Prohibitin Attenuates Colitis-Associated Tumorigenesis in Mice by Modulating P53 and STAT3 Apoptotic Responses

Published OnlineFirst August 6, 2012; DOI: 10.1158/0008-5472.CAN-12-0603

Cancer Molecular and Cellular Pathobiology Research

Prohibitin Attenuates Colitis-Associated Tumorigenesis in Mice 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

Abstract Although inflammatory bowel disease is associated with higher risk of colorectal cancer, the precise pathogenic mechanisms underlying this association are not completely understood. Prohibitin 1 (PHB), a protein implicated in the regulation of proliferation, apoptosis, and transcription, is decreased in intestinal inflammation. In this study, we have established a key function for PHB in mediating colitis-associated cancer. Wild-type and transgenic (Tg) mice specifically overexpressing PHB in intestinal epithelial cells were subjected to a classical two-stage protocol of colitis-associated carcinogenesis. In addition, wild-type and p53 null human cell models were used to assess PHB interaction with STAT3 and p53. Wild-type mice exhibited decreased mucosal PHB protein expression during colitis-associated carcinogenesis. Tg mice exhibited decreased susceptibility in a manner associated with increased apoptosis, p53, Bax, and Bad expression plus decreased Bcl-xL and Bcl-2 expression. PHB overexpression in wild-type but not p53 null human cells increased expression of Bax, Bad, and caspase-3 cleavage. In wild-type p53 cells, PHB overexpression decreased basal and interleukin-6-induced STAT3 activation and expression of the STAT3 responsive genes Bcl-xL and Bcl-2. PHB coimmunoprecipitated with phospho-STAT3 in addition to p53 in cultured cell lysates and colon mucosa. This is the first study to show interaction 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 in intestinal epithelial cells may offer a potential therapeutic approach to prevent colitis-associated carcinogenesis. Cancer Res; 72(22); 1–12. 2012 AACR.

Introduction of 7% after 20 years for UC and 8% for Crohn's disease (2). fl Inflammatory bowel disease (IBD) includes 2 major chronic Previous studies have shown that production of proin amma- intestinal disorders, Crohn's disease and ulcerative colitis (UC), tory cytokines in the lamina propria contribute to tumor fl which share related characteristics such as mucosal damage growth and cancer development during intestinal in amma- and diarrhea but have distinguishing clinical features. Evi- tion (3, 4). dence suggests that in addition to the widely accepted aberrant Prohibitin 1 (PHB) is an evolutionarily conserved, multi- mucosal immune response, nonimmune cells including epi- functional 32 kDa protein implicated in cellular processes thelial cells play an emerging role in the pathogenesis of disease including the regulation of cell cycle progression, apoptosis, – by modulating mucosal barrier integrity and homeostasis (1). and transcription (5 7). Expression of PHB is decreased in fl An association between IBD and colorectal cancer (CRC) has mucosal biopsies from UC and Crohn's disease af icted patients and in the dextran sodium sulfate (DSS) and inter- been well established with a cumulative risk of developing CRC leukin (IL)-10 / mouse models of colitis (8, 9). Proinflamma- tory cytokines such as TNFa and reactive oxygen species Authors' Affiliations: 1Department of Internal Medicine, Division of Gas- decrease expression of intestinal epithelial PHB in vivo and in troenterology, Baylor Research Institute, Baylor University Medical Center; vitro (8–10). Restoration of colonic epithelial PHB expression 2Department of Medicine, Veterans Affairs North Texas Health Care Sys- tem, University of Texas Southwestern Medical Center, Dallas, Texas; and using genetic manipulation [villin-PHB transgenic (Tg) mice] 3Gastroenterology Division, Department of Medicine and Department of or therapeutic delivery to the colon via nanoparticles or Oncology, the Sidney Kimmel Comprehensive Cancer Center and the Johns Hopkins School of Medicine, Baltimore, Maryland adenovirus protected mice from experimental colitis (11, 12). The role of PHB in cancer cell proliferation and/or tumor Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). suppression remains controversial especially because PHB expression is increased in many transformed cells and tumors Corresponding Author: Arianne L. Theiss, Division of Gastroenterology, – 250 Hoblitzelle, Baylor Research Institute, Baylor University Medical Cen- (13 19). Consensus binding sites for the oncoprotein Myc are ter, Dallas, TX 75246. Phone: 214-820-2752; Fax: 214-818-9292; E-mail: present in the PHB promoter and likely contribute to the [email protected] increased PHB levels in many tumors (20). Although somatic doi: 10.1158/0008-5472.CAN-12-0603 mutations in the PHB gene were observed in a few sporadic 2012 American Association for Cancer Research. breast cancers, none were identified in the ovary, hepatic, or

www.aacrjournals.org OF1

Kathiria et al.

