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Research Article

Isorhapontigenin (ISO) Inhibits Invasive BladderCancer Formation In Vivo and Human BladderCancer Invasion In Vitro by TargetingSTAT1/FOXO1 AxisGuosong Jiang1, Amy D.Wu1, Chao Huang1, Jiayan Gu2, Liping Zhang2,Haishan Huang2, Xin Liao1, Jingxia Li1, Dongyun Zhang1, Xingruo Zeng1,Honglei Jin1, Haojie Huang3, and Chuanshu Huang1

Abstract

Although our most recent studies have identified Isorhapon-tigenin (ISO), a novel derivative of stilbene that isolated from aChinese herb Gnetum cleistostachyum, for its inhibition ofhuman bladder cancer growth, nothing is known whetherISO possesses an inhibitory effect on bladder cancer invasion.Thus, we addressed this important question in current studyand discovered that ISO treatment could inhibit mouse-inva-sive bladder cancer development following bladder carcinogenN-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) exposurein vivo. We also found that ISO suppressed human bladdercancer cell invasion accompanied by upregulation of the fork-head box class O 1 (FOXO1) mRNA transcription in vitro.Accordingly, FOXO1 was profoundly downregulated in humanbladder cancer tissues and was negatively correlated withbladder cancer invasion. Forced expression of FOXO1 specif-ically suppressed high-grade human bladder cancer cell inva-sion, whereas knockdown of FOXO1 promoted noninvasive

bladder cancer cells becoming invasive bladder cancer cells.Moreover, knockout of FOXO1 significantly increased bladdercancer cell invasion and abolished the ISO inhibition of inva-sion in human bladder cancer cells. Further studies showed thatthe inhibition of Signal transducer and activator of transcrip-tion 1 (STAT1) phosphorylation at Tyr701 was crucial for ISOupregulation of FOXO1 transcription. Furthermore, this studyrevealed that metalloproteinase-2 (MMP-2) was a FOXO1downstream effector, which was also supported by dataobtained from mouse model of ISO inhibition BBN-inducedmouse-invasive bladder cancer formation. These findings notonly provide a novel insight into the understanding of mech-anism of bladder cancer's propensity to invasion, but alsoidentify a new role and mechanisms underlying the naturalcompound ISO that specifically suppresses such bladder cancerinvasion through targeting the STAT1–FOXO1–MMP-2 axis.Cancer Prev Res; 9(7); 567–80. �2016 AACR.

IntroductionBladder cancer is the sixth most common cancer in the

United States and the number one cause of death in patientswith urinary system malignancies. The incidence of bladdercancer has steadily risen in recent decades. It is estimated thatmore than 74,690 Americans will be diagnosed with bladdercancer and more than 15,580 will die of this disease in 2014

(1). Approximately 20% to 30% of bladder cancers are muscle-invasive, 50% of these patients die from metastasis within 2years of diagnosis, and the 5-year survival rate for metastaticbladder cancer is only 6% (2). The remaining 70% to 80% ofbladder cancers are diagnosed as non-muscular invasion, inwhich about 20% progress to muscle-invasive bladder cancerand have a 43% relative lower 5-year survival rate (2). Inaddition, bladder cancer is one of the most costly cancers asa result of necessary lifetime monitoring and treatment. Forthese reasons, the identification of new natural compounds thatspecifically suppress human bladder cancer invasion and defin-ing key molecules that are responsible for mediating humanbladder cancer invasion and metastasis are of extremely impor-tance for improving the clinical outcome of patients with thisdisease. Isorhapontigenin (ISO) is a novel derivative of stilbeneand is isolated and purified from a Chinese herb Gnetumcleistostachyum (3–5), which has been used for centuries astreatment for several cancers including bladder cancers. Recentwork by our group has demonstrated that ISO treatmentinduces cell-cycle arrest at G0–G1 phase and inhibits anchor-age-independent cell growth through inhibiting cyclin D1expression in both RT4 human noninvasive bladder cancercells and UMUC3 human-invasive bladder cancer cells (4).Our studies have also found that ISO exhibits the anticancer

1Nelson Institute of Environmental Medicine, New York UniversitySchool of Medicine, Tuxedo, New York. 2Zhejiang Provincial Key Lab-oratory for Technology and Application of Model Organisms, Schoolof Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang,China. 3Department of Biochemistry and Molecular Biology, MayoClinic College of Medicine, Rochester, Minnesota.

Note: Supplementary data for this article are available at Cancer PreventionResearch Online (http://cancerprevres.aacrjournals.org/).

A.D. Wu is a summer student from Cornwall Central High School, 10 DragonDrive, New Windsor, NY 12553.

Corresponding Author: Chuanshu Huang, New York University School ofMedicine, 57 Old Forge Road, Tuxedo, NY 10987. Phone: 845-731-3519; Fax:845-351-2320; E-mail: [email protected]

doi: 10.1158/1940-6207.CAPR-15-0338

�2016 American Association for Cancer Research.

CancerPreventionResearch

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Published OnlineFirst April 14, 2016; DOI: 10.1158/1940-6207.CAPR-15-0338

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activity accompanied by downregulating X-linked inhibitor ofapoptosis protein (XIAP), thus inducing apoptosis in T24Thuman-invasive bladder cancer cells in a relative high dose(5). Nevertheless, nothing is known whether ISO is able toinhibit bladder cancer invasion at noncytotoxic doses.

Forkhead box O (FOXO) proteins, which include FOXO1,FOXO3a, FOXO4, and FOXO6 in human, primarily functionas transcription factors in nucleus and act as tumor suppressors(6, 7). FOXO proteins are reported to upregulate negative reg-ulators of the G1–S transition of the cell cycle, such as p27KIP1,p21WAF1, and p130, and repress positive regulators, such as cyclinD1 and D2 (6). Overexpression of FOXO proteins also enhancesGadd45a and cyclin G2 promoter activity, resulting in cell-cycleG2–M arrest (8). Besides, activation of FOXOs triggers apoptosisthrough binding to the promoters of the proapoptotic gene, FasLand Bim, and inducing their expression (9–11). Moreover,FOXO1 has been found to inhibit prostate cancer cell migrationand invasion through binding to Runt-domain containing pro-tein Runx2 and repress its transcriptional activity (12). Expressionof a constitutive nuclear active form of FOXO1 significantlyinhibited matrix metalloproteinase-9 (MMP-9) activationinduced by EGF and prevented cell invasion in glioblastoma cells(13). Although themRNA and protein expression level of FOXO1are found tobe downregulated inhigh-grade and invasive bladdercancer, nothing is known about the roles, mechanisms, and theupstream regulator/downstream effectors of FOXO1 in humanbladder cancer invasion (14). Therefore, we addressed thesequestions and explored the potential FOXO1-related inhib-itory effects of ISO on human bladder cancer invasion in thepresent study both in vitro and in vivo.

Materials and MethodsPlasmids, antibodies, and other reagents

The FLAG-tagged human FOXO1 expression construct, FasLpromoter, and IGFBG-1 (3�IRS) promoter luciferase reporterswere constructed and used in previously studies (15). The shRNAsets for human FOXO1 were purchased from Open Biosystems(Thermo Fisher Scientific). The Clustered Regularly InterspacedShort Palindromic Repeats (CRISPR)/Cas9 system–specific target-ing FOXO1 was purchased from Applied Biological Materials(ABM) Inc. Human FOXO1 promoter luciferase reporter wascloned into the pGL3 luciferase assay vector and was kindlyprovided by Dr. Jean-Baptiste Demoulin (De Duve Institute,Catholic University of Louvain, BE-1200 Brussels, Belgium;ref. 16). A mutation of Signal transducer and activator of tran-scription 1 (STAT1)–binding site in the FOXO1 promoter wascreated using site-directed mutagenesis by the overlap extensionPCR method with mutagenic primers 50-ACAGAAACACTCGA-GAAGCGCCATCCAATAATAGAGATCCAAA-30 (sense) and 50-TTTGGATCTCTATTATTGGATGGCGCTTCTCGAGTGTTTCTGT-30

