Downregulation of SAFB Sustains the NF-kB Pathway by...

Biology of Human Tumors

Downregulation of SAFB Sustains the NF-kBPathway by Targeting TAK1 during theProgression of Colorectal CancerHong-Li Jiao1,2,3, Ya-Ping Ye1,2,3, Run-Wei Yang1,2,3, Hui-Ying Sun1,2,3,Shu-Yang Wang1,2,3, Yong-Xia Wang1,2,3, Zhi-Yuan Xiao1,2,3, Liu-Qing He1,2,3,Juan-Juan Cai1,2,3, Wen-Ting Wei1,2,3, Yan-Ru Chen1,2,3, Chun-Cai Gu1,2,3,Yue-Long Cai1,2,3, Yun-Teng Hu1,2,3, Qiu-Hua Lai1,2,3, Jun-Feng Qiu1,2,3,Li Liang1,2,3, Guang-Wen Cao4,Wen-Ting Liao1,2,3, and Yan-Qing Ding1,2,3

Abstract

Purpose: To investigate the role and the underlyingmechanismof scaffold attachment factor B (SAFB) in the progression ofcolorectal cancer (CRC).

Experimental Design: SAFB expression was analyzed in theCancer Outlier Profile Analysis of Oncomine and in 175 paraffin-embedded archived CRC tissues. Gene Ontology analyses wereperformed to explore themechanismof SAFB inCRCprogression.Western blot, RT-PCR, luciferase assay, and chromatin immuno-precipitation (ChIP) were used to detect the regulation of trans-forming growth factor-b–activated kinase 1 (TAK1) and NF-kBsignaling by SAFB. The role of SAFB in invasion, metastasis, andangiogenesis was investigated using in vitro and in vivo assays. Therelationship between SAFB and TAK1 was analyzed in CRCtissues.

Results: SAFB was downregulated in CRC tissues, and lowexpression of SAFB was significantly associated with an aggres-

sive phenotype and poorer survival of CRC patients. Thedownregulation of SAFB activated NF-kB signaling by target-ing the TAK1 promoter. Ectopic expression of SAFB inhibitedthe development of aggressive features and metastasis ofCRC cells both in vitro and in vivo. The overexpression of TAK1could rescue the aggressive features in SAFB-overexpressedcells. Furthermore, the expression of SAFB in CRC tissues wasnegatively correlated with the expression of TAK1- and NF-kB–related genes.

Conclusions:Our results show that SAFB regulated the activityof NF-kB signaling in CRC by targeting TAK1. This novel mech-anismprovides a comprehensive understanding of both SAFB andthe NF-kB signaling pathway in the progression of CRC andindicates that the SAFB–TAK1–NF-kB axis is a potential targetfor early therapeutic intervention in CRC progression. Clin CancerRes; 23(22); 7108–18. �2017 AACR.

IntroductionColorectal cancer (CRC), one of the most common malig-

nancies worldwide, is associated with high morbidity andmortality rates (1). Metastasis and recurrence, which increasethe incidence of complications and treatment failure, are the

major causes of CRC-related death (2). Therefore, an earlydiagnosis of CRC and effective interventions are of vital impor-tance. However, early diagnosis is infrequent, and the progno-sis is often difficult to determine despite advances in clinicalmanagement, histopathologic findings, and molecular markers(3). Thus, further studies of molecular markers that are able toassist with early diagnosis and treatment of metastatic CRC areurgently needed (4).

Scaffold attachment factor B (SAFB) is a nuclear matrixprotein that binds to the scaffold or the matrix attachmentregions of DNA elements (5). SAFB was initially found to inhibitthe activity of the heat shock protein 27 (HSP27) promoter bybinding to its TATA box in human breast cancer cells (6).Structurally, SAFB contains a DNA binding region in the SAFbox at its N-terminus, an RNA recognition motif, a nuclearlocalization signal at its center, and Glu/Arg-, Ser/Lys-, andGly-rich protein interaction regions at its C-terminus (7). Func-tionally, SAFB is localized within the nucleus where it partici-pates primarily in transcriptional repression by preventing theassembly of transcriptional complexes (7, 8). Genetic deletionof SAFB in embryonic fibroblasts causes upregulation of T-box2 and downregulation of p19ARF, suggesting that SAFB is impor-tant in the processes of cellular immortalization and transfor-mation (9). Additionally, SAFB has been reported to repress the

1Department of Pathology, Nanfang Hospital, Southern Medical University,Guangzhou, China. 2Department of Pathology, School of Basic Medical Sciences,Southern Medical University, Guangzhou, China. 3Guangdong Provincial KeyLaboratory of Molecular Tumor Pathology, Guangzhou, China. 4Department ofEpidemiology, Second Military Medical University, Shanghai, China.

