IKKa restoration via EZH2 suppression induces nasopharyngeal carcinoma differentiation

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1 Received Oct 3 Accepted 4 Mar 4 Published 7 Apr 4 DOI:.38/ncomms466 IKKa restoration via suppression induces nasopharyngeal carcinoma differentiation Min Yan,, Yan Zhang,,, Bin He,, Jin Xiang, Zi-feng Wang, Fei-meng Zheng, Jie Xu, Ming-yuan Chen, Yu-liang Zhu 3, Hai-jun Wen, Xiang-bo Wan 4, Cai-feng Yue, Na Yang, Wei Zhang, Jia-liang Zhang, Jing Wang, Yang Wang, Lian-hong Li, Yi-xin Zeng, Eric W.-F. Lam, Mien-Chie Hung 6,7 & Quentin Liu, Lack of cellular differentiation is a key feature of nasopharyngeal carcinoma (NPC), but it also presents as a unique opportunity for intervention by differentiation therapy. Here using RNA-seq profiling analysis and functional assays, we demonstrate that reduced IKKa expression is responsible for the undifferentiated phenotype of NPC. Conversely, overexpression of IKKa induces differentiation and reduces tumorigenicity of NPC cells without activating NF-kB signalling. Importantly, we describe a mechanism whereby directs IKKa transcriptional repression via H3K7 histone methylation on the IKKa promoter. The differentiation agent, retinoic acid, increases IKKa expression by suppressing - mediated H3K7 histone methylation, resulting in enhanced differentiation of NPC cells. In agreement, an inverse correlation between IKKa (low) and (high) expression is associated with a lack of differentiation in NPC patient samples. Collectively, these findings demonstrate a role for IKKa in NPC differentiation and reveal an epigenetic mechanism for IKKa regulation, unveiling a new avenue for differentiation therapy. Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine, Guangzhou 6, China. Institute of Cancer Stem Cell, First Affiliated Hospital Collaborative Innovation Center of Oncology, Dalian Medical University, Dalian 644, China. 3 Department of Radiotherapy, Zhongshan Affiliated Hospital of Sun Yat-sen University, Zhongshan 8, China. 4 Department of Medical Oncology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou 63, China. Division of Cancer, Department of Surgery and Cancer, Imperial College London, Hammersmith Hospital Campus, London W ONN, UK. 6 Department of Molecular and Cellular Oncology, The University of Texas M.D. Anderson Cancer Center, Houston, Texas 773, USA. 7 Graduate Institute of Cancer Biology and Center for Molecular Medicine, China Medical University, Taichung 44, Taiwan. These authors contributed equally to this work. Correspondence and requests for materials should be addressed to Q.L. ( liuq9@mail.sysu.edu.cn). NATURE COMMUNICATIONS :366 DOI:.38/ncomms466 & 4 Macmillan Publishers Limited. All rights reserved.

2 NATURE COMMUNICATIONS DOI:.38/ncomms466 Differentiation therapy holds great promise for cancer treatment and has yielded remarkable outcomes in certain type of cancers 6, but the molecular mechanisms involved remain elusive. Nasopharyngeal carcinoma (NPC) is a distinctive type of head and neck cancer. More than % of NPCs are pathologically diagnosed as Type III undifferentiated carcinomas. Patients with this diagnosis have a much higher mortality rate than those diagnosed with well-differentiated Type I-keratinizing squamous cell carcinomas (SCCs) 7. This clinical finding is consistent with the consensus view that lack of differentiation is an important hallmark of mammalian malignancy and progression 8,9. This aspect of cancer biology presents a unique therapeutic opportunity. IkB kinase a (IKKa), also known as IKK and conserved helix-loop-helix ubiquitous kinase, is one of the two catalytic subunits of the IKK complex. Although IKKa is involved in nuclear factor-kb (NF-kB) activation, recent studies have shown this kinase also functions as a molecular switch that controls epidermal differentiation independent of its kinase activity in NF-kB signalling 4. IKKa interacts with Smad/3-containing transcription-activating complexes in a TGFb-dependent manner and further induces the expression of Mad, Mad and Ovol, all of which encode negative regulators of Myc during keratinocyte differentiation. IKKa knockout mice display a hyperproliferative and undifferentiated epidermis, and IKKa-deficient keratinocytes fail to express terminal differentiation markers 6. One recent study reported that IKKa reduction deregulated the expression of oncogenes, tumour suppressors and stem cell regulators in K þ lung epithelial cells. The transition of IKKa low K þ p63 hi cells to tumour cells played a pivotal role in spontaneous SCC development 7. Conversely, overexpression of IKKa promotes epidermal differentiation, reduces keratinocyte proliferation and inhibits chemically induced SCC formation and progression. Notably, high-grade and poorly differentiated human SCCs display a loss of IKKa expression 8. What is not known is the precise underlying molecular events that mediate IKKa repression. Epigenetic regulation plays a central role in gene regulation and differentiation. Polycomb group proteins form polycomb repressive complexes (PRCs) that are crucial epigenetic gene silencers during embryonic development and adult somatic cell differentiation 9. PRC is originally identified as a silencer of Hox differentiation factors, a finding that has subsequently been expanded to dozens of other differentiation regulators. These include transcription factors of Gata, Sox, Fox, Pou and Pax families as well as components of the Wnt, TGF-b, Notch, FGF and retinoic acid signalling pathways. The PRC consists of mammalian homologues of the Drosophila melanogaster proteins, including the enhancer of zeste (E (Z)), suppressor of zeste (SU (Z) ) and extra sex combs. The enhancer of zeste homologue () is the catalytically active component of the PRC complex. It preferentially represses target genes that initiate differentiation and restrain proliferation by inducing trimethylation of lysine 7 and lysine 9 of histone H3 (ref. ). Indeed, genome-wide aberrant methylation has been observed in the majority of primary NPCs, indicating the importance of the epigenetic dysregulation during the process of NPC tumorigenesis 3. However, the potential role of in the regulation of cellular differentiation in NPC has not yet been elucidated. In the present study, we demonstrate that differentiation of NPC cells is impaired as a result of IKKa reduction. We show that the reduction in IKKa is caused by -mediated H3K7 histone methylation within the IKKa promoter. These new data establish an epigenetic mechanism by which represses IKKa expression and impairs tumour cell differentiation. Results Downregulated IKKa leads to undifferentiated NPC status. To identify the genes essential for NPC differentiation, we sequenced six RNA libraries