(antisense), and flanking primers 50-ATTGAGTAGAATTCCTCG-CGGCCGCC-30 (forward) and 50-TGGAAGATCTCTGCGCTGC-TGCCTGTTGAAT-30 (reverse). GFP-STAT1 Y701F was obtainedfrom Addgene. Human MMP-2 expression construct was kindlyprovided by Dr. Jian Cao (Department of Medicine, School ofMedicine, State University of New York at Stony Brook, StonyBrook, NY; ref. 17). Human MMP-2 promoter luciferase reporterwas a gift from Dr. Etty N. Benveniste (Department of CellBiology, The University of Alabama at Birmingham, Birmingham,Alabama; ref. 18). The antibodies against FOXO1, p-FOXO1,

FOXO4, PARP, STAT1, p-STAT1, STAT3, p-STAT3, and CREB werecommercially purchased from Cell Signaling Technology. Theantibodies against MMP-2, SOCS1, and GAPDH were boughtfrom Santa Cruz Biotechnology. ISO with purity greater than99.9% was purchased from Higher Biotech. ISO was dissolved indimethyl sulfoxide (DMSO; Sigma) to make a 20 mmol/L stocksolution, and the same concentration of DMSO was also madeand used as a vehicle control in all experiments.

Animal experimentsThe C57BL/6J male mice at age of 5 to 6 weeks were

randomly divided into three groups, 12 mice in each group,including vehicle-treated control group, N-butyl-N-(4-hydro-xybutyl) nitrosamine (BBN)-treated group, and BBN combinedwith ISO-treated group. Mice in BBN-treated group receivedBBN (0.05%) in drinking water for 20 weeks, whereas mice inBBN combined with ISO-treated group received BBN and ISO(150 mg/kg/day) in drinking water for 20 weeks. ISO was givento the mice in drinking water on the day of initial exposure toBBN and continued throughout the tumor induction period.Mice bladder tissues were excised and fixed overnight in 4%paraformaldehyde at 4�C. Fixed tissues were processed forparaffin embedding, and the serial 5-mm-thick sections werethen stained by hematoxylin and eosin (H&E) staining.

Cell culture and transfectionHuman-invasive bladder cancer cell line UMUC3 and

noninvasive bladder cancer cell line RT4 were provided byDr. Xue-Ru Wu (Departments of Urology and Pathology, NewYork University School of Medicine, New York, NY) in 2010, andwere described and used in our previous studies (4, 19). Thehuman metastatic bladder cancer cell line T24T, which was alineage-related lung metastatic variant of invasive bladder cancercell line T24 (20–22), was kindly provided by Dr. Dan Theodor-escu (Departments of Urology, University of Virginia, Charlottes-ville, VA; ref. 20) in 2010. All the cell lines were subjected to DNAtests and authenticated in our previous studies (4). The cell lineswere regularly authenticated on the basis of viability, recovery,growth, morphology, and chemical response as well and weremost recently confirmed 4 to 6months before use by using a shorttandem repeat method. UMUC3 cells weremaintained in DMEMsupplemented with 10% FBS (HyClone), 1% penicillin/strepto-mycin, and 2mmol/L L-glutamine (Life Technologies). T24T cellswere cultured inDMEM/Ham's F-12 (1:1 volume)mixedmediumsupplemented with 5% FBS, 1% penicillin/streptomycin, and2 mmol/L L-glutamine. RT4 cells were maintained in McCoy's5A supplemented with 10% FBS, 1% penicillin/streptomycin,and 2 mmol/L L-glutamine. Transfections were carried out usingPolyJet DNA In Vitro Transfection Reagent (SignaGen Laborato-ries) according to themanufacturer's instructions. The transfectedcells were then respectively selected with G418, hygromycin, orpuromycin (Life Technologies) for 4 to 6 weeks. Surviving cellswere pooled as stable mass transfectants as described in ourprevious studies (23, 24).

Human tissue specimensNinety-eight pairs of primary bladder cancer samples and

their paired adjacent normal bladder tissues were obtainedfrom patients who underwent radical cystectomy at Departmentof Urology of the Union Hospital of Tongji Medical College(Wuhan, China) between 2012 and 2015. All specimens were

Jiang et al.

Cancer Prev Res; 9(7) July 2016 Cancer Prevention Research568




immediately snap-frozen in liquid nitrogen after surgical remov-al. Histologic and pathologic diagnoses were confirmed, and thespecimens were classified by a certified clinical patho-logist according to the 2004World Health Organization Consen-sus Classification and Staging System for bladder neoplasms.All specimens were obtained with appropriate informed consentfrom the patients and a supportive grant obtained from theMedical Ethics Committee of China.

ATP cell viability assayCell viability was measured utilizing the CellTiter-Glo Lumi-

nescent Cell Viability Assay Kit (Promega Corp.) according tothe manufacturer's instructions as described in our previousstudies (25). Briefly, cells were plated in 96-well plates at adensity of 10,000 cells per well and allowed to adhere overnight.The cell culture medium was then replaced with 0.1% FBSDMEM and cultured for 12 hours. After ISO treatment for theindicated time and doses, 50 mL of CellTiter-Glo assay reagentwas added to each well. The contents were mixed on an orbitalshaker for 2 minutes to induce cell lysis and then incubated atroom temperature for 10 minutes to stabilize the luminescentsignal. Results were read on a microplate luminometer LB 96V(Berthold GmbH & Co. KG). Cell viability (%) was defined asthe relative absorbance of treated samples versus that of theuntreated control. All experiments were performed with six wellsfor each experiment and repeated at least three times.

In vitro cellular migration and invasion assaysIn vitro migration and invasion assays were conducted by

using Transwell chamber (for migration assay) or Transwellprecoated Matrigel chamber (for invasion assay) according tothe manufacturer's protocol (BD Biosciences) as describedpreviously (26, 27). Briefly, 700 mL of medium containing10% FBS (for UMUC3 and T24T cells) or 40% FBS (for RT4cells) was added to the lower chambers, whereas homoge-neous single-cell suspensions (5 � 104 cells/well) in 0.1% FBSmedium with or without ISO as indicated were added to theupper chambers. The Transwell plates were incubated in 5%CO2 incubator at 37�C for 24 hours, and thereafter werewashed by PBS, fixed with 4% formaldehyde, and stainedwith Giemsa stain. The nonmigration or noninvading cellswere scrapped off on the top of chamber. The migration andinvasion rates were quantified by counting the migration andinvaded cells at least three random fields under a light micro-scope (Olympus).

Western blottingWestern blot assay was assessed as previously described (28).

Briefly, cells were plated in 6-well plates and cultured in normalFBS medium until 70% to 80% confluence. The cells were thencultured in 0.1% FBS medium for 12 hours, followed bytreatment with different doses of ISO for the indicated time.The cells were washed once with ice-cold PBS, and cell lysateswere prepared with a lysis buffer [10mmol/L Tris-HCl (pH 7.4),1% SDS, and 1 mmol/L Na3VO4]. An equal amount (80 mg) oftotal protein from each cell lysate was subjected to Western blotwith the indicated antibody as described in previous studies(25, 26). Immunoreactive bands were detected by using thealkaline phosphatase-linked secondary antibody and ECFWestern blotting system (Amersham Biosciences). The images

were acquired by using Typhoon FLA 7000 imager (GEHealthcare).

Nuclear extract preparationNuclear extracts were prepared as previously described (23).

UMUC3 cells were seeded into 10-cm culture dishes at 70% to80% confluence, cultured in 0.1% FBS medium for 12 hours,and then treated either with vehicle (0.1% DMSO) or with10 mmol/L of ISO for 12 hours. The nuclear proteins wereextracted by the Nuclear/Cytosol Fractionation Kit (BioVisonTechnologies) following the manufacturer's protocols. EqualProtein concentrations were measured by a protein quantifica-tion assay kit (Bio-Rad). Nuclear extracts were stored at �80�Cbefore they were used.