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

H.-L. Jiao and Y.-P. Ye contributed equally to this article.

Corresponding Authors: Wen-Ting Liao, Department of Pathology, NanfangHospital and School of Basic Medical Sciences, Southern Medical University,Guangzhou 510515, China. Phone: 86-20-6164-8224; Fax: 86-20-6164-8224;E-mail: [email protected]: Yan-Qing Ding: Department of Pathology,Nanfang Hospital and School of Basic Medical Sciences, Southern MedicalUniversity, Guangzhou 510515, China. Phone: 86-20-6164-2148; Fax: 86-20-6164-2148; E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-17-0747

�2017 American Association for Cancer Research.

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human xanthine oxidoreductase gene (hXOR; ref. 10), which isinvolved in inflammation and tumorigenesis and is a source ofreactive oxygen species (11, 12). Low expression of SAFB isrelated to the metastasis of prostate cancer and poorer outcomesin patients with breast cancer, which is partly due to theunrestrained activities of the androgen receptor and estrogenreceptor a, respectively (13, 14). Our preliminary work and apublished microarray analysis found that SAFB expression wasdownregulated in CRC. However, the underlying mechanismsby which SAFB regulates the progression of CRC are largelyunknown.

The NF-kB signaling pathway has been shown to promotethe progression of different cancers, including breast cancer,prostate cancer, pancreatic cancer, and CRC (15–18). In CRC,cross-talk occurs between NF-kB and many other importantsignaling pathways, such as the RAS, AKT, and WNT signalingpathways. Together, these pathways form a complex networkthat regulates the progression of CRC (15, 19). Our prelimi-nary work and a published microarray analysis showed thatthe downregulation of SAFB expression increased the activa-tion of NF-kB signaling in CRC, but the molecular mechanismsby which SAFB regulates the activation of NF-kB signalingare unclear.

Here, we sought to investigate the potential role of SAFB in thetumorigenesis, invasion and metastasis of CRC and to discovernewmolecular mechanisms of how SAFB regulates the activationof NF-kB signaling. Our work may help to increase our under-standing of the precise role of SAFB in the progression of CRC,whichmay assist in the development of new therapeutic strategiesfor CRC.

Materials and MethodsPatients and tissue specimens

This study was conducted on 175 paraffin-embedded colo-rectal cancer samples, which were histopathologically andclinically diagnosed at Nanfang Hospital, Southern Medical

University. Prior patient consent and approval were obtainedfrom the Institutional Research Ethics Committee. Clinicalinformation regarding the samples is summarized in Supple-mentary Table S1 and in the Supplementary Materials andMethods. Ten CRC specimens and matched adjacent noncan-cerous mucosa tissues were frozen and stored in liquid nitrogenuntil further use.

Cell cultureThe human CRC cell lines SW480, Colo205, Ls174t, HT29,

HCT116, LoVo, HCT15, and SW620 were purchased from Amer-ican Type Culture Collection. SW620 and HT29 cells were cul-tured in DMEM medium (Gibco) with 10% FBS (Gibco) at37�C�C with 5% CO2. SW480, Colo205, Ls174t, HCT116, LoVo,and HCT15 cells were cultured in RPMI 1640 medium (Gibco)with 10% FBS (Gibco) at 37�C with 5% CO2.

Real-time quantitative PCR, Western blot, and IHCThe real-time quantitative PCR (RT-PCR), Western blotting

(WB) and IHC, were conducted according to previouslydescribed methods (20). Further details are provided in theSupplementary Materials and Methods section.

Transwell and luciferase assaysThe Transwell and luciferase assays were performed accord-

ing to previously described methods (21). Further details areprovided in the Supplementary Materials and Methods section.

Chicken chorioallantoic membrane assayA chicken chorioallantoic membrane (CAM) assay was per-

formed on the sixth day of development of fertilized chickeneggs (22). Further details are provided in the SupplementaryMaterials and Methods section.

Human umbilical vein endothelial cell tube formation assayFirst, 200 mL Matrigel was pipetted into each well of 24-well

plates and was allowed to polymerize for 30 minutes at 37�C.Human umbilical vein endothelial cells (HUVEC; 5 � 104) in200 mL of conditioned medium were added to each well andincubated at 37�C in 5% CO2 for 8 hours. Images were obtainedunder a 100 � bright-field microscope and the capillary tubeswere quantified by counting length.

