NF-�B Directly Regulates Fas Transcription to ModulateFas-mediated Apoptosis and Tumor Suppression*□S

Received for publication, February 24, 2012, and in revised form, May 9, 2012 Published, JBC Papers in Press, June 5, 2012, DOI 10.1074/jbc.M112.356279

Feiyan Liu‡§1, Kankana Bardhan‡1, Dafeng Yang‡, Muthusamy Thangaraju‡, Vadivel Ganapathy‡,Jennifer L. Waller‡‡, Georgia B. Liles¶�, Jeffrey R. Lee¶�, and Kebin Liu‡**2

From the Departments of ‡Biochemistry and Molecular Biology, ‡‡Biostatistics, and ¶Pathology, Georgia Health SciencesUniversity, Augusta, Georgia 30912, the §College of Life Sciences, Zhejiang University, Hangzhou, China, the �Charlie NorwoodVeterans Affairs Medical Center, Augusta, Georgia 30904, and the **Cancer Center, Georgia Health Sciences University,Augusta, Georgia 30912

Background: The functions of NF-�B in apoptosis and tumor development are controversial.Results: Fas functions as a tumor suppressor, and NF-�B directly binds to multiple sites in the Fas promoter region to regulateFas transcription.Conclusion: Canonical NF-�B is a Fas transcription activator, whereas alternate NF-�B is a Fas transcription repressor.Significance: Inhibition of NF-�B in cancer therapy might suppress Fas-mediated apoptosis to impair host immune cell-mediated tumor suppression.

Fas is amember of the death receptor family. Stimulation ofFas leads to induction of apoptotic signals, such as caspase 8activation, as well as “non-apoptotic” cellular responses,notably NF-�B activation. Convincing experimental datahave identified NF-�B as a critical promoter of cancer devel-opment, creating a solid rationale for the development ofantitumor therapy that suppresses NF-�B activity. On theother hand, compelling data have also shown that NF-�Bactivity enhances tumor cell sensitivity to apoptosis andsenescence. Furthermore, although stimulation of Fas acti-vates NF-�B, the function of NF-�B in the Fas-mediated apo-ptosis pathway remains largely undefined. In this study, weobserved that deficiency of either Fas or FasL resulted in sig-nificantly increased incidence of 3-methylcholanthrene-in-duced spontaneous sarcoma development in mice. Further-more, Fas-deficient mice also exhibited significantly greaterincidence of azoxymethane and dextran sodium sulfate-in-duced colon carcinoma. In addition, human colorectal cancerpatients with high Fas protein in their tumor cells had a lon-ger time before recurrence occurred. Engagement of Fas withFasL triggered NF-�B activation. Interestingly, canonicalNF-�Bwas found to directly bind to the FAS promoter. Block-ing canonical NF-�B activation diminished Fas expression,whereas blocking alternate NF-�B increased Fas expressionin human carcinoma cells. Moreover, although canonicalNF-�B protected mouse embryo fibroblast (MEF) cells fromTNF�-induced apoptosis, knocking out p65 diminished Fasexpression in MEF cells, resulting in inhibition of FasL-in-

duced caspase 8 activation and apoptosis. In contrast, knock-ing out p52 increased Fas expression inMEF cells. Our obser-vations suggest that canonical NF-�B is a Fas transcriptionactivator and alternate NF-�B is a Fas transcription repres-sor, and Fas functions as a suppressor of spontaneous sar-coma and colon carcinoma.

CD95 (also termed APO-1, Fas, tumor necrosis factor recep-tor superfamily member 6, or TNFRSF6) is a member of thedeath receptor family, a subfamily of the tumor necrosis factorreceptor superfamily. Binding to Fas by its physiological ligand,FasL, triggers receptor trimerization, followed by formation ofdeath-inducing signaling complex and subsequent apoptosis(1–3). Germ line and somatic mutations or deletions of Fas orFasL gene coding sequences in humans lead to autoimmunelymphoproliferative syndrome (4–8), suggesting a critical roleof the Fas-mediated apoptosis pathway in lymphocyte homeo-stasis and suppression of autoimmune diseases. Autoimmunelymphoproliferative syndrome patients also exhibited increasedrisk of both hematopoietic and non-hematopoietic cancers (4, 7,9). Furthermore, both the Fas and FasL gene promoters are poly-morphic, including aG toA substitution at�1377 bp and anA toG substitution at �670 bp at the Fas gene promoter and a C to Tsubstitution at �844 and a �124 A to G substitution at the FasLgene promoter. These polymorphisms diminish transcription fac-tor binding to theFas andFasLpromoter andFas/FasL expressionlevel and are also associated with increased risk of both hemato-poietic malignancies and non-hematopoietic carcinoma develop-ment in humans (10–15). These observations thus suggest thatFas functions not only in inhibition of human autoimmune dis-eases but also in suppression of cancer development in humans.Stimulation of the Fas receptor, however, has also been

shown to activate “non-apoptotic” signaling, notably NF-�Bactivation (16–18). In addition, it has been shown that loss ofFas inmousemodels of ovarian and liver cancers reduces tumorincidence and tumor sizes (19). These observations lead to the

* This work was supported, in whole or in part, by National Institutes of HealthGrant CA133085 (to K. L.). This work was also supported by American Can-cer Society Grant RSG-09-209-01-TBG (to K. L.) and the Siyuan Foundation(to F. L.).

