Prohibitin Attenuates Colitis-Associated Tumorigenesis in ... · Endoscopic assessment of polyp formation and polyp count Colonoscopy was done to assess polyp formation in mice by

Molecular and Cellular Pathobiology

Prohibitin Attenuates Colitis-Associated Tumorigenesis inMice by Modulating p53 and STAT3 Apoptotic Responses

Arwa S. Kathiria1, William L. Neumann2, Jennifer Rhees1, Erin Hotchkiss1, Yulan Cheng3,Robert M. Genta2, Stephen J. Meltzer3, Rhonda F. Souza2, and Arianne L. Theiss1

AbstractAlthough inflammatory bowel disease is associatedwith higher risk of colorectal cancer, the precise pathogenic

mechanisms underlying this association are not completely understood. Prohibitin 1 (PHB), a protein implicatedin the regulation of proliferation, apoptosis, and transcription, is decreased in intestinal inflammation. In thisstudy, we have established a key function for PHB in mediating colitis-associated cancer. Wild-type andtransgenic (Tg) mice specifically overexpressing PHB in intestinal epithelial cells were subjected to a classicaltwo-stage protocol of colitis-associated carcinogenesis. In addition, wild-type and p53 null human cell modelswere used to assess PHB interaction with STAT3 and p53. Wild-type mice exhibited decreased mucosal PHBprotein expression during colitis-associated carcinogenesis. Tg mice exhibited decreased susceptibility in amanner associated with increased apoptosis, p53, Bax, and Bad expression plus decreased Bcl-xL and Bcl-2expression. PHB overexpression in wild-type but not p53 null human cells increased expression of Bax, Bad, andcaspase-3 cleavage. In wild-type p53 cells, PHB overexpression decreased basal and interleukin-6-induced STAT3activation and expression of the STAT3 responsive genes Bcl-xL and Bcl-2. PHB coimmunoprecipitated withphospho-STAT3 in addition to p53 in cultured cell lysates and colon mucosa. This is the first study to showinteraction between PHB and STAT3 in vivo. In summary, our findings suggest that PHB protects against colitis-associated cancer by modulating p53- and STAT3-mediated apoptosis. Modulation of PHB expression inintestinal epithelial cells may offer a potential therapeutic approach to prevent colitis-associated carcinogenesis.Cancer Res; 72(22); 5778–89. �2012 AACR.

IntroductionInflammatory bowel disease (IBD) includes 2 major chronic

intestinal disorders, Crohn's disease and ulcerative colitis (UC),which share related characteristics such as mucosal damageand diarrhea but have distinguishing clinical features. Evi-dence suggests that in addition to thewidely accepted aberrantmucosal immune response, nonimmune cells including epi-thelial cells play an emerging role in the pathogenesis of diseaseby modulating mucosal barrier integrity and homeostasis (1).An association between IBD and colorectal cancer (CRC) hasbeenwell establishedwith a cumulative risk of developing CRC

of 7% after 20 years for UC and 8% for Crohn's disease (2).Previous studies have shown that production of proinflamma-tory cytokines in the lamina propria contribute to tumorgrowth and cancer development during intestinal inflamma-tion (3, 4).

Prohibitin 1 (PHB) is an evolutionarily conserved, multi-functional 32 kDa protein implicated in cellular processesincluding the regulation of cell cycle progression, apoptosis,and transcription (5–7). Expression of PHB is decreased inmucosal biopsies from UC and Crohn's disease afflictedpatients and in the dextran sodium sulfate (DSS) and inter-leukin (IL)-10�/� mouse models of colitis (8, 9). Proinflamma-tory cytokines such as TNFa and reactive oxygen speciesdecrease expression of intestinal epithelial PHB in vivo and invitro (8–10). Restoration of colonic epithelial PHB expressionusing genetic manipulation [villin-PHB transgenic (Tg) mice]or therapeutic delivery to the colon via nanoparticles oradenovirus protected mice from experimental colitis (11, 12).

The role of PHB in cancer cell proliferation and/or tumorsuppression remains controversial especially because PHBexpression is increased in many transformed cells and tumors(13–19). Consensus binding sites for the oncoprotein Myc arepresent in the PHB promoter and likely contribute to theincreased PHB levels in many tumors (20). Although somaticmutations in the PHB gene were observed in a few sporadicbreast cancers, none were identified in the ovary, hepatic, or

Authors' Affiliations: 1Department of Internal Medicine, Division of Gas-troenterology, Baylor Research Institute, Baylor UniversityMedical Center;2Department of Medicine, Veterans Affairs North Texas Health Care Sys-tem, University of Texas Southwestern Medical Center, Dallas, Texas; and3Gastroenterology Division, Department of Medicine and Department ofOncology, the Sidney Kimmel Comprehensive Cancer Center and theJohns Hopkins School of Medicine, Baltimore, Maryland

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Arianne L. Theiss, Division of Gastroenterology,250 Hoblitzelle, Baylor Research Institute, Baylor University Medical Cen-ter, Dallas, TX 75246. Phone: 214-820-2752; Fax: 214-818-9292; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-12-0603

�2012 American Association for Cancer Research.

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lung cancers examined (16). Previous studies have shown thatPHB has an antitumorigenic role in gastric, prostate, and livercancers (21–23). Despite the increasing number of studies onPHB, its role in CRC or colitis-associated cancer (CAC) has notbeen explored. Here, we used mice specifically overexpressingPHB in intestinal epithelial cells (IEC) to assess the role andmechanism of PHB in CAC.

Materials and MethodsAnimal modelsWild-type (WT) and villin-prohibitin Tg mice specifically

overexpressing prohibitin in IECs (12) were 9 weeks old at thebeginning of the experimental protocol. All mice weregrouped-housed in standard cages under a controlled temper-ature (25�C) and photoperiod (12-hour light–dark cycle) andwere allowed standard chow and tap water ad libitum. Allexperiments were approved by the Baylor Research InstituteInstitutional Animal Care and Use Committee.