Chromatin immunoprecipitationCells (2 � 106) in a 10-cm culture dish were treated with 1%

formaldehyde to cross-link proteins to DNA. The cell lysateswere subjected to ultrasound to shear the DNA into fragmentsbetween 200 and 1,000 bps. Equal aliquots of chromatinsupernatants were respectively incubated with 1 mg of anti-SAFB antibody (Abcam) and anti-IgG antibody (Millipore)overnight at 4�C with rotation. After the cross-links of theprotein/DNA complexes were reversed to free the DNA, RT-PCRwas performed. Further details are provided in the Supplemen-tary Materials and Methods section.

Orthotopic mouse metastatic modelA surgical orthotopic implantation mouse model of CRC was

established as previously described (23). Cells (2 � 106 permouse) were subcutaneously injected into the right dorsalflank of female BALB/c athymic nude mice (4–6 weeks of age,18–20 g), which were obtained from the Animal Center of

Translational Relevance

Scaffold attachment factor B (SAFB) has recently beenimplicated in the carcinogenesis and metastasis of prostatecancer and breast cancer. However, the biological functionsand underlying mechanisms of SAFB in the regulation of theprogression of colorectal cancer (CRC) are largely unknown.Our results showed that SAFB was downregulated in CRCtissues. Low expression of SAFB was significantly associatedwith poorer survival of patients with CRC. In addition, thedownregulation of SAFB enhanced the activity of the NF-kBsignaling pathway by relieving the transcriptional repressionof transforming growth factor-b–activated kinase 1 (TAK1).Exogenous expression of SAFB inhibited the aggressive fea-tures and metastases of CRC cells both in vitro and in vivo.Moreover, the overexpression of TAK1 in cells that overex-pressed SAFB restored the emergence of aggressive cellularfeatures. Taken together, we highlight a novel mechanism ofthe SAFB–TAK1–NF-kB axis in the regulation of CRC progres-sion, andourworkmayhelp promote the development of newtherapeutic strategies for CRC.

Downregulation of SAFB Promotes CRC Progression

www.aacrjournals.org Clin Cancer Res; 23(22) November 15, 2017 7109




Southern Medical University, Guangzhou, China. Two weekslater, the animals were sacrificed, and the tumors were excised.A portion of the tumor was fixed in 10% formaldehyde andparaffin-embedded, and then 5-mm sections were cut and sub-jected to IHC using an anti-CD31 antibody (Dako); othersections were stained with hematoxylin and eosin (H&E). Themicro-vessel density (MVD) was quantified by counting theproportion of CD31-positive cells. Another portion of tumorwas divided into small pieces approximately 1 mm in diameter.Surgical orthotopic implantation of the CRC tumor fragmentsonto the mesentery of the cecum was performed in nude miceafter anesthesia was administered. The mice were euthanized70 days after surgery, the individual organs were excised, andmetastases were observed by histological analysis.

5Z-7-OXO inhibitor treatmentWe treated SW480 cells with a TAK1 inhibitor (5Z-7-oxozeae-

nol, Tocris Cookson Ltd.) at a concentration of 0.5 mmol/L. Forin vivo treatment, mice received a daily oral dose of 100 mg/kg of5Z-7-OXO after the orthotopic implantation surgery.

Statistical analysisAll statistical analyses were performed using SPSS20.0 for

Windows. Statistical tests included the Fisher exact test, log-ranktest, c2 test, and Student t test. Bivariate correlations betweenstudy variables were calculated by the Spearman rank correlationcoefficients. Survival curves were plotted by the Kaplan–Meiermethod and were compared by the log-rank test. Data representthe mean � SD. P < 0.05 was considered statistically significant.

ResultsThe downregulation of SAFB was correlated with advancedprogression and poorer prognosis of CRC