□S This article contains supplemental Fig. S1 and Tables 1 and 2.1 Both authors contributed equally to this work.2 To whom correspondence should be addressed: Dept. of Biochemistry and

Molecular Biology, Georgia Health Sciences University, 1410 Laney WalkerBlvd., Augusta, GA 30912. Tel.: 706-721-9483; E-mail: Kliu@georgiahealth.edu.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 30, pp. 25530 –25540, July 20, 2012© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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proposal that Fas activity should be inhibited in cancer therapy(19). However, details of Fas-mediated “non-apoptotic” signal-ing pathways remain largely unknown (1). Importantly, al-though convincing experimental data have established NF-�Bas a tumor-promoting transcription factor (20), compellingrecent studies have started to shed light on the function ofNF-�B as a promoter of apoptosis and senescence (21–28).More importantly, it has been shown that NF-�B mediatesrecruitment of FADD and caspase 8 to the death-inducingsignaling complex to increase tumor cell sensitivity to Fas-mediated apoptosis in tumor cells (28). Furthermore, NF-�Bwas found to regulate TNF� and IFN-� expression to up-reg-ulate Fas expression (29). These studies thus demonstratethat NF-�B mediates tumor cell sensitivity to Fas-mediatedapoptosis.We report here that NF-�B directly regulates Fas transcrip-

tion in human colon carcinoma cells and inMEF cells. Our dataindicate that canonical NF-�B is a transcription activator of Fasand alternate NF-�B is a transcription repressor of Fas, and Fasfunctions as a tumor suppressor. Therefore, caution should beexercised in utilizing blockage ofNF-�Bactivity as a broad ther-apeutic strategy in cancer therapy.

EXPERIMENTAL PROCEDURES

Mice—The method of sarcoma induction using 3-methyl-cholanthrene (MCA)3 was as previously described (30). Briefly,MCA was used at a dose of 100 �g in 100 �l of peanut oil asdescribed previously (30) and was injected subcutaneously intoB6.MRL-Faslpr/J (n � 10), WT C57BL/6J (n � 10), CPt.C3-Faslgld/J (n � 23), and BALB/c (n � 14) mice. To induce coloncancer, the well established azoxymethane (AOM) and dextransodium sulfate (DSS) procedure was used (31). Briefly, AOM(10 mg/kg body weight) was injected intraperitoneally intoB6.MRL-Faslpr/J (n � 3) and WT C57BL/6J (n � 5) mice. Thedrinking water of the mice was replaced with DSS (2.25%) theday afterAOM injection for 1week. After four cycles ofwater (2weeks) and DSS (1 week), the mice were maintained with reg-ular water for 33 more days and examined for colon cancerdevelopment.Cells—All human tumor cell lines used in this study were

obtained from the American Type Culture Collection (ATCC)(Manassas, VA). p65 KO and the matched p65 WT MEF cellswere provided by Dr. Karen H. Vousden (NCI, National Insti-tutes of Health, Frederick, MD) (32). p52 KO and matched p52WTMEF cells were provided by Dr. Kenneth B. Marcu (StonyBrook University, New York) (33).Reagent—IKK�-KA and IKK�-KM plasmids were provided

by Warner C. Greene (University of California, San Francisco,CA) (34). Mega-Fas Ligand� (kindly provided by Drs. StevenButcher and Lars Damstrup, Topotarget A/S, Denmark) is arecombinant fusion protein that consists of three human FasLextracellular domains linked to a protein backbone comprising

the dimer-forming collagen domain of human adiponectin.The Mega-Fas ligand was produced as a glycoprotein in mam-malian cells using goodmanufacturing practice-compliant pro-cesses in Topotarget A/S (Copenhagen, Denmark). IFN-� andTNF� were obtained from R&D Systems (Minneapolis, MN).HumanColorectal Carcinoma Specimens—Colon cancer tis-

suemicroarray slides were provided by theNCI, National Insti-tutes ofHealth, CancerDiagnosis Program.The tissuemicroar-rays contain 341 stage I–IV colorectal cancer specimens andwere designed by NCI statisticians for high statistical power forexamination of associations of markers with tumor stage, clin-ical outcome, and other clinical-pathologic variables. Specimeninformation is listed in supplemental Table 1.Tumor Viability Assay—Tumor cells were seeded in 96-well

plates and cultured in the presence of IFN-�, FasL, or bothIFN-� and FasL for 3 days. The cell viability assay was carriedout using the MTT cell proliferation assay kit (ATCC).Immunohistochemistry—Tissues were fixed in formalin,

embedded in paraffin, sectioned, and stained with hematoxylinand eosin as described previously (35) using CD8 antibody(DAKO Corp.) at a 1:250 dilution and anti-Fas (C-20, SantaCruz Biotechnology, Inc., Santa Cruz, CA). Slides were coun-terstainedwith hematoxylin (Richard-Allan Scientific, Kalama-zoo, MI).RT-PCR Analysis—Total RNA was isolated from cells using