Induction of CAC in miceAge-matched male and female C57BL/6 WT and Tg mice

were intraperitoneally injected with 7.6 mg/kg azoxymethane(AOM; Sigma-Aldrich) on day 0. Colitiswas induced 7days afterAOM injection by oral administration of 3% (wt/vol) DSS(molecular weight, 50,000; MP Biomedicals) in drinking waterad libitum for 7 days followed by 14 days of recovery of wateralone. After recovery, a second cycle of DSS was repeated andmice were sacrificed after 3 weeks of recovery (day 56) aspreviously described (24). Three separate groups of mice wereused as controls: one group of mice was maintained withregular water (n ¼ 16 WT; n ¼ 10 Tg), a second group of micewas injected with AOM and given water for the remainder ofthe experiment (n ¼ 9 WT; n ¼ 7 Tg), and the third group ofmice was treated with DSS as described above without AOMinjection (n ¼ 20 WT; n ¼ 8 Tg). AOM alone representstreatment with the alkylating agent in the absence of inflam-mation and is expected to result in no tumor formation in WTmice at the dose used (25). DSS alone represents repeatedcycles of inflammation and was included to assess whetherinflammation alone caused dysplasia in these mice. The studywas powered in such a fashion that the control arms requiredless mice as death before the end of the study was notanticipated for these treatment groups. Body weight, stoolconsistency, and stool occult blood was monitored during theDSS treatment and recovery phases. Upon sacrifice, colon wasexcised from the ileocecal junction to anus, cut open longitu-dinally, and prepared for histologic evaluation.To assessmortality, the same protocol was followedwith the

exception of extending through 126 days or until mice devel-oped rectal prolapse and/or more than 20% body weight loss.Colons were assessed macroscopically for polyps using adissecting microscope.

Clinical score assessmentAssessment of body weight loss, stool consistency, and the

presence of occult/gross blood by a guaiac test (HemoccultSENSA; Beckman Coulter) were determined daily during DSS

administration to generate a clinical activity score as describedpreviously (12). During recovery periods, body weight wasmeasured every week for each group.

Endoscopic assessment of polyp formation and polypcount

Colonoscopy was done to assess polyp formation in miceby using the Coloview (Karl Storz Veterinary Endoscopy).Mice were euthanized with 1.5% to 2% isoflurane andapproximately 3 cm of the colon proximal to the anus wasvisualized after inflation of the colon with air. Mice werethen sacrificed by cervical dislocation after euthanizing withisoflurane. Colon was excised from ileocecal junction toanus, washed with 0.9% NaCl, cut open longitudinally andpreserved in 10% neutral-buffered formalin. The number andsize of the polyps were determined using a Jenko dissectingmicroscope.

Histopathology scoringColons fixed in 10% neutral buffered formalin were Swiss-

rolled, embedded in paraffin, sectioned at 5 mm, and stainedwith hematoxylin and eosin stain for histopathologic exami-nation of polyps and adenocarcinoma (neoplasia). Scoring wascarried out in a blind fashion by 2 qualified pathologists. Scorewas given on the basis of these criteria: normal (score 0), low-grade dysplasia (score 1), high-grade dysplasia (score 2), intra-mucosal adenocarcinoma (score 3), and invasive adenocarci-noma (score 4).

ImmunohistochemistryParaffin-embedded sections (5 mm) of colon were analyzed

for Ki67 staining as a marker of cell proliferation as previouslydescribed (12). A minimum of 15 crypts with normal morphol-ogy were counted for Ki67-positive cells per section.

Terminal deoxynucleotidyl transferase-mediated dUTPnick end labeling staining

Immunofluorescent terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining wascarried out to measure apoptosis from paraffin-embeddedsections using the In Situ Cell Death Detection Kit as describedby the manufacturer (Roche). Nuclei were stained with 40, 6-diamidino-2-phenylindole to count total cells per crypt. Aminimum of 10 crypts with normal morphology were countedper section.

p53 mutational analysesTumor tissue was microdissected from paraffin-embedded

sections (7 mm) of Swiss-rolled mouse colon. Genomic DNAwas isolated from microdissected tissues using the QIAampDNAFFPETissue purification kit (Qiagen). The PCRampliconswere generated and sequenced as previously described (26).See the Supplementary Materials and Methods section forprimer sequences.

Human tissue samplesMatching normal and tumor tissues were obtained at the

time of surgical resection from patients with one or more

Prohibitin Functions as a Tumor Suppressor in CAC

www.aacrjournals.org Cancer Res; 72(22) November 15, 2012 5779




UC-associated colorectal neoplasms, consisting of adenocar-cinomas or dysplasias. Normal control samples consisted ofcolonic normal mucosa adjacent to tumors or ileal mucosa.Inflamed UC tissues were obtained from patients withoutevident dysplasia. All tissues were grossly dissected free ofnormal surrounding tissue, and parallel sections were used forhistologic characterization. Tissue collection was approved bypatients according to Institutional Review Board guidelines.

Total RNA extraction and quantitative real-time PCRTotal RNAwas extracted for human tissues using the RNeasy

kit (Qiagen). Quantitative real-time PCR was carried out asdescribed previously (11). See the Supplementary Materialsand Methods section for primer sequences.

Cell culture and transfectionCaco2-BBE cells were used to assess the interaction of PHB

with STAT3. As Caco2-BBE cells have mutated p53, WTHCT116 human CRC cells were used to assess PHB interactionwith p53. All cell lines were obtained from the American TypeCulture Collection. Cells were grown and transfected as pre-viously described (Kathiria and colleagues; submitted forpublication).

g-Irradiationg-Irradiation was used to induce DNA damage in WT and

p53�/� HCT116 cells (27) after 72 hours of transfection withpEGFPN1 vector or pEGFPN1-PHB. Cells were irradiated byusing 137Cs g-irradiator at 6.5 cGy/s for 32 seconds for a total of209.4 cGy. Cells were harvested 24 hours after g-irradiation forsubsequent assays.

Protein extraction, Western blot analysis, andimmunoprecipitation

Mucosal strippings from Tg andWTmice were obtained forWestern blot analysis as described previously (12). Totalprotein was isolated from cultured cells as described previ-ously (Kathiria and colleagues; submitted for publication).Antibodies used were mouse monoclonal PHB (Thermo Fish-er), mouse monoclonal GFP, p53, Bcl-2, Bcl-xL and Bad andrabbit polyclonal BAX, p-Bad (ser 155), STAT3 (Santa CruzBiotechnology), rabbit polyclonal proliferating cell nuclearantigen (PCNA) antibody (Abcam), rabbit polyclonal cas-pase-3, p53, pSTAT3 (Cell Signaling Technology), rabbit poly-clonal p53 upregulated modulator of apoptosis (PUMA), andmouse monoclonal anti-b-actin (Sigma-Aldrich).