lung cancers examined (16). Previous studies have shown that administration to generate a clinical activity score as described PHB has an antitumorigenic role in gastric, prostate, and liver previously (12). During recovery periods, body weight was cancers (21–23). Despite the increasing number of studies on measured every week for each group. PHB, its role in CRC or colitis-associated cancer (CAC) has not been explored. Here, we used mice specifically overexpressing Endoscopic assessment of polyp formation and polyp PHB in intestinal epithelial cells (IEC) to assess the role and count mechanism of PHB in CAC. Colonoscopy was done to assess polyp formation in mice by using the Coloview (Karl Storz Veterinary Endoscopy). Materials and Methods Mice were euthanized with 1.5% to 2% isoflurane and approximately 3 cm of the colon proximal to the anus was Animal models visualized after inflation of the colon with air. Mice were Wild-type (WT) and villin-prohibitin Tg mice specifically then sacrificed by cervical dislocation after euthanizing with overexpressing prohibitin in IECs (12) were 9 weeks old at the isoflurane. Colon was excised from ileocecal junction to beginning of the experimental protocol. All mice were anus, washed with 0.9% NaCl, cut open longitudinally and grouped-housed in standard cages under a controlled temper- preserved in 10% neutral-buffered formalin. The number and ature (25C) and photoperiod (12-hour light–dark cycle) and size of the polyps were determined using a Jenko dissecting were allowed standard chow and tap water ad libitum. All microscope. experiments were approved by the Baylor Research Institute Institutional Animal Care and Use Committee. Histopathology scoring Colons fixed in 10% neutral buffered formalin were Swiss- Induction of CAC in mice rolled, embedded in paraffin, sectioned at 5 mm, and stained Age-matched male and female C57BL/6 WT and Tg mice with hematoxylin and eosin stain for histopathologic exami- were intraperitoneally injected with 7.6 mg/kg azoxymethane nation of polyps and adenocarcinoma (neoplasia). Scoring was (AOM; Sigma-Aldrich) on day 0. Colitis was induced 7 days after carried out in a blind fashion by 2 qualified pathologists. Score AOM injection by oral administration of 3% (wt/vol) DSS was given on the basis of these criteria: normal (score 0), low- (molecular weight, 50,000; MP Biomedicals) in drinking water grade dysplasia (score 1), high-grade dysplasia (score 2), intra- ad libitum for 7 days followed by 14 days of recovery of water mucosal adenocarcinoma (score 3), and invasive adenocarci- alone. After recovery, a second cycle of DSS was repeated and noma (score 4). mice were sacrificed after 3 weeks of recovery (day 56) as previously described (24). Three separate groups of mice were Immunohistochemistry used as controls: one group of mice was maintained with Paraffin-embedded sections (5 mm) of colon were analyzed n ¼ n ¼ regular water ( 16 WT; 10 Tg), a second group of mice for Ki67 staining as a marker of cell proliferation as previously was injected with AOM and given water for the remainder of described (12). A minimum of 15 crypts with normal morphol- n ¼ n ¼ the experiment ( 9 WT; 7 Tg), and the third group of ogy were counted for Ki67-positive cells per section. mice was treated with DSS as described above without AOM n ¼ n ¼ injection ( 20 WT; 8 Tg). AOM alone represents Terminal deoxynucleotidyl transferase-mediated dUTP fl treatment with the alkylating agent in the absence of in am- nick end labeling staining mation and is expected to result in no tumor formation in WT Immunofluorescent terminal deoxynucleotidyl transferase- mice at the dose used (25). DSS alone represents repeated mediated dUTP nick end labeling (TUNEL) staining was fl cycles of in ammation and was included to assess whether carried out to measure apoptosis from paraffin-embedded fl in ammation alone caused dysplasia in these mice. The study sections using the In Situ Cell Death Detection Kit as described was powered in such a fashion that the control arms required by the manufacturer (Roche). Nuclei were stained with 40,6- less mice as death before the end of the study was not diamidino-2-phenylindole to count total cells per crypt. A anticipated for these treatment groups. Body weight, stool minimum of 10 crypts with normal morphology were counted consistency, and stool occult blood was monitored during the per section. DSS treatment and recovery phases. Upon sacrifice, colon was excised from the ileocecal junction to anus, cut open longitu- p53 mutational analyses dinally, and prepared for histologic evaluation. Tumor tissue was microdissected from paraffin-embedded To assess mortality, the same protocol was followed with the sections (7 mm) of Swiss-rolled mouse colon. Genomic DNA exception of extending through 126 days or until mice devel- was isolated from microdissected tissues using the QIAamp oped rectal prolapse and/or more than 20% body weight loss. DNA FFPE Tissue purification kit (Qiagen). The PCR amplicons Colons were assessed macroscopically for polyps using a were generated and sequenced as previously described (26). dissecting microscope. See the Supplementary Materials and Methods section for primer sequences. Clinical score assessment Assessment of body weight loss, stool consistency, and the Human tissue samples presence of occult/gross blood by a guaiac test (Hemoccult Matching normal and tumor tissues were obtained at the SENSA; Beckman Coulter) were determined daily during DSS time of surgical resection from patients with one or more

OF2 Cancer Res; 72(22) November 15, 2012 Cancer Research

Prohibitin Functions as a Tumor Suppressor in CAC

UC-associated colorectal neoplasms, consisting of adenocar- comparisons using Bonferroni post hoc tests. A P value less cinomas or dysplasias. Normal control samples consisted of than 0.05 was considered statistically significant in all analyses. colonic normal mucosa adjacent to tumors or ileal mucosa. fl In amed UC tissues were obtained from patients without Results evident dysplasia. All tissues were grossly dissected free of fi normal surrounding tissue, and parallel sections were used for IEC-speci c PHB overexpression decreases colonic histologic characterization. Tissue collection was approved by tumorigenesis in a mouse model of CAC patients according to Institutional Review Board guidelines. Previous studies have shown that PHB has an antitumorigenic role in gastric, prostate, and liver cancers (21–23). We Total RNA extraction and quantitative real-time PCR used the AOM DSS mouse model to study the role of PHB in Total RNA was extracted for human tissues using the RNeasy CAC. Body weight was measured weekly as one parameter to kit (Qiagen). Quantitative real-time PCR was carried out as assess the severity of disease. WT and Tg mice given water only described previously (11). See the Supplementary Materials throughout the experiment and mice injected with AOM and Methods section for primer sequences. followed by water alone showed similar body weight gain over the 8-week protocol (Supplementary Fig. S1A) and did not Cell culture and transfection develop polyps as previously reported for this dose of AOM Caco2-BBE cells were used to assess the interaction of PHB (25). WT mice given 2 cycles of DSS without AOM injection lost with STAT3. As Caco2-BBE cells have mutated p53, WT more body weight following DSS administration and recovered HCT116 human CRC cells were used to assess PHB interaction less weight compared with Tg mice over the 8-week protocol fi with p53. All cell lines were obtained from the American Type (Supplementary Fig. S1B), similar to our previous ndings (12). Culture Collection. Cells were grown and transfected as pre- Although both WT and Tg AOM DSS-treated mice lost weight fi viously described (Kathiria and colleagues; submitted for after the rst administration of DSS, Tg mice gained body publication). weight more rapidly during the recovery phase and maintained body weight better than WT mice throughout the remainder of g-Irradiation the study (Fig. 1A). g-Irradiation was used to induce DNA damage in WT and Stool consistency, stool occult blood, and body weight loss p53 / HCT116 cells (27) after 72 hours of transfection with were monitored following DSS administration to generate a pEGFPN1 vector or pEGFPN1-PHB. Cells were irradiated by clinical disease score. AOM DSS-treated Tg mice showed a fi using 137Cs g-irradiator at 6.5 cGy/s for 32 seconds for a total of decreased clinical score after the rst administration of DSS 209.4 cGy. Cells were harvested 24 hours after g-irradiation for compared with WT mice, but this did not reach statistical fi fi subsequent assays. signi cance. Tg mice exhibited a signi cantly lower clinical score after the second administration of DSS (Fig. 1B). Tg mice Protein extraction, Western blot analysis, and given DSS without AOM injection had lower clinical scores immunoprecipitation after DSS administration compared with WT mice (Supple- fi Mucosal strippings from Tg and WT mice were obtained for mentary Fig. S1C), similar to our previous ndings (12). Western blot analysis as described previously (12). Total The overall survival rate for AOM DSS-treated Tg mice was protein was isolated from cultured cells as described previ- 82% (14 of 17 mice) compared with 60% for WT (12 of 20 mice) ously (Kathiria and colleagues; submitted for publication). (Fig. 1C). Tg mice given DSS without AOM injection exhibited n ¼ Antibodies used were mouse monoclonal PHB (Thermo Fish- no mortality throughout the 8-week protocol ( 8) compared er), mouse monoclonal GFP, p53, Bcl-2, Bcl-xL and Bad and with 60% survival rate for WT mice (12 of 20 mice; Supple- fi rabbit polyclonal BAX, p-Bad (ser 155), STAT3 (Santa Cruz mentary Fig. S1D). Upon sacri ce the entire colon from ileo- Biotechnology), rabbit polyclonal proliferating cell nuclear cecal junction to anus was excised and assessed for number antigen (PCNA) antibody (Abcam), rabbit polyclonal cas- and size of polyps. Tg mice showed considerably fewer polyps pase-3, p53, pSTAT3 (Cell Signaling Technology), rabbit poly- with the majority being smaller than 3 mm in diameter clonal p53 upregulated modulator of apoptosis (PUMA), and compared with WT mice (Fig. 1D). Figure 1E shows represen- mouse monoclonal anti-b-actin (Sigma-Aldrich). tative photos of excised colons and endoscopic images of polyp PHB was immunoprecipitated from 0.6 mg total protein formation in WT and Tg mice following AOM-DSS treatment. lysates from HCT116 or Caco2-BBE cells or 0.20 mg total After polyps were counted and measured, colons were Swiss- protein lysates from mouse mucosa with 1 mg mouse anti- rolled and assessed for histopathologic score by a trained PHB, anti-p53, or anti-pSTAT3 antibody and 30 mL 50% protein pathologist. WT mice had a higher occurrence of high-grade A sepharose beads (GE Healthcare). Blots were incubated with dysplasia and adenocarcinoma compared with Tg mice, which the rabbit p53 antibody or PHB antibody, respectively. Omis- exhibited more low-grade dysplasia and normal morphology sion of primary antibody during the immunoprecipitation was (Fig. 1F). Collectively, these results suggest that PHB Tg mice carried out as a negative control. show less susceptibility to CAC than WT mice.