TRIzol (Invitrogen) and used for semiquantitative and real-time RT-PCR analysis of gene expression as described (36, 37).The PCR primer sequences are listed in supplemental Table 2.Western Blotting Analysis—Cytosol and mitochondrion-en-

riched fractions were prepared as described previously (38).Western blotting analysis was performed as described previ-ously (35). Anti-cleaved caspase 8 was obtained from R&D Sys-tems. Anti-�-actin was obtained from Sigma.Apoptosis Assays—Cells were either stained with propidium

iodide (PI) (Trevigen, Gaithersburg, MD) or PI plus AnnexinV-Alexa Fluor 647 (Biolegend) and analyzed by flow cytometry.Cell Surface Fas Protein Analysis—Human tumor cells were

stained with anti-human Fas mAb (Biolegend). MEFs werestained with anti-mouse Fas mAb (Biolegend). Isotype-matched control IgGs (Biolegend) were used as negative con-trols. The stained cells were analyzed by flow cytometry.Chromatin Immunoprecipitation (ChIP)—ChIP assays were

carried out according to protocols fromUpstate Biotechnology,Inc. (Lake Placid, NY) as described previously (36). Immuno-precipitation was performed using anti-pSTAT1 antibody(Santa Cruz Biotechnology, Inc.) and anti-p50 antibody (SantaCruz Biotechnology, Inc.), respectively, followed by pull-downwith agarose-protein A beads (Upstate Biotechnology, Inc.).PCR primers are listed in supplemental Table 2.Electrophoresis Mobility Shift Assay (EMSA) of NF-�B

Activation—NF-�B activation was analyzed using an NF-�Bprobe (AGT TGA GGG GAC TTT CCC AGG C; Santa CruzBiotechnology, Inc.) and probes with NF-�B consensus se-quences of the Fas promoter region (supplemental Table 2) asdescribed previously (35). Briefly, the end-labeled probes wereincubated with nuclear extracts for 20 min at room tempera-ture. For specificity controls, unlabeled probe or mutant probewas added to the reaction at a 1:100 molar excess. Anti-p65,

3 The abbreviations used are: MCA, 3-methylcholanthrene; AOM, azoxymeth-ane; DSS, dextran sodium sulfate; MTT, 3-(4,5-dimethylthiazol-2)-2,5-di-phenyltetrazolium bromide; PI, propidium iodide; MEF, mouse embryofibroblast; GAS, � activation site; CTL, cytotoxic T lymphocyte; MFI, meanfluorescence intensity.

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-p52, and -p50 antibodies (Santa Cruz Biotechnology, Inc.)were also included to identify NF-�B-specific DNA binding.DNA-protein complexes were separated by electrophoresis in6% polyacrylamide gels and identified using a PhosphorImagerscreen (GE Healthcare), and the images were acquired using aStorm 860 imager (GE Healthcare).Statistical Analyses—All statistical analysis was performed

using SAS 9.2, and statistical significance was assessed using an� level of 0.05. �2 or Fisher exact tests were used to examinedifferences in tumor stage, node stage, metastasis stage, andsummary stage by Fas stain intensity (bright versus dim), CD8Tcell infiltration level (low versus high), and a combination of Fasstain intensity and CD8 T cell infiltration level. Kaplan-Meiersurvival analysis was used to examine differences in time torecurrence by Fas stain intensity, CD8 T cell infiltration level,and a combination between Fas stain intensity and CD8 T cellinfiltration level. A log rank test was used to assess differencesin survival between the groups.

RESULTS

Loss of Fas or FasL Function Leads to Increased SpontaneousSarcoma Development—Because membrane-bound FasL onlyinduces Fas-mediated apoptosis and membrane-bound FasL is

primarily expressed on activated lymphocytes (39, 40), it is thuscritical to examine Fas function in a tumor microenvironmentwhere tumors are immunogenic and lymphocyte infiltrates arepresent. The MCA-induced spontaneous sarcoma is an immu-nogenic mouse tumor model, and the host lymphocytes areactively involved in anti-tumor immunity in sarcoma-bearinghosts (30). MCAwas injected into Faslpr (B6.MRL-Faslpr/J) andFasgld (CPt.C3-Faslgld/J) mice, respectively, and examined forspontaneous sarcoma development. Ninety percent (9 of 10) ofFaslpr mice and 100% (23 of 23) of Fasgld mice developed highgrade sarcoma (Fig. 1,A andC), respectively, whereas only 20%(2 of 10) of WT Fas C57BL/6J mice and 71% (10 of 14) of FasWTBALB/cmice developed tumors (Fig. 1,B andD). Althoughthe tumor sizes were not significantly different between Faslprmice andWTC57BL/6J controlmice (Fig. 1B), tumors in Fasgldmice grew significantly faster than inWTBALB/c control mice(Fig. 1D). As a complementary mouse tumor model, we alsoused the AOM-DSS-induced spontaneous colon carcinomamouse model to examine the function of Fas in tumor devel-opment. Faslpr and C57BL/6J mice were treated with AOM-DSS and observed for spontaneous colon carcinoma devel-opment. AOM-DSS induced a higher colon cancer incidencein Faslpr mice as compared with the WT C57BL/6J control