PHB was immunoprecipitated from 0.6 mg total proteinlysates from HCT116 or Caco2-BBE cells or 0.20 mg totalprotein lysates from mouse mucosa with 1 mg mouse anti-PHB, anti-p53, or anti-pSTAT3 antibody and 30 mL 50% proteinA sepharose beads (GE Healthcare). Blots were incubated withthe rabbit p53 antibody or PHB antibody, respectively. Omis-sion of primary antibody during the immunoprecipitation wascarried out as a negative control.

Statistical analysisValues are expressed as mean � SEM. Statistical analysis

was conducted using 2-way ANOVA and subsequent pairwise

comparisons using Bonferroni post hoc tests. A P value lessthan 0.05was considered statistically significant in all analyses.

ResultsIEC-specific PHB overexpression decreases colonictumorigenesis in a mouse model of CAC

Previous studies have shown that PHB has an antitumori-genic role in gastric, prostate, and liver cancers (21–23). Weused the AOM DSS mouse model to study the role of PHB inCAC. Body weight was measured weekly as one parameter toassess the severity of disease. WT and Tgmice givenwater onlythroughout the experiment and mice injected with AOMfollowed by water alone showed similar body weight gain overthe 8-week protocol (Supplementary Fig. S1A) and did notdevelop polyps as previously reported for this dose of AOM(25). WTmice given 2 cycles of DSS without AOM injection lostmore bodyweight following DSS administration and recoveredless weight compared with Tg mice over the 8-week protocol(Supplementary Fig. S1B), similar to our previous findings (12).Although both WT and Tg AOM DSS-treated mice lost weightafter the first administration of DSS, Tg mice gained bodyweightmore rapidly during the recovery phase andmaintainedbody weight better thanWTmice throughout the remainder ofthe study (Fig. 1A).

Stool consistency, stool occult blood, and body weight losswere monitored following DSS administration to generate aclinical disease score. AOM DSS-treated Tg mice showed adecreased clinical score after the first administration of DSScompared with WT mice, but this did not reach statisticalsignificance. Tg mice exhibited a significantly lower clinicalscore after the second administration of DSS (Fig. 1B). Tg micegiven DSS without AOM injection had lower clinical scoresafter DSS administration compared with WT mice (Supple-mentary Fig. S1C), similar to our previous findings (12).

The overall survival rate for AOM DSS-treated Tg mice was82% (14 of 17 mice) compared with 60% for WT (12 of 20 mice)(Fig. 1C). Tg mice given DSS without AOM injection exhibitednomortality throughout the 8-week protocol (n¼ 8) comparedwith 60% survival rate for WT mice (12 of 20 mice; Supple-mentary Fig. S1D). Upon sacrifice the entire colon from ileo-cecal junction to anus was excised and assessed for numberand size of polyps. Tg mice showed considerably fewer polypswith the majority being smaller than 3 mm in diametercompared with WT mice (Fig. 1D). Figure 1E shows represen-tative photos of excised colons and endoscopic images of polypformation in WT and Tg mice following AOM-DSS treatment.After polyps were counted and measured, colons were Swiss-rolled and assessed for histopathologic score by a trainedpathologist. WT mice had a higher occurrence of high-gradedysplasia and adenocarcinoma compared with Tgmice, whichexhibited more low-grade dysplasia and normal morphology(Fig. 1F). Collectively, these results suggest that PHB Tg miceshow less susceptibility to CAC than WT mice.

PHB Tg mice are less susceptible to CAC and mortalityTo assess mortality and cancer development, the same

protocol was followedwith the exception of extending through

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Cancer Res; 72(22) November 15, 2012 Cancer Research5780




126 days or until mice developed rectal prolapse and/or>20% body weight loss. WT mice showed greater mortalitythan Tg mice throughout the study. The difference inmortality was especially pronounced after the formation ofpolyps, which occurs at approximately 8 weeks (Supple-mentary Fig. S2A). Tg mice showed significantly less totalnumber of polyps, and although rare, a significantly lessnumber of large polyps (>6 mm in diameter) compared withWT mice (Supplementary Fig. S2B). Mice that survived from8 through 18 weeks were assessed for histopathologic scoreas animals that died before 8 weeks did not yet developpolyps. As shown in Supplementary Fig. S2C, the majority ofWT mice developed adenocarcinoma (invasive adenocarci-

noma: n ¼ 2, intramucosal adenocarcinoma: n ¼ 7) com-pared with 5 Tg mice (intramucosal adenocarcinoma). Themajority of Tg mice had low- or high-grade dysplasia.Supplementary Fig. S2D shows representative gross anato-my of colon from 3 WT and Tg mice, which also reflects thenumber and size of polyps. These findings suggest that IEC-specific PHB overexpression improves mortality associatedwith CAC.

IEC-specific PHB overexpression does not affect cellproliferation during CAC

Alteration in cell proliferation can contribute to tumorigen-esis (28). Ki67 immunohistochemical staining and PCNA were

Figure 1. IEC-specific PHBoverexpression reduces colonictumorigenesis in a mouse model ofCAC. Mice were intraperitoneallyinjected with azoxymethane (AOM)and 7 days later treated with 2 cyclesof 3%DSS, eachcycle consistingof 7days DSS followed by 14 days ofwater recovery, and sacrificed on day56 after AOM injection. A, percentchange in body weight during eachweek of the CAC protocol. B,clinical score following DSStreatment. End first cycle DSS, n¼ 13WT; n ¼ 14 Tg. End second cycleDSS, n ¼ 12 WT; n ¼ 14 Tg. C,percent survival. P ¼ 0.146. D,number of polyps and sizedistribution of polyps as determinedusing a dissecting microscope. E,representative gross anatomy of thecolon (top) and representativecolonoscopy images (bottom) at theend of the CAC protocol. F,histopathology scores. �, P < 0.05 vs.AOM DSS-treated WT.

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assessed to examine the effect of PHB overexpression on cellproliferation in CAC. Both WT and Tg mice showed increasedKi67 staining following AOM-DSS treatment (Fig. 3A, middlepanels) compared with mice given water (Supplementary Fig.S3A, top panels and Supplementary Fig. S3B). There was nosignificant difference in the percent Ki67-positive cells percrypt in normal epithelium (Supplementary Fig. S3A, middlepanels and Supplementary Fig. S3B) or areas of dysplasia(Supplementary Fig. S3A, bottom panels) betweenWT and Tgmice treated with AOM DSS. Similar results were corrobo-rated by PCNA Western blot analysis. PCNA proteinexpression was increased in both WT and Tg mice followingAOM-DSS treatment compared with mice given water (Sup-plementary Fig. S3C and S3D). There was no significantdifference in PCNA protein expression between WT and Tgmice treated with AOM DSS. These results suggest that WTand Tg mice exhibited increased cell proliferation after AOMDSS, and that IEC-specific PHB overexpression does not alter

cell proliferation induced by AOM-DSS treatment or incolonic tumors/polyps.