from three paired NPC tumours (T, 3T, 3T) as well as adjacent non-tumour (N, 3N, 3N) tissues. Differential expression profiling of 3,63 deregulated genes was displayed as a heat map (Fig. a), where the genes were listed in descending order according to Baggerley s test analysis. Thirteen genes (3/ 84) related to epithelial differentiation were downregulated in all three NPC patient samples compared with paired adjacent nontumour tissues. Deregulation of seven candidate genes (7/3) was further confirmed by real-time quantitative PCR analysis (Fig. b), and they included ATF, HES, IKKa, KLF4, MAPK6, MAPK3 and NFATC4. We next explored the potential developmental role of these seven genes in the well-differentiated CNE cell line by gene silencing using sirnas. As shown in the left panel of Fig. c, these sirnas effectively downregulated mrna expression of each of these seven genes. More importantly, western blot analysis showed that sirna-mediated repression of either IKKa or NFATC4 significantly reduced expression of the epithelial cell differentiation markers involucrin and CK8 (Fig. c right panel). In contrast, both IKKa and NFATC4 markedly increased expression of the squamous metaplasia dedifferentiation marker CK3. Subsequent experiments focused on IKKa because downregulation of IKKa mrna level was also detected with five other pairs of primary NPC when compared with adjacent normal nasopharyngeal epithelium (- to -fold, Fig. d left panel). Moreover, IKKa expression was also enriched in the normal nasopharyngeal epithelial cell line (NP69, -fold) as well as the well-differentiated NPC cell line (CNE, 44-fold) compared with several poorly differentiated NPC cell lines (CNE, SUNE and HONE, Fig. d right panel). These data suggest that lack of IKKa expression is related to the undifferentiated state of NPC cells. IKKa induces differentiation and decreases tumorigenicity. To explore whether overexpression of IKKa inhibits proliferation of NPC cells and leads to their differentiation, we ectopically introduced either a control vector or wild-type IKKa in CNE cells. Forced expression of IKKa in poorly differentiated CNE cells led to marked morphologic changes that were comparable to well-differentiated NP69 or CNE cells. These changes were histologically characterized as a slender and fusiform phenotype (Fig. a and Supplementary Fig. ). Moreover, expression of involucrin and CK8 was increased, whereas CK3 expression was reduced (Fig. b). Similar results with forced expression of IKKa were observed in two other poorly differentiated NPC cell lines (HONE and SUNE, Supplementary Fig. a). In addition, CNE-IKKa cells exhibited the downregulation of vimentin and upregulation of E-cadherin (Fig. b), indicative of NPC cell differentiation. Both cell number and colony formation assays showed that cell growth was significantly suppressed following overexpression of IKKa in CNE cells (Fig. c,d). In addition, overexpression of IKKa in CNE cells increased the G population, reduced cyclin D and cyclin E expression (Supplementary Fig. b,c), decreased the ratio of Ki67-positive cells and increased the proportion of senescence-associated (SA) b-gal-positive cells (Fig. e,f). Furthermore, overexpression of IKKa in CNE cells did not reduce the IkBa expression (Fig. g, upper left panel), which inhibited NF-kB activity. Forced expression of IKKa also did not induce p6 nuclear translocation (Fig. g, lower panel) and NF-kB target gene IL6, IL8 transcription (Fig. g, upper right panel). Given the phenotypic and functional changes we observed in CNE cells that overexpress IKKa cells in vitro, we next tested NATURE COMMUNICATIONS :366 DOI:.38/ncomms466 & 4 Macmillan Publishers Limited. All rights reserved.

3 NATURE COMMUNICATIONS DOI:.38/ncomms466 Colour Key 4 Value T/N 3T/3N 3T/3N Relative mrna expression (log ) 3 4 T 3T 3T 3N N 3N ATF ETS HES KLF4 MAPK MAPK6 MAPK3 NCOA6 NFATC4 NOTCH RX SMAD4 Relative mrna expression ATF HES KLF4 MAPK6 MAPK3 sirna NFATC4 3 siatf sihes si siklf4 simapk6 simapk sinfatc4 Involucrin CK8 CK3 GAPDH Relative expression N T Relative expression Tissue samples NP69 CNE CNE HONE SUNE Figure IKKa downregulation is responsible for the undifferentiated phenotype of NPC. (a) An unbiased RNA-seq expression profiling heat map between normal nasopharyngeal tissues (N, 3N, 3N) and NPC tissues (T, 3T, 3T). High and low expression levels are indicated in red and blue, respectively. (b) The mrna levels of 3 candidate genes in the three paired primary samples (N, T, 3N, 3T, 3N and 3T) were verified with real-time PCR. (c) Repression of target genes was measured by real-time PCR (left panel). Involucrin, CK8 and CK3 levels in sirnas transient-transfected CNE cells were analysed by western blot (right panel). (d) The mrna level of IKKa in NPC specimens (left panel) and cell lines (right panel) was detected by real-time PCR. T: NPC tissues, N: paired normal nasopharyngeal tissues (n ¼ 3). NP69 is an immortalized normal human nasopharyngeal epithelial cell line. CNE is a well-differentiated NPC cell line, whereas CNE, HONE and SUNE are poorly differentiated NPC cell lines. Po., Po. (two-tailed Student s t-tests). Error bars represent mean±s.d. whether IKKa overexpression affected tumorigenicity in vivo by using a xenograft tumour model. As shown in Fig. h, distinct tumour masses were detected as early as days in mice injected subcutaneously with CNE-vector cells. In contrast, tumour masses could not be detected in mice injected with CNE-IKKa even 4 days later (Supplementary Table ). Similarly, welldifferentiated CNE cells had a markedly lower tumorigenic activity compared with poorly differentiated CNE cells (Supplementary Table ). In addition, the mouse tumours were examined with cell differentiation markers by immunohistochemical analysis. The tumours of mice injected with CNE- IKKa cells showed markedly higher staining of IKKa, CK8 and involurcin compared with those injected with CNE-vector control cells (Supplementary Fig. 3). Taken together, these results suggest that IKKa induces differentiation and reduces tumorigenicity of NPC cells, which are not mediated through NF-kB signalling activation. IKKa is repressed by -mediated promoter H3 methylation. To elucidate the mechanism that is responsible for the downregulation of IKKa in NPC, we examined the promoter region of IKKa and identified consensus polycomb response elements 4 (Fig. 3a). This finding raised the possibility that PRC might be involved in the repression of IKKa and led us to examine the expression and activity of PRC in NPC. We found high levels of the PRC component as well as modification in NPC compared with normal cells. Specifically, NATURE COMMUNICATIONS :366 DOI:.38/ncomms & 4 Macmillan Publishers Limited. All rights reserved.