To investigate the role of SAFB in CRC progression, we firstexamined the expression of SAFB in the public database Onco-mine (www.oncomine.com), which provides a Cancer OutlierProfile Analysis (COPA) for 20 different types of cancer (Supple-mentary Fig. S1A, left). Among 176 unique analyses of statisticalsignificance, 124 data sets showed low expression of SAFB (Sup-plementary Fig. S1A, left), which was also demonstrated in 8(72%) of 11 CRC data sets (Supplementary Fig. S1A, right). Geneset enrichment analysis (GSEA; ref. 24) using expression datafrom the primary colorectal cancers GSE13294 and GSE35896suggested that low expression of SAFB was associated with theupregulation of CRC-related gene sets (Supplementary Fig. S1B).Wemeasured the expression levels of SAFB protein andmRNA in10 pairs of primary CRC tissues and matched adjacent normaltissues. Consistent with these results, a relatively lower expressionof SAFB was found in CRC tissues compared with the matchedadjacent normal mucosa (Fig. 1A and B). Furthermore, theexpression of SAFBwasmeasured in 175 paraffin-embedded CRCtissues by IHC. Analyses of the results indicated that a lowerexpression of SAFB was significantly correlated with poorer dif-ferentiation, more advanced Duke's stage, increased TNM stage,and poorer survival of patients (Fig. 1C and D; SupplementaryTable S1). Kaplan–Meier survival analyses of five published CRCdata sets (GSE39582, GSE17538, GSE14333, GSE17536, andGSE17537) revealed the same relationship (Fig. 1D; Supplemen-tary Fig. S1C). Taken together, these results suggest that SAFBmaybe a prognostic biomarker for CRC.

Downregulation of SAFB enhanced the activity of NF-kBpathway signaling

Analyses of SAFB-regulated gene signatures via GSEA andGO analyses revealed that lower expression of SAFB was pos-itively correlated with an enrichment of NF-kB signaling path-way gene signatures (GSE13294, GSE35896, and GSE15548;Fig. 2A; Supplementary Fig. S2A). Genes upregulated by SAFB-silencing showed enrichment for the GO terms "double-strand-ed DNA binding," "transcription factor binding," and "NF-kBbinding" (Supplementary Fig. S2B). These results suggestedthat SAFB contributes to the activation of NF-kB signalingthrough DNA binding. To further validate these results, weestablished stable SAFB-overexpressing and SAFB-knockdownCRC cell lines (Fig. 2B; Supplementary Fig. S2C–S2E). Asshown in Fig. 2C, the overexpression of SAFB significantlyreduced NF-kB luciferase activity, but the knockdown of SAFBenhanced NF-kB luciferase activity. The same trend wasobserved for phosphorylated levels of IKKb, p65 (Ser 536),and IkBa (Fig. 2D). In addition, the nuclear translocation ofp65 was inhibited by SAFB overexpression (Fig. 2E; Supple-mentary Fig. S2F and S2G). Furthermore, an analysis of anumber of NF-kB downstream genes revealed that the silencingof SAFB significantly upregulated the expression of NF-kBdownstream genes (Fig. 2F). These results indicate that down-regulation of SAFB enhances the activity of NF-kB signaling.

Downregulation of SAFB enhanced the activity of the NF-kBsignaling pathway by relieving transcriptional repressionof TAK1

To further test the mechanism of how SAFB inhibits NF-kBsignaling, we quantified the levels of phosphorylated and totalprotein involved in this pathway. It was found that SAFB regulatedthe phosphorylationof IKKb andp65, but it did not affect the totalexpression levels of these proteins (Fig. 2E). We then tested howSAFB affected proteins upstream of the NF-kB signaling pathway,including TAK1, RIP, FLOT, and CYLD. RT-PCR results showedthat TAK1, RIP, and FLOT were affected by SAFB downregulation(Figs. 2F and 3A). WB showed that only TAK1 was significantlyaffected by SAFB downregulation (Fig. 3B; SupplementaryFig. S3A). Thus, we concluded that TAK1 might be a key targetof SAFB. To verify this hypothesis, we first determined the expres-sion of TAK1 from information in the public database. The resultsshowed that the expression of TAK1 in colorectal cancer wassignificantly higher than that in normal intestinal mucosa (Sup-plementary Fig. S3B and S3C). Next, we verified this hypothesisthrough reexpression of TAK1 in SAFB-overexpressing cells and bythe knockdown or inhibition of TAK1 in SAFB-silenced cells.NF-kB luciferase reporter assays revealed that the reexpression ofTAK1 rescued the activation of NF-kB signaling (Fig. 3C). Con-versely, the knockdown of TAK1 or the treatment of cells with theTAK1 inhibitor 5Z-7-oxozeaenol (5Z-7-OXO) significantly inhib-ited the NF-kB signaling pathway (Fig. 3D). Chromatin immuno-precipitation (ChIP) assays revealed that endogenous SAFB proteinwas bound to the first E-box (P1) region of the TAK1 promoter(Fig. 3E). Moreover, SAFB inhibition activated the wild-type TAK1promoter but did not affect the mutant promoter (Fig. 3F).