Statistical analysis PHB Tg mice are less susceptible to CAC and mortality Values are expressed as mean SEM. Statistical analysis To assess mortality and cancer development, the same was conducted using 2-way ANOVA and subsequent pairwise protocol was followed with the exception of extending through

www.aacrjournals.org Cancer Res; 72(22) November 15, 2012 OF3

Kathiria et al.

A 120 * B 115 * 110 * 7 WT 105 * * 6 Tg 100 5

percentage 95 WT AOM DSS 4 * 90 Tg AOM DSS 3

Body weight change in Body weight change 2 85 1

012345678wk Total clinical score 0 Figure 1. IEC-specific PHB wk: 25 overexpression reduces colonic AOM tumorigenesis in a mouse model of Sacrifice End I DSS End II DSS Start DSS End I DSS1-wk recov End II DSS1-wk recov2-wk recov CAC. Mice were intraperitoneally Start II DSS injected with azoxymethane (AOM) and 7 days later treated with 2 C 100 WT AOM DSS D cycles of 3% DSS, each cycle Tg AOM DSS consisting of 7 days DSS followed 90 12 WT by 14 days of water recovery, and 14/17 Tg 80 10 * sacrificed on day 56 after AOM 8 injection. A, percent change in body 70 * 6 weight during each week of the 60 12/20 Percent survival CAC protocol. B, clinical score 4 fi 50 following DSS treatment. End rst 2 * n ¼ n ¼ 012345678wk polyps Number of cycle DSS, 13 WT; 14 Tg. 0 End second cycle DSS, n ¼ 12 WT; n ¼ P ¼ AOM <1-3 3-6 mm Total 14 Tg. C, percent survival.

Sacrifice mm 0.146. D, number of polyps and size Start DSS End I DSS1-wk recov End II DSS1-wk recov2-wk recov Start II DSS distribution of polyps as determined using a dissecting E F microscope. E, representative WT Tg * gross anatomy of the colon (top) 3 and representative colonoscopy images (bottom) at the end of the 2 CAC protocol. F, histopathology scores. , P < 0.05 vs. AOM DSS- 1 treated WT.

Histopath scores 0 WT Tg

126 days or until mice developed rectal prolapse and/or noma: n ¼ 2, intramucosal adenocarcinoma: n ¼ 7) com- >20% body weight loss. WT mice showed greater mortality pared with 5 Tg mice (intramucosal adenocarcinoma). The than Tg mice throughout the study. The difference in majority of Tg mice had low- or high-grade dysplasia. mortality was especially pronounced after the formation of Supplementary Fig. S2D shows representative gross anato- polyps, which occurs at approximately 8 weeks (Supple- my of colon from 3 WT and Tg mice, which also reflects the mentary Fig. S2A). Tg mice showed significantly less total number and size of polyps. These findings suggest that IEC- number of polyps, and although rare, a significantly less specific PHB overexpression improves mortality associated number of large polyps (>6 mm in diameter) compared with with CAC. WT mice (Supplementary Fig. S2B). Mice that survived from 8 through 18 weeks were assessed for histopathologic score IEC-specific PHB overexpression does not affect cell as animals that died before 8 weeks did not yet develop proliferation during CAC polyps. As shown in Supplementary Fig. S2C, the majority of Alteration in cell proliferation can contribute to tumorigen- WT mice developed adenocarcinoma (invasive adenocarci- esis (28). Ki67 immunohistochemical staining and PCNA were

OF4 Cancer Res; 72(22) November 15, 2012 Cancer Research

Prohibitin Functions as a Tumor Suppressor in CAC

AOM DSS AOM DSS A Water normal epithelium dysplasia

Figure 2. IEC-speciﬁc PHB overexpression is associated with Tg increased apoptosis during CAC. A, ﬂuorescent micrograph of TUNEL staining (green) of colonic sections from untreated (water) and AOM-DSS treated WT and PHB Tg mice. Sections were stained with DAPI to B C Water AOM DSS visualize nuclei (blue). Scale bars, 100 1.2 * WT Tg WT Tg mm. B, number of TUNEL-positive 1.0 Mouse # 1 2 1 2 1 2 1 2 cells per crypt in normal epithelium. , P < 0.05; n ¼ 3 for water; n ¼ 9for 0.8 * Caspase-3 35 kDa AOM DSS. C, representative Western 0.6 WT blots showing colonic mucosal Tg Cleaved 19 kDa caspase-3 cleaved caspase-3 levels and b-actin 0.4 cells per crypt cells per as a loading control. D, cumulative 0.2 mean SEM relative to WT water. , No. of TUNEL-positive β-Actin 42 kDa P < 0.05 vs. WT AOM DSS; n 5 per 0.0 treatment. Water AOM DSS D 3.5 * 3.0 2.5 -actin β 2.0 WT 1.5 Tg 1.0 0.5 Cleaved caspase-3 normalized to 0.0 Water AOM DSS

assessed to examine the effect of PHB overexpression on cell cell proliferation induced by AOM-DSS treatment or in proliferation in CAC. Both WT and Tg mice showed increased colonic tumors/polyps. Ki67 staining following AOM-DSS treatment (Fig. 3A, middle panels) compared with mice given water (Supplementary Fig. IEC-specific PHB overexpression is associated with S3A, top panels and Supplementary Fig. S3B). There was no increased apoptosis during CAC significant difference in the percent Ki67-positive cells per Suppression of apoptosis plays an important role in tumor- crypt in normal epithelium (Supplementary Fig. S3A, middle igenesis by allowing genetically compromised cells to proliferate panels and Supplementary Fig. S3B) or areas of dysplasia (29). TUNEL staining and Western blot analysis of cleaved (Supplementary Fig. S3A, bottom panels) between WT and Tg caspase-3 levels were used to assess IEC apoptosis during CAC. mice treated with AOM DSS. Similar results were corrobo- WT and Tg mice exhibited increased number of TUNEL-positive rated by PCNA Western blot analysis. PCNA protein cells per crypt after AOM-DSS treatment (Fig. 2A and B). Tg mice expression was increased in both WT and Tg mice following exhibited a higher number of TUNEL-positive cells compared AOM-DSS treatment compared with mice given water (Sup- with WT mice after AOM-DSS treatment in normal epithelium plementary Fig. S3C and S3D). There was no significant (Fig. 2A and B). Figure 2C and D corroborate the TUNEL difference in PCNA protein expression between WT and Tg immunofluorescence staining by showing increased protein mice treated with AOM DSS. These results suggest that WT expression of cleaved caspase-3 in Tg mice compared with WT and Tg mice exhibited increased cell proliferation after AOM mice with CAC. Thus, IEC-specific PHB overexpression may DSS, and that IEC-specific PHB overexpression does not alter suppress tumor development by promoting IEC apoptosis.

www.aacrjournals.org Cancer Res; 72(22) November 15, 2012 OF5

Kathiria et al.