FIGURE 1. Loss of Fas function increased spontaneous sarcoma and colon carcinoma development. A, MCA was injected into WT (n � 10) and Faslpr (n �10) mice subcutaneously and analyzed for sarcoma development 102 days later. Shown are live sarcoma in a tumor-bearing mouse (left) and H&E-stainedtumor section (right). B, tumor incidence (left) and size (right). Columns, mean; error bars, S.D. C, MCA was injected into WT (n � 14) and Fasgld (n � 23) mice andanalyzed for sarcoma development. Shown are live sarcoma in a tumor-bearing mice (top left), an H&E-stained section of the sarcoma (bottom), and tumorincidence (top right). D, tumor growth kinetics in WT (left) and Fasgld mice (right). MCA-induced sarcomas as shown in C were measured for sizes over time.E, AOM-DSS-induced colon carcinoma in WT and Faslpr mice. WT (n � 5) and Faslpr (n � 3) mice were treated with AOM and DSS as described under“Experimental Procedures” and examined for colon cancer development. Morphology of tumor-bearing colon tissues are shown (top). The tumor nodulenumber was counted and is presented at the bottom. Columns, mean; error bars, S.D. **, p � 0.01. F, histological analysis of the AOM-DSS-induced coloncarcinoma as shown in E. The tumor sections were stained with H&E. a, normal colon tissue; b, adenoma; c, adenocarcinoma.

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mice (Fig. 1, E and F). Taken together, our data suggest thatFas functions as a suppressor of spontaneous sarcoma andcolon carcinoma.Correlation between Fas Level and Cancer Recurrence in

Human Colorectal Cancer Patients—Fas is frequently silencedin human cancer, especially in metastatic cancer cells (41), sug-gesting that human cancer cellsmight use loss of Fas as ameansto advance the disease. To determine the relationship betweenFas expression level and colorectal cancer development inhumans, we used an immunohistochemical approach to exam-ine Fas protein level in a large cohort of human colorectal can-cer specimens (n � 341). Analysis of Fas protein levels in these341 CRC patients revealed that the average time to cancerrecurrence for patients with a high Fas level in the tumor cells is93.82 months, whereas that time is only 47.4 months forpatients with low Fas protein level in the tumor cells (Table 1).However, due to the large variations in times to recurrenceamong patients, the difference was not statistically significant(p � 0.35). The level of CD8� T cell infiltration in the tumor ispositively correlated with decreased recurrence (Table 1) (p �0.0001). No correlation between Fas level and tumor stages wasobserved.Because Fas receptor alone does not initiate cellular signals

and FasL is the physiological ligand of Fas (42), that is primarily

FIGURE 2. Correlation between Fas protein level, CD8� T cells, and cancerrecurrence in human colorectal cancer patients. Tissue microarray slidescontaining human colorectal cancer specimens (n � 341, supplemental Table1) were stained for tumor-infiltrating CD8� T cells and Fas protein level. Thestained specimens were then graded and statistically analyzed for correlationwith cancer recurrence. Each variable is indicated by colored lines in the plot.

TABLE 1Kaplan-Meier survival on recurrence

Variable Level

Meanrecurrence

timea S.D.Log rank test

p value

monthsFas protein Bright 93.82 3.59

Dim 47.4 1.98 0.3496CD8� T cells High 113.79 3.37

Low 55.83 2.26 0.0001Fas protein/CD8� T cell Bright/Low 59.91 2.72

Dim/Low 36.86 2.46 0.0479Bright/High 114.39 4.50Dim/High 41.48 1.30 0.4876

a Length of time (in months) from date of cancer diagnosis to first recurrence orlast verified recurrence-free.

FIGURE 3. FasL specifically induces tumor cell growth inhibition throughFas receptor in vitro. A, eight human colon carcinoma cell lines were incu-bated with IFN-�, FasL, or both IFN-� and FasL for 3 days and analyzed forgrowth inhibition by MTT assays. The growth rate of untreated cells was set at100%. % Cell Growth, percentage of cell growth as measured by MTT assays oftreated cells over untreated cells. Columns, mean; bars, S.D. B, multiple typesof human cancer cells were analyzed for sensitivity to FasL-induced apoptosisas in A. The cell lines are indicated below the plot. C, FasL induces tumor cellgrowth inhibition specifically through the Fas receptor. Tumor cell lines wereestablished from sarcomas derived from tumor-bearing WT (n � 2) and Faslpr

(n � 5) mice as shown in Fig. 1A and incubated with IFN-�, FasL, or both IFN-�and FasL for 3 days and analyzed for growth inhibition by MTT assay as in A.D, loss of FasL in tumor cells does not affect tumor cell sensitivity to FasL-induced apoptosis. Tumor cell lines were established from tumor-bearing WT(n � 4) and Fasgld (n � 4) mice as shown in Fig. 1C and analyzed by MTT assayas in A.