IEC-specific PHB overexpression is associated withincreased apoptosis during CAC

Suppression of apoptosis plays an important role in tumor-igenesis by allowing genetically compromised cells toproliferate(29). TUNEL staining and Western blot analysis of cleavedcaspase-3 levels were used to assess IEC apoptosis during CAC.WTandTgmice exhibited increasednumber ofTUNEL-positivecells per crypt after AOM-DSS treatment (Fig. 2AandB). Tgmiceexhibited a higher number of TUNEL-positive cells comparedwith WT mice after AOM-DSS treatment in normal epithelium(Fig. 2A and B). Figure 2C and D corroborate the TUNELimmunofluorescence staining by showing increased proteinexpression of cleaved caspase-3 in Tg mice compared with WTmice with CAC. Thus, IEC-specific PHB overexpression maysuppress tumor development by promoting IEC apoptosis.

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Figure 2. IEC-specific PHBoverexpression is associated withincreased apoptosis during CAC.A, fluorescent micrograph ofTUNEL staining (green) of colonicsections from untreated (water) andAOM-DSS treated WT and PHB Tgmice. Sections were stained withDAPI to visualize nuclei (blue).Scale bars, 100 mm. B, number ofTUNEL-positive cells per crypt innormal epithelium. �,P < 0.05; n¼ 3for water; n ¼ 9 for AOM DSS. C,representative Western blotsshowing colonic mucosal cleavedcaspase-3 levels and b-actin as aloading control. D, cumulativemean � SEM relative to WT water.�, P < 0.05 vs. WT AOM DSS; n � 5per treatment.

Kathiria et al.





IEC-specific overexpression of PHB is associated withaltered levels of p53, PUMA, Bax, and p-Bad during CACGiven the effect of PHB overexpression on apoptosis during

CAC, we examined levels of p53, a tumor suppressor, and itsdownstream targets PUMA and Bax as well as the activationof the proapoptotic protein Bad. p53 regulates cell survivalthrough induction of cell cycle arrest or apoptosis. Our pre-vious study showed that PHB protein levels are decreased inmucosal biopsies of active inflammation from Crohn's diseaseafflicted patients and in theDSS and IL-10�/�mousemodels ofcolitis (9). To determine whether mucosal PHB levels areaffected during CAC, Western blot analysis was carried outon total protein isolated from mucosal stippings from WTmice. Figure 3A shows that mucosal PHB protein levels were

decreased in WT mice after AOM-DSS treatment comparedwithwater controlWTmice (water¼ 1.0� 0.09 vs. AOMDSS¼0.75 � 0.06, P < 0.05). Tg mice exhibited higher expression ofmucosal PHB protein levels compared with WT mice whengiven water or AOM-DSS treatment (Fig. 3A). Mucosal PHBprotein expression in Tg mice was similar to our previousstudies using these animals (12). Although induction of CACincreased p53 protein expression in both WT and Tg micecompared with water control mice, p53 levels were signifi-cantly higher in Tg mice compared with WT mice after AOM-DSS treatment (Fig. 3B). Sequencing of exons 5 to 8 of p53 fromDNA isolated from areas of dysplasia from 5WT and 5 Tgmicefound no mutations. PUMA, a central mediator of the p53apoptotic response and suppressor of intestinal tumorigenesis

Figure 3. IEC-specificoverexpression of PHB is associatedwith altered levels of p53, PUMA,Bax, and activation of Bad.Representative Western blots of totalprotein isolated from colonicmucosal strippings were probed withanti-PHB (A), anti-p53 (B), anti-PUMA(C), anti-Bax (D), and anti-phospho-Bad and anti-Bad (E). Anti-b-actinwas used as a loading control.Histograms show cumulative meanband densitometry � SEM,respectively. n � 5 per treatment.�, P < 0.05; ��, P < 0.01.

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in mice (30), was increased only in Tg mice during CAC (Fig.3C). Similarly, the proapoptotic protein Bax was also increasedin Tg mice after AOM-DSS treatment (Fig. 3D). Phosphoryla-tion of Bad at serine 155 inhibits the proapoptotic function ofBad by disrupting its binding to antiapoptotic Bcl-2 (31).Western blot analysis showed that Tg mice exhibited lessphosphorylation of Bad at serine 155 compared with WT miceduring CAC (Fig. 3E), suggesting that in Tgmice Bad retains itsproapoptotic function. Together, these data suggest that CACin PHB Tg mice is associated with increased expression of p53,PUMA, and Bax and decreased Bad phosphorylation.

PHB-induced altered expression of PUMA, Bax, and Badand requires p53 signaling in HCT116 cells

HCT116 colorectal carcinoma cells were used as an in vitromodel to understand the underlying role of PHB inmodulating

p53 activation and apoptosis. WT and p53�/� HCT116 cellswere transiently transfected with GFP-tagged PHB (GFP PHB)or vector alone and downstream targets of p53 were measuredby Western blot analysis. WT HCT116 cells overexpressingPHB (Fig. 4A, lane 2) showed increased PUMA and Bax anddecreased phospho-Bad protein expression compared withvector-transfected cells (lane 1). Cleaved caspase-3, a markerof apoptosis, was also increased in PHB overexpressing cells(Fig. 4A, lane 2 vs. lane 1). p53�/� HCT116 cells showed nochange in PUMA, Bax, phospho-Bad, or cleaved caspase-3protein expression after PHB transfection (Fig. 4A, lane 6)compared with vector-transfected cells (lane 5), suggestingthat p53 is required for PHB-induced alteration of theseproteins. WT and p53�/� HCT116 cells were exposed tog-irradiation to induce DNA damage. Following g-irradiation,PHB overexpressing WT HCT116 cells showed significantly

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Figure 4. PHB-induced alteredexpression of PUMA, Bax, and Badrequires p53 signaling in HCT116cells and PHB interacts with p53 inHCT116 cells and in colonmucosa.A, WT and p53�/� HCT116 cellswere transiently transfected withpEGFPN1 vector or pEGFPN1-PHB (GFP-PHB) and subjected tog-irradiation to induce DNAdamage 72 hours posttransfection.Total protein was collected 24hours after g-irradiation. Westernblots were probed with anti-PUMA,anti-Bax, anti-phospho-Bad, anti-Bad, and anti-caspase-3. Blotswere subsequently probed withanti-GFP to ensure transfectionefficiency and anti-p53 to ensurep53 knockout. Anti-b-actin wasused as a loading control.Histograms show cumulative meanband densitometry � SEM. n ¼ 3per treatment. �, P < 0.05. PHB (B)or p53 (C) was immunoprecipitatedfrom total HCT116 WT cell lysatesoverexpressing vector orGFP-PHBand subjected to g-irradiation toinduce DNA mutation 72 hoursposttransfection. Total protein wascollected 24 hours afterg-irradiation. Immunoprecipitateswere separated by SDS-PAGE andmembranes were immunoblottedfor p53 or PHB expression.Omission of primary antibodyduring the immunoprecipitationwas carried out as a negativecontrol. D, PHB wasimmunoprecipitated from colonmucosal protein lysates fromwater- andAOMDSS-treatedmice,followed by immunoblot for p53expression. IB, immunoblotting; IP,immunoprecipitation.

