant plasmids. Western blot shows that TROG/PPARg/DNLS, not C139A, reduced MUC1-C protein levels (Supplementary Figures 6a and b), suggesting that TROG/PPARg-reduced MUC1-C protein levels were independent of PPARg transcriptional activity. To further detect whether TROG could enhance the interaction of PPARg with MUC1-C, immunoprecipitation analysis was performed. Our results show that TROG significantly increased the binding of PPARg to MUC1-C (Figure 5b) and MUC1-C ubiquitination & 2013 Macmillan Publishers Limited
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Figure 2. PPARg induces MUC1-C degradation. (a) HT29, MCF-7 and LS 174T cells were treated with or without MG132 (20 mM) for 6 h. Cell lysates were subjected to western blot. (b) HT29 or MCF-7 cells were transfected with control shRNA or PPARg shRNA for 48 h. Cell lysates were subjected to western blot. (c) MCF-7 cells were transfected with vector or Flag-PPARg for 36 h. Cell lysates were subjected to western blot. (d) HEK293T cells were transfected with vector, his-MUC1-C or his-MUC1-C together with Flag-PPARg for 36 h. Cell lysates were subjected to western blot. MCF-7 cells (e) or HEK293T cells (f ) were transfected with plasmids as indicated for 36 h. Cells were treated with or without MG132 (20 mM) for 6 h before cell lysis. Cell lysates were subjected to western blot. (g) HT29 cells were transfected pcDNA3 (vector) or PPARg plasmids as indicated. MUC1-C gene expression was assayed by reverse transcriptase–PCR (RT–PCR). (h) Control or PPARg-silenced HT29 cells were treated with cycloheximide (CHX, 30 mg/ml) for 0, 6 and 12 h to inhibit de novo protein synthesis and harvested MUC1-C for western blot. The percent MUC1-C protein remaining at each time point was calculated accordingly. (i) HEK293T cells were transfected with his-MUC1 with Flag-PPARg or pcDNA3 (vector). After 36 h, cells were treated with CHX (30 mg/ml) for 0, 4 and 8 h to inhibit de novo protein synthesis and harvested for western blot. The percent MUC1-C protein remaining at each time point was calculated accordingly.
(Figure 5c), suggesting that activation of PPARg increased its E3 ubiquitin ligase activity, leading to TROG-induced MUC1-C protein degradation (Figure 5d). PPARg inhibits MUC1-C-mediated cell proliferation MUC1-C oncoprotein induces anchorage-independent growth.19–21 Our results have demonstrated that PPARg induced MUC1-C proteasome-dependent degradation, we next determine whether PPARg could terminate MUC1-C-mediated cell proliferation. Silenced PPARg led to increased cell proliferation (Figure 6a) and colony formation by using soft agar analysis (Figure 6b). As TROG reduced MUC1-C protein levels, it is not surprising & 2013 Macmillan Publishers Limited
that liganded PPARg decreased cell proliferation (Supplementary Figure 7). Our data show that K134 of MUC1-C was a critical lysine residue for PPARg-mediated MUC1-C degradation (Figure 4), further analysis shows that PPARg did not inhibit MUC1-C/K134R-mediated cell proliferation (Figure 6c), colony formation (Figure 6d). Taken together, these results suggest that PPARg terminated MUC1-C-mediated cell proliferation by inducing its degradation. DISCUSSION Lots of studies show that aberrant MUC1 expression is thought to be associated with colon, breast, ovarian, lung and pancreatic Oncogene (2013) 1 – 7
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Figure 3. PPARg induces MUC1-C ubiquitination. (a) HT29 cells were treated with or without MG132 (20 mM) for 6 h. Cell lysates were subjected to denatured immunoprecipitation and western blot. (b) HT29 cells were transfected with control shRNA or PPARg shRNA for 36 h. Cell lysates were subjected to denatured immunoprecipitation and western blot. Cells were treated with MG132 (20 mM) for 6 h before cell lysis. (c) HEK293T cells were transfected with his-MUC1-C plasmids together with or without PPARg for 36 h. Cells were treated with or without MG132 (20 mM) for 6 h before cell lysis. Cell lysates were subjected to denatured Ni-NTA pull-down and western blot. (d) In vitro ubiquitination of MUC1-C analysis was performed (see experimental procedures) in the reaction buffer containing UBCH3, MUC1-C (20 mg) and 10 ng PPARg (WT or C139A), as indicated. Reactions were incubated at 30 1C for 2 h, resolved by SDS–PAGE, and the ubiquitinated products were detected with MUC1-C antibody. (e) HEK293T cells were transfected with plasmids as indicated. Cell lysates were subjected to denatured Ni-NTA pulldown and western blot. Cells were treated with MG132 (20 mM) for 6 h before cell lysis. (f ) HEK293T cells were transfected with his-MUC1-C together with PPARg or its mutant plasmids, as indicated. Cell lysates were subjected to denatured Ni-NTA pull-down and western blot. Cells were treated with MG132 (20 mM) for 6 h before cell lysis.