IEC-specific overexpression of PHB is associated with decreased in WT mice after AOM-DSS treatment compared altered levels of p53, PUMA, Bax, and p-Bad during CAC with water control WT mice (water ¼ 1.0 0.09 vs. AOM DSS ¼ Given the effect of PHB overexpression on apoptosis during 0.75 0.06, P < 0.05). Tg mice exhibited higher expression of CAC, we examined levels of p53, a tumor suppressor, and its mucosal PHB protein levels compared with WT mice when downstream targets PUMA and Bax as well as the activation given water or AOM-DSS treatment (Fig. 3A). Mucosal PHB of the proapoptotic protein Bad. p53 regulates cell survival protein expression in Tg mice was similar to our previous through induction of cell cycle arrest or apoptosis. Our pre- studies using these animals (12). Although induction of CAC vious study showed that PHB protein levels are decreased in increased p53 protein expression in both WT and Tg mice mucosal biopsies of active inflammation from Crohn's disease compared with water control mice, p53 levels were signifi- afflicted patients and in the DSS and IL-10 / mouse models of cantly higher in Tg mice compared with WT mice after AOM- colitis (9). To determine whether mucosal PHB levels are DSS treatment (Fig. 3B). Sequencing of exons 5 to 8 of p53 from affected during CAC, Western blot analysis was carried out DNA isolated from areas of dysplasia from 5 WT and 5 Tg mice on total protein isolated from mucosal stippings from WT found no mutations. PUMA, a central mediator of the p53 mice. Figure 3A shows that mucosal PHB protein levels were apoptotic response and suppressor of intestinal tumorigenesis

* A Water AOM DSS 2.0 * ** WTTg WT Tg 1.5 Mouse # 1 2 1 2 1 2 1 2 WT PHB 32 kDa 1.0 β -Actin Tg β-Actin 42 kDa 0.5 PHB normalized to PHB normalized 0.0 Water AOM DSS B Water AOM DSS 4.0 WTTg WT Tg * Mouse # 1 2 1 2 1 2 1 2 3.0 WT 2.0 * p53 53 kDa Tg β -Actin

to 1.0 Figure 3. IEC-speciﬁc β-Actin 42 kDa p53 normalized overexpression of PHB is 0.0 associated with altered levels of Water AOM DSS p53, PUMA, Bax, and activation of C Water AOM DSS 2.0 Bad. Representative Western blots WTTg WT Tg * of total protein isolated from 1.5 Mouse # 1 2 1 2 1 2 1 2 colonic mucosal strippings were probed with anti-PHB (A), anti-p53 23 kDa 1.0 WT PUMA β -Actin (B), anti-PUMA (C), anti-Bax (D),

to Tg β-Actin 42 kDa 0.5 and anti-phospho-Bad and anti- Bad (E). Anti-b-actin was used as a PUMA normalized PUMA 0.0 loading control. Histograms show Water AOM DSS cumulative mean band Water AOM DSS densitometry SEM, respectively. D 2.0 WTTg WT Tg * n 5 per treatment. P P Mouse # 1 2 1 2 1 2 1 2 1.5 , < 0.05; , < 0.01.

Bax 20 kDa β -Actin 1.0 WT

to Tg β 0.5

-Actin 42 kDa Bax to normalized

0.0 Water AOM DSS E Water AOM DSS WTTg WT Tg 1.5 * Mouse # 1 2 1 2 1 2 1 2 p-Bad 1.0 (ser 155) 25 kDa WT Tg Bad 25 kDa 0.5 to total Bad p-Bad normalized β-Actin 42 kDa 0.0 Water AOM DSS

OF6 Cancer Res; 72(22) November 15, 2012 Cancer Research

Prohibitin Functions as a Tumor Suppressor in CAC

in mice (30), was increased only in Tg mice during CAC (Fig. p53 activation and apoptosis. WT and p53 / HCT116 cells 3C). Similarly, the proapoptotic protein Bax was also increased were transiently transfected with GFP-tagged PHB (GFP PHB) in Tg mice after AOM-DSS treatment (Fig. 3D). Phosphoryla- or vector alone and downstream targets of p53 were measured tion of Bad at serine 155 inhibits the proapoptotic function of by Western blot analysis. WT HCT116 cells overexpressing Bad by disrupting its binding to antiapoptotic Bcl-2 (31). PHB (Fig. 4A, lane 2) showed increased PUMA and Bax and Western blot analysis showed that Tg mice exhibited less decreased phospho-Bad protein expression compared with phosphorylation of Bad at serine 155 compared with WT mice vector-transfected cells (lane 1). Cleaved caspase-3, a marker during CAC (Fig. 3E), suggesting that in Tg mice Bad retains its of apoptosis, was also increased in PHB overexpressing cells proapoptotic function. Together, these data suggest that CAC (Fig. 4A, lane 2 vs. lane 1). p53 / HCT116 cells showed no in PHB Tg mice is associated with increased expression of p53, change in PUMA, Bax, phospho-Bad, or cleaved caspase-3 PUMA, and Bax and decreased Bad phosphorylation. protein expression after PHB transfection (Fig. 4A, lane 6) compared with vector-transfected cells (lane 5), suggesting PHB-induced altered expression of PUMA, Bax, and Bad that p53 is required for PHB-induced alteration of these and requires p53 signaling in HCT116 cells proteins. WT and p53 / HCT116 cells were exposed to HCT116 colorectal carcinoma cells were used as an in vitro g-irradiation to induce DNA damage. Following g-irradiation, model to understand the underlying role of PHB in modulating PHB overexpressing WT HCT116 cells showed signiﬁcantly

* 2.5 * Figure 4. PHB-induced altered A Lane: 1 2 3 4 5 6 7 8 2.0 expression of PUMA, Bax, and Bad − −−− 1.5 * requires p53 signaling in HCT116 p53: + ++ + V cells and PHB interacts with p53 in γ-Irradiation: −−− ++− + + 1.0

to actin PHB HCT116 cells and in colon mucosa. −−−− 0.5 GFP-PHB: +++ + A, WT and p53 / HCT116 cells were PUMA normalized 0.0 PUMA transiently transfected with 23 kDa WT* WT p53 p53 pEGFPN1 vector or pEGFPN1-PHB 2.0 * (GFP-PHB) and subjected to Bax 20 kDa g-irradiation to induce DNA damage 1.5 * p-Bad 25 kDa 72 hours posttransfection. Total 1.0 V protein was collected 24 hours after Bad 25 kDa actin PHB g-irradiation. Western blots were 0.5 probed with anti-PUMA, anti-Bax, GFP-PHB 59 kDa