4 NATURE COMMUNICATIONS DOI:.38/ncomms Involucrin CK8 CK3 E-cadherin Vimentin GAPDH E-cadherin/DAPI Vimentin/DAPI Cell number ( ) h 4 h 48 h 7 h Colony formation ability % 3 Ki67/DAPI Ki67-positive cells % , 3 3 p6.3. IκBα GAPDH Relative mrna expression IL6 TNF-α IL8 Tumour volume (mm 3 ) Days after tumour implantation p6/dapi β-gal-positive cells % Figure Overexpression of IKKa induces differentiation in vitro and decreases tumorigenicity in vivo. (a) Phase contrast images of CNE cells expressing either control vector (pbabe) or pbabe-ikka. (b) Western blot and immunofluorescence analysis with indicated antibodies in control (vector) and IKKa-overexpressed CNE cells. (c f) IKKa-overexpressed and control (vector) CNE cells were subjected to determining the number of cells (n ¼ 3), Po., two-tailed Student s t-tests (c), colony formation (n ¼ 3), Po., two-tailed Student s t-tests (d), immunofluorescence staining of Ki67 (n ¼ 3), Po., two-tailed Student s t-tests (e) and SA-b-Gal staining (n ¼ ), Po., two-tailed Student s t-tests (f). (g) IKKa-overexpressed and control (vector) CNE cells were subjected to western blot, immunofluorescence staining of p6 and real-time PCR analysis. TNF-a was used as a positive control. (h) IKKa-overexpressed and control (vector) CNE cells (3 cells per mouse) were injected into nude mice subcutaneously, and tumour volume was determined (n ¼ 9), Po., two-tailed Student s t-tests. Scale bars, mm (a,f); mm (b, e and g). Error bars represent mean±s.d. both and were highly elevated in the poorly differentiated CNE, HONE and SUNE NPC cell lines compared with the normal nasopharyngeal epithelial cell line NP69 as well as well-differentiated NPC cells CNE (Fig. 3b). Next, we performed chromatin immunoprecipitation (CHIP) analysis to investigate whether was associated with the IKKa locus. As shown in Fig. 3c, bound to the GAGAG motif within the IKKa promoter where and 4 NATURE COMMUNICATIONS :366 DOI:.38/ncomms466 & 4 Macmillan Publishers Limited. All rights reserved.

5 NATURE COMMUNICATIONS DOI:.38/ncomms466 PRE within the promoter CAGCCTTTAAAT... GAGAG... GAGAG GGGCCTTCTCGC PHO Motif GAGAG Motif GAGAG Motif PHO Motif 7 7 Relative promoter activity NP69 CNE CNE HONE SUNE si si- H3 GAPDH β-actin si- 7 7 GAPDH H3 si Input IgG SUZ H3 Relative levels of promoter occupancy -WT -H694A -R73K β-actin β-actin Input IgG CHIP Relative levels of promoter occupancy CHIP Relative levels of promoter occupancy CHIP IgG Re-CHIP IgG Re-CHIP /IgG / SUZ -WT Input IgG si IgG -WT -H694A -R73K -H694A -R73K CHIP Figure 3 IKKa is repressed by -mediated H3K7 histone methylation within the IKKa promoter. (a) Illustration of Polycomb response element (PRE) within the IKKa promoter. The sequence alignment of PRE within the IKKa promoter is shown (green letter: core DNA motifs of PRE, black bar: region amplified in CHIP-PCR). (b) Expressions of and in NPC cell lines were measured by western blot analysis. (c) Products of CHIP using an antibody and Re-CHIP using an antibody were subjected to PCR and real-time PCR in CNE cells. Data in the bottom panel are expressed as the fold of enrichment relative to the original input DNA. The relative levels of IKKa promoter occupancy were examined by real-time PCR and calculated using DCt value. (d,e) CNE cells were transfected with si for 4 h and subjected to western blot (d, upper), RT PCR (d, lower) and CHIP assay (n ¼ 3), Po., two-tailed Student s t-tests (e). (f) CNE cells treated with si for 4 h and subjected to dual-luciferase reporter assays. Data are shown as relative IKKa promoter activity (n ¼ 3), Po., Po., two-tailed Student s t-tests. (g,h) CNE cells expressing vector (pbabe), pbabe--wild-type (-WT), pbabe--h694a (-H694A) or pbabe--r73k (-R73K) were analysed by western blot (g, upper), RT PCR (g, lower) and CHIP with the indicated antibodies (n ¼ 7), Po., Po., two-tailed Student s t-tests (h). The relative levels of IKKa promoter occupancy were examined by real-time PCR and calculated using DCt value. Error bars represent mean±s.d. NATURE COMMUNICATIONS :366 DOI:.38/ncomms466 & 4 Macmillan Publishers Limited. All rights reserved.

6 NATURE COMMUNICATIONS DOI:.38/ncomms466 were co-occupied, as revealed by CHIP and Re-CHIP assays. Then, by using sirna to knock down, we found that was reduced at the same time that both mrna and protein expressions of IKKa were increased (Fig. 3d). In addition, the PRC complex proteins and SUZ, as well as, bound the IKKa promoter DNA much less efficiently in sirna-treated CNE cells compared with si-control cells (Fig. 3e). A dual-luciferase reporter assay established that transcriptional activity of IKKa increased significantly in si- treated CNE cells compared with control cells (Fig. 3f). Moreover, we transiently transfected either wild type (WT) or mutants (H694A, a SET domain mutant, and R73K, a C-terminus mutant related to retain the ability to methylate histone H3 ) cdna in CNE cells. RT-PCR and western blot assays showed that IKKa expression was reduced in -WT cells compared with empty vector-transfected cells. In contrast, overexpression of the methyl-transferase mutant -H694A increased IKKa expression while reducing levels compared with -WT and vector control (Fig. 3g). Consistently, CHIP assays showed that -WT increased, but -H694A mutant reduced, H3K7 histone methylation within the IKKa promoter when compared with vector control (Fig. 3h). -R73K mutant, which retained the histone methyl-transferase (HMT) activity, showed a slight nonsignificant decrease in IKKa expression compared with vector control (Fig. 3g). CHIP assays showed that the -R73K mutant retained the H3K7 methylation within the IKKa promoter comparable to vector control, but less efficient than that in -WT-expressing CNE cells (Fig. 3h). Collectively, these data provide evidence that H3K7 histone methylation mediated by, which requires the intact SET domain, is involved in silencing IKKa transcription. Recent studies strongly suggest that the establishment of the basic DNA methylation profile might be mediated through histone modification 6. We thus analysed the methylation status of IKKa promoter by methylation-specific PCR (MSP). The CpG islands of IKKa gene were heavily methylated in NPC cell lines and samples compared with those in the non-cancer nasopharyngeal cell line and tissues (Supplementary Fig. 4a,b). To explore whether is correlated with the DNA methylation of IKKa promoter, we performed the MSP assay in CNE cells silenced by sirna. Results showed that the methylated (M) IKKa promoter was reduced, whereas the unmethylated (U) promoter was increased in si-treated CNE cells (Supplementary Fig. 4c). These results suggested that -mediated histone methylation may contribute to DNA methylation in the IKKa promoter. modulates differentiation and tumorigenicity of NPC. These new findings suggested that silencing might induce differentiation in NPC. To test this notion, we used sirnas to suppress endogenous in CNE, HONE and SUNE cells. The results showed that silencing of caused an increase in levels of IKKa, involucrin and CK8 and decrease of CK3 (Fig. 4a and Supplementary Fig. ). Suppression of in CNE cells also led to increased E-cadherin and reduced vimentin expression (Fig. 4a), as well as morphological changes in CNE cells (Fig. 4b), similar to IKKa-induced NPC cell differentiation. To further test our hypothesis, we then stably expressed - shrna in CNE cells (referred to as sh cells). Compared with CNE cells expressing a control shrna, expression of protein was markedly decreased in sh CNE cells (Fig. 4c). As