Kathiria et al.





increased PUMA, Bax, and cleaved caspase-3 protein expres-sion whereas phospho-Bad levels were decreased (Fig. 4A, lane4) compared with vector-transfected cells (lane 3). PHB over-expressing WT cells exposed to g-irradiation exhibited afurther increase in Bax and cleaved caspase-3 and decreasein phospho-Bad compared with PHB overexpressing cells leftuntreated (Fig. 4A, lane 4 vs. lane 2). g-Irradiation did not alterexpression of PUMA, Bax, phospho-Bad, or cleaved caspase-3in p53�/�HCT116 cells regardless of PHB transfection (Fig. 4A,lanes 7 and 8).

PHB interacts with p53 in HCT116 cells and in colonmucosaTo determine whether PHB interacts with p53 in IECs, PHB

was immunoprecipitated from total cell lysates from WTHCT116 cells overexpressing vector or PHB and exposed tog-irradiation to induce DNA damage. Following immunopre-cipitation and SDS-PAGE, membranes were immunoblottedfor p53 and subsequently PHB expression. p53 coimmunopre-cipitated with PHB during basal conditions and after g-irra-diation (Fig. 4B and C). To determine whether PHB interactswith p53 in vivo, we conducted coimmunoprecipitation experi-ments for PHB and p53 in colon mucosal lysates from water-and AOM DSS-treated mice. p53 coimmunoprecipitated withPHB in lystates from both WT and Tg mice (Fig. 4D), withincreased levels in Tg mice (Fig. 4D). Collectively, these resultssuggest that PHB interacts with p53 in cultured human CRCcells and in colon mucosa.

PHB interacts with phospho-STAT3 and modulatesSTAT3 responsive genes, Bcl-xL, and Bcl-2 in vivo andin vitroThe IL-6/STAT3 signaling pathway is known to play a

pathogenic role in intestinal inflammation and colonic cancerthrough the induction of the antiapoptotic genes Bcl-xL andBcl-2 (32). Caco2-BBE cells, which are responsive to IL-6, showdecreased basal and IL-6-induced STAT3 activation, anddecreased Bcl-xL and Bcl-2 protein expression when PHB isoverexpressed (Fig. 5A). Coimmunoprecipitation experimentsusing Caco2-BBE cells treated with IL-6 suggest that PHBinteracts with phospho-STAT3 (Fig. 5B and C). To determinewhether PHB interacts with STAT3 in vivo, we conductedcoimmunoprecipitation experiments for PHB and phospho-STAT3 in colon mucosal lysates from water- and AOM DSS-treated mice. Phospho-STAT3 coimmunoprecipitated withPHB in lystates from Tg mice (Fig. 5D). Induction of CACincreased protein expression of STAT3 responsive genes Bcl-2and Bcl-xL in both WT and Tg mice compared with watercontrol mice. Bcl-2 and Bcl-xL levels were significantlydecreased in Tg mice compared with WT mice after AOM-DSS treatment (Fig. 5E).

PHB mRNA expression is decreased in inflamed UC andUC-associated dysplasia and inversely correlates withSTAT3 and p53 mRNA expressionQuantitative real-time PCR analysis of total RNA isolated

frommucosal samples from patients with inflamed UC or UC-associated adenocarcinoma/dysplasia revealed that PHB

expression is significantly decreased compared with matchednormal samples (Fig. 6). p53 expression was increased ininflamed UC and UC-associated dysplasia tissues, howeverthis increase did not achieve statistical significance in theinflamed tissue samples. STAT3 expression was markedlyincreased in inflamed UC and UC-associated dysplasia butdid not achieve statistical significance.

DiscussionThe specificmechanism bywhich IBD leads to CRC is poorly

understood, but it is clear that chronic mucosal inflammationplays a causative role in the transition to adenocarcinoma. Ourprevious studies have shown that restoration of mucosal PHBcan ameliorate experimental colitis (11, 12). For this reason, wechose to use the AOM DSS model to assess the role of PHB inCAC. Here, we show that in mice with CAC, IEC-specific PHBoverexpression suppresses colonic neoplastic formation inassociation with increased rates of apoptosis. IEC-specificoverexpression of PHB was associated with altered levels ofp53, PUMA, Bax, p-Bad, Bcl-xL, and Bcl-2 during CAC. Weshow, for the first time, that PHB interacts with phospho-STAT3 in addition to p53 in human colon cancer cells andcolon mucosa, thereby modulating the apoptotic response(Fig. 7).

IBD patients with longstanding colitis require surveillancecolonoscopy after 8 years of disease duration because of thehigh risk of cancer development (33). Several features makeCAC distinct from sporadic colon cancer. CAC is often ana-plastic, broadly infiltrating, rapidly growing, develops in flatdysplastic tissue, and occurs at a younger age. While geneticalterations are similar to those in sporadic colon cancer, thefrequency and sequence of these events differ in CAC (34). Lossof p53 function is an early critical event in colitis-associatedneoplasia in humans in the pathway from dysplasia to cancer(35). Furthermore, aberrent IL-6/STAT3 signaling is associatedwith IBD and CAC (32). Activated STAT3 is enhanced in CRCpatients and has been shown to induce the antiapoptoticproteins Bcl-2 and Bcl-xL (36). Inhibition of STAT3 signalinginduces apoptosis of CRC cells via the mitochondrial pathwayby modulating Bcl-2 and Bcl-xL (37). Here, we show that inmice with CAC, PHB suppresses colonic tumor formation inassociation with increased rates of apoptosis. In human coloncancer cells, PHB interacts with p53 and phospho-STAT3 andmodulates their downstream apoptotic proteins. These find-ings suggest that PHB protects against CAC bymodulating p53and STAT3 apoptotic responses.