Bax normalized to 0.0 anti-phospho-Bad, anti-Bad, and WT WT p53 p53 anti-caspase-3. Blots were GFP 27 kDa 1.4 * subsequently probed with anti-GFP 1.2 * ﬁ 1.0 to ensure transfection ef ciency and Caspase-3 35 kDa anti-p53 to ensure p53 knockout. 0.8 * V 0.6 Anti-b-actin was used as a loading Cleaved 19 kDa PHB

to total Bad 0.4 control. Histograms show cumulative caspase-3 0.2 mean band densitometry SEM. n ¼ p-Bad normalized 0.0 P p53 53 kDa 3 per treatment. , < 0.05. PHB (B) or WT WT p53 p53 p53 (C) was immunoprecipitated * β * from total HCT116 WT cell lysates -Actin 42 kDa 7 overexpressing vector or GFP-PHB 6 5 and subjected to g-irradiation to B IP: PHB induce DNA mutation 72 hours 4 V posttransfection. Total protein was γ-Irradiation: −− ++ 3 * PHB collected 24 hours after g-irradiation. 2 GFP-PHB: −−++Neg ctl

normalized to actin normalized to 1 Immunoprecipitates were separated Cleaved Caspase-3 by SDS-PAGE and membranes were 0 IB: p53 53 kDa γ immunoblotted for p53 or PHB -Irradiation:––+ + p53: + + –– expression. Omission of primary IB: PHB 32 kDa antibody during the immunoprecipitation was carried out as a negative control. D, PHB was C IP: p53 D AOM immunoprecipitated from colon IP: PHB Water DSS mucosal protein lysates from water- γ-Irradiation: −− ++

and AOM DSS-treated mice, WT Tg WT Tg Neg ctl GFP-PHB: −−++Neg ctl followed by immunoblot for p53 expression. IB, immunoblotting; IP, IB: PHB 32 kDa IB: p53 53 kDa immunoprecipitation. IB: p53 53 kDa IB: PHB 32 kDa

www.aacrjournals.org Cancer Res; 72(22) November 15, 2012 OF7

Kathiria et al.

increased PUMA, Bax, and cleaved caspase-3 protein expres- expression is significantly decreased compared with matched sion whereas phospho-Bad levels were decreased (Fig. 4A, lane normal samples (Fig. 6). p53 expression was increased in 4) compared with vector-transfected cells (lane 3). PHB over- inflamed UC and UC-associated dysplasia tissues, however expressing WT cells exposed to g-irradiation exhibited a this increase did not achieve statistical significance in the further increase in Bax and cleaved caspase-3 and decrease inflamed tissue samples. STAT3 expression was markedly in phospho-Bad compared with PHB overexpressing cells left increased in inflamed UC and UC-associated dysplasia but untreated (Fig. 4A, lane 4 vs. lane 2). g-Irradiation did not alter did not achieve statistical significance. expression of PUMA, Bax, phospho-Bad, or cleaved caspase-3 in p53 / HCT116 cells regardless of PHB transfection (Fig. 4A, lanes 7 and 8). Discussion The specific mechanism by which IBD leads to CRC is poorly PHB interacts with p53 in HCT116 cells and in colon understood, but it is clear that chronic mucosal inflammation mucosa plays a causative role in the transition to adenocarcinoma. Our To determine whether PHB interacts with p53 in IECs, PHB previous studies have shown that restoration of mucosal PHB was immunoprecipitated from total cell lysates from WT can ameliorate experimental colitis (11, 12). For this reason, we HCT116 cells overexpressing vector or PHB and exposed to chose to use the AOM DSS model to assess the role of PHB in g-irradiation to induce DNA damage. Following immunopre- CAC. Here, we show that in mice with CAC, IEC-specific PHB cipitation and SDS-PAGE, membranes were immunoblotted overexpression suppresses colonic neoplastic formation in for p53 and subsequently PHB expression. p53 coimmunopre- association with increased rates of apoptosis. IEC-specific cipitated with PHB during basal conditions and after g-irra- overexpression of PHB was associated with altered levels of diation (Fig. 4B and C). To determine whether PHB interacts p53, PUMA, Bax, p-Bad, Bcl-xL, and Bcl-2 during CAC. We with p53 in vivo, we conducted coimmunoprecipitation experi- show, for the first time, that PHB interacts with phospho- ments for PHB and p53 in colon mucosal lysates from water- STAT3 in addition to p53 in human colon cancer cells and and AOM DSS-treated mice. p53 coimmunoprecipitated with colon mucosa, thereby modulating the apoptotic response PHB in lystates from both WT and Tg mice (Fig. 4D), with (Fig. 7). increased levels in Tg mice (Fig. 4D). Collectively, these results IBD patients with longstanding colitis require surveillance suggest that PHB interacts with p53 in cultured human CRC colonoscopy after 8 years of disease duration because of the cells and in colon mucosa. high risk of cancer development (33). Several features make CAC distinct from sporadic colon cancer. CAC is often ana- PHB interacts with phospho-STAT3 and modulates plastic, broadly infiltrating, rapidly growing, develops in flat STAT3 responsive genes, Bcl-xL, and Bcl-2 in vivo and dysplastic tissue, and occurs at a younger age. While genetic in vitro alterations are similar to those in sporadic colon cancer, the The IL-6/STAT3 signaling pathway is known to play a frequency and sequence of these events differ in CAC (34). Loss pathogenic role in intestinal inflammation and colonic cancer of p53 function is an early critical event in colitis-associated through the induction of the antiapoptotic genes Bcl-xL and neoplasia in humans in the pathway from dysplasia to cancer Bcl-2 (32). Caco2-BBE cells, which are responsive to IL-6, show (35). Furthermore, aberrent IL-6/STAT3 signaling is associated decreased basal and IL-6-induced STAT3 activation, and with IBD and CAC (32). Activated STAT3 is enhanced in CRC decreased Bcl-xL and Bcl-2 protein expression when PHB is patients and has been shown to induce the antiapoptotic overexpressed (Fig. 5A). Coimmunoprecipitation experiments proteins Bcl-2 and Bcl-xL (36). Inhibition of STAT3 signaling using Caco2-BBE cells treated with IL-6 suggest that PHB induces apoptosis of CRC cells via the mitochondrial pathway interacts with phospho-STAT3 (Fig. 5B and C). To determine by modulating Bcl-2 and Bcl-xL (37). Here, we show that in whether PHB interacts with STAT3 in vivo, we conducted mice with CAC, PHB suppresses colonic tumor formation in coimmunoprecipitation experiments for PHB and phospho- association with increased rates of apoptosis. In human colon STAT3 in colon mucosal lysates from water- and AOM DSS- cancer cells, PHB interacts with p53 and phospho-STAT3 and treated mice. Phospho-STAT3 coimmunoprecipitated with modulates their downstream apoptotic proteins. These find- PHB in lystates from Tg mice (Fig. 5D). Induction of CAC ings suggest that PHB protects against CAC by modulating p53 increased protein expression of STAT3 responsive genes Bcl-2 and STAT3 apoptotic responses. and Bcl-xL in both WT and Tg mice compared with water The best-characterized function of PHB is as a chaperone control mice. Bcl-2 and Bcl-xL levels were significantly involved in the stabilization of mitochondrial proteins thereby decreased in Tg mice compared with WT mice after AOM- maintaining normal mitochondrial function and morphology DSS treatment (Fig. 5E). (reviewed in ref. 38). In most cell types studied, including IECs, PHB is predominantly localized to the mitochondria (9). PHB mRNA expression is decreased in inflamed UC and However, it has been localized to the cell membrane, cyto- UC-associated dysplasia and inversely correlates with plasm, or the nucleus in some cell types (6, 39, 40). Although STAT3 and p53 mRNA expression PHB expression is increased in many transformed cells and Quantitative real-time PCR analysis of total RNA isolated tumors (13–19), in our study, colonic mucosal PHB protein from mucosal samples from patients with inflamed UC or UC- expression was reduced after the development of colitis-asso- associated adenocarcinoma/dysplasia revealed that PHB ciated tumors in WT mice. PHB expression was decreased in