Reverse transcription PCR and qRT-PCRTotal RNA was extracted with TRIzol reagent (Invitrogen

Corp.), and cDNAs were synthesized with the SuperScript IIIFirst-Strand Synthesis System for RT-PCR (Invitrogen Corp.). Apair of oligonucleotides (Forward: 50-GATG ATCTTGAGGC-TGTTGTC-30 and Reverse: 50-CAGGGCTGCTTTTAACTCTG-30)were used to amplify human gapdh cDNA as loading control.The human foxo1 cDNA fragments were amplified by a pair ofhuman foxo1-specific PCR primers (Forward: 50-AACCT GGCAT-TACAGTTGGCC-30; Reverse: 50-AAATGCAGGAGGCATGACTAC-GT-30). The flag-foxo1 fragments were amplified by primers 50-ACAAGGACGACGATGACAAGGG-30 (Forward) and 50-GGCCG-AGTTGGACTGGCTAAA-30 (Reverse). The human mmp-2 cDNAfragments were amplified by 50-CAAGTGGGACAAGAACCAGA-30

(Forward) and 50-CCA AAGTTGATCATGATGTC-30 (Reverse). Thehuman mmp-9 cDNA fragments were amplified by 50-GGGACG-CAGACATCGTCAC-30 (Forward) and 50-TCGTCATCGTCGAAA-TGGC-30 (Reverse). The PCR products were separated on 2%agarose gels and stained with ethidium bromide. The images werevisualized and scanned with UV lights with FluorChem SP imag-ing system (Alpha Innotech Inc.) as described previously (29).The Quantitative reverse transcription PCR (qRT-PCR) analysiswas carried out using the SYBR Green PCR Kit (Qiagen) and the7900HT Fast Real-time PCR system (Applied Biosystems).

Luciferase assayAs described in our previous studies (4, 5), dual-luciferase

reporter assay was performed by using the luciferase assaysystem (Promega Corp.). Briefly, Human FasL promoter,IGFBG-1 (3�IRS) promoter, FOXO1 promoter, and MMP-2promoter luciferase reporters were transfected into the indicat-ed human bladder cancer cells, respectively. After ISO treat-ment, cells were extracted with passive lysis buffer [25 mmol/LTris-phosphate (pH 7.8), 2 mmol/L EDTA, 1% Triton X-100,and 10% glycerol]. The luciferase activity was measured witha microplate luminometer LB 96V (Berthold GmbH & Co. KG).The Renilla luciferase signal was normalized to the internalfirefly luciferase transfection control.

Immunohistochemistry paraffin of mice bladder tissuesMice bladder tissues were immunostained by antibodies

specific against FOXO1 (Cell Signaling Technology) andMMP-2 (Santa Cruz Biotechnology), respectively. The resultantimmunostaining images were captured using the AxioVisionRel.4.6 computerized image analysis system (Carl Zeiss). Pro-tein expression levels were analyzed by calculating the

ISO Inhibits Bladder Cancer Invasion by Inducing FOXO1

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integrated optical density per stained area (IOD/area) usingImage-Pro Plus version 6.0 (Media Cybernetics). Briefly, theIHC-stained sections were evaluated at 400-fold magnifica-tions, at least 5 representative staining fields of each sectionwere analyzed to calculate the optical density based on typicalphotographs that had been captured.

Statistical methodsAssociations between categorical variables were assessed using

the c2 test. The Student t test was utilized to compare continuousvariables, summarized as mean � SD, between different groups.Paired t test was performed to compare the difference betweenpaired tissues using real-time PCR analyze. P < 0.05 was consid-ered statistically.

ResultsISO treatment inhibited BBN-induced mouse-invasive bladdercancer formation in vivo and human bladder cancer invasionin vitro

ISO has been shown to inhibit growth and induce apoptosisin human bladder cancer cells in our recent studies (4, 5). BBNis a well-characterized bladder carcinogen for its induction of100% invasive bladder cancer in mouse model (30). To explorewhether ISO exhibit bladder cancer invasion, we first employedBBN-induced invasive bladder cancer mouse model and exam-ined the effects of ISO on BBN-induced mouse-invasive bladdercancer formation. As shown in Table 1 and Fig. 1A, none of thevehicle-treated control mice developed bladder cancer, whereas

Table 1. Effect of ISO treatment on BBN-induced mouse-invasive bladder cancer formation

GroupNumberof mice Carcinogen Treatment

Number (%) ofpapillomas

Number (%) of noninvasivebladder cancer

Number (%) of invasivebladder cancer

1 12 Vehicle only Vehicle only 0 (0) 0 (0) 0 (0)2 12 BBN Vehicle only 0 (0) 0 (0) 12 (100)a

3 12 BBN ISO 7 (58.3) 3 (25) 2 (16.7)b

aSignificant difference between vehicle control group and BBN-treated group (P < 0.05).bSignificant difference between BBN-treated group and BBN combined with ISO-treated group (P < 0.05).

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Figure 1.ISO inhibited bladder cancer invasion in vivo and in vitro. A, mice were divided into vehicle-treated control group (n ¼ 12), BBN-treated group (n ¼ 12),and BBN combined with ISO-treated group (n ¼ 12). H&E staining was performed, and the representative images of each group were shown. HumanUMUC3 (B) and T24T (C) bladder cancer cells were cultured in chamber or precoated Matrigel chamber and treated with medium containing either vehicleor indicated concentration of ISO for 24 hours. The cells were then fixed and stained. The invasion and migration rates were quantified by countingthe relative migrated (Transwell) and invaded cells at least three random fields under a light microscope. D, UMUC3 cells were treated with mediumcontaining either vehicle or 10 mmol/L ISO up to 36 hours for cell viability assessment by ATPase assay. E, T24T cells were treated with mediumcontaining either vehicle or 20 mmol/L ISO for the indicated time. Cell viability was also evaluated by ATPase assay. Results are the mean � SD oftriplicates. Symbol "�" indicates a significant difference between vehicle- and ISO-treated group (P < 0.05).

Jiang et al.





BBN induced 100% (12/12) high-grade muscle-invasive blad-der cancers formation. Interestingly, only 16.7% (2/12) of theBBN-treated mice developed high-grade muscle-invasive blad-der cancer while ISO was administrated, with 7 cases of pap-illomas and 3 cases of low-grade non–muscle-invasive bladdercancers, demonstrating a novel biologic activity of ISO as anefficient drug that targets at stage of invasive bladder cancerdevelopment in vivo (P < 0.05). Subsequently, two human-invasive bladder cancer cell lines UMUC3 and T24T wereemployed for Transwell cancer cell invasion assay in the pres-ence of relative low dosages, 10 mmol/L and 20 mmol/L, ofISO treatment, according to the dosages applied in cell prolif-eration analyses described previously (4, 5). As shownin Fig. 1B and C, the relative invasion rates of UMUC3 andT24T cells were reduced by 63.1% and 61.2% after 10 mmol/Land 20 mmol/L ISO treatment, respectively, in comparison withthe vehicle control, whereas ISO did not show observed effectson the bladder cancer cell migration under same experimentalconditions. To further exclude any possible involvementof cellular toxicity, the cells were treated with the same dosagesof ISO as shown in Fig. 1D and E for different time points from6 hours to 36 hours, and cell survival rates were analyzed byATPase assay. As expected, the selected dosages of ISO did notshow any observable cellular toxicity to either UMUC3(Fig. 1D) or T24T (Fig. 1E) cells at the time point (24 hours)when cell migration and invasion were measured. Takentogether, these data, for the first time, demonstrate that ISOis a new natural compound that can specifically inhibit mouse-invasive bladder cancer development in vivo following BBNexposure and suppress bladder cancer cell invasion in vitro.