The best-characterized function of PHB is as a chaperoneinvolved in the stabilization of mitochondrial proteins therebymaintaining normal mitochondrial function and morphology(reviewed in ref. 38). In most cell types studied, including IECs,PHB is predominantly localized to the mitochondria (9).However, it has been localized to the cell membrane, cyto-plasm, or the nucleus in some cell types (6, 39, 40). AlthoughPHB expression is increased in many transformed cells andtumors (13–19), in our study, colonic mucosal PHB proteinexpression was reduced after the development of colitis-asso-ciated tumors in WT mice. PHB expression was decreased in






mucosal samples from human patients with inflamed UC orUC-associated adenocarcinoma/dysplasia. Proinflammatorycytokines such as TNFa and reactive oxygen species decreaseexpression of intestinal epithelial PHB in vivo and in vitro (8–10). Thus, it is likely that PHB levels are decreased in the colonicmucosa following AOM-DSS treatment due to the inflamma-tory process. Tg mice with forced overexpression of PHBin IECs exhibited no inflammation-induced loss of PHBexpression and showed decreased susceptibility to CAC andmortality.

PHB has been reported to have an antitumorigenic rolein prostate cancer (21), gastric cancer (23), and liver cancer(22). Mice with hepatocyte-specific deletion of PHB exhib-ited liver injury, fibrosis, oxidative stress, and hepatocellu-lar carcinoma developing by 8 months of age (22). Mito-chondrial abnormalities in hepatocytes are evident by 3weeks of age including no cristae formation (22). Knock-

down of PHB expression by siRNA in Caco2-BBE IECscaused mitochondrial depolarization and increased intra-cellular reactive oxygen species (41). Interestingly, mito-chondrial dysfunction is a common feature of cancer cellsand multiple studies suggest that mitochondrial chaperoneproteins, including PHB, modulate tumorigenesis (reviewedin ref. 42).

A functional role for PHB as a regulator of transcriptionhas become evident due to its interactions with p53, E2F,and Rb (6, 7, 43). Here, we show that in addition to p53, PHBinteracts with STAT3 in vitro and in vivo. Studies in breastcancer, prostate cancer, and retinal pigment epithelial celllines indicate that PHB increases the transcriptional activityand prevents denaturation of p53, thereby regulating apo-ptosis (44–46). Our study showed that PHB suppressescolonic tumor formation in association with increased ratesof apoptosis and p53 apoptotic responses. Sequencing of

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Figure 5. PHB interacts withphospho-STAT3 and modulatesSTAT3 responsive genes, Bcl-xLand Bcl-2 in vivo and in vitro. A,representative Western blots ofpSTAT3, Bcl-2, Bcl-xL proteinexpression in Caco2-BBE cellsstably overexpressing pEGFPN1-PHB (GFP-PHB) or empty vectorand treated with 100 ng/mL IL-6basolaterally for 1 hour. Blotswere subsequently probedwith anti-GFP to ensuretransfection efficiency. B and C,coimmunoprecipitation of PHBand pSTAT3 using cell lysatesdescribed in A. D, PHB wasimmunoprecipitated from colonmucosal protein lysates fromwater- and AOM DSS-treatedmice, followed by immunoblotfor pSTAT3 expression. E,representative Western blots oftotal protein isolated from colonicmucosal strippings were probedwith anti-Bcl-2, anti-Bcl-xL, anti-b-actin (loading control).Histograms show mean banddensitometry � SEM. n � 5 pertreatment. �, P < 0.05.

Kathiria et al.





exons 5, 6, 7, and 8 of p53 from DNA isolated from areas ofdysplasia from 5 WT and 5 Tg mice found no mutations.Unlike human UC-associated cancers, DSS-induced colontumors in mice do not commonly harbor p53 mutations (26).Therefore, it is likely that the increased p53 in Tg mice isfunctional, unlike the increased, but mutant p53, in humanIBD patients. PHB interaction with phospho-STAT3 wasassociated with decreased Bcl-xL and Bcl-2 expression incultured IECs and colon mucosa. The localization of PHBin the mitochondria and nucleus makes it an ideal candidateto modulate the mitochondrial apoptotic pathway and tran-scription activation. Further studies will assess the mecha-nism of PHB modulation of STAT3 signaling. PHB interactswith p53 and STAT3 in colon mucosa from mice treatedwith DSS alone without AOM injection (Supplementary Fig.S1E), suggesting interaction is not dependent upon thepresence of tumors. Furthermore, Tg mice treated with DSSalone without AOM injection exhibited altered mucosalp53, PUMA, Bcl-2, and Bcl-xL protein expression (Supple-mentary Fig. S1F). Because the development of CAC isdependent on the severity of chronic inflammation, PHBtransgene expression likely decreases the occurrence of CACby decreasing the severity of DSS-induced inflammation andenhancing p53-mediated apoptosis and inhibiting STAT3induction of antiapoptotic genes during tumor formation.Early loss of PHB expression during inflammation may makegenetically compromised IECs more resistant to cell death,thereby promoting tumorigenesis.In conclusion, PHB suppresses colonic tumor formation

in association with increased rates of apoptosis in micewith CAC. PHB interacts with p53 and phospho-STAT3 in

IECs in vivo and in vitro and modulates downstreamapoptotic responses. Reduced levels of PHB during chronicintestinal inflammation may be an underlying factor pro-moting tumorigenesis. Modulation of PHB expression inIECs represents a potential therapeutic approach to pre-vent colitis-associated tumorigenesis.

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

Authors' ContributionsConception and design: A.S. Kathiria, A.L. TheissDevelopment of methodology: A.S. Kathiria, W.L. NeumannAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A.S. Kathiria, W.L. Neumann, Y.Cheng, S.J. Meltzer, A.L. TheissAnalysis and interpretation of data (e.g., statistical analysis, bio-statistics, computational analysis): A.S. Kathiria, R.M. Genta, R.F. Souza,A.L. TheissWriting, review, and/or revision of the manuscript: A.S. Kathiria, W.L.Neumann, S.J. Meltzer, R.F. Souza, A.L. TheissAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): J. Rhees, E. Hotchkiss, R.M. Genta, S.J.MeltzerStudy supervision: A.L. TheissInterpreting pathology slides: R.M. Genta

AcknowledgmentsThe authors thank the late Dr. Shanthi V. Sitaraman from Emory Uni-

versity, Atlanta, GA for her mentoring role with this project; Dr. Ajay Goelfrom Baylor Research Institute, Baylor University Medical Center, Dallas, TXfor providing the HCT116 cell line as well as Bax, Bcl-2, and Bcl-xL antibodies;Dr. Linda A. Feagins from Veterans Affairs North Texas Health Care System,University of Texas Southwestern Medical Center, Dallas, TX for helpfulscientific discussions. The authors also thank Dr. Shinichi Matsumoto, AnaRahman, Jeff SoRelle, and Mazhar Kanak for their assistance with tissuesectioning through the Baylor Islet Cell Transplant Program, Baylor ResearchInstitute as well as Dr. Steven Phillips from Baylor Research Institute for hisassistance with the g-irradiator.