OF8 Cancer Res; 72(22) November 15, 2012 Cancer Research

Prohibitin Functions as a Tumor Suppressor in CAC

ABIL-6: − + IP: PHB − PHB: −−++ IL-6: +

pSTAT3 92 kDa PHB: −−++Neg ctl 3TATSp :BI 3TATSp 29 aDk STAT3 92 kDa IB: STAT3 92 kDa Bcl-2 Figure 5. PHB interacts with 26 kDa phospho-STAT3 and modulates IB: PHB 32 kDa STAT3 responsive genes, Bcl-xL and Bcl-xL 30 kDa Bcl-2 in vivo and in vitro.A, C IP: STAT3 representative Western blots of GFP-PHB 59 kDa pSTAT3, Bcl-2, Bcl-xL protein IL-6: − + expression in Caco2-BBE cells stably GFP-PHB: −−++Neg ctl overexpressing pEGFPN1-PHB GFP 27 kDa (GFP-PHB) or empty vector and IB: PHB 32 kDa treated with 100 ng/mL IL-6 β-Actin 42 kDa basolaterally for 1 hour. Blots IB: pSTAT3 92 kDa were subsequently probed with anti-GFP to ensure transfection IB: STAT3 92 kDa ﬁ ef ciency. B and C, D E coimmunoprecipitation of PHB Water AOM DSS and pSTAT3 using cell lysates described in A. D, PHB was AOM WTTg WT Tg immunoprecipitated from colon IP: PHB Water DSS Mouse # 1 2 1 2 1 2 1 2

mucosal protein lysates from water- WT Tg WT Tg Neg ctl and AOM DSS-treated Bcl-2 26 kDa mice, followed by immunoblot :BI 3TATSp 29 aDk for pSTAT3 expression. E, Bcl-xL 30 kDa representative Western blots of total :BI BHP 23 aDk protein isolated from colonic mucosal strippings were probed with β-Actin 42 kDa anti-Bcl-2, anti-Bcl-xL, anti-b-actin (loading control). Histograms show mean band densitometry SEM. n Bcl-2 Bcl-xL 5 per treatment. , P < 0.05. 2.5 * 2.5 * 2.0 2.0 -actin β 1.5 1.5 WT WT 1.0 1.0 Tg Tg 0.5 0.5 Band densitometry Band densitometry normalized to 0.0 0.0 Water12AOM Water12AOM DSS DSS

mucosal samples from human patients with inflamed UC or down of PHB expression by siRNA in Caco2-BBE IECs UC-associated adenocarcinoma/dysplasia. Proinflammatory caused mitochondrial depolarization and increased intra- cytokines such as TNFa and reactive oxygen species decrease cellular reactive oxygen species (41). Interestingly, mito- expression of intestinal epithelial PHB in vivo and in vitro (8– chondrial dysfunction is a common feature of cancer cells 10). Thus, it is likely that PHB levels are decreased in the colonic and multiple studies suggest that mitochondrial chaperone mucosa following AOM-DSS treatment due to the inflamma- proteins, including PHB, modulate tumorigenesis (reviewed tory process. Tg mice with forced overexpression of PHB in ref. 42). in IECs exhibited no inflammation-induced loss of PHB A functional role for PHB as a regulator of transcription expression and showed decreased susceptibility to CAC and has become evident due to its interactions with p53, E2F, mortality. and Rb (6, 7, 43). Here, we show that in addition to p53, PHB PHB has been reported to have an antitumorigenic role interacts with STAT3 in vitro and in vivo. Studies in breast in prostate cancer (21), gastric cancer (23), and liver cancer cancer, prostate cancer, and retinal pigment epithelial cell (22). Mice with hepatocyte-specific deletion of PHB exhib- lines indicate that PHB increases the transcriptional activity ited liver injury, fibrosis, oxidative stress, and hepatocellu- and prevents denaturation of p53, thereby regulating apo- lar carcinoma developing by 8 months of age (22). Mito- ptosis (44–46). Our study showed that PHB suppresses chondrial abnormalities in hepatocytes are evident by 3 colonictumorformationinassociation with increased rates weeks of age including no cristae formation (22). Knock- of apoptosis and p53 apoptotic responses. Sequencing of

www.aacrjournals.org Cancer Res; 72(22) November 15, 2012 OF9

Kathiria et al.

exons 5, 6, 7, and 8 of p53 from DNA isolated from areas of dysplasia from 5 WT and 5 Tg mice found no mutations. IL-6 Other cytokines Unlike human UC-associated cancers, DSS-induced colon tumors in mice do not commonly harbor p53 mutations (26). Therefore, it is likely that the increased p53 in Tg mice is functional, unlike the increased, but mutant p53, in human IBD patients. PHB interaction with phospho-STAT3 was DNA damage associated with decreased Bcl-xL and Bcl-2 expression in JAKs cultured IECs and colon mucosa. The localization of PHB in the mitochondria and nucleus makes it an ideal candidate STAT3 PHB p53 to modulate the mitochondrial apoptotic pathway and transcription activation. Further studieswillassessthemecha- Bcl-xL Bax nism of PHB modulation of STAT3 signaling. PHB interacts Bcl-2 PUMA with p53 and STAT3 in colon mucosa from mice treated with DSS alone without AOM injection (Supplementary Fig. S1E), suggesting interaction is not dependent upon the Apotosis presence of tumors. Furthermore, Tg mice treated with DSS alone without AOM injection exhibited altered mucosal p53, PUMA, Bcl-2, and Bcl-xL protein expression (Supple- Figure 7. Model of PHB modulation of p53 and STAT3 apoptotic mentary Fig. S1F). Because the development of CAC is responses. PHB induces apoptosis in cells with DNA damage during dependent on the severity of chronic inflammation, PHB inflammation through inhibition of STAT3-induced Bcl-xL and Bcl-2 and induction of p53 signaling via activation of Bax and PUMA. Reduced PHB transgene expression likely decreases the occurrence of CAC fl fl expression in in amed tissues may make genetically compromised by decreasing the severity of DSS-induced in ammation and intestinal epithelial cells more resistant to cell death, thereby promoting enhancing p53-mediated apoptosis and inhibiting STAT3 tumorigenesis. induction of antiapoptotic genes during tumor formation. Early loss of PHB expression during inflammation may make IECs in vivo and in vitro and modulates downstream genetically compromised IECs more resistant to cell death, apoptotic responses. Reduced levels of PHB during chronic thereby promoting tumorigenesis. intestinal inflammation may be an underlying factor pro- In conclusion, PHB suppresses colonic tumor formation moting tumorigenesis. Modulation of PHB expression in in association with increased rates of apoptosis in mice IECs represents a potential therapeutic approach to pre- with CAC. PHB interacts with p53 and phospho-STAT3 in vent colitis-associated tumorigenesis.