ISO induced FOXO1 protein expression andFOXO1-dependent transcriptional activity in humanbladder cancer cells

FOXO1 has been proposed as a potential prognostic markerfor bladder cancer since an increased foxo1 mRNA expression isassociated with the reduced bladder cancer disease progressionand a significantly prolonged survival of the patients (14). Toelucidate the mechanisms underlying ISO inhibitory effecton human bladder cancer, we investigated whether ISO couldregulate the expression of FOXO1 in human-invasive bladdercancer cell lines, UMUC3, and T24T. As shown in Fig. 2A, ISOtreatment induced FOXO1 protein expression in a dose-depen-dent manner in UMUC3 cells. Treatment of UMUC3 cells with10 mmol/L ISO also resulted in a gradual induction in FOXO1protein level over various time points (Fig. 2B). Consistently,similar FOXO1 protein expression profile was also observed inthe metastatic human bladder cancer T24T cells upon ISOtreatment (Fig. 2C). It was noted that ISO treatments at doseof 10 mmol/L for UMUC3 cells and 20 mmol/L for T24T cellscould markedly induce FOXO1 protein expression (Fig. 2A–C)with a profoundly specific inhibition of cancer cell invasionwithout affecting their migration (Fig. 1C and D). To unravelthe function of ISO-induced FOXO1 protein, we determineFOXO1 protein location and its dependent transcriptionalactivation in ISO-treated cells. As shown in Fig. 2D, FOXO1protein was almost all located in nuclear fraction, while it wasnot observable in cytoplasmic fraction. Consistent with itslocation, ISO treatment remarkably enhanced FOXO1-depen-dent IGFBG-1 promoter and FasL promoter transcription activ-ities (Fig. 2E and F), strongly demonstrating that ISO-induced

FOXO1 proteins are not only located in nuclear, but alsofunctional in human bladder cancer cells.

FOXO1 was downregulated in human bladder cancer tissuesand acted as an effective suppressor of invasion of humanbladder cancer cells

To find out the clinical relevance of therapeutic effect of ISOin human bladder cancer, we evaluated FOXO1 expression in98 pairs of bladder cancer tissues and their adjacent normalbladder tissues surgically removed from patients who werediagnosed with bladder cancers. As shown in Fig. 3A, a pro-found reduction of foxo1 mRNA expression was observed inhuman bladder cancer tissues with an overall average 2.4-foldlower relative foxo1 mRNA level in comparison with theiradjacent normal bladder tissues (P < 0.001). Moreover, asignificant negative relationship was observed between foxo1expression and bladder cancer grade, invasion, as well as the

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Figure 2.ISO treatment upregulated FOXO1 expression and enhanced FOXO1-dependent transcriptional activity in human bladder cancer cells. UMUC3(A & B) and T24T cells (C) were treated with medium containing eithervehicle or ISO as the indicated concentrations for 12 hours (A and C) ortreated with medium containing either vehicle or 10 mmol/L ISO for 6 to 18hours as indicated (B). The whole-cell lysates were used for Westernblotting. GAPDH was used as protein loading control. D, the cell nuclearand cytoplasm fractions of UMUC3 cells treated with 10 mmol/L ISO for12 hours were subjected to SDS-PAGE and immunoblotted with theindicated specific antibodies. The expressions of PARP and GAPDH wererespectively used as markers for nuclear and cytoplasm fractions,respectively. E, UMUC3 cells stably transfected with IGFBG-1 promoter–driven luciferase reporter were treated with 10 mmol/L of ISO for theindicated times. Luciferase activity was determined by the Dual-LuciferaseReporter Assay System. F, UMUC3 cells were stably transfected with FasLpromoter–driven luciferase reporter construct, and were treated with10 mmol/L of ISO for the indicated times to determine the promotertranscriptional activity. Results are the mean � SD of triplicates.Symbol "�" indicates a significant difference between vehicle- andISO-treated group (P < 0.05).






lymph nodes metastasis, respectively (P < 0.05, Table 2).Consistent with mRNA expression, a significantly decreasedFOXO1 protein expression was also observed in human-inva-sive bladder cancer tissues (Fig. 3B). To clarify the relation of

FOXO1 protein to bladder cancer invasion, UMUC3 and T24Tcells were stably transfected with exogenous Flag-FOXO1. Asshown in Fig. 3C–E, ectopic expression of FOXO1 specificallyattenuated the cell invasion with no significant inhibition ofcell migration in both high-grade UMUC3 and T24T bladdercancer cells. On the other hand, knockdown of FOXO1(shFOXO1-4 and shFOXO1-6; Fig. 3F) increased invasivenesswithout affecting migration of noninvasive RT4 bladder cancercells (Fig. 3G). These results are consistent to demonstrate thatdownregulation of FOXO1 expression plays an important rolein the bladder cancer invasion, and the FOXO1 induction byISO treatment might be crucial for its inhibitory effect onhuman bladder cancer invasion.

ISO upregulated FOXO1 expression by increasing its mRNAtranscription in human bladder cancer cells

It is well-known that FOXO1 expression can be regulatedthrough its nuclear exportation and degradation (6). To addresswhether ISO treatment alternates FOXO1 protein degradation,both transfectants of UMUC3(vector) and UMUC3(Flag-FOXO1) were employed. As shown in Fig. 4A and B, ISOtreatment only increased endogenous FOXO1 protein expres-sion inUMUC3(vector) cells, whereas it did not affect exogenous

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shFOXO1-6

shFOXO1-6

Nonsense

Figure 3.FOXO1 was downregulated in human bladder cancer tissues and acted as a regulator of human bladder cancer cell invasion. Total RNA and proteinlysates were prepared from human normal (N) and paired cancerous (T) tissues among 98 patients diagnosed with bladder cancers, and subjected toqRT-PCR and Western blotting analyses for determining FOXO1 mRNA (A) and protein (B) expression profiles, respectively. Flag-FOXO1 expressionconstruct was used to stably transfect into UMUC3 and T24T cells, respectively. The stable transfectants, UMUC3(Flag-FOXO1) and T24T(Flag-FOXO1),were identified by Western blotting (C) and then used for determination of their abilities of cell invasion and migration as compared with their vectorcontrol transfectants (D & E) as described in the "Materials and Methods" section. F, RT4 cells were stably transfected with nonsense shRNA or twoFOXO1 shRNA constructs (shFOXO1-4 and shFOXO1-6), respectively, and the knockdown efficiency of FOXO1 protein was evaluated by Western blot. G,FOXO1 stably knockdown transfectants, RT4(shFOXO1-4) and RT4(shFOXO1-6), as well as Nonsense transfectant RT4(Nonsense) were then used fordetermination of their migration and invasion abilities. Results are the mean � SD of triplicates. Symbol "�" indicates a significant differencebetween vector control and FOXO1 overexpression group (P < 0.05). Symbol "#" indicates a significant difference between NONSENSE transfectantand shRNA transfectants (P < 0.05).

Table 2. Correlation between foxo1 mRNA level and clinicopathologic factors

Foxo1expression

Parameters Group Total Low High P value

Gender Male 79 60 19Female 19 13 6 0.561

Age (years) <55 17 14 3�55 81 59 22 0.548

Histologic grade Low 32 19 13High 66 54 12 0.017a

Tumor stage Tis, Ta, T1 39 22 17T2, T3, T4 59 51 8 0.001a

Tumor size <3.0 cm 40 31 9 0.570�3.0 cm 58 42 16

Tumor multiplicity Unifocal 25 19 6Multifocal 73 54 19 0.841

Lymph nodes metastasis Absent 65 45 20Present 33 30 3 0.008a

aP < 0.05.

Jiang et al.





Flag-FOXO1 expression, excluding the possibility that FOXO1protein induction by ISO is at protein degradation level. We,therefore, next evaluated the mRNA levels of foxo1 in the samebladder cancer cells. Consistent with the results obtained fromprotein levels, endogenous foxo1 mRNA expression wasmarkedly upregulated upon ISO treatment in both UMUC3 andT24T cells (Fig. 4C–E), whereas the flag-tagged exogenous flag-foxo1 mRNA was not affected by ISO treatment (Fig. 4F and G).These results suggest that ISO-induced FOXO1 expression maybe regulated at mRNA transcription level. To test this notion, thefoxo1 promoter–driven luciferase reporter was transfected intoUMUC3 cells, and foxo1 promoter activity was significantlyupregulated in a time-dependent manner upon ISO treatment(Fig. 4H). Taken together, these data strongly reveal that ISO-induced FOXO1 protein expression is regulated at mRNA tran-scriptional level in human bladder cancer cells.