Downregulation of SAFB promoted the aggressiveness of CRCcells through the SAFB–TAK1–NF-kB axis

To analyze the functional relationship between SAFB andTAK1, we overexpressed TAK1 in SAFB-overexpressing cells,

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Figure 1.

Downregulation of SAFB was correlated with advanced progression and poor prognosis in CRC. A, WB for SAFB expression in 10 human CRC tissues (T) andmatched adjacent normal tissues (N) from the same patient. a-Tubulin was used as a loading control. Expression levels were normalized to those ofa-tubulin (left). Ratio (N/T) of SAFB mRNA expression in 10 primary CRC tissues and adjacent normal tissues from the same patient, as determined by RT-PCR(right). Expression levels were normalized to those of GAPDH (right). Error bars represent the mean � SD calculated from 3 parallel experiments.B, IHC staining indicated that SAFB protein expression was reduced in human CRC (well to poorly differentiated) compared with normal intestinalepithelium. C, Statistical analyses of the average MOD of SAFB staining between normal intestinal tissues and CRC specimens with different degrees ofdifferentiation, T classification, N classification, and M classification. D, Kaplan–Meier analyses of the outcome of CRC patients with low versus highexpression of SAFB according to our data (left), the GSE39582 data sets (middle), and the GSE17538 data sets (right) (P < 0.05, log-rank test).�� , P < 0.01.






Figure 2.

Downregulation of SAFB enhanced the activity of NF-kB pathway signaling. A, GSEA plot showed that low expression of SAFB was positively correlatedwith the NF-kB pathway (BIOCARTA_NFKB_PATHWAY, GILMORE_NFKB_PATHWAY, SCHOEN_NFKB_SIGNALING) in published CRC patient geneexpression profiles (NCBI/GEO/GSE13294, n ¼ 155, and GSE35896, n ¼ 62) and in published gene expression profiles of MCF-7 breast cancer cells (GSE15548,n ¼ 6). B, Ectopic expression of SAFB mRNA in HCT116, SW620, SW480, and HCT15 CRC cell lines as detected by RT-PCR. The mRNA expression levelswere normalized to those of GAPDH. Error bars represent the mean � SD calculated from 3 parallel experiments. �� , P < 0.01. C, NF-kB luciferasereporter activity was analyzed in the indicated cells. Error bars represent the mean � SD of 3 independent experiments, �� P < 0.01. D, WB analyses of p-p65,total p65, p-IkBa, and total IkBa, p-IKKb, and total IKKa/b expression in the indicated cells. a-Tubulin was used as a loading control. E, WB fornuclear p65 and cytoplasmic p65 in the indicated cells. LaminB1 was used as a nuclear loading control, and a-tubulin was used as a cytoplasmicloading control. F, RT-PCR analyses indicated an apparent overlap between NF-kB–dependent gene expression and SAFB-regulated gene expression.The pseudo colors represent the intensity scale for SAFB-shRNA versus the scrambled vector, generated by a log2 transformation.

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knocked down TAK1 using two TAK1 shRNAs, and suppressedTAK1 using a TAK1 inhibitor (5Z-7-OXO). We found thatoverexpression of TAK1 in SAFB-overexpressing cells signifi-cantly rescued NF-kB signaling activity as well as the invasive

and metastatic phenotypes of the cells (Fig. 4A and C, top;Supplementary Fig. S4A). Conversely, inhibition of TAK1 byshRNAs or the inhibitor 5Z-7-OXO attenuated NF-kB signalingactivity as well as the invasive and metastatic phenotypes of

Figure 3.

Downregulation of SAFB enhanced the activity of the NF-kB signaling pathway by relieving transcriptional repression of TAK1. A, Expression of TAK1 mRNAin the indicated cells. Expression levels were normalized to those of GAPDH. B, WB analyses of TAK1 and p-p65 in the indicated cells. a-Tubulin wasused as a loading control. C and D, NF-kB luciferase reporter activity was analyzed (top), and WB was used to detect TAK1 expression in the indicated cells(bottom). a-Tubulin was used as a loading control. E, Schematic depiction of the TAK1 promoter with several SAFB binding sites (blue rectangles), asindicated, and the SAFB binding motif in the P1 proximal promoter and its mutant containing altered nucleotides in P1 (top). ChIP analysis of SAFB bindingto the TAK1 promoter in SW480 cells. Primers against the �524 to �438 base pairs in the promoter region (P1) showed significant enrichment afternormalization to the GAPDH control (bottom). RT-PCR experiments were performed. F, Relative expression of a WT TAK1 promoter–driven luciferasereporter in vector control or SAFB-knockdown CRC cells (left) and the relative expression of WT or MUT TAK1 promoter–driven luciferase reporters inSAFB-knockdown CRC cells (right). Error bars represent the mean � SD of 3 independent experiments; � , P < 0.05; �� , P < 0.01.