cancers,1 which is involved in the MUC1 C-terminal subunit (MUC1-C, cytoplasmic tail) as a putative receptor to trigger intracellular signaling pathway.2,3 Although the GO-203, an inhibitor of MUC1-C, can terminate part of the MUC1-C signal in breast cancer cells,22 the cellular physical inhibitor and mechanism of MUC1-C pathway are still unclear. Here we first found that MUC1-C undergoes proteasome-dependent degradation, because the proteasome inhibitor (MG132) treatment led to increased MUC1-C protein levels in cancer cells, which was consistent with MG132 treatment resulting in MUC1-C ubiquitination. These interesting findings suggest that MUC1-C protein can be degraded by proteasome-dependent signal. Many RING finger domains simultaneously bind ubiquitination enzymes and their substrates, and hence function as E3 ubiquitin ligases.23,24 Ubiquitination in turn targets the substrate protein for degradation.16,23–25 Although we have demonstrated that PPARg induced MUC1-C ubiquitination, however, it is still unclear what kind of E3 ubiquitin ligase targets MUC1-C and induces it degradation. Western blot shows that PPARg is expressed in HT29, LS 174T and MCF-7 cells, which is the new identified E3 ubiquitin ligase;16 therefore, PPARg could be associated with MUC1-C proteasome-dependent degradation. As expected, silenced PPARg significantly increased MUC1-C protein levels as well as reduced MUC1-C ubiquitination. In contrast, overexpression of PPARg led to decreased MUC1-C protein levels and MUC1-C ubiquitination. Further analysis shows that PPARg interacted with MUC1-C to trigger its ubiquitination and degradation, but previous report shows that PPARg agonist TROG reduces IL-8 expression (a pro-inflammatory cytokine) by increasing MUC1 gene expression in AGS cells.26,27 However, Oncogene (2013) 1 – 7
MUC1 N-terminal subunit is shed from the cell surface3 in response to luminal signals, leaving the MUC1 C-terminal subunit (MUC1-C, cytoplasmic tail) as a putative receptor to trigger intracellular signal.2,3 In addition, MUC1-C increases NFkB activation by interaction with IKK or NFkB/p65, and NFkB activation leads to increased pro-inflammatory gene expression (IL-8 and so on),16,28 therefore it is unclear that the mechanism of MUC1 inhibits pro-inflammatory response,26,27 maybe this phenomenon occurs only in AGS cells. Other studies show that PPARg induces Muc1 gene expression in murine trophoblasts,17 but that it has no effect on the MUC1 gene expression in human uterine epithelial cell lines (HES and HEC-1A) and T47D human breast cancer cells,18 suggesting that PPARg regulates the murine Muc1 and human MUC1 promoters in opposite manners, which is possible considering the differential responsiveness of the mouse and human promoter regions;18 therefore, it is not surprising that overexpression of PPARg had no effect on human MUC1-C gene levels (Figure 2g). Although PPARg is a transcriptional factor, it is bound to cytoplasmic MUC1-C, and deletion of the nuclear signal location of PPARg still induced MUC1-C degradation, suggesting that PPARginduced MUC1-C degradation was independent of its transcriptional activity. PPARg has an essential role in several diseases, and thus it is not surprising that its expression is low in colon cancer patients.29 Similarly, PPARg agonists inhibit carcinogen-induced colon tumor growth, suggesting that PPARg can function as a tumor suppressor.30,31 Some important classes of synthetic agonists of PPARg (TROG, PIOG) can activate PPARg by binding its ligand-binding domain;32,33 here we found that PPARg agonists such as TROG and PIOG significantly increased the binding of & 2013 Macmillan Publishers Limited
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Figure 4. K134 of MUC1-C is required for PPARg-mediated MUC1-C degradation. (a) Construct of MUC1-C and lysine sites. (b) HEK293T cells were co-transfected with F-PPARg plasmids together with MUC1-C or mutants. Cell lysates were subjected to denatured Ni-NTA pull-down and western blot. Cells were treated with MG132 (20 mM) for 6 h before cell lysis. (c) HEK293T cells were co-transfected with F-PPARg plasmids together with MUC1-C or mutants for 36 h. Cell lysates were subjected to western blot. (d) HEK293T cells were transfected with his-MUC1-C or K134R. After 36 h, cells were treated with cycloheximide (30 mg/ml) for 0, 10 and 16 h to inhibit de novo protein synthesis and harvested for western blot. The percent MUC1-C protein remaining at each time point was calculated accordingly.
Figure 5. Liganded PPARg enhances its activity. (a) HT29 cells were treated with DMSO, PIOG and TROG (100 mM) for 1 h. Cell lysates were subjected to western blot. (b) HT29 cells were treated with PIOG or TROG (100 mM) for 10 min. Cell lysates were subjected to immunoprecipitation and western blot. (c) HT29 cells were treated with or without TROG (100 mM) for 15 min. Cell lysates were subjected to denatured immunoprecipitation and western blot. (d) HT29 cells were treated with TROG (100 mM) for 1 h. Cells were treated with cycloheximide (30 mg/ml) for 0, 4 and 6 h to inhibit de novo protein synthesis and harvested for western blot. The percent MUC1-C protein remaining at each time point was calculated accordingly.
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6 Western blot and immunoprecipitation Subcellular fractionation and immunoprecipitation were performed as described previously.16,28 For denatured immunoprecipitation, cell extracts were heated at 95 1C for 5 min in the presence of 1% SDS to disrupt the non-covalent protein interactions. The SDS was diluted and the proteins were subjected to immunoprecipitation and western blot. The samples were subjected to 10–20% gradient SDS–PAGE, transferred to a nitrocellulose membrane, then probed by western blot analysis with the indicated antibody and developed by using an ECL Kit.