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FIGURE 4. IFN-� and TNF� up-regulate Fas expression through pSTAT1 and NF-�B binding to the Fas promoter in human colon carcinoma cells. A, Fasis silenced in metastatic human colon carcinoma cells. SW480 and SW620 cells were analyzed for cell surface Fas protein level by flow cytometry (top) and FasmRNA by semi-quantitative (bottom left) and real-time RT-PCR analysis (bottom right). Gray-filled area, IgG isotype control; solid line, Fas-specific staining.Column, mean; bar, S.D. B, IFN-� and TNF� cooperate to up-regulate Fas expression in metastatic human colon carcinoma cells. SW620 cells were treated withIFN-�, TNF�, or both IFN-� and TNF� and stained with Fas-specific mAb for cell surface Fas level. Gray-filled area, IgG isotype control staining; solid line,Fas-specific staining. C, FAS promoter structure. The number below the bar indicates nucleotide number relative to the FAS transcription initiation site. The ChIPPCR regions are indicated above the bar. The GAS and NF-�B-binding consensus sequence elements are also indicated. D, ChIP assays of pSTAT1 associationwith the FAS promoter (top). SW620 cells were either untreated or treated with IFN-� for 4 h and used for ChIP. PCR amplification was conducted with FASpromoter sequence-specific primers as shown in C. The primer sequences are listed in supplemental Table 2. Bottom, EMSA of pSTAT1 binding to the GASelements of the human FAS promoter. Probe 1, GAS1; Probe 2, GAS 2, as in C. The probe sequences are listed in supplemental Table 2. E, NF-�B binds to the FASpromoter DNA. SW620 cells were either untreated or treated with TNF� for 30 min. ChIP assays of canonical NF-�B association with the FAS promoter (top) wereperformed to determine the protein-DNA interactions with p50-specific antibody. IgG was used as antibody negative control. Genomic DNA (gDNA) was usedas a PCR positive control. Bottom, EMSA of NF-�B binding to the NF-�B consensus sequence (C) of the human FAS promoter. The probes correspond to theNF-�B consensus sequences as listed in C and are listed in supplemental Table 2. P, positive control NF-�B probe (Santa Cruz).

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expressed on activated lymphocytes, particularly CD8� T cells(40), we analyzed CD8� T cell infiltration level in these tumorspecimens. As expected, CTL infiltration level is positively cor-related with decreased recurrence (p � 0.0001) (43, 44).Although no correlation was observed between Fas level andrecurrence time in patients with high levels of CD8� T cellinfiltration, patients with high Fas protein levels in the tumorcells generally have a longer time before recurrence occurswhen CD8� T cell infiltration level in their tumor cells is low(Table 1 and Fig. 2) (p � 0.048). The mean recurrence time is59.91 months for patients with a high Fas protein level and36.86 months for patients with a low Fas protein level, respec-tively (Table 1).FasL Induces Tumor Growth Inhibition in Vitro—To deter-

mine whether FasL induces apoptosis (45) or promotes tumorcell growth (19), eight human colon carcinoma cell lines werecultured in the presence of FasL in vitro and measured for cellgrowth. Because certain tumor cells are resistant to apoptosisand IFN-� sensitizes tumor cells to apoptosis induction (35),IFN-� treatment was included for each cell line. It is clear thatFasL inhibited the growth of all nine cell lines in vitro (Fig. 3A).Next, nine human cancer cell lines of six types of cancers,including sarcoma (PLS-1, MPNST724, and SW116), hepa-toma (HepG2), ovarian carcinoma (IGROV1 and SKOV3), lungcarcinoma (A549), mammary carcinoma (MCF7), and prostatecancer (DU145), were tested for their sensitivity to FasL-in-duced apoptosis. All nine cell lines are sensitive to FasL-in-duced apoptosis (Fig. 3B). Furthermore, IFN-� increased thesensitivity of six cell lines to FasL-induced apoptosis (Fig. 3B).Our data thus indicate that Fas-FasL interaction suppresseshuman cancer cell growth in vitro.

To determine the specificity of FasL-induced apoptosis, wefirst established sarcoma cell lines fromWT and Faslpr tumorsderived from the tumor-bearing mice, as shown in Fig. 1, andexamined the response of these tumor cell lines to FasL. Incu-bation of FasL with WT sarcoma tumor cells induced tumorcell growth inhibition, whereas Faslpr tumor cells exhibited noresponse to the FasL treatment (Fig. 3C). Next, we establishedsarcoma cell lines fromWT and Fasgld mice and examined thesensitivity of these cell lines to FasL. Both WT and Fasgld sar-coma cell lines are sensitive to FasL-mediated growth inhibi-tion (Fig. 3D). In summary, our data suggest that FasL inducestumor cell growth inhibition specifically through the Fasreceptor.IFN-� and TNF� Up-regulate Fas Expression through

pSTAT1 and NF-�B Binding to the FAS Promoter—Humanmetastatic colon carcinoma cells often exhibit diminished Fas