SAFB-silenced CRC cells (Fig. 4B and C, bottom; Supplemen-tary Fig. S4B).

The tubule formation and chicken CAM assays revealed thatoverexpression of SAFB strongly inhibited the formation oftubules by HUVECs and inhibited angiogenesis in CAM; bothof these effects were increased after SAFB was silenced (Fig. 4Dand E; Supplementary Fig. S4C–S4F). Additionally, TAK1reversed the effects of SAFB on tubule formation and angio-genesis (Fig. 4D and E; Supplementary Fig. S4C–S4F), suggest-ing that SAFBmodulates angiogenesis through the regulation ofTAK1 in CRC. Taken together, TAK1 and SAFB are involved in

the regulation of NF-kB signaling and the development ofaggressive cellular features of CRC.

Upregulation of SAFB inhibited metastasis and angiogenesisin CRC through the SAFB–TAK1–NF-kB axis

Next, exogenous SAFB was overexpressed in cells to test theeffects of SAFB upregulation on metastasis and angiogenesisin CRC through the SAFB–TAK1–NF-kB axis. The overexpres-sion of SAFB in HCT116 cells decreased the number oftumors along the intestines and the frequency of liver meta-stasis (Fig. 5A and C, left; Supplementary Fig. S5A). SAFB

Figure 4.

Downregulation of SAFB contributed to the aggressive features of CRC cells through the SAFB-TAK1-NF-kB axis. A, WB for nuclear p65 and totalp65 expression in the indicated cells. LaminB1 was used as the nuclear loading control. B, WB for nuclear p65 and total p65 expression in the indicated cellswith different treatments. LaminB1 served as a nuclear loading control whereas a-tubulin served as a cytoplasmic loading control. C, The numbers ofmigrating CRC cells were evaluated by the Matrigel-coated Boyden chamber invasion assay. D, HUVEC tube formation after stimulation withconditioned media from the indicated cells. E, Representative images of the blood vessels formed in the CAM assay after stimulation withconditioned media from the indicated cells; �� , P < 0.01.

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overexpression also extended the overall survival time of nudemice injected with the CRC cell lines (Fig. 5C, right). Incontrast, the overexpression of TAK1 significantly increased thefrequency of liver metastases (Fig. 5A and C, left; Supplemen-tary Fig. S5A) and shortened the overall survival time of thenude mice (Fig. 5C, right). Conversely, inhibition of TAK1 byshRNAs or the inhibitor 5Z-7-OXO attenuated the metastaticpotential of SAFB-silenced SW480 cells (Fig. 5B and D, left;Supplementary Fig. S5B), while the overall survival of the micewas extended (Fig. 5D, right). Additionally, SAFB-overexpres-sing tumors had a higher micro-vascular density (indicated byCD31-positive cells) than control tumors, while SAFB-silenced

tumors had a lower micro-vascular density compared withcontrols. Micro-vascular density was increased by the reexpres-sion of TAK1 and was decreased by TAK1 knockdown (Fig. 5Eand F; Supplementary Fig. S5C and S5D).

Expression of SAFB in CRC tissue was negatively correlatedwith that of TAK1 and NF-kB–related genes

Finally, we examined whether activation of the SAFB–TAK1–NF-kB axis identified in our CRC cell models is also evident inclinical CRC tissues. As shown in Supplementary Fig. S6A,correlation studies using the public database revealed a nega-tive correlation between SAFB and TAK1 in the GSE17538

Figure 5.

Upregulation of SAFB inhibited metastasis and angiogenesis in CRC through the SAFB–TAK1–NF-kB axis. In the orthotropic transplantation assay in thececum, mice were orthotopically transplanted with the indicated cells (n ¼ 10 in each group). A and B, The representative gross images of theintestines and livers from different experimental groups are shown. Sections of the liver were stained with H&E. Arrows indicate the metastases in theintestines and livers. C and D, Box-scatter plots show the number of metastatic nodules in the liver as observed in each group (left) and the overallsurvival time of each group (log-rank test, P < 0.05) (right). E and F, H&E (top) and IHC (bottom) staining illustrate angiogenesis in the nude mice. The microvessel density was evaluated according to CD31 positivity. Error bars represent the mean � SD of 3 independent experiments; � , P < 0.05; �� , P < 0.01.