expected, suppression of markedly reduced proliferation of CNE cells (Fig. 4d). In addition, silencing of in CNE cells caused a significant reduction in colony number (Fig. 4e). Finally, we subcutaneously injected CNE cells with either -shrna or control shrna into nude mice and evaluated tumour growth. In contrast to control CNE cells, which formed large tumours, sh CNE cells displayed greatly reduced tumour growth (Fig. 4f). In addition, immunohistochemical analysis revealed increased levels of IKKa, CK8, involucrin and E-cadherin with decreased vimentin staining in tumours of mice injected with sh CNE cells, compared with those injected with control cells (Fig. 4g). Derepression of IKKa by retinoic acid depends on inhibition. Retinoic acid () is currently in use or being evaluated for several human cancers as a differentiation agent. Here we tested the hypothesis that differentiation therapy with would regulate pathways upstream of IKKa. As expected, we found that increased IKKa mrna and protein levels in a dose-dependent manner (Fig. a). More importantly, decreased both and expression. CHIP analysis then revealed that binding of and SUZ with the endogenous IKKa promoter region was significantly reduced in treated CNE cells (Fig. b). Consistently, in the presence of, H3K7 histone methylation was also decreased within the IKKa promoter. Finally, by using a dual-luciferase reporter assay, we found that increased transcriptional activity of IKKa in CNE cells (Fig. c). To investigate whether downregulation of by is essential for IKKa expression and differentiation, we then overexpressed (WT and T487A mutant) in CNE cells 7,8. Consistent with this hypothesis, western blot analysis showed that was unable to enhance IKKa, involucrin or CK8 expression in both -WT- and -T487A-expressing cells (Fig. d). In contrast, increased IKKa, involucrin and CK8 expression in the control vector-expressing cells. In addition, -induced repression of CK3 in vector cells was abrogated in -WT and -T487A cells. Furthermore, in response to, slender and fusiform morphological changes were observed in control cells but not in -WT- or -T487A-expressing cells (Fig. e). Colony formation assays showed that proliferation of control cells was severely suppressed in the presence of. In contrast, there was no significant reduction in cell proliferation in -treated -WT- and -T487A-expressing cells (Fig. f). These results establish that restores IKKa expression by reducing - mediated H3K7 methylation in NPC cells. promotes NPC differentiation by inducing IKKa expression. Next, we explored the possibility that would induce differentiation in NPC cells. As expected, western blot showed that increased IKKa protein in CNE, HONE and SUNE cells (Fig. 6a and Supplementary Fig. 6a). These three poorly differentiated NPC cell lines incubated with displayed further induction of differentiation, as determined by increased levels of involucrin and CK8, and reduced CK3 expression. In addition, treatment also increased E-cadherin and reduced vimentin in CNE cells, as shown in the analysis by western blot and immunofluorescence staining (Fig. 6a). These changes were similar to those observed in CNE-IKKa cells. Moreover, treatment with caused morphological changes indicative of differentiation, suppressed proliferation, colony formation and the number of Ki67-positive cells, and increased the proportion of SA b-gal-positive CNE cells (Fig. 6b d, Supplementary Fig. 6b,c). To further characterize -induced differentiation, we performed an air liquid interface three-dimensional (3D) culture assay (Fig. 6e). CNE cells formed a multi-layered tissue when cultured at an air liquid interface, as described previously 9. Immunohistochemical staining showed that the supra basal layer 6 NATURE COMMUNICATIONS :366 DOI:.38/ncomms466 & 4 Macmillan Publishers Limited. All rights reserved.

7 -.8 3 Involucrin 4.. CK si E-cadherin/DAPI. si Vimentin /DAPI.4 CK3 Vimentin.6. β-actin. sh. shctrl h 4 h 48 h 7 h shctrl sh sh 3. Colony formation ability % 3. shctrl.6 Cell number () 3 shctrl 3. shctrl 4. E-cadherin 3 sh sh,8 β-actin, CK8 shctrl sh, 9 Involucrin Tumour volume (mm3) si EZ H si EZ H trl si C - NATURE COMMUNICATIONS DOI:.38/ncomms Days after tumour implantation E-cadherin sh Vimentin shctrl Figure 4 Suppression of in CNE cells induces differentiation in vitro and decreases tumorigenicity in vivo. (a) Expression of indicated proteins was analysed by western blot and immunofluorescence in si-treated CNE cells. (b) Morphology of si-treated CNE cells is shown by phase contrast photomicrographs. Scale bars, mm. (c) Western blot assays showed that expression is suppressed in sh CNE cells. (d,e) Proliferation of sh or sh-control CNE cells was analysed using both cell number (n ¼ 3), Po., two-tailed Student s t-tests (d), colony formation assays (n ¼ 3), Po., two-tailed Student s t-tests (e). (f) Mice were injected with sh or sh-control CNE cells subcutaneously ( 6 cells per mouse). Tumour volume was determined as described in Methods (n ¼ 7 9), Po., two-tailed Student s t-tests. (g) Immunohistochemical analysis of tumours from mice injected with CNE-sh cells or control cells with IKKa, CK8, involucrin, E-cadherin and vimentin antibodies. Representative immunohistochemical images are shown. Scale bars, mm (a); mm (b), mm (g). Error bars represent mean±s.d. NATURE COMMUNICATIONS :366 DOI:.38/ncomms466 & 4 Macmillan Publishers Limited. All rights reserved. 7

8 NATURE COMMUNICATIONS DOI:.38/ncomms H GAPDH.9.9 Input IgG SUZ CHIP β-actin WT -T487A Involucrin tro CK8 CK3 C on R A β-actin -WT -T487A -T487A Colony formation ability % -WT l Relative promoter activity EZ H SU Z H 3K 7 m e3 on. R A.3 C.6 tro l Relative levels of promoter occupancy Ig G WT T487A Figure Derepression of IKKa by depends on inhibition. (a) CNE cells were treated with increasing doses (,, and 3 mm) of for 48 h and subjected to western blot (up panel) and RT-PCR analysis (lower panel). (b,c) CNE cells were incubated with (3 mm) for 48 h and subjected to CHIP (n ¼ 3), Po., Po., two-tailed Student s t-tests (b), dual-luciferase reporter assays (n ¼ 3), Po., two-tailed Student s t-tests (c). (d f) CNE cells expressing pcdna3.- (WT or T487A) or vector (pcdna3.) were cultured with (3 mm) or control (DMSO) for 48 h and subjected to western blot analysis (d), phase contrast imaging (e), colony formation assay (n ¼ 3), Po., two-tailed Student s t-tests (f). Error bars represent mean±s.d. Scale bars, mm (e). cells expressed higher levels of involucrin (apical marker) in the -treated group (Fig. 6e, right panel) as compared with the DMSO-treated control group (Fig. 6e, middle panel). Moreover, in the DMSO control tissue, the basal markers CK3 and vimentin were diffusely expressed throughout all cell layers. In contrast, CK3 and vimentin were mainly restricted to the basal layer in -treated tissue, which was similar to that observed in clinical non-cancer nasopharyngeal specimens (Fig. 6e, left panel). 8 To determine the effects of on tumorigenicity in vivo, we established subcutaneous flank xenograft tumours using CNE, HONE and SUNE cells in nude mice. As shown in Fig. 6f and Supplementary Fig. 7a,b, treatment (i.g. mg kg per days) resulted in a significant inhibition of tumour growth compared with control mice as indicated by decreased sizes of tumours (n ¼ 8). In addition, CNE and SUNE cells pretreated with for 4 to days showed significantly reduced tumorigenic capacity than control cells (Fig. 6g and NATURE COMMUNICATIONS :366 DOI:.38/ncomms466 & 4 Macmillan Publishers Limited. All rights reserved.