Ni-NTA purification and pull-down For native Ni-NTA purification or pull-down, cells were washed with cold PBS and lysed in lysis buffer (50 mM NaH2PO4, 300 mM NaCl containing protease inhibitors (PMSF, Aprotinin, Leupeptin, E46), pH 8.0), sonicated and spun down. The supernatant was loaded onto a Ni-NTA-agarose column (QIAGEN, Pudong, Shanghai, China) or pull-down, sequentially washed with 30 bed volumes (V) of buffer A (10 mM Tris-HCl, pH 8.0, 50 mM NaH2PO4, 10 mM imidazole) twice, 30V of buffer B (10 mM Tris-HCl, pH 6.3, 50 mM NaH2PO4, 10 mM imidazole) twice and eluted with 250 mM imidazole, pH 8.0. For denaturing Ni-NTA pull-down, cells were washed with cold PBS and lysed in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 8 M Urea, PH 8.0), and spun down and the supernatant was subjected to Ni-NTA pull-down. Sequentially beads were washed with buffer A twice and then buffer B twice, and finally eluted with 250 mM imidazole, pH 8.0.
Figure 6. PPARg terminates MUC1-C-mediated cell proliferation. (a) Cell proliferation was analyzed for the stable knockdown of PPARg or control shRNA in HT29 cells (means±s.e.m., n ¼ 4). (b) Soft agar analysis of the stable knockdown of PPARg or control shRNA in HT29 cells, and the colonies enumerated per well (means±s.e.m., n ¼ 4). (c) Cell proliferation was analyzed for the stably expressing pcDNA3 (vector), MUC1-C, MUC1-C þ PPARg or K134R þ PPARg plasmids cells (means±s.e.m., n ¼ 4). (d) Soft agar analysis of the stably expressing different plasmid cells, and the colonies enumerated per well (means±s.e.m., n ¼ 4).
PPARg to MUC1-C, resulting in MUC1-C degradation and inhibition of cell proliferation. Taken together, PPARg induced MUC1-C ubiquitination and degradation, leading to termination of MUC1C-mediated cell proliferation.
MATERIALS AND METHODS Cell lines, plasmids and antibodies The human colonic adenocarcinoma cells such as HT29, MCF-7, LS 174T and HEK293T were obtained from the ATCC (Manassas, VA, USA). hisMUC1-C cDNA (complimentary DNA) was cloned into pCMV or Flag-PPARg cDNA was cloned into pcDNA3 vector, respectively. Plasmids were mutated by the site-directed mutagenesis method, and all the plasmids were identified by DNA sequencing. PPARg shRNA and MUC1-C monoclonal antibody (MH1 (CT2), corresponding to SSLSYTNPAVAATSANL from the cytoplasmic tail of MUC1) were obtained from Thermo Fisher Scientific, Waltham, MA, USA. Plasmids were transfected by LipfectAMINE2000 according to the manufacturer’s instructions (Invitrogen, Changning, Shanghai, China). Monoclonal PPARg, Ubiquitin (P4D1) antibodies, polyclonal his and Flag antibodies were obtained from Santa Cruz, Santa Cruz, CA, USA. Secondary antibodies were obtained from Jackson Immunoresearch (West Grove, PA, USA).
Metabolic labeling Cells were placed for 30 min in cysteine- and methionine-free medium. At the end of this period, 35S-radiolabeled methionine and cysteine were added to the medium for 1 h, and then replaced with regular medium supplemented with excess unlabeled methionine (2 mM) and cysteine (2 mM). Whole-cell lysates were then prepared at the indicated time points, and the samples were subjected to immunoprecipitation with MUC1-C antibody. The recovered material at the end of the immunoprecipitation was resolved by SDS–PAGE, and MUC1-C was detected by autoradiography. Oncogene (2013) 1 – 7
In vitro ubiquitination assay PPARg or MUC1-C cDNA was subcloned into PGEX-6P-1 vector. GST–PPARg or GST–MUC1-C was expressed in Escherichia coli strain BL21. The recombinant protein was purified by glutathione-conjugated sepharose beads (QIAGEN). MUC1-C ubiquitination assay contained 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, ATP-regenerating system, 0.2 mM dithiothreitol, 50 mM ZnCl2, 0.1 mM E1 (Boston Biochem, Cambridge, MA, USA), 0.4 mM UBCH3, 5 mg Ub, 10 mg MUC1-C and 10 ng PPARg (WT or C139A mutant). Reactions were incubated at 30 1C for 2 h, the ubiquitinated products were detected by western blot with MUC1-C antibody.
In vitro binding analysis GST–PPARg fusion protein was immobilized on glutathione–agarose beads in buffer (25 mM HEPES (pH 7.5), 6 mM NaCl and 0.2% NP-40) for 30 min at 4 1C, and then in vitro translated proteins (MUC1-C) were added and incubated for another 2 h. Adsorbates to glutathione-conjugated beads were analyzed by western blot.
Soft agar assay A total of 1 105 cells were suspended in 0.3% Noble agar in 10% FBS with DMEM and antibiotics, and was layered over 0.5% agar in 60-mm dishes and cultured for two weeks at 37 1C under 5% CO2. Dishes were stained with 0.05% crystal violet for 2 h at room temperature and colonies were counted in the entire dish.