FIGURE 5. NF-�B is a Fas transcription activator in human colon carci-noma cells. A, FasL induces NF-�B activation in human colon carcinoma cells.Nuclear extracts were prepared from SW480 cells and analyzed for NF-�Bactivity using EMSA with NF-�B consensus sequence-containing DNA probes.Anti-p65 and anti-p50 antibodies were used to identify the canonical NF-�B-probe binding (left). Mutant probe was used as a negative control. Nuclearextracts were also prepared from SW480 cells treated with FasL for the indi-cated time and analyzed for NF-�B activation using EMSA (right). B, blockingNF-�B activation diminishes Fas expression in human colon carcinoma cells.SW480 cells were stably transfected with control vector, an IKK� dominantnegative mutant (IKK�-KA), or an IKK� dominant negative mutant (IKK�-KM)and analyzed for Fas protein level on the cell surface. Gray-filled area, IgG

isotype control staining; solid thin line, Fas level in SW480. In vector cells, thesolid bold line shows Fas protein level in SW480.IKK�-KA cells, and the dottedthin line shows Fas protein level in SW480.IKK�-KM cells. The Fas MFI wasquantified and is presented in the right panel. Columns, mean; error bars, S.D.C and D, blocking canonical NF-�B activation results in loss of response oftumor cells to TNF�-mediated Fas up-regulation. SW480.Vector, SW480.IKK�-KA, and SW480.IKK�-KM cells were treated with TNF� and analyzed for Fasprotein level on the cell surface (C) and mRNA level by real-time RT-PCR (D).Gray-filled area, isotype control staining; solid line, untreated cells; dotted line,TNF�-treated cells. The Fas MFI was quantified and is presented in the rightpanel. Columns, mean; bars, S.D. For real-time RT-PCR analysis of the FasmRNA level, the relative Fas mRNA level in SW480.Vector cells was set at 1.

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expression (41) (Fig. 4A). It has been shown that IFN-� andTNF�, cytokines secreted by senescent tumor cells and acti-vated immune cells, can up-regulate Fas expression (29, 46, 47).Indeed, IFN-� and TNF� synergistically up-regulated Fas inhuman colon carcinoma cells (Fig. 4B). Consistent withincreased Fas expression, the metastatic colon carcinoma cellsbecame sensitive to FasL-induced apoptosis (supplemental Fig.S1). To determine whether IFN-�-activated pSTAT1 andTNF�-activated NF-�B directly bind to the FAS promoter toactivate FAS transcription, we performed ChIP assays of thehuman FAS gene promoter from �4000 to �1000 relative tothe FAS gene transcription initiation site. Immunoprecipita-tion with pSTAT1- and p50-specific antibodies revealed thatboth pSTAT1 and NK-�B bind to multiple regions of the FASgene promoter chromatin in the human colon carcinoma cells(Fig. 4, D and E).

Analysis of the human FAS promoter regionwithMacVectoridentified two potential pSTAT1 binding consensus � activa-tion site (GAS) elements and at least five potential NF-�B-bind-ing consensus sequences (Fig. 4C). We next performed EMSAand observed that pSTAT1 binds to both GAS elements (Fig.4D). EMSA also showed that canonical NF-�B binds to all fiveNF-�B consensus sequences at the FAS promoter region inhuman colon carcinoma cells (Fig. 4E). In summary, our datademonstrated that pSTAT1 andNF-�Bdirectly bind to the FASpromoter in human colon carcinoma cells, and we havemapped the precise binding sites in the human FAS promoterregion.NF-�B Regulates FAS Transcription in Human Colon Carci-

noma Cells—TNF� is a potent NF-�B inducer. The aboveobservations that TNF� up-regulates Fas expression suggestthat NF-�B might activate Fas expression. To test this hypoth-esis, Fas-high SW480 cells were stably transfected with thedominant negative IKK� mutant (IKK�-KA) to block thecanonical NF-�B pathway. EMSA revealed that canonicalNF-�B is activated in SW480 cells, and FasL enhances canoni-

cal NF-�B activation (Fig. 5A). Flow cytometry analysis indi-cated that blocking canonical NF-�B activation significantlydecreases Fas protein level on the cell surface (Fig. 5B) and FasmRNA level in the cells (Fig. 5D). Furthermore, blockingcanonical NF-�B activation also inhibited tumor cell responseto TNF� to activate Fas (Fig. 5, C and D). In contrast, blockingalternate NF-�B activation with the dominant negative IKK�mutant (IKK�-KM) increased cell surface Fas protein level (Fig.5B) and Fas mRNA level (Fig. 5D) in human colon carcinomacells. In addition, blocking alternate NF-�B activation does notalter tumor cell response to TNF� to activate Fas (Fig. 5, C andD). Together, these data suggest that canonical NF-�B is a FAStranscription activator and alternate NF-�B is a FAS transcrip-tion repressor.NF-�B Is Also a Fas Transcription Activator in MEF Cells—