Disclosure of Potential Conﬂicts of Interest ﬂ 28 No potential con icts of interest were disclosed.

26 Authors' Contributions Conception and design: A.S. Kathiria, A.L. Theiss Development of methodology: A.S. Kathiria, W.L. Neumann 6 Acquisition of data (provided animals, acquired and managed patients, * provided facilities, etc.): A.S. Kathiria, W.L. Neumann, Y.Cheng, S.J. Meltzer, A. L. Theiss 4 * Analysis and interpretation of data (e.g., statistical analysis, bio- statistics, computational analysis): A.S.Kathiria,R.M.Genta,R.F.Souza,

mRNA fold difference mRNA * A.L. Theiss 2 Writing, review, and/or revision of the manuscript: A.S. Kathiria, W.L. Neumann, S.J. Meltzer, R.F. Souza, A.L. Theiss 0 Administrative, technical, or material support (i.e., reporting or orga- nizing data, constructing databases): J. Rhees, E. Hotchkiss, R.M. Genta, S.J. UC UC UC Meltzer Study supervision: A.L. Theiss Normal Normal Normal Interpreting pathology slides: R.M. Genta UC-dysplasia UC-dysplasia UC-dysplasia Acknowledgments PHB STAT3 p53 The authors thank the late Dr. Shanthi V. Sitaraman from Emory Uni- versity, Atlanta, GA for her mentoring role with this project; Dr. Ajay Goel from Baylor Research Institute, Baylor University Medical Center, Dallas, TX Figure 6. PHB mRNA expression is decreased in inflamed ulcerative for providing the HCT116 cell line as well as Bax, Bcl-2, and Bcl-xL antibodies; colitis and ulcerative colitis-associated dysplasia and inversely Dr. Linda A. Feagins from Veterans Affairs North Texas Health Care System, University of Texas Southwestern Medical Center, Dallas, TX for helpful correlates with STAT3 and p53 mRNA expression. Quantitative real- scientific discussions. The authors also thank Dr. Shinichi Matsumoto, Ana timePCRanalysisofPHB,STAT3,andp53intotalRNAisolated Rahman, Jeff SoRelle, and Mazhar Kanak for their assistance with tissue fl from patients with in amed ulcerative colitis (UC) or UC-associated sectioning through the Baylor Islet Cell Transplant Program, Baylor Research adenocarcinoma/dysplasia. n ¼ 8normal;n ¼ 5 UC-dysplasia; n ¼ 5 Institute as well as Dr. Steven Phillips from Baylor Research Institute for his UC. , P < 0.05 vs. normal. assistance with the g-irradiator.

OF10 Cancer Res; 72(22) November 15, 2012 Cancer Research

Prohibitin Functions as a Tumor Suppressor in CAC

Grant Support advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate This work was supported by NIH grants K01-DK085222 (A.L. Theiss) and R01- this fact. 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 the Received February 16, 2012; revised June 6, 2012; accepted June 26, 2012; payment of page charges. This article must therefore be hereby marked published OnlineFirst August 6, 2012.