Statistical analysis Data are expressed as the mean±s.e.m. Statistical comparison was carried out with one-way analysis of variance (ANOVA) and Dunnett’s test. Significance was defined as Po0.05.
CONFLICT OF INTEREST The authors declare no conflict of interest
ACKNOWLEDGEMENTS This work was supported by Jiangsu Province Natural Science Foundation (SBK201320232) and Start Fund of University of JiangSu (12JDG071).
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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)
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ORIGINAL ARTICLE
PPARg E3 ubiquitin ligase regulates MUC1-C oncoprotein stability Y Hou, J Gao, H Xu, Y Xu, Z Zhang, Q Xu and C Zhang MUC1-C oncoprotein is associated with colon, breast, ovarian, lung and pancreatic cancers. MUC1-C interacts with intracellular proteins to elicit signaling cascades that induce cell proliferation and tumor growth. Here we report that peroxisome proliferatoractivated receptor gamma (PPARg), an E3 ubiquitin ligase, is an inhibitor of MUC1-C-mediated cell proliferation. PPARg does so by binding to and inducing MUC1-C proteasome-dependent degradation that was independent of PPARg transcriptional activity. Lys134 residue was found to be critically important for PPARg-mediated MUC1-C degradation, as it terminated MUC1-C-mediated cell proliferation. These findings demonstrate PPARg induces MUC1-C ubiquitination and degradation that is critical to terminate MUC1-C signaling pathway-elicited cancer. Oncogene advance online publication, 2 December 2013; doi:10.1038/onc.2013.504 Keywords: PPARg; MUC1-C; ubiquitin; degradation; ubiquitination; cancer
INTRODUCTION MUC1 is expressed on the aptical surface of epithelial cells, such as the mammary gland, gastrointestinal, respiratory, urinary and reproductive tracts. Although MUC1 has an important role in protection and lubrication of epithelial surfaces, however, aberrant expression of MUC1 is often associated with colon, breast, ovarian, lung and pancreatic cancers.1 MUC1 N-terminal subunit is shed from the cell surface, leaving the MUC1 C-terminal subunit (MUC1-C, cytoplasmic tail) to elicit signaling cascades.2,3 ZAP-70 (zeta-chainassociated protein kinase 70), PKCd (protein kinase C d), GSK-3b (glycogen synthase kinase 3 beta), c-Src (c-Src tyrosine kinase) or Lck (lymphocyte-specific protein tyrosine kinase) can induce MUC1-C phosphorylation to trigger downstream pathway.3 As oncoprotein, MUC1-C interacts with IKK (the IkB kinase), NFkB/p65, Stat3 (Signal transducer and activator of transcription 3), p53 or BAX4–9 to activate downstream pathway associated with tumor growth. MUC1-C interacts with several cellular proteins to regulate tumorigenesis,1–9 but the intracellular inhibitor of MUC1-C is still unclear. Peroxisome proliferator-activated receptor gamma (PPARg) is a critical regulator of inflammation, adipocyte differentiation, glucose homeostasis and tumorigenesis,10,11 it has been implicated in the pathology of numerous diseases including cancer12,13 and can repress pro-inflammatory genes via transrepression and transcriptional squelching.14,15 Our previous report shows that PPARg E3 ubiquitin ligase induces NFkB/p65 degradation, leading to termination of NFkB activation.16 Other reports show that PPARg increases Muc1 gene expression in murine trophoblasts,17 but it has no effect on human MUC1 gene expression.18 However, it is unclear that the interaction of PPARg with MUC1-C, here we found that PPARg induced MUC1-C degradation, which terminated MUC1-C-elicited pathway. RESULTS PPARg interacts with MUC1-C To determine the interaction of PPARg with MUC1-C, the immunoprecipitation analysis was performed by using HT29 cell
lysates. Our results show that PPARg was directly bound to MUC1C (Figure 1a), which was further demonstrated by in vitro binding analysis (Figure 1b). The interaction of PPARg/MUC1-C complex was confirmed by Ni-NTA pull-down assay that showed PPARg was bound to MUC1-C (Figure 1c). Further analysis shows PPARg was bound to MUC1-C in the cytoplasm and nucleus (Figure 1d). To determine the specific region(s) of PPARg interacts with MUC1C, we generated mutants of PPARg. The results show that the 401– 505 fragment of PPARg was required for binding to MUC1-C (Figure 1e, Supplementary Figure 1).