The above observation is a surprising one because canonicalNF-�B is often considered a tumor promoter (20, 48, 49), andwe showed here that canonical NF-�B is a FAS transcriptionactivator. To determine whether this is a general phenomenon,we examined Fas level inWTandp65KOMEFcells. It is knownthat knocking down p65 diminishes Bcl-xL expression and sen-sitizes MEF cells to TNF�-induced apoptosis (50). It has alsobeen shown that inhibition of NF-�B activity strongly enhancesTNF�-mediated apoptosis (28). Indeed, p65KOMEF cells havedecreased Bcl-xL level (Fig. 6A) and increased sensitivity toTNF�-induced apoptosis (Fig. 6, B and C). However, althoughknocking out p52 also down-regulated Bcl-xL (Fig. 6D), in con-trast to p65 KO MEF cells, p52 KO MEF cells exhibited noincreased sensitivity to TNF�-induced apoptosis as comparedwith their matched p52 WTMEF cells (Fig. 6, E and F).Next, we analyzed p65 and p52 MEF cells to FasL-induced

apoptosis. Consistent with what was observed in human coloncarcinoma cells, we observed that both Fas protein level andmRNA level are dramatically lower in p65 KO MEF cells ascompared with p65 WT MEF cells (Fig. 7A). Like the humancolon carcinoma cells, the p65WTMEF cells are responsive to

FIGURE 6. Sensitivity of WT and NF-�B KO MEF cells to TNF�-induced apoptosis. A–C, p65 WT and p65 KO MEF cells. A, RT-PCR analysis of Bcl-xL mRNA level.B, cells were cultured in the absence or presence of IFN-� or TNF� overnight, stained with Annexin V and PI, and analyzed by flow cytometry. The number in eachbox indicates percentage of Annexin V- and PI-double positive cells. C, quantification of apoptotic cell death. Percentage apoptosis was calculated as thepercentage of Annexin V- and PI-positive cells in the presence of IFN-� or TNF� minus the percentage of Annexin V and PI positive in the absence of IFN-� orTNF�. Columns, mean; bars, S.D. D–F, p52 WT and p52 KO MEF cells. D, RT-PCR analysis of Bcl-xL mRNA level. E, cells were cultured in the absence or presenceof IFN-� or TNF� and analyzed for apoptosis as in B. F, quantification of apoptotic cell death as in C.

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IFN-� and TNF� to up-regulate Fas expression (Fig. 7B). Thep65 KO MEF cells are also responsive to IFN-� to up-regulateFas (Fig. 7B), but due to massive cell death after exposure toTNF� (Fig. 6B), p65KOMEF cells could not be analyzed for Fasexpression level after TNF� treatment. FasL induced caspase 8activation (Fig. 7C) and apoptosis in the WT MEF cells (Fig.7D). Consistent with diminished Fas expression, p65 KO MEFcells failed to respond to FasL to activate caspase 8 (Fig. 7C) and

acquired a resistant phenotype to FasL-induced apoptosis (Fig.7D).In contrast to p65 KO cells, p52 KO MEF cells exhibited

increased cell surface Fas protein andmRNA level as comparedwith the matched p52 WT MEF cells (Fig. 7E). Both p52 WTand p52 KO MEF cells responded to IFN-� and TNF� to up-regulate Fas expression (Fig. 7F). Furthermore, both p52 WTand p52 KO MEF cells are highly sensitive to FasL-induced

FIGURE 7. Canonical NF-�B is a Fas transcription activator, and alternate NF-�B is a Fas transcription repressor in MEF cells. A–D, p65 WT and p65 KO MEFcells. A, cell surface Fas protein (left) and mRNA (right) levels. Gray-filed area, IgG isotype control staining. The Fas MFI was quantified and is presented in themiddle panel. Columns, mean; bars, S.D. B, responses of WT and p65 KO MEF cells to IFN-� and TNF� in Fas up-regulation. WT and p65 KO MEF cells were treatedwith IFN-�, TNF�, or both IFN-� and TNF� and analyzed for Fas protein level as in A. Gray-filled area, isotype control staining. The Fas protein staining levels incells of various treatments are represented by colored lines as indicated. The Fas MFI of WT MEF cells as shown in the top panel was quantified and is presentedin the bottom panel. C, caspase 8 activation in WT and p65 KO MEF cells. Cells were treated with FasL for the indicated times, and cytosol fractions were preparedfor the Western blotting analysis of cleaved caspase 8. D, sensitivity of WT and p65 KO MEF cells to FasL-induced apoptosis. Cells were cultured in the absenceor presence of FasL overnight and stained with Annexin V and PI. The percentage of FasL-induced cell death was calculated as the percentage of Annexin- andPI-positive cells in the presence of FasL (�FasL) minus the percentage that were Annexin V- and PI-positive in the absence of FasL (�FasL) and is presented inthe right panel. Column, mean; bar, S.D. E–G, p52 WT and p52 KO MEF cells. E, cell surface Fas protein (left) and mRNA (right) levels. Gray-filed area, IgG isotypecontrol staining. The Fas MFI was quantified and is presented in the middle panel. Columns, mean; bars, S.D. F, responses of WT and p52 KO MEF cells to IFN-�and TNF� in Fas up-regulation. Cells were treated with IFN-�, TNF�, or both IFN-� and TNF� and analyzed for Fas protein level. Gray-filled area, isotype controlstaining. The Fas protein staining levels in cells of various treatments were represented by colored lines as indicated. The Fas MFI of WT and p21 KO MEF cellsas shown in the top panel was quantified and is presented in the bottom panel. Columns, mean; bars, S.D. G, sensitivity of WT and p52 KO MEF cells toFasL-induced apoptosis. Apoptosis was analyzed as in D.