9 NATURE COMMUNICATIONS DOI:.38/ncomms Involucrin CK3 CK8 E-cadherin Vimentin GAPDH Vimentin/DAPI E-cadherin/DAPI Ki67 / DAPI Ki67-positive cells % β-gal-positive cells % 3 3 Vimentin CK3 Involucrin Non-Ca NPC cell (3D culture) + Tumour volume (mm 3 ),4,, mg kg d Days after tumour implantation Tumour volume (mm 3 ) 6 - h Days after tumour implantation Left: - h Right: control Figure 6 promotes differentiation in poorly differentiated NPC cells by inducing IKKa expression. (a) CNE cells were treated with in increasing doses (,, and 3 mm) for 48 h and subjected to western blot and immunofluorescence analysis. (b) Morphology of treated (3 mm 48 h) or control (DMSO) CNE cells are shown by phase contrast photomicrographs. (c,d) CNE cells were treated with (3 mm) or control (DMSO) and subjected to immunofluorescence staining for Ki67 (n ¼ 3), Po., two-tailed Student s t-tests (c), SA-b-Gal staining (n ¼ 3), Po., two-tailed Student s t-tests (d). (e) Immunohistochemistry of involucrin, vimentin and CK3 in air liquid interface 3D cultured CNE cells (control (DMSO): middle panel, ( mm): right panel) and non-cancer specimens (left panel). (f) Mice were injected with CNE cells subcutaneously ( 6 cells per mouse). At days following injection, mice were treated with ( mg kg (d), i.g.) or water (control). Tumour volumes were calculated as described in the Methods (n ¼ 8), Po., two-tailed Student s t-tests. (g) CNE cells were incubated with (3 mm) or control (DMSO) for h and injected subcutaneously ( 6 cells per mouse, n ¼ ). Tumour volumes were determined as described in the Methods (n ¼ ), Po., two-tailed Student s t-tests. Scale bars, mm (a,c); mm(b,d,e). Error bars represent mean±s.d. NATURE COMMUNICATIONS :366 DOI:.38/ncomms & 4 Macmillan Publishers Limited. All rights reserved.

10 NATURE COMMUNICATIONS DOI:.38/ncomms466 Supplementary Fig. 7c). Immunohistochemistry revealed that treatment with resulted in induction of IKKa, CK8 and involucrin levels in tumour cells compared with control vehicle treatment (Supplementary Fig. 7d). To determine whether IKKa is required for -induced differentiation, we knocked down IKKa expression using sirna before treatment in CNE cells. These experiments showed that increased expression of both involucrin and CK8 in control cells treated with a scrambled sirna (si-control, Supplementary Fig. 8a). However, induction of involucrin and CK8 was significantly inhibited in cells transfected with siikka. In addition, -induced repression of CK3 in si-control cells was abrogated in siikka cells. Moreover, we observed differentiation-associated morphological changes in si-control cells in response to, but not in -treated siikka cells (Supplementary Fig. 8b). In the presence of, the colonyforming ability of si-control cells was severely inhibited. In contrast, there was no significant difference in growth of treated IKKa repression cells (Supplementary Fig. 8c). Low IKKa and high correlate with undifferentiated NPC. Collectively, all of these new data suggest that - mediated repression of IKKa expression contributes to both tumorigenesis and differentiation blockage in NPC. We therefore examined the expression of in the primary human NPC samples and found that was significantly higher in tumour samples compared with the paired adjacent normal nasopharyngeal epithelium (Supplementary Fig. 9). Next, we used immunohistochemistry to determine expression of IKKa and in primary human NPC and non-cancer nasopharyngeal samples. Clinical features of the patients are summarized in Supplementary Table. The best cutoff points for parameters were determined by receiver operating characteristic curve analysis. A total of nine noncancer nasopharyngeal mucositis and 87 NPC samples were evaluated. A significant portion of non-cancer nasopharyngeal samples (6/9, 66.7%) and differentiated NPC specimens (9/3, 69.%) showed strong IKKa staining, but only a minority (6/74, 3.%) of undifferentiated NPC samples displayed high IKKa staining (Fig. 7a and Table ). In addition, all non-cancer specimens showed -negative staining and only a few (3/3, 3.%) of pathologically differentiated NPC specimens displayed - positive staining. In contrast, was significantly elevated in a majority (63/74, 8.%) of undifferentiated NPC specimens. Analysis by the Fisher s exact test showed that IKKa negatively and positively correlated with undifferentiated status in NPC tissues (Table ). Furthermore, the median score of IKKa expression in undifferentiated NPC specimens was markedly lower compared with the differentiated NPC and non-cancer tissues, whereas expression had the opposite tendency (Fig. 7b). Finally, we detected high expression in 4 of (76.4%) specimens with low IKKa expression, indicating a significant negative correlation between IKKa and expression (Table ). Discussion These new data provide compelling evidence to suggest the application of differentiation therapy as a therapeutic strategy for patients with NPC by revealing several novel findings: () a reduction of IKKa is involved in the undifferentiated status of NPC, () ectopic expression of IKKa in poorly differentiated NPC cells induces cellular differentiation in vitro and reduces tumorigenicity in vivo, (3) repression of IKKa by -induced H3K7 methylation within the IKKa promoter ultimately blocks cellular differentiation, (4) restoration of IKKa by promotes differentiation in NPC through inhibition of and () IKKa negatively and positively correlates with undifferentiated status in human primary NPC specimens. These data demonstrate a previously unknown but a critical pathway for the blockage of NPC cell differentiation, which is caused by - mediated epigenetic silencing of IKKa. These data provide a sound and rational biochemical basis to explore IKKa as a novel target for differentiation therapy in NPC. In the present study, we found that poorly differentiated CNE, HONE and SUNE cells, exhibiting regular polygonal shape, expressed high level of CK3 and low levels of involucrin and CK8. Overexpression of IKKa and inhibition of, as well as treatment, resulted in fusiform morphological change, reduction in CK3 and increase in involucrin and CK8. These phenotypes were compatible with the well-differentiated NP69 and CNE cells. In addition, the induction of nasopharyngeal squamous metaplasia characterized as loss of CKs 7, 8 and 8, coupled with increased expression of CKs, and 3, which is closely related to the histogenesis of NPC 3,3. CK3 is also identified as a squamous metaplasia marker in other SCCs that originated from columnar epithelium 3 3. Meanwhile, involucrin is a frequently referred marker for epithelial differentiation 36,37. The induction of both CK8 and involucrin coupled with reduction of CK3 is therefore considered as markers indicating differentiation of NPC cells. However, the CK expression patterns are complicated in NPC 38,39. Furthermore, morphological change of squamous carcinoma cells into fibroblast-like fusiform was associated with the