PPARg induces MUC1-C proteasome-dependent degradation Figure 2a shows that MUC1-C protein undergoes degradation in the cancer cells (HT29, MCF-7 or LS 174T cells) with the expression of endogenous PPARg, because cells were treated with MG132 (proteasome inhibitor) resulting in inhibition of MUC1-C degradation. To further detect the relevance of PPARg with MUC1-C degradation, HT29 cells were transfected with PPARg shRNA (short hairpin RNA). The results show that silenced PPARg led to increased MUC1-C protein levels (Figure 2b). Similar results were observed that overexpression of PPARg decreased endogenous MUC1-C protein levels in HT29 cells (Figure 2c) and exogenous MUC1-C protein levels in HEK293T cells (Figure 2d). In contrast, cells were treated with MG132, resulting in inhibition of MUC1-C protein degradation (Figures 2e and f). Figure 2g shows that overexpression of PPARg had no effect on MUC1-C mRNA (messenger RNA) levels and PPARg-reduced cytoplasmic MUC1-C protein levels (Supplementary Figure 2), suggesting that PPARgreduced MUC1-C protein levels were not involved in its transcriptional activity. To determine the regulation of PPARg on MUC1-C protein stability under basal state, PPARg shRNA-silenced HT29 cells were treated with cycloheximide to inhibit protein synthesis and observed a rapid decrease in MUC1-C protein halflife (Figure 2h), which was consistent with pulse-chase analysis (Supplementary Figure 3a). In contrast, overexpression of PPARg significantly decreased the half-life of MUC1-C protein (Figure 2i),
Institute of Life Science, JiangSu University, Zhenjiang, China. Correspondence: Dr Y Hou, Institute of Life Science, JiangSu University, Zhenjiang, JiangSu Province 212013, China. E-mail: [email protected] Received 22 June 2013; revised 14 October 2013; accepted 18 October 2013
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Figure 1. PPARg binds to MUC1-C. (a) HT29 cell lysates were subjected to immunoprecipitation and western blot. (b) GST pull-down assay in which GST–PPARg bound to glutathione–agarose beads was incubated with recombinant MUC1-C. (c) HEK293T cells were transfected with his-MUC1-C, together with or without PPARg plasmids, as indicated for 36 h. Cell lysates were subjected to Ni-NTA pull-down and western blot. Cells were treated with MG132 (20 mM) for 6 h before cell lysis. (d) The cytoplasmic or nuclear extracts of HT29 were subjected to immunoprecipitation and western blot. (e) Left panel shows the constructs of PPARg or mutant. Right panel shows that HT29 cells were transfected with Flag-PPARg or mutant plasmids as indicated. Cell lysates were subjected to immunoprecipitation and western blot. Cells were treated with MG132 (20 mM) for 6 h before cell lysis.
which was further demonstrated by pulse-chase analysis (Supplementary Figure 3b). These findings suggest that PPARg induced MUC1-C proteasome-dependent degradation. PPARg induces MUC1-C ubiquitination HT29 cells treated with MG132 led to increased MUC1-C ubiquitination (Figure 3a) and inhibit MUC1-C degradation under basal state (Figure 2a). To detect the effect of PPARg on MUC1-C ubiquitination, HT29 cells were transfected with PPARg shRNA. The results show that silenced PPARg significantly decreased MUC1-C ubiquitination (Figure 3b). In contrast, overexpression of PPARg significantly induced MUC1-C ubiquitination (Figure 3c), which was further demonstrated by in vitro ubiquitination analysis (Figure 3d). As C139 is the activity site of PPARg E3 ubiquitin ligase,16 PPARg/C139A mutant did not induce MUC1-C ubiquitination (Figures 3d and e) and degradation (Supplementary Figure 4). To determine whether PPARg-induced MUC1-C degradation was independent of its transcriptional activity, the nuclear location signal (NLS; 184–189 amino acids) of PPARg was deleted.16 Our data show that PPARg-DNLS was still able to induce MUC1-C ubiquitination (Figure 3f) and degradation (Supplementary Figure 4). These results show that PPARg-induced MUC1-C degradation was independent of its transcriptional activity. Deleted 401–505 fragment of PPARg did not induce MUC1-C ubiquitination (Supplementary Figure 5a) and degradation (Supplementary Figure 5b), which was consistent with the 401–505 fragment of PPARg binding to MUC1-C (Figure 1e, Supplementary Figure 1). Oncogene (2013) 1 – 7
We have demonstrated that PPARg induced MUC1-C ubiquitination, although it is still unclear whether the polyubiquitin targets the lysine site of MUC1-C; to detect this, lysine residues of MUC1-C (Figure 4a) were replaced with arginine, and the mutant plasmids were co-transfected with PPARg into HEK293T cells. Western blot analysis showed that Lys134R of MUC1-C significantly inhibited PPARg-mediated MUC1-C ubiquitination (Figure 4b) and degradation (Figures 4c and d), suggesting that lys134 of MUC1-C was critical for PPARg-mediated MUC1-C ubiquitination and degradation. Liganded PPARg leads to increased MUC1-C degradation PPARg is activated by its ligands to enhance its E3 ligase activity;16 therefore, we next determine whether the activation of PPARg by its ligands would affect MUC-C protein levels. Figure 5a