FOXO1 expression was required for ISO inhibition ofbladder cancer cell invasion

To address the contribution of FOXO1 induction by ISO toits inhibition of bladder cancer invasion, we transfected variousFOXO1 shRNAs into UMUC3 and T24T, respectively, and the

stable FOXO1 knockdown transfectants were identified byWestern blotting as shown in Fig. 5A and B. The stable trans-fectants, UMUC3(shFOXO1-4), UMUC3(shFOXO1-6), T24T(shFOXO1-4), and T24T(shFOXO1-6), were used to determineinvasion and migration abilities as compared with their Non-sense control transfectants, UMUC3(Nonsense), and T24T(Nonsense), respectively. As shown in Fig. 5C and Supplemen-tary Fig. S1A and S1B, knockdown of FOXO1 by its shRNAs(shFOXO1-4 and shFOXO1-6) showed no effects on cell migra-tion, but significantly promoted invasion in both UMUC3 andT24T cells (Fig. 5D; Supplementary Fig. S1A and S1B), whichconsistently support that FOXO1 protein exhibits specific inhi-bition of cancer invasion. Importantly, suppression of FOXO1protein expression markedly reduced ISO inhibition of cellinvasion in both UMUC3 and T24T cells (Fig. 5E; Supplemen-tary Fig. S1A and S1B). To further verify the role of FOXO1in ISO suppression of bladder cancer invasion, CRISPR/Cas9systems were used to knockout FOXO1 gene in both UMUC3and T24T cells. The single-clone stable FOXO1 knockout trans-fectants, UMUC3(KO FOXO1, clone 2) and T24T(KO FOXO1,clone 5), were selected for our studies (Fig. 5F and G). Asshown in Fig. 5H and Supplementary Fig. S2A and S2B, bladdercancer migration was not affected upon FOXO1 knockout,whereas the invasion of UMUC3 and T24T cells was furtherincreased (Fig. 5I and Supplementary Fig. S2A and S2B). More-over, knockout of FOXO1 successfully diminished the effectsof ISO on bladder cancer invasion (Fig. 5J; SupplementaryFig. S2A and S2B). These results greatly demonstrate thatFOXO1 protein induction by ISO is crucial for its inhibitionof bladder cancer invasion.

ISO treatment enhanced FOXO1 transcription throughinhibition of STAT1 phosphorylation at Tyr701

The above results demonstrate that ISO inhibition of hu-man bladder cancer invasion was dependent on FOXO1induction via upregulating FOXO1 gene promoter transcrip-tion activity. To identify the transcription factor that is respon-sible for ISO upregulation of FOXO1 transcription, TFANSFACTranscription Factor Binding Sites Software (Biological Data-base) was used for bioinformatics analysis of the FOXO1promoter region. The results revealed that an approximate800-bp promoter region of the human FOXO1 gene containedthe putative DNA-binding sites of various transcription fac-tors, including CREB, FOXO4, STAT1, and STAT3 (Fig. 6A).We next examined the changes in both expression and thenuclear translocation of these factors following ISO treat-ment for 12 hours. As shown in Fig. 6B, treatment of UMUC3cells with ISO resulted in a dramatic inhibition of STAT1phosphorylation at Tyr701 in a dose-dependent manner with-out effecting total protein expression, whereas there was only aslightly effect on phosphorylation of STAT3 and expressionof FOXO4 and CREB. The results obtained from distributionsof the transcription factors between nuclear and cytoplasmfractions consistently indicated that ISO treatment mainlytargeted on STAT1 phosphorylation at Tyr701 (Fig. 6C).To investigate the role of STAT1 phosphorylation in upregula-tion of FOXO1 by ISO, a dominant-negative mutant formof STAT1(GFP-STAT1 Y701F) was ectopically expressed inUMUC3 cells, and the dominant-negative effect was con-firmed by its inhibition of a STAT1-regulated SOCS1 expres-sion (ref. 31; Fig. 6D). The results showed that transfection of

UMUC3

foxo1 (106 bp)gapdh (258 bp)

0 2.5 5 10 ISO (µmol/L)

foxo1 (106 bp)

gapdh (258 bp)

UMUC30 3 6 9 Time (h)

FOXO1

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GAPDH

Flag

GAPDH

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0 10 0 10 ISO (µmol/L)foxo1 (106 bp)gapdh (258 bp)

T24T0 20 ISO (µmol/L)

0 10 20 0 10 20 ISO (µmol/L)

UMUC3(Vector)

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T24T(Vector)

T24T (Flag-FOXO1)

gapdhflag-foxo1

gapdhflag-foxo1

F

E

G

0 10 20 0 10 20 ISO (µmol/L)

Figure 4.ISO upregulated FOXO1 expression at mRNA transcriptional level inhuman bladder cancer cells. UMUC3(Vector) and UMUC3(Flag-FOXO1; A)or T24T(Vector) and T24T(Flag-FOXO1; B) cells were treated with mediumcontaining either vehicle or ISO as the indicated concentrations for12 hours. The cell extracts were subjected to Western blotting fordetermination of ISO on protein expression of FOXO1 and Flag-FOXO1as indicated. UMUC3 (C and D) and T24T (E) cells were treated withISO for 6 hours at indicated concentrations (C and E) or 10 mmol/L ISO forthe indicated time (D), and foxo1 mRNA expression was determined byRT-PCR. The gapdh mRNA level was used as loading control. UMUC3(Vector) and UMUC3(Flag-FOXO1; F) or T24T (Vector) and T24T(Flag-FOXO1; G) cells were treated with medium containing either vehicleor ISO as the indicated concentrations for 6 hours. The Flag-Foxo1 mRNAlevels were evaluated by RT-PCR as indicated. H, UMUC3 cells stablytransfected with Foxo1 promoter–driven luciferase reporter were treatedwith 10 mmol/L ISO for the indicated times. Luciferase activity wasevaluated by the Dual-Luciferase Reporter Assay System. Results are themean � SD of triplicates. Symbol "�" indicates a significant differencebetween vehicle- and ISO-treated group (P < 0.05).






dominant-negative STAT1 markedly enhanced ISO-inducedFOXO1 protein expression (Fig. 6D) as well as FOXO1 pro-moter–driven transcription activity (Fig. 6E), indicating thatsuppression of STAT1 activity could mimic ISO treatment. Inthe contrast, mutation of STAT1-binding site effectively dimin-ished the increased FOXO1 promoter activity induced by ISO(Fig. 6F). Thus, these data suggest that phosphorylated STAT1at Tyr701 can bind to the FOXO1 promoter to inhibit itstranscription, and ISO-induced upregulation of FOXO1 tran-scription is specifically mediated through ISO suppression ofSTAT1 phosphorylation at Tyr701.