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Figure 6. PHB mRNA expression is decreased in inflamed ulcerativecolitis and ulcerative colitis-associated dysplasia and inverselycorrelates with STAT3 and p53 mRNA expression. Quantitative real-time PCR analysis of PHB, STAT3, and p53 in total RNA isolatedfrom patients with inflamed ulcerative colitis (UC) or UC-associatedadenocarcinoma/dysplasia. n ¼ 8 normal; n ¼ 5 UC-dysplasia; n ¼ 5UC. �, P < 0.05 vs. normal.

IL-6

Other cytokines

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Bcl-xL

Bcl-2

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Figure 7. Model of PHB modulation of p53 and STAT3 apoptoticresponses. PHB induces apoptosis in cells with DNA damage duringinflammation through inhibition of STAT3-induced Bcl-xL and Bcl-2 andinduction of p53 signaling via activation of Bax andPUMA. ReducedPHBexpression in inflamed tissues may make genetically compromisedintestinal epithelial cells more resistant to cell death, thereby promotingtumorigenesis.






Grant SupportThis work was supported by NIH grants K01-DK085222 (A.L. Theiss) and R01-

CA133012 (S.J. Meltzer) and funds from the Baylor Research Institute (A.L.Theiss).

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.

Received February 16, 2012; revised June 6, 2012; accepted June 26, 2012;published OnlineFirst August 6, 2012.

References1. Abraham C, Cho JH. Inflammatory bowel disease. N Engl J Med

2009;361:2066–78.2. Gillen CD, Walmsley RS, Prior P, Andrews HA, Allan RN. Ulcerative

colitis and Crohn's disease: a comparison of the colorectal cancer riskin extensive colitis. Gut 1994;35:1590–2.

3. Grivennikov S, Karin E, Terzic J, Mucida D, Yu GY, Vallabhapurapu S,et al. IL-6 and Stat3 are required for survival of intestinal epithelial cellsand development of colitis-associated cancer. Cancer Cell 2009;15:103–13.

4. PopivanovaBK,KitamuraK,WuY,KondoT, KagayaT,KanekoS, et al.Blocking TNF-alpha in mice reduces colorectal carcinogenesis asso-ciated with chronic colitis. J Clin Invest 2008;118:560–70.

5. McClung JK, Jupe ER, Liu XT, Dell'Orco RT. Prohibitin: potential role insenescence, development, and tumor suppression. Exp Gerontol1995;30:99–124.

6. Wang S, Fusaro G, Padmanabhan J, Chellappan SP. Prohibitin co-localizes with Rb in the nucleus and recruits N-CoR and HDAC1 fortranscriptional repression. Oncogene 2002;21:8388–96.

7. Wang S, Zhang B, Faller DV. Prohibitin requires Brg-1 and Brm for therepression of E2F and cell growth. EMBO J 2002;21:3019–28.

8. Hsieh SY, Shih TC, Yeh CY, Lin CJ, Chou YY, Lee YS. Comparativeproteomic studies on the pathogenesis of human ulcerative colitis.Proteomics 2006;6:5322–31.

9. Theiss AL, Idell RD, Srinivasan S, Klapproth JM, Jones DP, Merlin D,et al. Prohibitin protects against oxidative stress in intestinal epithelialcells. FASEB J 2007;21:197–206.

10. Theiss AL, Jenkins AK, Okoro NI, Klapproth JM, Merlin D, SitaramanSV. Prohibitin inhibits tumor necrosis factor alpha-induced nuclearfactor-kappa B nuclear translocation via the novel mechanism ofdecreasing importin alpha3 expression. Mol Biol Cell 2009;20:4412–23.

11. Theiss AL, Laroui H, Obertone TS,Chowdhury I, ThompsonWE,MerlinD, et al. Nanoparticle-based therapeutic delivery of prohibitin to thecolonic epithelial cells ameliorates acutemurine colitis. InflammBowelDis 2011;17:1163–76.

12. Theiss AL, Vijay-Kumar M, Obertone TS, Jones DP, Hansen JM,Gewirtz AT, et al. Prohibitin is a novel regulator of antioxidant responsethat attenuates colonic inflammation in mice. Gastroenterology2009;137:199–208, 08 e1–6.

13. Tsai HW, Chow NH, Lin CP, Chan SH, Chou CY, Ho CL. The signif-icance of prohibitin and c-Met/hepatocyte growth factor receptor inthe progression of cervical adenocarcinoma. Hum Pathol 2006;37:198–204.

14. Ren HZ, Wang JS, Wang P, Pan GQ, Wen JF, Fu H, et al. Increasedexpression of prohibitin and its relationship with poor prognosis inesophageal squamous cell carcinoma. Pathol Oncol Res 2010;16:515–22.

15. Kang X, Zhang L, Sun J, Ni Z,MaY, Chen X, et al. Prohibitin: a potentialbiomarker for tissue-based detection of gastric cancer. J Gastroen-terol 2008;43:618–25.

16. Sato T, Sakamoto T, TakitaK, SaitoH,OkuiK,NakamuraY. Thehumanprohibitin (PHB) gene family and its somatic mutations in humantumors. Genomics 1993;17:762–4.

17. Nan Y, Yang S, Tian Y, Zhang W, Zhou B, Bu L, et al. Analysis of theexpression protein profiles of lung squamous carcinoma cell usingshot-gun proteomics strategy. Med Oncol 2009;26:215–21.

18. WuTF,WuH,WangYW,ChangTY,ChanSH, Lin YP, et al. Prohibitin inthe pathogenesis of transitional cell bladder cancer. Anticancer Res2007;27:895–900.

19. Gregory-Bass RC, Olatinwo M, Xu W, Matthews R, Stiles JK, ThomasK, et al. Prohibitin silencing reverses stabilization of mitochondrial

integrity and chemoresistance in ovarian cancer cells by increasingtheir sensitivity to apoptosis. Int J Cancer 2008;122:1923–30.