shows that HT29 cells treated with troglitazone (TROG) or pioglitazone (PIOG) led to decreased MUC1-C protein levels. To further analyze whether liganded PPARg-reduced MUC1-C protein levels were independent of its transcriptional activity, HEK293T cells were transfected with his-MUC1-C with PPARg or its mutant plasmids. Western blot shows that TROG/PPARg/DNLS, not C139A, reduced MUC1-C protein levels (Supplementary Figures 6a and b), suggesting that TROG/PPARg-reduced MUC1-C protein levels were independent of PPARg transcriptional activity. To further detect whether TROG could enhance the interaction of PPARg with MUC1-C, immunoprecipitation analysis was performed. Our results show that TROG significantly increased the binding of PPARg to MUC1-C (Figure 5b) and MUC1-C ubiquitination & 2013 Macmillan Publishers Limited
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Figure 2. PPARg induces MUC1-C degradation. (a) HT29, MCF-7 and LS 174T cells were treated with or without MG132 (20 mM) for 6 h. Cell lysates were subjected to western blot. (b) HT29 or MCF-7 cells were transfected with control shRNA or PPARg shRNA for 48 h. Cell lysates were subjected to western blot. (c) MCF-7 cells were transfected with vector or Flag-PPARg for 36 h. Cell lysates were subjected to western blot. (d) HEK293T cells were transfected with vector, his-MUC1-C or his-MUC1-C together with Flag-PPARg for 36 h. Cell lysates were subjected to western blot. MCF-7 cells (e) or HEK293T cells (f ) were transfected with plasmids as indicated for 36 h. Cells were treated with or without MG132 (20 mM) for 6 h before cell lysis. Cell lysates were subjected to western blot. (g) HT29 cells were transfected pcDNA3 (vector) or PPARg plasmids as indicated. MUC1-C gene expression was assayed by reverse transcriptase–PCR (RT–PCR). (h) Control or PPARg-silenced HT29 cells were treated with cycloheximide (CHX, 30 mg/ml) for 0, 6 and 12 h to inhibit de novo protein synthesis and harvested MUC1-C for western blot. The percent MUC1-C protein remaining at each time point was calculated accordingly. (i) HEK293T cells were transfected with his-MUC1 with Flag-PPARg or pcDNA3 (vector). After 36 h, cells were treated with CHX (30 mg/ml) for 0, 4 and 8 h to inhibit de novo protein synthesis and harvested for western blot. The percent MUC1-C protein remaining at each time point was calculated accordingly.
(Figure 5c), suggesting that activation of PPARg increased its E3 ubiquitin ligase activity, leading to TROG-induced MUC1-C protein degradation (Figure 5d). PPARg inhibits MUC1-C-mediated cell proliferation MUC1-C oncoprotein induces anchorage-independent growth.19–21 Our results have demonstrated that PPARg induced MUC1-C proteasome-dependent degradation, we next determine whether PPARg could terminate MUC1-C-mediated cell proliferation. Silenced PPARg led to increased cell proliferation (Figure 6a) and colony formation by using soft agar analysis (Figure 6b). As TROG reduced MUC1-C protein levels, it is not surprising & 2013 Macmillan Publishers Limited
that liganded PPARg decreased cell proliferation (Supplementary Figure 7). Our data show that K134 of MUC1-C was a critical lysine residue for PPARg-mediated MUC1-C degradation (Figure 4), further analysis shows that PPARg did not inhibit MUC1-C/K134R-mediated cell proliferation (Figure 6c), colony formation (Figure 6d). Taken together, these results suggest that PPARg terminated MUC1-C-mediated cell proliferation by inducing its degradation. DISCUSSION Lots of studies show that aberrant MUC1 expression is thought to be associated with colon, breast, ovarian, lung and pancreatic Oncogene (2013) 1 – 7
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Figure 3. PPARg induces MUC1-C ubiquitination. (a) HT29 cells were treated with or without MG132 (20 mM) for 6 h. Cell lysates were subjected to denatured immunoprecipitation and western blot. (b) HT29 cells were transfected with control shRNA or PPARg shRNA for 36 h. Cell lysates were subjected to denatured immunoprecipitation and western blot. Cells were treated with MG132 (20 mM) for 6 h before cell lysis. (c) HEK293T cells were transfected with his-MUC1-C plasmids together with or without PPARg for 36 h. Cells were treated with or without MG132 (20 mM) for 6 h before cell lysis. Cell lysates were subjected to denatured Ni-NTA pull-down and western blot. (d) In vitro ubiquitination of MUC1-C analysis was performed (see experimental procedures) in the reaction buffer containing UBCH3, MUC1-C (20 mg) and 10 ng PPARg (WT or C139A), as indicated. Reactions were incubated at 30 1C for 2 h, resolved by SDS–PAGE, and the ubiquitinated products were detected with MUC1-C antibody. (e) HEK293T cells were transfected with plasmids as indicated. Cell lysates were subjected to denatured Ni-NTA pulldown and western blot. Cells were treated with MG132 (20 mM) for 6 h before cell lysis. (f ) HEK293T cells were transfected with his-MUC1-C together with PPARg or its mutant plasmids, as indicated. Cell lysates were subjected to denatured Ni-NTA pull-down and western blot. Cells were treated with MG132 (20 mM) for 6 h before cell lysis.