Figure 6.

Expression of SAFB in CRC tissue was negatively correlated with that of TAK1 and NF-kB–related genes. A, The SAFB expression level was negativelyassociated with TAK1 expression in 78 CRC and 2 normal intestinal epithelium tissue arrays. Three representative cases are shown. Original magnification,�200. Correlation analysis of the average MOD of SAFB and TAK1 staining in 80 specimens. B, Kaplan–Meier analysis of the prognostic significanceof the expression of TAK1 in patients with CRC from the published gene expression profile GSE17538 (log-rank test, P < 0.05). C, SAFB and TAK1,MMP9, IL6, VEGF, and IL8 mRNA expression in 10 fresh human CRC samples. Correlation analysis of the mRNA expression of SAFB and TAK1 (D), andMMP9, IL6, VEGF, and IL8 (E) in 10 fresh human CRC samples. Error bars represent the mean � SD of 3 parallel experiments. F, Model: SAFBdownregulation induces TAK1 expression and activates the NF-kB signaling pathway, ultimately leading to an aggressive CRC phenotype.

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(r¼�0.171, P¼ 0.023) and GSE39582 data sets (r¼�0.169, P< 0.001). Furthermore, an analysis of 78 CRC tissue chipspecimens by IHC revealed that the expression of SAFB wasinversely correlated with that of TAK1 (Fig. 6A, r ¼ �0.437, P <0.001 and Supplementary Fig. S6B). Moreover, cohort studydata from the GEO database revealed that both low expressionof SAFB and high expression of TAK1 were associated withpoorer survival of patients (GSE17538, n ¼ 177, P ¼ 0.022 andP ¼ 0.020 for SAFB and TAK1, respectively; Fig. 1D, right;Fig. 6B). SAFB was consistently negatively correlated with thelevels of TAK1 mRNA and protein in 10 cases of clinical CRCtissues (Fig. 6C and D, r ¼ �0.671, P ¼ 0.033 and Supple-mentary Fig. S6C). In addition, SAFB mRNA levels were sig-nificantly correlated with TAK1 mRNA levels in 40 CRC biop-sies from Fig. 1B (Supplementary Fig. S6D; r ¼ �0.585, P <0.001). Moreover, in the same 10 cases described in Fig. 6C, themRNA levels of SAFB were also negatively correlated with thoseof MMP9, IL6, VEGF, and IL8 (Fig. 6E, r ¼ �0.743, P ¼ 0.014;r ¼ �0.723, P ¼ 0.018; r ¼ �0.724, P ¼ 0.018; and r ¼ �0.637,P ¼ 0.047, respectively). These data further support the notionthat the downregulation of SAFB in CRC induces TAK1 expres-sion and thus activates the NF-kB signaling pathway, promot-ing the expression of MMP9, IL6, IL8, and VEGF-C; this ulti-mately leads to poorer CRC outcomes (Fig. 6F).

DiscussionCRC progression is a multistep process that requires the accu-

mulation of a driver mutation and abnormal expression of manymarkers that are involved in signal transduction pathways (25).This study revealed that decreased expression of SAFB was signif-icantly associated with aggressive cellular characteristics of CRC(e.g., poor cellular differentiation, advanced Dukes stage,advanced TNM stage) and with a poorer outcome of patients,suggesting that SAFB might be a tumor suppressor and a prog-nosticmarker of CRCprogression. Consistent with our results, thedownregulation of SAFB has been reported to be responsible forthe metastasis of prostate cancer and poorer outcomes in patientswith breast cancer (13, 14).

As a nuclearmatrix proteinwithmultiple domains, SAFB servesas a transcription factor that regulates the expression of targetgenes and the activity of many signaling pathways that playimportant roles in various cellular processes (7, 8, 26). Interest-ingly, we discovered that the downregulation of SAFB was corre-lated with the activation of the NF-kB signaling pathway. NF-kBplays a pivotal role in the stroma formation of colonic adeno-matous polyps as well as the carcinogenesis (27, 28) and metas-tasis of CRC (29). The NF-kB family contains p65 (RelA), RelB,c-Rel, p50/p105 (NF-kB1), and p52/p100 (NF-kB2), whileNF-kBitself consists of p65 and one of the other homodimers orheterodimers (30). In its silent state, the NF-kB dimers areretained in the cytoplasm where they bind to inhibitory kBs(IkB), whereas in its stimulated state, IkBs induce proteasomaldegradation after they are phosphorylated by activated IkBkinases (IKKa/b; ref. 31). The NF-kB dimers are activated byvarious other modifications and ultimately translocate into thenucleus where they regulate the expression of inflammation-related and oncogenic genes (31). Our study revealed that SAFBdecreased the levels of phosphorylated p65 and IKKb, decreasedthe nuclear translocation of p65, and regulated invasion, metas-tasis and angiogenesis in CRC both in vitro and in vivo. These

results further indicate that the downregulation of SAFBpromotesthe progression of CRC via the regulation of NF-kB signalingpathway activity.