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apoptosis (Fig. 7G). In summary, our data indicate that 1)canonical NF-�B, but not alternate NF-�B, protects MEF cellsfrom TNF�-induced apoptosis; and 2) canonical NF-�B is ageneral Fas transcription activator, and alternative NF-�B is aFas transcription repressor.

DISCUSSION

Although extensive data have shown that Fas functions as atumor suppressor in both human and mouse tumor models (7,10–13, 15, 51–57), the role of Fas as a tumor promoter has alsobeen reported, and blocking Fas activity for cancer therapy hasbeen proposed (19). The apparently contrasting observationsmight be due to the mouse tumor models and cells used in thestudies. Fas is a cell surface receptor, and it alone does notgenerate cellular signaling. It is the FasL that binds to Fas toinitiate the Fas-mediated signaling pathways. It has been pro-posed that membrane-bound FasL only induces Fas-mediatedapoptosis, whereas sFasL triggers non-apoptotic signaling path-ways (19, 42). Membrane-bound FasL is primarily expressed onactivated lymphocytes. Therefore, activation and infiltration oftumor-specific lymphocytes, primarily tumor-specific CD8�

CTLs in the tumor microenvironment, might be essential for Fasfunction. If the tumors are non-immunogenic or immune sup-pressive, tumor-specific immunecellsmay thennotbeactivated toinfiltrate into the tumor microenvironment. Studies have shownthat human colorectal tumors are immunogenic (43, 44), whereasMCA-induced sarcoma is highly immunogenic (30), Using theseimmunogenic spontaneous colon carcinoma and sarcomamousemodels, we demonstrated here that Fas functions as a suppressorof tumor development under physiological conditions.Our finding that Fas functions as a tumor suppressor is fur-

ther supported by our data from human colorectal cancer spec-imens. We demonstrated that patients with high Fas proteinlevels in their tumor cells have a longer time before recurrenceoccurs when tumor-infiltrating CTL levels are low in theirtumors (Fig. 2). It is known that the perforin-dependent cyto-toxicity is the dominant anti-tumor cytotoxic effector mecha-nism (58); therefore, the perforin cytotoxicity alone might besufficient to suppress tumor development when CTL level ishigh (51). Our data suggest that in the tumor microenviron-ment with limited CTL infiltration, Fas-mediated tumor cellapoptosis might play a critical role in CTL-mediated tumorsuppression in human colorectal cancer.Constitutive NF-�B activation often promotes oncogenesis,

providing a strong rationale for anticancer strategies thatinhibit NF-�B signaling (48, 49). Indeed, as reported in the lit-erature and observed in this study, canonical NF-�B protectsMEF cells fromTNF�-induced apoptosis (28, 50) (Fig. 6). How-ever, we observed that canonical NF-�B functions in an oppos-ingway inmediating FasL-induced apoptosis. Thus, our findingrevealed that canonical NF-�B is a general Fas transcriptionactivator to promote Fas-mediated apoptosis. Therefore, incontexts where prosurvival signals derive from other onco-genes, canonicalNF-�B activitymight instead promote apopto-sis (47, 59, 60). Our finding is consistent in principlewith recentobservations that NF-�B signaling enhances tumor cell sensi-tivity to Fas-mediated apoptosis and to cytotoxic chemother-apy, thereby exerting a tumor-suppressor function (23–28).

Our observation that alternate NF-�B is a Fas transcriptionrepressor in both human colon carcinoma cells andMEF cells isan interesting one (Figs. 5 and 7). Alternate NF-�B is oftensequentially activated after the canonical NF-�B (61). There-fore, it is possible that alternate NF-�B might function as a Fastranscription repressor to turn off canonical NF-�B-activatedFas transcription to prevent sustained Fas transcription activa-tion. Our initial study did not identify direct interactionbetween alternate NF-�B interaction and the Fas promoter inhuman colon carcinoma cells (Fig. 5A). Therefore, determina-tion of how alternative NF-�B represses Fas transcriptionrequires further study. Nevertheless, our data suggest that atherapeutic strategy to inhibit NF-�B signaling should take intoconsideration that such inhibition may confer tumor cell resis-tance to Fas-mediated apoptosis, thereby suppressing the Fas-mediated apoptosis of the host cancer immune surveillancesystem.

Acknowledgments—We thank Kimberly Smith for excellent technicalassistance in immunohistochemical staining of tumor tissues.We alsothank Drs. Butcher and Lars Damstrup for providing Mega-FasL.

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NF-�B Regulates Fas Transcription

25540 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 30 • JULY 20, 2012

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Ganapathy, Jennifer L. Waller, Georgia B. Liles, Jeffrey R. Lee and Kebin LiuFeiyan Liu, Kankana Bardhan, Dafeng Yang, Muthusamy Thangaraju, Vadivel

and Tumor SuppressionB Directly Regulates Fas Transcription to Modulate Fas-mediated ApoptosisκNF-

doi: 10.1074/jbc.M112.356279 originally published online June 5, 20122012, 287:25530-25540.J. Biol. Chem. 

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