cancers,1 which is involved in the MUC1 C-terminal subunit (MUC1-C, cytoplasmic tail) as a putative receptor to trigger intracellular signaling pathway.2,3 Although the GO-203, an inhibitor of MUC1-C, can terminate part of the MUC1-C signal in breast cancer cells,22 the cellular physical inhibitor and mechanism of MUC1-C pathway are still unclear. Here we first found that MUC1-C undergoes proteasome-dependent degradation, because the proteasome inhibitor (MG132) treatment led to increased MUC1-C protein levels in cancer cells, which was consistent with MG132 treatment resulting in MUC1-C ubiquitination. These interesting findings suggest that MUC1-C protein can be degraded by proteasome-dependent signal. Many RING finger domains simultaneously bind ubiquitination enzymes and their substrates, and hence function as E3 ubiquitin ligases.23,24 Ubiquitination in turn targets the substrate protein for degradation.16,23–25 Although we have demonstrated that PPARg induced MUC1-C ubiquitination, however, it is still unclear what kind of E3 ubiquitin ligase targets MUC1-C and induces it degradation. Western blot shows that PPARg is expressed in HT29, LS 174T and MCF-7 cells, which is the new identified E3 ubiquitin ligase;16 therefore, PPARg could be associated with MUC1-C proteasome-dependent degradation. As expected, silenced PPARg significantly increased MUC1-C protein levels as well as reduced MUC1-C ubiquitination. In contrast, overexpression of PPARg led to decreased MUC1-C protein levels and MUC1-C ubiquitination. Further analysis shows that PPARg interacted with MUC1-C to trigger its ubiquitination and degradation, but previous report shows that PPARg agonist TROG reduces IL-8 expression (a pro-inflammatory cytokine) by increasing MUC1 gene expression in AGS cells.26,27 However, Oncogene (2013) 1 – 7
MUC1 N-terminal subunit is shed from the cell surface3 in response to luminal signals, leaving the MUC1 C-terminal subunit (MUC1-C, cytoplasmic tail) as a putative receptor to trigger intracellular signal.2,3 In addition, MUC1-C increases NFkB activation by interaction with IKK or NFkB/p65, and NFkB activation leads to increased pro-inflammatory gene expression (IL-8 and so on),16,28 therefore it is unclear that the mechanism of MUC1 inhibits pro-inflammatory response,26,27 maybe this phenomenon occurs only in AGS cells. Other studies show that PPARg induces Muc1 gene expression in murine trophoblasts,17 but that it has no effect on the MUC1 gene expression in human uterine epithelial cell lines (HES and HEC-1A) and T47D human breast cancer cells,18 suggesting that PPARg regulates the murine Muc1 and human MUC1 promoters in opposite manners, which is possible considering the differential responsiveness of the mouse and human promoter regions;18 therefore, it is not surprising that overexpression of PPARg had no effect on human MUC1-C gene levels (Figure 2g). Although PPARg is a transcriptional factor, it is bound to cytoplasmic MUC1-C, and deletion of the nuclear signal location of PPARg still induced MUC1-C degradation, suggesting that PPARginduced MUC1-C degradation was independent of its transcriptional activity. PPARg has an essential role in several diseases, and thus it is not surprising that its expression is low in colon cancer patients.29 Similarly, PPARg agonists inhibit carcinogen-induced colon tumor growth, suggesting that PPARg can function as a tumor suppressor.30,31 Some important classes of synthetic agonists of PPARg (TROG, PIOG) can activate PPARg by binding its ligand-binding domain;32,33 here we found that PPARg agonists such as TROG and PIOG significantly increased the binding of & 2013 Macmillan Publishers Limited
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Figure 4. K134 of MUC1-C is required for PPARg-mediated MUC1-C degradation. (a) Construct of MUC1-C and lysine sites. (b) HEK293T cells were co-transfected with F-PPARg plasmids together with MUC1-C or mutants. Cell lysates were subjected to denatured Ni-NTA pull-down and western blot. Cells were treated with MG132 (20 mM) for 6 h before cell lysis. (c) HEK293T cells were co-transfected with F-PPARg plasmids together with MUC1-C or mutants for 36 h. Cell lysates were subjected to western blot. (d) HEK293T cells were transfected with his-MUC1-C or K134R. After 36 h, cells were treated with cycloheximide (30 mg/ml) for 0, 10 and 16 h to inhibit de novo protein synthesis and harvested for western blot. The percent MUC1-C protein remaining at each time point was calculated accordingly.
Figure 5. Liganded PPARg enhances its activity. (a) HT29 cells were treated with DMSO, PIOG and TROG (100 mM) for 1 h. Cell lysates were subjected to western blot. (b) HT29 cells were treated with PIOG or TROG (100 mM) for 10 min. Cell lysates were subjected to immunoprecipitation and western blot. (c) HT29 cells were treated with or without TROG (100 mM) for 15 min. Cell lysates were subjected to denatured immunoprecipitation and western blot. (d) HT29 cells were treated with TROG (100 mM) for 1 h. Cells were treated with cycloheximide (30 mg/ml) for 0, 4 and 6 h to inhibit de novo protein synthesis and harvested for western blot. The percent MUC1-C protein remaining at each time point was calculated accordingly.
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6 Western blot and immunoprecipitation Subcellular fractionation and immunoprecipitation were performed as described previously.16,28 For denatured immunoprecipitation, cell extracts were heated at 95 1C for 5 min in the presence of 1% SDS to disrupt the non-covalent protein interactions. The SDS was diluted and the proteins were subjected to immunoprecipitation and western blot. The samples were subjected to 10–20% gradient SDS–PAGE, transferred to a nitrocellulose membrane, then probed by western blot analysis with the indicated antibody and developed by using an ECL Kit.