FOXO1 mediated ISO inhibition of bladder cancer invasionthrough downregulation of MMP-2 expression

Extensive studies have identified MMPs as the key playersin cancer cell invasion by degrading cellular matrix compo-nents and the basement membrane (32). Elevated MMP-2levels have been reported to be of independent prognosticvalue in patients with bladder cancers (33), whereas the otheressential MMP member, such as MMP-9 expression, is not

correlated with tumor grade, stage, or overall survival ofthe patients with bladder cancer (34). Our previous studyhas also confirmed that upregulation of MMP-2, but notMMP-9, participated in mediation of bladder cancer invasion(22). To determine whether MMP-2 is associated with ISOinhibition of human bladder cancer invasion, we evaluatedthe effect of ISO on MMP-2 protein expression. The resultsshowed that ISO treatment profoundly inhibited MMP-2protein expression in both UMUC3 and T24T cells (Fig. 7Aand B). Moreover, MMP-2 mRNA level and its promoter-driven luciferase reporter activity were also attenuated byISO treatment (Fig. 7C and D), suggesting that thedownregulation of MMP-2 expression by ISO occurred attranscriptional level. Meanwhile, the mRNA level of MMP-9was not affected upon ISO treatment (Fig. 7C). To test thepotential relation of FOXO1 induction to MMP-2 inhibitionfollowing ISO treatment in bladder cancer cells, we nextevaluated the effects of FOXO1 overexpression and knock-down on MMP-2 expression in T24T cells. As shown in Fig. 7E,enforced expression of FOXO1 impaired MMP-2 expression,

Nonsense shFOXO1-4shFOXO1-6



Vehicle ISO (10 µmol/L) Vehicle ISO (20 µmol/L)

FOXO1

GAPDH

B

FOXO1

GAPDH

A

C1.21.00.80.60.40.2

0

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EUMUC3 T24T UMUC3 T24T

##

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##

Vector C1 C2 C3 C4 C5 C6

FOXO1

GAPDH

KO FOXO1Vector C1 C2 C3 C4 C5 C6

KO FOXO1

FOXO1

GAPDH

UM

UC

3

T24

T

1.21.00.80.60.40.2

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##1.2

1.00.80.60.40.2

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&

&1.21.00.80.60.40.2

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VectorKO FOXO1

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UMUC3 T24T

1.21.00.80.60.40.2

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JVehicleISO (10 µm)

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*

Nonsense

shFOXO1-1

shFOXO1-2

shFOXO1-3

shFOXO1-4

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shFOXO1-6

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UMUC3T24T

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ISO (20 µmol/L)

Vehicle


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Nonsense

shFOXO1-4

shFOXO1-6Nonsense

shFOXO1-4

Vector

KO FOXO1Vector

KO FOXO1

shFOXO1-6

Figure 5.Knockdown and knockout of FOXO1 reversed ISO inhibition of bladder cancer cell invasion. A–E, UMUC3 (A) and T24T (B) cells were stably transfectedwith nonsense shRNA or a set of six various FOXO1 shRNA (shFOXO1) constructs, respectively. The knockdown efficiency of FOXO1 protein wasassessed by Western blotting. FOXO1 stably knockdown transfectants, including UMUC3(shFOXO1-4), UMUC3(shFOXO1-6), T24T(shFOXO1-4), and T24T(shFOXO1-6), as well as Nonsense transfectants were then used for determination of their cell migration (C) and invasion (D and E) in the presence ofeither vehicle or the indicated concentration of ISO treatment for 24 hours. F–J, CRISPR/Cas9 systems were then applied to knockout FOXO1 gene,and the single-clone stable FOXO1 knockout transfectants, UMUC3(KO FOXO1, C1-C6) and T24T(KO FOXO1, C1-C6), were identified by Western blot(F and G). FOXO1 stably knockout transfectants, including UMUC3(KO FOXO1, clone5) and T24T(KO FOXO1, clone2), as well as Vector controltransfectants were used for determination of their cell migration (H) and invasion (I and J) in the presence of either vehicle or the indicatedconcentration of ISO treatment for 24 hours. Results are the mean � SD of triplicates. Symbol "#" indicates a significant difference between Nonsensetransfectant and shRNA transfectants (P < 0.05). Symbol "&" indicates a significant difference between scramble vector control and FOXO1 knockouttransfectants (P < 0.05). Symbol "�" indicates a significant difference between vehicle- and ISO-treated group (P < 0.05).

Jiang et al.





whereas knockdown of FOXO1 upregulated the basal levelsof MMP-2 protein and mRNA and effectively reversed ISOdownregulation of MMP-2 expression (Fig. 7F). Moreover, theinhibition of STAT1 phosphorylation at Tyr701 by ISO wascomparable between T24T(Nonsense) and T24T(shFOXO1)transfectants. Accordingly, the basal level of MMP-2 promoteractivity in T24T(shFOXO1) transfectant was also significantlyincreased in comparison with T24T(Nonsense) transfectant,and ISO inhibition of MMP-2 promoter activity was impairedin T24T(shFOXO1) transfectant (Fig. 7G). It was important tonote that ectopic expression of MMP-2 (Fig. 7H) in T24T cellsdid not affect cell migration (Fig. 7I; Supplementary Fig. S3),but specifically enhanced the basal level of cell invasion (Fig.7J; Supplementary Fig. S3), while it effectively attenuated ISOinhibition of T24T cell invasion (Fig. 7K; SupplementaryFig. S3). These results revealed that MMP-2 acted as FOXO1downstream being responsible for ISO inhibition of bladdercancer invasion, and this novel finding was greatly supportedby our results obtained from BBN-induced mouse-invasivebladder cancers in vivo showing that ISO treatment down-regulated FOXO1 and upregulated MMP-2 expression inmouse bladder tissues (Fig. 8A–C). Taken together, our resultsdemonstrate that FOXO1 mediates ISO inhibition of MMP-2transcription and protein expression, by which it inhibitsbladder cancer invasion in vitro and invasive bladder cancerdevelopment in vivo as diagramed in Fig. 7L.

Discussion

Muscular invasion of bladder cancer causes 100% death andrepresents amajor therapeutic challenge of this disease. Therefore,an endeavor to identify new anticancer compounds with stronginhibition of cancer invasion and understand the mechanismsunderlying their inhibitory effect onbladder cancer invasion is thekey step to discover the unmet future medicines against theinvasive malignant disease. ISO is a new derivative isolated froma Chinese herb Gnetum Cleistostachyum that has been used to treatbladder cancer for many years (3). Our recent in vivo pharmaco-kinetic study shows that the concentration of ISO in serum canreach to 47.9 mmol/L without any observed toxicity to experi-mental mice (4). Here, our results obtained from in vivo animalstudies show that ISO treatment inhibits BBN-induced mouse-invasive bladder cancer formation, for first time demonstratingthe chemopreventive effects of ISO to the best of our knowledge.We also find that ISO at in vivo–relevant concentrations of 10 to20 mmol/L specifically represses bladder cancer invasion by pro-moting FOXO1 transcription through inhibition of STAT1 phos-phorylation at Tyr701. Our study also identify that MMP-2 is adownstream target of FOXO1 for its mediation of ISO inhibitionof bladder cancer invasion. Our results clearly demonstrate thatthe natural compound ISO specifically inhibits human bladdercancer invasion through targeting STAT1–FOXO1–MMP-2 axis,which is completely distinct from its induction of cancer cell

SOCS1

0 10 0 10 ISO (µmol/L)

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STAT1 Y701F

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oter

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0 12 18 24Time (h)

WT Mut

#

*

*

*

*

Figure 6.ISO promoted FOXO1 transcription by inhibition of STAT1 phosphorylation at Tyr701. A, schematic representation of transcription factor binding sites ofhuman FOXO1 gene promoter. B, UMUC3 cells were treated with either vehicle or ISO as the indicated concentrations for 12 hours. Expression ofthe related transcription factors in the whole-cell lysates was determined by Western blotting, and GAPDH was used as protein loading control. C, theUMUC3 cells were treated with either vehicle or 10 mmol/L of ISO for 12 hours. The cell extracts were used to isolate cell nuclear and cytoplasmfractions and then subjected to Western blotting with the specific antibodies as indicated. PARP and GAPDH were used as markers for nuclearand cytoplasm fractions, respectively. D, UMUC3 cells were stably transfected with dominant-negative STAT1, GFP-STAT1 Y701F, and the stabletransfectants were treated with either vehicle or 10 mmol/L ISO for 12 hours. The expression of FOXO1 and SOCS1 was determined by Westernblotting. GAPDH was used as protein loading control. E, FOXO-1 promoter–driven luciferase reporter was transfected into UMUC3(EGFP) andUMUC3(STAT1 Y701F) cells, and the stable transfectants were then treated with 10 mmol/L of ISO for the indicated times. Luciferase activity wasevaluated by the Dual-Luciferase Reporter Assay System. F, UMUC3 cells were stably transfected with FOXO-1 promoter–driven luciferase or theSTAT1-binding site mutant, and the stable transfectants were treated with 10 mmol/L of ISO for 18 hours. Dual-Luciferase Reporter Assay Systemwas performed to determine the Luciferase activity. Results are the mean � SD of triplicates. Symbol "�" indicates a significant difference betweenUMUC3(EGFP-vector) and UMUC3(GFP-STAT1 Y701F) in the same time point upon ISO treatment (P < 0.05). Symbol "#" indicates a significantdifference between vehicle control and ISO-treated group (P < 0.05).






apoptosis and inhibition of cancer cell anchorage-independentgrowth. Prevention and therapeutic intervention by phytochem-icals represents a newer dimension in cancer management. Sily-marin, a mixture of flavonoids isolated mainly from milk thistle,has been extensively studied in preclinical models as well asclinically for its efficacy against variety of cancers includingbladder cancer, and it has been shown to effectively inhibit cellproliferation, angiogenesis, epithelial mesenchymal transition,metastasis, as well as promote cell apoptosis (35, 36). WhetherISO exhibits other anticancer effects apart from targeting cellgrowth, apoptosis, and invasion deserves further investigation.