dedifferentiation in some epithelial cell types 4. More established differentiation markers, such as E-cadherin and vimentin, were thus measured in IKKa-overexpressed cells. A pattern of increased E-cadherin and decreased vimentin, along with the changes of CKs and involucrin, pointed to a phenotype of differentiation in the IKKa-overexpressed NPC cells. Previous studies have shown that IKKa plays an important role in epidermal differentiation in mice,,4. Deletion of IKKa results in a hyperproliferative and undifferentiated epidermis. Moreover, IKKa / keratinocytes do not respond to differentiation-inducing signals. Reintroduction of WT or kinase-inactive IKKa induced keratinocyte terminal differentiation and inhibited hyperproliferation both in vitro and in vivo 4,4. These functions of IKKa are consistent with our findings that IKKa is a pivotal differentiation regulator in NPC, and overexpression of IKKa can reverse the poorly differentiated state of NPC cells by reducing proliferation and inducing differentiation. More importantly, re-expression of IKKa also reduces the tumorigenicity of NPC cells in vivo. In the present study, we found that overexpression of IKKa in the poorly differentiated CNE cell line resulted in a higher ability to reduce the tumorigenicity compared with knockdown of or treatment. We reasoned that this finding is probably due to the reason that IKKa is the critical downstream effector activating differentiation process. Either inhibition or stimulation was among the important upstream regulators to induce IKKa expression and subsequent differentiation. Moreover, using MSP analysis, the CpG islands of IKKa gene were heavily methylated in NPC cell lines and clinical specimens, compared with those in the non-cancer nasopharyngeal cell line and tissues. These results were consistent with the recent opinion that DNA methylation and histone modification cooperate to achieve silencing of target genes 6. Thus, these results suggested that IKKa plays a key role in inducing differentiation and reducing tumorigenicity of NPC cells. The mechanism for the regulation of IKKa is not yet clearly understood, especially during tumorigenesis. MicroRNAs have been reported to be the negative regulators for IKKa. MiR-3, mir-a, mir-6 and mir-3b reduce transcription of IKKa during macrophage differentiation and interleukin-7-associated NATURE COMMUNICATIONS :366 DOI:.38/ncomms466 & 4 Macmillan Publishers Limited. All rights reserved.

11 NATURE COMMUNICATIONS DOI:.38/ncomms466 Non-Ca Differentiated Undifferentiated 7 score 4 3 score Undifferentiated Differentiated Non-Ca Undifferentiated Differentiated Non-Ca PRC SUZ EED K7 OFF PRC SUZ EED K7 ON AT promoter Undifferentiated promoter Differentiated Figure 7 Low IKKa and high correlate with undifferentiated status in NPC tissues. (a) Human primary NPC and non-cancer nasopharyngeal specimens (n ¼ 96) were analysed by immunohistochemical staining with IKKa and antibodies. Consecutive sections from three representative cases are shown. Scale bars, mm. (b) Expression levels of IKKa and were compared between undifferentiated, differentiated NPC and non-cancer group. Scores were determined as described in the Methods. (c) Model depicting the mechanism of -mediated epigenetic silencing of IKKa. In undifferentiated NPC cells, the PRC complex proteins and SUZ are bound to the IKKa promoter where the (filled circles) protein is co-occupied and IKKa transcription is suppressed. In the presence of, is suppressed, which causes a reduction in H3K7 methylation (open circles). This leads to IKKa transcription that then triggers cellular differentiation. Table Expression patterns of IKKa and in consecutive sections from NPC and non-cancer tissues. Group n IKKa ( ) ( þ ) 3 ( ) 3 ( þ ) 4 7 Undifferentiated (64.9%) 6 (3.%) (4.9%) 63 (8.%) Differentiated 3 4 (3.8%) 9 (69.%) (76.9%) 3 (3.%) Non-Ca 9 3 (33.3%) 6 (66.7%) 9 (.%) (.%) P ¼.3 o. IKKa, IkB kinase a; NPC, nasopharyngeal carcinoma. ( ): negative-low, ( þ ): positive-high. Fisher s exact test. NATURE COMMUNICATIONS :366 DOI:.38/ncomms466 & 4 Macmillan Publishers Limited. All rights reserved.

12 NATURE COMMUNICATIONS DOI:.38/ncomms466 Table Correlation between IKKa and levels in human primary NPC and non-cancer specimens. Non-Cancer and NPC tissues samples ( ) 3 ( þ ) 4 7 Total IKKa ( ) 3 4 ( þ ) Total IKKa, IkB kinase a; NPC, nasopharyngeal carcinoma. P ¼., Pearson s w -test. autoimmune inflammation 4,. The p63 transcription factor, which functions as a tumour suppressor, has been found to induce IKKa expression in epithelial development and primary keratinocytes directly or indirectly 44,4. Here, we demonstrate a mechanism for the downregulation of IKKa, which is associated with the poor differentiation status of NPC. Recent studies demonstrate that epigenetic regulation has a crucial function in establishing and maintaining the pattern of gene expression. Polycomb group proteins epigenetically mediate transcriptional silencing, which is involved in the maintenance of embryonic and adult stem cells and is implicated in cancer development 9. Downregulation of IKKa expression occurs in human SCCs of the skin and lung as well as the head and neck 8, Suppression of IKKa is associated with dedifferentiation, invasion and progression of carcinoma. Here we report that suppression of caused a significant increase in IKKa mrna as well as protein in NPC cells. This effect was accompanied by a reduction in the level of H3K7 methylation and an increase in transactivation of the IKKa promoter. In addition, in -silenced CNE cells, the PRC complex proteins and SUZ, as well as, showed lower occupancies at the IKKa promoter. Furthermore, overexpression of an -H694A mutant, which is HMT inactive, resulted in significantly increased IKKa expression. In contrast, R73K, another SET domain mutant of that retains HMT activity, resulted in similar IKKa expression as vector control. Overexpression of -WT, however, significantly decreased IKKa expression. These results establish a mechanism where reduction of IKKa in poorly differentiated CNE cells is a consequence of -mediated epigenetic silencing. These new findings open avenues for differentiation therapy in NPC through targeting the -IKKa axis. A recent study showed that an T487A mutant resulted in enhancement of HMT activity 7. Interestingly, although overexpression of both -WT and -T487A prevented -induced differentiation in CNE cells, we did not observe significantly stronger inhibition of differentiation using - T487A, as compared with that with -WT. We reasoned that the high HMT activity of -WT was sufficient to block induced demethylation within the IKKa promoter, thus retaining cells in undifferentiated status. The objective of traditional chemotherapy or radiation therapy is to directly induce cell death. In contrast, differentiation therapy attempts to reactivate endogenous differentiation programmes in cancer cells to induce tumour cellular maturation and loss of the tumour phenotypes. A