Ni-NTA purification and pull-down For native Ni-NTA purification or pull-down, cells were washed with cold PBS and lysed in lysis buffer (50 mM NaH2PO4, 300 mM NaCl containing protease inhibitors (PMSF, Aprotinin, Leupeptin, E46), pH 8.0), sonicated and spun down. The supernatant was loaded onto a Ni-NTA-agarose column (QIAGEN, Pudong, Shanghai, China) or pull-down, sequentially washed with 30 bed volumes (V) of buffer A (10 mM Tris-HCl, pH 8.0, 50 mM NaH2PO4, 10 mM imidazole) twice, 30V of buffer B (10 mM Tris-HCl, pH 6.3, 50 mM NaH2PO4, 10 mM imidazole) twice and eluted with 250 mM imidazole, pH 8.0. For denaturing Ni-NTA pull-down, cells were washed with cold PBS and lysed in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 8 M Urea, PH 8.0), and spun down and the supernatant was subjected to Ni-NTA pull-down. Sequentially beads were washed with buffer A twice and then buffer B twice, and finally eluted with 250 mM imidazole, pH 8.0.
Figure 6. PPARg terminates MUC1-C-mediated cell proliferation. (a) Cell proliferation was analyzed for the stable knockdown of PPARg or control shRNA in HT29 cells (means±s.e.m., n ¼ 4). (b) Soft agar analysis of the stable knockdown of PPARg or control shRNA in HT29 cells, and the colonies enumerated per well (means±s.e.m., n ¼ 4). (c) Cell proliferation was analyzed for the stably expressing pcDNA3 (vector), MUC1-C, MUC1-C þ PPARg or K134R þ PPARg plasmids cells (means±s.e.m., n ¼ 4). (d) Soft agar analysis of the stably expressing different plasmid cells, and the colonies enumerated per well (means±s.e.m., n ¼ 4).
PPARg to MUC1-C, resulting in MUC1-C degradation and inhibition of cell proliferation. Taken together, PPARg induced MUC1-C ubiquitination and degradation, leading to termination of MUC1C-mediated cell proliferation.
MATERIALS AND METHODS Cell lines, plasmids and antibodies The human colonic adenocarcinoma cells such as HT29, MCF-7, LS 174T and HEK293T were obtained from the ATCC (Manassas, VA, USA). hisMUC1-C cDNA (complimentary DNA) was cloned into pCMV or Flag-PPARg cDNA was cloned into pcDNA3 vector, respectively. Plasmids were mutated by the site-directed mutagenesis method, and all the plasmids were identified by DNA sequencing. PPARg shRNA and MUC1-C monoclonal antibody (MH1 (CT2), corresponding to SSLSYTNPAVAATSANL from the cytoplasmic tail of MUC1) were obtained from Thermo Fisher Scientific, Waltham, MA, USA. Plasmids were transfected by LipfectAMINE2000 according to the manufacturer’s instructions (Invitrogen, Changning, Shanghai, China). Monoclonal PPARg, Ubiquitin (P4D1) antibodies, polyclonal his and Flag antibodies were obtained from Santa Cruz, Santa Cruz, CA, USA. Secondary antibodies were obtained from Jackson Immunoresearch (West Grove, PA, USA).
Metabolic labeling Cells were placed for 30 min in cysteine- and methionine-free medium. At the end of this period, 35S-radiolabeled methionine and cysteine were added to the medium for 1 h, and then replaced with regular medium supplemented with excess unlabeled methionine (2 mM) and cysteine (2 mM). Whole-cell lysates were then prepared at the indicated time points, and the samples were subjected to immunoprecipitation with MUC1-C antibody. The recovered material at the end of the immunoprecipitation was resolved by SDS–PAGE, and MUC1-C was detected by autoradiography. Oncogene (2013) 1 – 7
In vitro ubiquitination assay PPARg or MUC1-C cDNA was subcloned into PGEX-6P-1 vector. GST–PPARg or GST–MUC1-C was expressed in Escherichia coli strain BL21. The recombinant protein was purified by glutathione-conjugated sepharose beads (QIAGEN). MUC1-C ubiquitination assay contained 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, ATP-regenerating system, 0.2 mM dithiothreitol, 50 mM ZnCl2, 0.1 mM E1 (Boston Biochem, Cambridge, MA, USA), 0.4 mM UBCH3, 5 mg Ub, 10 mg MUC1-C and 10 ng PPARg (WT or C139A mutant). Reactions were incubated at 30 1C for 2 h, the ubiquitinated products were detected by western blot with MUC1-C antibody.
In vitro binding analysis GST–PPARg fusion protein was immobilized on glutathione–agarose beads in buffer (25 mM HEPES (pH 7.5), 6 mM NaCl and 0.2% NP-40) for 30 min at 4 1C, and then in vitro translated proteins (MUC1-C) were added and incubated for another 2 h. Adsorbates to glutathione-conjugated beads were analyzed by western blot.
Soft agar assay A total of 1 105 cells were suspended in 0.3% Noble agar in 10% FBS with DMEM and antibiotics, and was layered over 0.5% agar in 60-mm dishes and cultured for two weeks at 37 1C under 5% CO2. Dishes were stained with 0.05% crystal violet for 2 h at room temperature and colonies were counted in the entire dish.
Statistical analysis Data are expressed as the mean±s.e.m. Statistical comparison was carried out with one-way analysis of variance (ANOVA) and Dunnett’s test. Significance was defined as Po0.05.
CONFLICT OF INTEREST The authors declare no conflict of interest
ACKNOWLEDGEMENTS This work was supported by Jiangsu Province Natural Science Foundation (SBK201320232) and Start Fund of University of JiangSu (12JDG071).
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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)
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