Our recent studies have revealed that ISO effectively attenu-ates transcription factor–specific protein 1 (Sp1) expressionand transactivation (4, 5), which results in the decreasedbindings of Sp1 to promoter region of its regulated genes,cyclin D1 and XIAP, in turn leading to the downregulation ofthese gene expressions and consequently resulting in suppres-

sion of cancer cell anchorage-independent growth and induc-tion of apoptosis in bladder cancer cells (4, 5). Although ourgroup has reported above mechanism underlying anticancereffects of ISO compound, evaluation of whether ISO is a newagent with strong activity for inhibition of cancer invasion andunderstanding the potential mechanism involved in its inhi-bition of bladder cancer invasion are of tremendous impor-tance for ISO potentially therapeutic clinical application. In thepresent study, we demonstrate that ISO at relevant applicableconcentrations shows a great inhibition of bladder cancerinvasion in vitro and mouse-invasive bladder cancer develop-ment in vivo. Accordingly, treatment of ISO significantly pro-motes FOXO1 expression, whereas suppression of FOXO1expression completely abolishes ISO inhibition of bladdercancer invasion in both UMUC3 and T24T cells. Thus, ISO isan effective therapeutic agent for inhibiting bladder cancerinvasion, and upregulation of FOXO1 plays a critical role in

VectorMMP-2

Vehicle ISO (20 µmol/L)

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gapdhmmp-2

MMP-2

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I J K

gapdhmmp-9

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ISO (20 µmol/L)Vector

MMP-2Vector

MMP-2

Nonsense

shFOXO1-6

Figure 7.ISO inhibited bladder cancer cell invasion through downregulation of MMP-2 in a FOXO1-dependent manner. UMUC3 (A) and T24T (B) cells were treatedwith either vehicle or ISO as the indicated concentrations for 18 hours. The protein level of MMP-2 was determined by Western blotting, and GAPDHwas used as protein loading control. C, T24T cells were treated with either vehicle or the indicated concentrations of ISO for 18 hours. The MMP-2 and MMP-9mRNA levels were evaluated by RT-PCR, and gapdh mRNA was used as the internal loading control. D, T24T cells were stably transfected with MMP-2promoter–driven luciferase reporter, and the stable transfectants were treated with 20 mmol/L of ISO for the indicated times. Dual-Luciferase ReporterAssay System was used to detect the Luciferase activity. Results are the mean � SD of triplicates. Symbol "�" indicates a significant differencebetween vehicle control and ISO-treated groups (P < 0.05). E, the expression of FOXO1 in T24T(Vector) and T24T(Flag-FOXO1) stable transfectantswas detected by Western blotting. F, T24T(shFOXO1-6) and T24T(Nonsense) cells were treated with vehicle or 20 mmol/L of ISO for 18 hours. Celllysates were subjected to Western blotting with the specific antibodies indicated. G, T24T(shFOXO1-6) and T24T(Nonsense) cells were transfectedwith MMP-2 promoter–driven luciferase reporter, and the stable transfectants were then treated with 20 mmol/L of ISO for the indicated times, and cells werethen subjected to determine luciferase activity using Dual-Luciferase Reporter Assay. Results are the mean � SD of triplicates. Symbol "#" indicates asignificant difference between T24T(shFOXO1-6) and T24T(Nonsense) cells in the same time point upon ISO treatment (P < 0.05). H, MMP-2 wasstably transfected into T24T cells, and the stable transfectant was identified by verification of ectopic expression of MMP-2 protein by Western blotting.The MMP-2 stable transfectant and its vector control transfectant were subjected to cell migration (I) and invasion (J and K) assay in the presence ofeither vehicle or 20 mmol/L of ISO for 24 hours. Results are the mean � SD of triplicates. Symbol "#" indicates a significant difference between Vectortransfectant and MMP-2 stable transfectant (P < 0.05). L, the proposed schematic for the cascade underlying ISO inhibition of human bladder cancercell invasion through downregulation of MMP-2 in FOXO1-dependent manner.

Jiang et al.





this biologic effect. It is highlighted in the previous reports thatactivated EGFR signaling serves as a potential therapeutic targetfor muscle-invasive bladder cancers (37, 38). Given thatFOXO1 can restore chemosensitivity to anti–EGFR-based ther-apies both in vitro and in vivo (39), the combination of ISO withanti–EGFR-based therapy may result in improved treatmentresponse through upregulation of FOXO1 expression. FOXO1has been found to inhibit invasion of other cancers, such asprostate cancer and glioblastoma (12, 13), indicating thattreatment of ISO may result in similar therapeutic effects inthese cancers. Collectively, our present study provides a valu-able information for the design of more effective strategies forutilizing ISO or for the synthesis of other novel conformation-constrained derivatives to treat bladder cancers and othercancers.

FOXO1, which primarily functions as transcription factor,regulates a large spectrum of cancer-related gene expressionand thus plays important biologic roles as tumor suppressorin regulating cell-cycle arrest, apoptosis, DNA damage repair,and/or oxidative stress resistance (6, 7). Nevertheless, the

FOXO1 signaling in regulation of cell cancer cell invasion isdifferential, depending on the cancer types and cellular con-text. Forced expression of FOXO1 has been reported to sup-press Runx2-promoted prostate cancer cell migration andinvasion (12). Inhibition of FOXO1 nuclear exclusion pre-vents the metastatic factor MMP-9 activation and cell invasioninduced by EGF in glioblastoma (13). However, FOXO1 hasbeen shown to directly regulate transcription of MMP-1 andis thought to promote breast cancer cell metastasis (40).Moreover, FOXO1 is required for keratinocyte migration andwound healing, which involves upregulation of TGFb1 andits downstream targets (41). To explore the role of FOXO1in bladder cancer invasion, the expression of FOXO1 is exam-ined in human bladder cancer tissues as well as the adjacentnormal bladder tissues surgically removed from 98 patientsdiagnosed with bladder cancers. We find that FOXO1 wasprofoundly downregulated in human bladder cancer tissues,and a significant negative relationship was found betweenfoxo1 mRNA expression and bladder cancer invasion, which isconsistently supported by the previous study that FOXO1expression is reduced in the progressed bladder cancer tissuesas compared with nonprogressed patients (14). Moreover, wedemonstrate that ectopic expression of FOXO1 suppressescancer cell invasion in both UMUC3 and T24T human-inva-sive bladder cancer cells. Furthermore, knockdown of FOXO1further enhances bladder cancer invasion in the absence ofISO treatment, whereas it impairs ISO inhibition of bladdercancer invasion. Thus, our results demonstrate that down-regulation of FOXO1 expression contributes to bladder cancerinvasion and that ISO-induced FOXO1 expression plays acrucial role in its inhibition of bladder cancer invasion.