References 1. Abraham C, Cho JH. Inflammatory bowel disease. N Engl J Med integrity and chemoresistance in ovarian cancer cells by increasing 2009;361:2066–78. their sensitivity to apoptosis. Int J Cancer 2008;122:1923–30. 2. Gillen CD, Walmsley RS, Prior P, Andrews HA, Allan RN. Ulcerative 20. Coates PJ, Nenutil R, McGregor A, Picksley SM, Crouch DH, Hall PA, colitis and Crohn's disease: a comparison of the colorectal cancer risk et al. Mammalian prohibitin proteins respond to mitochondrial stress in extensive colitis. Gut 1994;35:1590–2. and decrease during cellular senescence. Exp Cell Res 2001;265: 3. Grivennikov S, Karin E, Terzic J, Mucida D, Yu GY, Vallabhapurapu S, 262–73. et al. IL-6 and Stat3 are required for survival of intestinal epithelial cells 21. Dart DA, Spencer-Dene B, Gamble SC, Waxman J, Bevan CL. Manip- and development of colitis-associated cancer. Cancer Cell 2009;15: ulating prohibitin levels provides evidence for an in vivo role in andro- 103–13. gen regulation of prostate tumours. Endocr Relat Cancer 2009;16: 4. Popivanova BK, Kitamura K, Wu Y, Kondo T, Kagaya T, Kaneko S, et al. 1157–69. Blocking TNF-alpha in mice reduces colorectal carcinogenesis asso- 22. Ko KS, Tomasi ML, Iglesias-Ara A, French BA, French SW, Ramani K, ciated with chronic colitis. J Clin Invest 2008;118:560–70. et al. Liver-specific deletion of prohibitin 1 results in spontaneous liver 5. McClung JK, Jupe ER, Liu XT, Dell'Orco RT. Prohibitin: potential role in injury, fibrosis, and hepatocellular carcinoma in mice. Hepatology senescence, development, and tumor suppression. Exp Gerontol 2010;52:2096–108. 1995;30:99–124. 23. Liu T, Tang H, Lang Y, Liu M, Li X. MicroRNA-27a functions as an 6. Wang S, Fusaro G, Padmanabhan J, Chellappan SP. Prohibitin co- oncogene in gastric adenocarcinoma by targeting prohibitin. Cancer localizes with Rb in the nucleus and recruits N-CoR and HDAC1 for Lett 2009;273:233–42. transcriptional repression. Oncogene 2002;21:8388–96. 24. Fukata M, Chen A, Vamadevan AS, Cohen J, Breglio K, Krishnareddy 7. Wang S, Zhang B, Faller DV. Prohibitin requires Brg-1 and Brm for the S, et al. Toll-like receptor-4 promotes the development of colitis- repression of E2F and cell growth. EMBO J 2002;21:3019–28. associated colorectal tumors. Gastroenterology 2007;133:1869–81. 8. Hsieh SY, Shih TC, Yeh CY, Lin CJ, Chou YY, Lee YS. Comparative 25. Clapper ML, Cooper HS, Chang WC. Dextran sulfate sodium-induced proteomic studies on the pathogenesis of human ulcerative colitis. colitis-associated neoplasia: a promising model for the development Proteomics 2006;6:5322–31. of chemopreventive interventions. Acta Pharmacol Sin 2007;28: 9. Theiss AL, Idell RD, Srinivasan S, Klapproth JM, Jones DP, Merlin D, 1450–9. et al. Prohibitin protects against oxidative stress in intestinal epithelial 26. Chang WC, Coudry RA, Clapper ML, Zhang X, Williams KL, Spittle CS, cells. FASEB J 2007;21:197–206. et al. Loss of p53 enhances the induction of colitis-associated neo- 10. Theiss AL, Jenkins AK, Okoro NI, Klapproth JM, Merlin D, Sitaraman plasia by dextran sulfate sodium. Carcinogenesis 2007;28:2375–81. SV. Prohibitin inhibits tumor necrosis factor alpha-induced nuclear 27. Bunz F, Hwang PM, Torrance C, Waldman T, Zhang Y, Dillehay L, et al. factor-kappa B nuclear translocation via the novel mechanism of Disruption of p53 in human cancer cells alters the responses to decreasing importin alpha3 expression. Mol Biol Cell 2009;20: therapeutic agents. J Clin Invest 1999;104:263–9. 4412–23. 28. Terzic J, Grivennikov S, Karin E, Karin M. Inflammation and colon 11. Theiss AL, Laroui H, Obertone TS, Chowdhury I, Thompson WE, Merlin cancer. Gastroenterology 2010;138:2101–14, e5. D, et al. Nanoparticle-based therapeutic delivery of prohibitin to the 29. Huerta S, Goulet EJ, Livingston EH. Colon cancer and apoptosis. Am J colonic epithelial cells ameliorates acute murine colitis. Inflamm Bowel Surg 2006;191:517–26. Dis 2011;17:1163–76. 30. Qiu W, Carson-Walter EB, Kuan SF, Zhang L, Yu J. PUMA suppresses 12. Theiss AL, Vijay-Kumar M, Obertone TS, Jones DP, Hansen JM, intestinal tumorigenesis in mice. Cancer Res 2009;69:4999–5006. Gewirtz AT, et al. Prohibitin is a novel regulator of antioxidant response 31. Downward J. How BAD phosphorylation is good for survival. Nat Cell that attenuates colonic inflammation in mice. Gastroenterology Biol 1999;1:E33–5. 2009;137:199–208, 08 e1–6. 32. Atreya R, Neurath MF. Signaling molecules: the pathogenic role of the 13. Tsai HW, Chow NH, Lin CP, Chan SH, Chou CY, Ho CL. The signif- IL-6/STAT-3 trans signaling pathway in intestinal inflammation and in icance of prohibitin and c-Met/hepatocyte growth factor receptor in colonic cancer. Curr Drug Targets 2008;9:369–74. the progression of cervical adenocarcinoma. Hum Pathol 2006;37: 33. Neumann H, Vieth M, Langner C, Neurath MF, Mudter J. Cancer risk in 198–204. IBD: how to diagnose and how to manage DALM and ALM. World J 14. Ren HZ, Wang JS, Wang P, Pan GQ, Wen JF, Fu H, et al. Increased Gastroenterol 2011;17:3184–91. expression of prohibitin and its relationship with poor prognosis in 34. Feagins LA, Souza RF, Spechler SJ. Carcinogenesis in IBD: potential esophageal squamous cell carcinoma. Pathol Oncol Res 2010;16: targets for the prevention of colorectal cancer. Nat Rev Gastroenterol 515–22. Hepatol 2009;6:297–305. 15. Kang X, Zhang L, Sun J, Ni Z, Ma Y, Chen X, et al. Prohibitin: a potential 35. Brentnall TA, Crispin DA, Rabinovitch PS, Haggitt RC, Rubin CE, biomarker for tissue-based detection of gastric cancer. J Gastroen- Stevens AC, et al. Mutations in the p53 gene: an early marker of terol 2008;43:618–25. neoplastic progression in ulcerative colitis. Gastroenterology 16. Sato T, Sakamoto T, Takita K, Saito H, Okui K, Nakamura Y. The human 1994;107:369–78. prohibitin (PHB) gene family and its somatic mutations in human 36. Lassmann S, Schuster I, Walch A, Gobel H, Jutting U, Makowiec F, tumors. Genomics 1993;17:762–4. et al. STAT3 mRNA and protein expression in colorectal cancer: effects 17. Nan Y, Yang S, Tian Y, Zhang W, Zhou B, Bu L, et al. Analysis of the on STAT3-inducible targets linked to cell survival and proliferation. J expression protein profiles of lung squamous carcinoma cell using Clin Pathol 2007;60:173–9. shot-gun proteomics strategy. Med Oncol 2009;26:215–21. 37. Du W, Hong J, Wang YC, Zhang YJ, Wang P, Su WY, et al. Inhibition of 18. Wu TF, Wu H, Wang YW, Chang TY, Chan SH, Lin YP, et al. Prohibitin in JAK2/STAT3 signaling induces colorectal cancer cell apoptosis via the pathogenesis of transitional cell bladder cancer. Anticancer Res mitochondrial pathway. J Cell Mol Med. 2011 Nov 2. [Epub ahead of 2007;27:895–900. print]. 19. Gregory-Bass RC, Olatinwo M, Xu W, Matthews R, Stiles JK, Thomas 38. Artal-Sanz M, Tavernarakis N. Prohibitin and mitochondrial biology. K, et al. Prohibitin silencing reverses stabilization of mitochondrial Trends Endocrinol Metab 2009;20:394–401.

www.aacrjournals.org Cancer Res; 72(22) November 15, 2012 OF11

Kathiria et al.

39. Rastogi S, Joshi B, Fusaro G, Chellappan S. Camptothecin induces 43. Joshi B, Rastogi S, Morris M, Carastro LM, DeCook C, Seto E, et al. nuclear export of prohibitin preferentially in transformed cells Differential regulation of human YY1 and caspase 7 promoters by through a CRM-1-dependent mechanism. J Biol Chem 2006;281: prohibitin through E2F1 and p53 binding sites. Biochem J 2007;401: 2951–9. 155–66. 40. Sharma A, Qadri A. Vi polysaccharide of Salmonella typhi targets the 44. Chander H, Halpern M, Resnick-Silverman L, Manfredi JJ, Germain D. prohibitin family of molecules in intestinal epithelial cells and sup- Skp2B attenuates p53 function by inhibiting prohibitin. EMBO Rep presses early inﬂammatory responses. Proc Natl Acad Sci U S A 2009;11:220–5. 2004;101:17492–7. 45. Fusaro G, Dasgupta P, Rastogi S, Joshi B, Chellappan S. Prohibitin 41. Kathiria AS, Butcher LD, Feagins LA, Souza RF, Boland CR, Theiss AL. induces the transcriptional activity of p53 and is exported from the Prohibitin 1 modulates mitochondrial stress-related autophagy in nucleus upon apoptotic signaling. J Biol Chem 2003;278:47853–61. human colonic epithelial cells. PLoS One 2012;7:e31231. 46. Chander H, Halpern M, Resnick-Silverman L, Manfredi JJ, Germain D. 42. Czarnecka AM, Campanella C, Zummo G, Cappello F. Mitochondrial Skp2B overexpression alters a prohibitin-p53 axis and the transcrip- chaperones in cancer: from molecular biology to clinical diagnostics. tion of PAPP-A, the protease of insulin-like growth factor binding Cancer Biol Ther 2006;5:714–20. protein 4. PLoS One 2011;6:e22456.

OF12 Cancer Res; 72(22) November 15, 2012 Cancer Research

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

Arwa S. Kathiria, William L. Neumann, Jennifer Rhees, et al.

Cancer Res Published OnlineFirst August 6, 2012.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-12-0603

Supplementary Access the most recent supplemental material at: Material http://cancerres.aacrjournals.org/content/suppl/2012/08/06/0008-5472.CAN-12-0603.DC1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/early/2012/11/08/0008-5472.CAN-12-0603. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.