In this study, the downregulation of SAFB enhanced theactivity of the NF-kB pathway, but the specific molecularmechanisms by which SAFB regulates the activation of NF-kBsignaling require further discussion. Notably, TAK1 is a keyregulator of the canonical NF-kB signaling pathway (32), as thesuccessful activation of signals induced by stimuli that bind toreceptors largely depends on TAK1 (33, 34). TAK1 has also beenreported to activate the NF-kB-inducing kinase that is respon-sible for the activation of the noncanonical NF-kB signalingpathway (35). Moreover, TAK1 plays critical roles in the metas-tasis of breast cancer and CRC to the lungs (36, 37). Withrespect to the regulation of TAK1, previous studies were focusedon posttranslational modifications, including phosphoryla-tion, ubiquitination, methylation, and acetylation (38–40).However, little is known about the transcriptional regulationof TAK1 in CRC. As a transcriptional repressor, SAFB wasdiscovered to target the TATA box of the HSP27 promoter andthe E-box of the hXOR promoter, inhibiting the expression ofboth (6, 10). In the current study, through both in vitro and invivo experiments, we showed that SAFB targeted the first E-boxof the TAK1 promoter and inhibited its activity, which led tothe suppression of the NF-kB signaling pathway during CRCinvasion, metastasis, and angiogenesis. Overall, we highlight anovel mechanism of regulation by the TAK1–NF-kB axis, whichis mediated by the transcriptional repressor SAFB during CRCprogression.

In summary, this study demonstrates that SAFB regulates theactivity of the NF-kB signaling pathway in CRC via the targetingof TAK1. Together, these data implicate the SAFB–TAK1–NF-kBaxis as a potential target for early therapeutic intervention todecrease the progression of CRC.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: W.-T. Liao, Y.-Q. DingDevelopment of methodology: H.-L. Jiao, Y.-P. Ye, S.-Y. WangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): Z.-Y. Xiao, L.-Q. He, J.-J. Cai, Y.-R. Chen, J.-F. Qiu,L. LiangAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R.-W. Yang, H.-Y. Sun, Y.-X. Wang, W.-T. Wei,C.-C. Gu, Q.-H. Lai, G.-W. CaoWriting, review, and/or revision of the manuscript: H.-L. Jiao, Y.-P. Ye,R.-W. Yang, H.-Y. Sun, S.-Y. Wang, W.-T. Wei, Q.-H. Lai, W.-T. LiaoAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): H.-L. Jiao, Y.-P. Ye, S.-Y. Wang, Y.-L. Cai,Y.-T. Hu, G.-W. CaoStudy supervision: H.-L. Jiao, Y.-P. Ye, S.-Y. Wang, W.-T. Liao, Y.-Q. Ding

Grant SupportThis work was supported by the National Basic Research Program of

China (973 program, 2015CB554002), Project of the National NaturalScience Foundation of China supported by the NSFC-Guangdong Joint Fund(U1201226), the National Natural Science Foundation of China (81402277,81472313, 81172055, 81472710 and 81402375), the Postdoctoral ScienceFoundation of China (2016M592511), the Guangdong Provincial NaturalScience Foundation of China (S2012010009643, 2014A030313283,2016A030310395, 2016A030310392), the Science and Technology Inno-vation Foundation of Guangdong Higher Education (CXZD1016), the Key






Program of the National Natural Science Foundation of Guangdong(2010B031500012), and the Guangzhou Science & Technology Plan Project(201300000056).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedMarch 22, 2017; revised July 17, 2017; accepted September 7, 2017;published OnlineFirst September 14, 2017.

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2017;23:7108-7118. Published OnlineFirst September 14, 2017.Clin Cancer Res Hong-Li Jiao, Ya-Ping Ye, Run-Wei Yang, et al.

during the Progression of Colorectal CancerTAK1B Pathway by Targeting κ Sustains the NF-SAFBDownregulation of

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