FOXO1 itself is subject to multiple levels of regulation viavarious signaling pathways, whereas regulation of its post-translational modifications and subcellular localization hasbeen intensively investigated (7). Phosphorylation of FOXO1by protein kinases, such as Akt and cyclin-dependent kinase 2(CDK2), results in nuclear export, reduced DNA-binding abil-ity, and degradation of this transcription factor (42, 43).Although hyperactivation of the PI3K-Akt cascade is a com-mon feature in many cancers, including bladder cancer (44),FOXO1 was still mainly located in the nuclear of bladdercancer cells as demonstrated in the present study, suggestingposttranslational modification by AKT probably is not a majorplayer in the ISO regulation of FOXO1 expression in bladdercancer cells. This notion is greatly supported by the resultsshowing that the exogenous FOXO1 protein is not affected byISO treatment. Meanwhile, the expressions of endogenousprotein and mRNA, as well as promoter activity of FOXO1,are significantly induced upon ISO treatment, demonstratingthat the increased nuclear-located FOXO1 was regulated atmRNA transcriptional level.

STAT1 is a cytoplasmic protein that is activated via phos-phorylation at Tyr701 by IFNs and other cell signals in lym-phocytes (45). Following activation, STAT1 is translocated tobind specific regulatory sequences to activate or repress tran-scription of its targeted genes (45). As IFN-induced STAT1signaling is primarily implicated in mediating immune antivi-ral and/or antipathogen functions, suppression of cell prolif-eration, and the induction of apoptosis in lymphocytes, whichis the concept of STAT1 as a tumor-suppressive function(45, 46). Nevertheless, the tumor suppressor recognition of

FOXO1 MMP-2

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Figure 8.ISO reversed the BBN-induced downregulation of FOXO1 andupregulation of MMP-2 in mice. Mice were divided into vehicle-treatedcontrol group (n ¼ 12), BBN-treated group (n ¼ 12), and BBN combinedwith ISO-treated group (n ¼ 12). A, immunohistochemistry paraffinstaining by antibodies specific against FOXO1 and MMP-2 wasperformed. FOXO1 (B) and MMP-2 (C) protein expression levels wereanalyzed by calculating the integrated IOD/area using Image-Pro Plusversion 6.0. Results are the mean � SD of 12 mice in each group.Symbol "�" indicates a significant difference between vehicle controlgroup and BBN-treated group (P < 0.05). Symbol "#" indicates asignificant difference between BBN-treated group and BBN combinedwith ISO-treated group (P < 0.05).






STAT1 has been shattered by emerging evidence that constitu-tively activated STAT1 signaling is involved in the resistanceto DNA-damaging therapeutic agents of epithelial cancers(45–47). In line with these observations, muscle-invasive blad-der cancer tissues were reported to be characterized by consti-tutively nuclear expression of phosphorylated STAT1 (48).STAT1 has been thought to exhibit a negative regulatory effecton FOXO1 transcription in pancreatic b cells (49); however,this notion has never been explored in any cancer cells. Inaddition, nothing is known about the mechanism underlyingSTAT1 regulation of FOXO1 transcription. In the presentstudy, we demonstrate that ISO treatment increases FOXO1transcription accompanied with repression of STAT1 phosphor-ylation at Tyr701 in bladder cancer cells. The inhibition ofSTAT1 activity by overexpression of dominant-negative STAT1could mimic the biologic effects of ISO treatment, whereas themutation of STAT1-binding site in FOXO1 promoter regionreversed the increased FOXO1 transcription activity inducedby ISO treatment. These results reveal a novel function ofphosphorylated STAT1 at Tyr701 for its direct binding toFOXO1 promoter resulting in suppression of FOXO1 transcrip-tion, which is also the mechanisms for ISO upregulation ofFOXO1 transcription and expression, and in turn inhibitingbladder cancer invasion in vitro and mouse-invasive bladdercancer formation in vivo and providing new information forusing ISO as a new agent targeting STAT1 and FOXO1 for thebladder cancer therapy.

FOXO proteins could not only act as transcription factorsin the nucleus through sequence-specific interaction withDNA-binding sites, but can also cooperate with or titrate awayspecific transcription factors or cofactors, and subsequentlyactivate or repress the transcription of the downstream genesthat lack consensus binding sequences for FOXO factors (50).FOXO1 has been found to physically interact with transcrip-tion factor Runx2 and downregulate the transcription ofRunx2-targeted endogenous genes, such as the osteopontin(OP), VEGF, and MMP-13, thereby inhibiting prostate cancercell migration and invasion (12). The present study shows thattranscription of MMP-2, a key player in many cancer cellinvasion, is downregulated upon ISO treatment. Moreover,knockdown of FOXO1 upregulates the basal expression levelof MMP-2 and effectively reverses ISO inhibition of MMP-2expression and bladder cancer invasion as well, suggestingthat FOXO1 plays a negative role in regulation of MMP-2transcription. Accordingly, the inhibition of BBN-inducedmice-invasive bladder cancer formation by ISO is accompa-nied by upregulation of FOXO1 and downregulation of MMP-2 expression. As MMP-2 promoter lacks consensus bindingsite for FOXO1, we anticipate that FOXO1 probably repressesMMP-2 transcription indirectly through interacting with otherspecific transcription factors. Further elucidating of thishypothesis is currently undergoing in our laboratory.

In conclusion, we demonstrate that FOXO1 acts as a tumorsuppressor by inhibiting bladder cancer invasion, and thenovel derivative anticancer drug ISO specifically represses

BBN-induced invasive bladder cancer formation in vivo andhuman bladder cancer invasion in vitro through upregulationof FOXO1 expression at transcription level. Moreover, theSTAT1-binding site in FOXO1 promoter is critical for itssuppression of FOXO1 transcription, and ISO-induced FOXO1expression is mediated through dephosphorylation of STAT1at Tyr701. In addition, downregulation of MMP-2 mRNAtranscription by FOXO1 is responsible for ISO inhibitionof bladder cancer invasion. These findings not only providea novel insight into understanding of underlying mechanismof bladder cancer's propensity to invasion, but also identifynatural compound ISO acting as a new agent for inhibition ofinvasive bladder cancer formation, which could be used forpreventive strategies or to prevent recurrence and progressafter Transurethral Resection of Bladder Tumor of non–mus-cle-invasive tumors.

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

Authors' ContributionsConception and design: G. Jiang, C. HuangDevelopment of methodology: G. Jiang, A.D. Wu, C. Huang, L. Zhang, H. JinAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): G. Jiang, A.D. Wu, C. Huang, J. Gu, L. Zhang, J. Li,D. Zhang, H. JinAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): G. Jiang, C. Huang, J. Gu, L. Zhang, H. Huang,X. Zeng, H. Jin, C. HuangWriting, review, and/or revision of themanuscript:G. Jiang, X. Liao, C. HuangAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): J. Li, C. HuangStudy supervision: H. Huang, C. HuangOther (provided key reagents for experiments and inputs for study):H. Huang

AcknowledgmentsThe authors thank Dr. Jean-Baptiste Demoulin from Catholic University

of Louvain for generous gift of human FOXO1 promoter luciferase reporter;Dr. Jian Cao from State University of New York at Stony Brook for the gift ofhuman MMP-2 expression construct; and Dr. Etty N. Benveniste from TheUniversity of Alabama at Birmingham for providing human MMP-2 promoterluciferase reporter.

Grant SupportThis work was partially supported by grants from NIH/NCI CA177665

(to C. Huang), CA165980 (to C. Huang), CA112557 (to C. Huang), NIH/NIEHS ES000260 (to C. Huang), Natural Science Foundation of China(NSFC81229002; to C. Huang), and Key Project of Science and TechnologyInnovation Team of Zhejiang Province (2013TD10; to C. Huang), as well asWenzhou Science and Technology Bureau (Y20150008; to H. Huang).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received September 16, 2015; revised March 3, 2016; accepted March 29,2016; published OnlineFirst April 14, 2016.

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2016;9:567-580. Published OnlineFirst April 14, 2016.Cancer Prev Res Guosong Jiang, Amy D. Wu, Chao Huang, et al. STAT1/FOXO1 Axis

by TargetingIn Vitro and Human Bladder Cancer Invasion In VivoIsorhapontigenin (ISO) Inhibits Invasive Bladder Cancer Formation

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isorhapontigenin (iso) inhibits invasive bladder cancer ...€¦ · 2–m arrest (8). besides,...

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