20. Coates PJ, Nenutil R, McGregor A, Picksley SM, Crouch DH, Hall PA,et al. Mammalian prohibitin proteins respond to mitochondrial stressand decrease during cellular senescence. Exp Cell Res 2001;265:262–73.

21. Dart DA, Spencer-Dene B, Gamble SC, Waxman J, Bevan CL. Manip-ulating prohibitin levels provides evidence for an in vivo role in andro-gen regulation of prostate tumours. Endocr Relat Cancer 2009;16:1157–69.

22. Ko KS, Tomasi ML, Iglesias-Ara A, French BA, French SW, Ramani K,et al. Liver-specific deletion of prohibitin 1 results in spontaneous liverinjury, fibrosis, and hepatocellular carcinoma in mice. Hepatology2010;52:2096–108.

23. Liu T, Tang H, Lang Y, Liu M, Li X. MicroRNA-27a functions as anoncogene in gastric adenocarcinoma by targeting prohibitin. CancerLett 2009;273:233–42.

24. Fukata M, Chen A, Vamadevan AS, Cohen J, Breglio K, KrishnareddyS, et al. Toll-like receptor-4 promotes the development of colitis-associated colorectal tumors. Gastroenterology 2007;133:1869–81.

25. Clapper ML, Cooper HS, Chang WC. Dextran sulfate sodium-inducedcolitis-associated neoplasia: a promising model for the developmentof chemopreventive interventions. Acta Pharmacol Sin 2007;28:1450–9.

26. ChangWC, Coudry RA, Clapper ML, Zhang X,Williams KL, Spittle CS,et al. Loss of p53 enhances the induction of colitis-associated neo-plasia by dextran sulfate sodium. Carcinogenesis 2007;28:2375–81.

27. Bunz F, Hwang PM, Torrance C,Waldman T, Zhang Y, Dillehay L, et al.Disruption of p53 in human cancer cells alters the responses totherapeutic agents. J Clin Invest 1999;104:263–9.

28. Terzic J, Grivennikov S, Karin E, Karin M. Inflammation and coloncancer. Gastroenterology 2010;138:2101–14, e5.

29. Huerta S, Goulet EJ, Livingston EH. Colon cancer and apoptosis. Am JSurg 2006;191:517–26.

30. QiuW, Carson-Walter EB, Kuan SF, Zhang L, Yu J. PUMA suppressesintestinal tumorigenesis in mice. Cancer Res 2009;69:4999–5006.

31. Downward J. How BAD phosphorylation is good for survival. Nat CellBiol 1999;1:E33–5.

32. Atreya R, Neurath MF. Signaling molecules: the pathogenic role of theIL-6/STAT-3 trans signaling pathway in intestinal inflammation and incolonic cancer. Curr Drug Targets 2008;9:369–74.

33. Neumann H, Vieth M, Langner C, Neurath MF, Mudter J. Cancer risk inIBD: how to diagnose and how to manage DALM and ALM. World JGastroenterol 2011;17:3184–91.

34. Feagins LA, Souza RF, Spechler SJ. Carcinogenesis in IBD: potentialtargets for the prevention of colorectal cancer. Nat Rev GastroenterolHepatol 2009;6:297–305.

35. Brentnall TA, Crispin DA, Rabinovitch PS, Haggitt RC, Rubin CE,Stevens AC, et al. Mutations in the p53 gene: an early marker ofneoplastic progression in ulcerative colitis. Gastroenterology1994;107:369–78.

36. Lassmann S, Schuster I, Walch A, Gobel H, Jutting U, Makowiec F,et al. STAT3mRNAandprotein expression in colorectal cancer: effectson STAT3-inducible targets linked to cell survival and proliferation. JClin Pathol 2007;60:173–9.

37. DuW, Hong J,Wang YC, Zhang YJ, Wang P, SuWY, et al. Inhibition ofJAK2/STAT3 signaling induces colorectal cancer cell apoptosis viamitochondrial pathway. J Cell Mol Med. 2011 Nov 2. [Epub ahead ofprint].

38. Artal-Sanz M, Tavernarakis N. Prohibitin and mitochondrial biology.Trends Endocrinol Metab 2009;20:394–401.

Kathiria et al.





39. Rastogi S, Joshi B, Fusaro G, Chellappan S. Camptothecin inducesnuclear export of prohibitin preferentially in transformed cellsthrough a CRM-1-dependent mechanism. J Biol Chem 2006;281:2951–9.

40. Sharma A, Qadri A. Vi polysaccharide of Salmonella typhi targets theprohibitin family of molecules in intestinal epithelial cells and sup-presses early inflammatory responses. Proc Natl Acad Sci U S A2004;101:17492–7.

41. Kathiria AS, Butcher LD, Feagins LA, Souza RF, Boland CR, Theiss AL.Prohibitin 1 modulates mitochondrial stress-related autophagy inhuman colonic epithelial cells. PLoS One 2012;7:e31231.

42. Czarnecka AM, Campanella C, Zummo G, Cappello F. Mitochondrialchaperones in cancer: from molecular biology to clinical diagnostics.Cancer Biol Ther 2006;5:714–20.

43. Joshi B, Rastogi S, Morris M, Carastro LM, DeCook C, Seto E, et al.Differential regulation of human YY1 and caspase 7 promoters byprohibitin through E2F1 and p53 binding sites. Biochem J 2007;401:155–66.

44. Chander H, Halpern M, Resnick-Silverman L, Manfredi JJ, Germain D.Skp2B attenuates p53 function by inhibiting prohibitin. EMBO Rep2009;11:220–5.

45. Fusaro G, Dasgupta P, Rastogi S, Joshi B, Chellappan S. Prohibitininduces the transcriptional activity of p53 and is exported from thenucleus upon apoptotic signaling. J Biol Chem 2003;278:47853–61.

46. Chander H, Halpern M, Resnick-Silverman L, Manfredi JJ, Germain D.Skp2B overexpression alters a prohibitin-p53 axis and the transcrip-tion of PAPP-A, the protease of insulin-like growth factor bindingprotein 4. PLoS One 2011;6:e22456.






2012;72:5778-5789. Published OnlineFirst August 6, 2012.Cancer Res Arwa S. Kathiria, William L. Neumann, Jennifer Rhees, et al. Modulating p53 and STAT3 Apoptotic ResponsesProhibitin Attenuates Colitis-Associated Tumorigenesis in Mice by

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