majority of NPCs arise from the mucosal epithelium of the nasopharynx, which makes surgical resection a challenging operation. Radiotherapy is the main treatment for this type of cancer, but radiotherapy often induces many undesirable side effects. NPC is a unique type of carcinoma because more than % of the NPCs in Southern China are undifferentiated. This unique characteristic makes NPCs an excellent model to define mechanisms for differentiation therapy. Several earlier studies showed that inhibits NPC cell growth, but the underlying mechanisms are not well understood,. Here, we used as a well-accepted and canonical differentiation therapy agent to demonstrate that it has the capability to induce NPC cell differentiation. It does so by abrogating -mediated epigenetic repression of IKKa. Although additional experiments will be required to further assess the therapeutic efficacy of in NPC patients, our data provide important implications for exploring a novel strategy of differentiation therapy in NPC. In earlier studies, has been reported to act via R/RXR signalling,3. More recently, has also been showed to be involved in epigenetic regulation. For example, in primary blasts from leukaemia patients, PML-Ra recruits PRC and DNMTs to specific target genes, an effect that is reversed by treatment 4. In accordance with these data, here we report that the binding of, SUZ and to the IKKa promoter DNA is reduced significantly following treatment. In addition, decreased protein in NPC cells. The specific mechanism for -reduced expression in NPC cells remains to be investigated. One recent study showed that mir- 4 downregulated by targeting the 3 UTR during -induced differentiation in pluripotent embryonic stem cells. Although mir-6a, mir- and mir-98 have been suggested to be involved in regulation in NPC 6, the mechanism remains poorly defined. Future work should address whether -mediated suppression is associated with microrna regulation. In summary, poor differentiation is a hallmark of solid tumours and is strongly related with uncontrolled growth of tumours and poor prognosis of patients. In this study, we have demonstrated that IKKa plays a pivotal and essential role in NPC differentiation and that -mediated repression of IKKa is a novel epigenetic regulatory mechanism for IKKa reduction in NPC. These data suggest a promising molecular mechanism for treatment of solid tumours and greatly strengthens the case for differentiation therapy in cancer treatment. Methods Cell lines and culture conditions. The NP69 cell line was obtained from professor Wenlin Huang (Sun Yat-sen University, Guangzhou, China). CNE, CNE, HK-, HONE and SUNE cell lines were obtained from Dr Chaonan Qian (Sun Yat-sen University, Guangzhou, China). The immortalized normal human nasopharyngeal epithelial cell line NP69 was maintained in keratinocyte/serum-free medium (Invitrogen). The NPC cell lines CNE, CNE, HK-, HONE and SUNE were maintained in RPMI 64 (Invitrogen) supplemented with % fetal bovine serum (Hyclone). The cells were incubated at 37 C in a humidified chamber containing % CO. Plasmid constructs and transfection. The plasmids encoding human (wild type, WT) and human IKKa were generated by PCR amplification and subcloned into the pbabe-puro expression vector (Invitrogen). mutant constructs (H694A), (R73K) were developed using the QuickChange Site directed mutagenesis kit (Stratagene) according to the manufacturer s instructions. The mutations were chosen based on a previously published study. The plasmids pcdna- (WT) and pcdna- (T487A) were provided by Mien-Chie Hung (University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA) 7,8. Expression plasmids were transfected into cells using Lipofectamine (Invitrogen) according to the manufacturer s instructions. The primers for gene cloning were as follows (Invitrogen): IKKa-F -ATTTGAATTCGCCACCA TGGAGCGGCCCCCGGGGC-3,IKKa-R -ACGCGTCGACTCATTCTGTTA ACCAACTCC-3, -WT-F -AAGGATCCATGGGCCAGACTGGGA AGA-3, -WT-R -GAGTCGACTCAAGGGATTTCCATTTCTC-3, - H694A-F -CAAAATTCGTTTTGCAAATGCTTCGGTAAATCCAAACTGC-3, -H694A-R -GCAGTTTGGATTTACCGAAGCATTTGCAAAACGAATT TTG-3, -R73K-F -CGAAGAGCTGTTTTTTGATTACAAGTACAGCCA GGCTGATGCCC-3, -R73K-R -GGGCATCAGCCTGGCTGTACTTGT AATCAAAAAACAGCTCTTCG-3. Gene knockdown by short-hairpin RNA. Knockdown of genes was performed with the specific shrnas delivered by a lentiviral system purchased from NATURE COMMUNICATIONS :366 DOI:.38/ncomms466 & 4 Macmillan Publishers Limited. All rights reserved.

13 NATURE COMMUNICATIONS DOI:.38/ncomms466 Sigma-Aldrich Corp. according to the instruction manual. In brief, to generate the lentivirus containing specific shrna, 93T cells were co-transfected with. mg pmd.g and 7. mg pspax compatible packaging plasmids and mg of plko. plasmid bearing the specific shrna for 4 h. The cultured medium containing lentivirus was collected and stored at 8 C as aliquots for further use. To deliver the specific shrna construct, approximately % confluent cells were infected with the lentivirus bearing specific shrna in growth medium containing 8 mgml polybrene and incubated at 37 C for 4 h. Afterwards, cells were subcultured and selected with mgml puromycin. The shrna constructs targeting the interested gene and referring to the sequence is: (NM_446.3): TRCN474 (Insert Sequence: -CCGGGCTAGGTTAATTGGGACCAAACTCGAGTTTGGT CCCAATTAACCTAGCTTTTTG-3 ). The MISSION Non-Target shrna control vector SHC (Insert Sequence: -CCGGCAACAAGATGAAGAGCACCAAC TCGAGTTGGTGCTCTTCATCTTGTTGTTTTT-3 ) that contains a shrna insert that does not target human or mouse genes was used as a negative control. Antibodies. The following antibodies were used: involucrin (abcam ab4), CK8, CK3, vimentin, IkBa and p6 (Epitomics 3-, 73-, 77-, 3- and 46-, respectively), GAPDH and b-actin (proteintech 64--Ig and 68- -Ig), IKKa (Santa Cruz sc-78), cyclin D, cyclin E, and Ki67 (BD 48, 6, 6666 and 6969, respectively), (Millipore 7-449), Histone H3, E-cadherin and SUZ (Cell Signaling Technology 4499, 3 and 3737), Horseradish peroxidase-conjugated goat anti-mouse and goat anti-rabbit IgG (Pierce, 3 and 6), Alexa-488 and Alexa-46 (Invitrogen Molecular Probes, A8 and A36). Real-time RT PCR. Total RNA was extracted by using TRIzol reagent (Invitrogen), which was used to generate cdna by using SuperScript III RT (Invitrogen) with an oligo-dt primer. Real-time RT-PCR was performed using Platinum SYBR Green qpcr SuperMix (Invitrogen) as recommended by the manufacturer. The primers used are listed in Supplementary Table 3. GAPDH was used as the internal control. Small interfering RNA transfection. RNA oligonucleotides duplexes to target genes were synthesized. Cells were seeded onto six-well plates at 6 h before transfection. In each well, nm or nm of sirna and ml of Lipofectamine were added to Opti-MEM, mixed and then added to the cells. After transfection of sirnas for 4 or 48 h, RNAi efficiency was determined by real-time RT-PCR and western blot. The sirnas were purchased from GenePharma company and the sequences were as follows: si-- -AAGAGGTTCAGACG AGCTGAT-3, si-- -AAGACTCTGAATGCAGTTGCT-3, si-ikka- -GCAGGCUCUUUCAGGGACATT-3, si-ikka- -CAAAGAAGCUGACAA UACU-3. Immunofluorescence staining. Cells were fixed in % paraformaldehyde at room temperature for min and permeabilized in.% Triton X- in PBS for min. Slides were incubated with the primary antibody for 6 min. The following antibodies were used: E-cadherin (: dilution), vimentin (: dilution), Ki67 (: dilution) and p6 (: dilution). Immune complexes were stained with the secondary antibody conjugated to Alexa-488 or Alexa-46 (Molecular Probes, Invitrogen, : dilution). Nuclei were stained with DAPI (Sigma-Aldrich) and viewed with an Olympus IX7 microscope. SA-b-Gal staining. Cultured cells were washed in PBS and SA-b-Gal activity was detected using senescence b-gal staining kit (Sigma-Aldrich) according to the manufacturer s directions. Air liquid interface 3D cultured assay. Cells were plated on top of a Matrigel gel containing fibroblasts (HLF cells, ATCC). After 3 days, cells were exposed to air and fed only through bottom surface, which remained in contact with the culture medium. After 4 days of culture, gels were fixed in formalin, embedded into paraffin, sectioned and subjected to immunohistochemical staining. The following antibodies were used: involucrin (: dilution), CK3 (:8 dilution) and vimentin (: dilution). Cell cycle analysis. Single-cell suspensions were fixed in ice-cold 7% ethanol for 3 min, labelled with ml propidium iodide ( mgml, Sigma-Aldrich) for at least min in the dark at 37 C and analysed directly on a Beckon Dickinson FACScan flow cytometer (Oxford, UK). Colony formation assay. Approximately, cells were seeded into six-well plates in triplicate and incubated for 4 days. Colonies were stained with crystal violet and counted. Cell lysis and western blot analysis. Cells were lysed on ice in RIPA buffer. Protein concentration was determined by using the Bradford dye method. Equal amounts of cell extracts were subjected to electrophoresis in % gradient SDS- PAGE gels and then transferred to nitrocellulose membranes (Millipore) for antibody blotting. The following antibodies were used: involucrin (:, dilution), CK8 (:, dilution), CK3 (:, dilution), GAPDH (:, dilution), IKKa (: dilution), E-cadherin (:, dilution), vimentin (:, dilution), Cyclin D (:, dilution), Cyclin E (:, dilution), IkBa (:, dilution), (:, dilution), (:, dilution), Histone H3 (:, dilution) and b-actin (:, dilution). Horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG (Pierce, :, dilution) was used as a secondary antibody. Proteins were visualized with a Super Signal West Pico chemiluminescence kit (Pierce). Uncropped scans of the most important blots used in the manuscript were shown in Supplementary Fig.. Chromatin Immunoprecipitation. CHIP assays were performed according to the manufacturer s protocol (EZ-Magna ChIP kit, Millipore). Antibodies used for CHIP assay included, SUZ (Cell Signaling Technology) and (Millipore). For sequential CHIP (Re-CHIP), the first elute immunoprecipitated by anti- was further immunoprecipitated by anti- and IgG antibodies. The percentage of bound DNA was quantified against the original DNA input. The primers used for the amplification of the precipitated DNA fragments were (position: 89/ 76 within IKKa promoter): -AATACAGGAGAGAC TGGGCTGCTTT-3 and -GGGAGGGCTGAACGGAACCACAATG-3. Immunohistochemical staining and statistical analysis. The paraffin-embedded tissue blocks were sectioned for immunohistochemical staining 7. Paraffinembedded tissue specimens were sectioned, deparaffinized in xylene and rehydrated. Antigenic retrieval was processed with sodium citrate. The sections were then incubated in H O (3%) for min, blocked in % bovine serum albumin for 6 min and incubated with an anti- antibody (:4 dilution) and anti-ikka antibody (: dilution) at 4 C overnight. After incubation with the secondary antibody for 6 min, specimens were incubated with H O -diaminobenzidine until the desired stain intensity was developed. Sections were then counterstained with haematoxylin, dehydrated and mounted. Staining intensity and extent of and IKKa expression were graded as follows: negative (score ), bordering (score ), weak (score ), moderate (score 3) and strong (score 4). Extent of staining was also grouped into quintiles according to the percentage of high-staining cells in the field: negative (score ), r% (score ), 6 % (score ), 7% (score 3) and 76 % (score 4). All immunohistochemical staining was evaluated and scored by at least two independent pathologists. Luciferase reporter assay. Cells were plated at a density of /well in 4-well plates. After 4 h, cells were transfected with IKKa promoter-driven luciferase constructs (pgl3-ikka, kindly provide by Professor Melino G, 4 ) or control (pgl3-basic) luciferase constructs using Lipofectamine according to the manufacturer s instruction. Twenty-four hours after transfection, cells were harvested. Firefly and Renilla luciferase activities were measured using a dual luciferase kit (Promega). The firefly luciferase data for each sample were normalized based on transfection efficiency as measured by Renilla luciferase activity. Mouse xenograft assay. Four- to six-week-old BALB/c athymic nude mice (nu/ nu, male and female) were used with each experimental group consisting of 9 mice. In brief, mice were subcutaneously injected with cells (3 B 7 / mouse). For assay using, days after injection, the group received ( mg kg (d) ) and the control group received equal volume of water. Tumours were measured perpendicular dimensions using calipers. Volumes were estimated using the formula (a b)/, where a is the shorter of the two dimensions and b is the longer one 8.TheP-value is a comparison between the control and treatment groups at the final time point when the tumours were removed from the mice. Care of experimental animals was approved by Institutional Animal Care and Use Committee of Sun-Yat-Sen University and in accordance with national guidelines for the care and maintenance of laboratory animals. Tissue samples. All clinical specimens used for RNA-Seq, real-time PCR, western blot and immunohistochemical analysis were collected from NPC patients at Sun Yat-sen University Cancer Center. Patients consent and approval from Sun Yatsen University Cancer Center Institute Research Ethics Committee were obtained for the use of these clinical materials. RNA-seq and accession number. RNA-Seq, data generation and normalization were performed on the Illumina Cluster Station and Illumina HiSeq System at BGI and Guangzhou Genedenovo Corp. The RNA-seq data described herein have been deposited in the National Center for Biotechnology Information Sequence Read Archive ( under accession no. S64. Methylation-specific PCR. Bisulphite DNA modification was performed according to the manufacturer s instructions (Active Motif). Primer sequences of NATURE COMMUNICATIONS :366 DOI:.38/ncomms & 4 Macmillan Publishers Limited. All rights reserved.