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Published OnlineFirst August 24, 2010; DOI: 10.1158/0008-5472.CAN-10-1075

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apeutics, Targets, and Chemical Biology

inoflavone, a Ligand of the Aryl Hydrocarbon Receptor,

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bits HIF-1α Expression in an AhR-Independent Fashion

Terzuoli1,2, Maura Puppo1,3, Annamaria Rapisarda1, Badarch Uranchimeg1, Liang Cao4,
ika M. Burger5, Marina Ziche2, and Giovanni Melillo1
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inoflavone (AF), the active component of a novel anticancer agent (AFP464) in phase I clinical trials, is aof the aryl hydrocarbon receptor (AhR). AhR dimerizes with HIF-1β/AhR, which is shared with HIF-1α,scription factor critical for the response of cells to oxygen deprivation. To address whether pharmaco-ctivation of the AhR pathway might be a potential mechanism for inhibition of HIF-1, we tested theof AF on HIF-1 expression. AF inhibited HIF-1α transcriptional activity and protein accumulation incells. However, inhibition of HIF-1α by AF was independent from a functional AhR pathway. Indeed, AFed HIF-1α expression in AhR100 cells, in which the AhR pathway is functionally impaired, yet did notcytotoxicity, providing evidence that these effects are mediated by distinct signaling pathways. More-F was inactive in MDA-MB-231 cells, yet inhibited HIF-1α in MDA-MB-231 cells transfected with theA1 gene. AF inhibited HIF-1αmRNA expression by ∼50%. Notably, actinomycin-D completely abrogatedility of AF to downregulate HIF-1α mRNA, indicating that active transcription was required for thetion of HIF-1α expression. Finally, AF inhibited HIF-1α protein accumulation and the expression oftarget genes in MCF-7 xenografts. These results show that AF inhibits HIF-1α in an AhR-independent
HIF-1
fashion, and they unveil additional activities of AF that may be relevant for its further clinical development.Cancer Res; 70(17); 6837–48. ©2010 AACR.

activatumortion, mHIF-αand iHIF-1therapAm

a prodidly co

duction

oxia, a decrease in oxygen levels, is a hallmark of solids. The response of mammalian cells to hypoxia is me-, at least in part, by a family of transcription factorsas hypoxia inducible factors (HIF; ref. 1). HIF-1 is a

dimer consisting of a constitutively expressed β sub-nd an oxygen regulated α subunit (2), which is ubiqui-d and degraded under normoxic conditions (3). Inst, under hypoxic conditions, the HIF-α subunit is sta-

slocates to the nucleus, in which it dimerizesso known as AhR nuclear translocator) and

cific pcytotoset ofThe sewith iceptorclearxenobtarget1A1 (pathwto becells (Sev

betwewhethbe a vstoodin vitr

s: 1Tumor Hypoxia Laboratory, Science Applicationsoration-Frederick, Inc., National Cancer Institute atick, Maryland; 2Department of Molecular Biology,a, Siena, Italy; 3G. Gaslini Institute, Laboratory of, Genoa, Italy; 4Molecular Targets Core, Geneticsor Cancer Research, National Cancer Institute,nd; and 5Barbara Ann Karmanos Cancer Institute,

ry data for this article are available at Cancer Researchrres.aacrjournals.org/).

Puppo contributed equally to this work.

uthor: Giovanni Melillo, Developmental Therapeuticsypoxia Laboratory, Building 432, Room 218, SAIC-tional Cancer Institute at Frederick, Frederick, MD1-846-5050; Fax: 301-846-6081; E-mail: melillog@

5472.CAN-10-1075

ssociation for Cancer Research.

ls.org

Research. on January 14, 20cancerres.aacrjournals.org ed from

tes the transcription of genes involved in key steps ofigenesis, including angiogenesis, metabolism, prolifera-etastasis, and differentiation (4). Overexpression ofhas been reported in >70% of human cancers (5–11)s associated with poor patient prognosis, makingan attractive target for the development of novel cancereutics (12).inoflavone (AF; NSC 686288) is the active component ofrug (AFP464) in phase I clinical trials. AFP464 is rap-nverted to AF, in plasma or tissue culture, by nonspe-lasma esterases. AF has a unique COMPARE pattern ofxicity in the NCI60 (13, 14), with activity only in a sub-cell lines, including MCF-7 breast cancer cells (15–18).nsitivity of cancer cell lines to AF has been associatedts ability to act as a ligand of the aryl hydrocarbon re-(AhR), which upon dimerization with HIF-1β/AhR nu-

translocator activates transcription by binding to theiotic response element (XRE) in the promoters ofgenes, including but not limited to cytochrome P450CYP1A1). Indeed, the presence of a functional AhRay and the induction of CYP1A1 expression by AF seemessential for its antiproliferative activity in MCF-718–20).eral studies have suggested the existence of a cross-talken the AhR and HIF-1 pathways (21–26). However,er pharmacologic activation of the AhR pathway mayiable approach to inhibit HIF-1 remains poorly under-
. We show that AF inhibits HIF-1α expression, botho and in MCF-7 xenografts, in an AhR-independent
6837

19. © 2010 American Association for Cancer

http://cancerres.aacrjournals.org/

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n. Notably, AF partially inhibited HIF-1αmRNA expres-et almost completely blocked HIF-1α protein accumu-. The ability of actinomycin-D to completely reverttion of HIF-1α mRNA expression by AF is consistenthe existence of a transcription-dependent pathway thategulate HIF-1α mRNA expression and its translation.

rials and Methods

nes and reagentsan cell lines were maintained in RPMI 1640 supple-d with L-glutamine and 5% heat-inactivated fetal bo-erum (Hyclone), and grown at 37°C in 5% CO2 andnt oxygen (normoxia). Hypoxia was achieved in an

2 400 hypoxic workstation (Ruskinn Technology) deliv-1% oxygen in 5% CO2 at 37°C. AhR-deficient MCF-7AhR100; ref. 27) were kindly provided by Dr. David[STB, National Cancer Institute (NCI), Frederick, MD].

MB-231 cells stably transfected with a SULT1A1 cDNA,SULT1A1 (28), were kindly provided by Dr. David C.(Wadsworth Center, Albany, NY). All cell lines were ob-from the Developmental Therapeutics Program andalidated according to information provided on the De-ental Therapeutics Program Web site (http://dtp.nci.

v/branches/btb/characterizationNCI60.html). AF (NSC) was from the Drug Synthesis and Chemistry Branch,pmental Therapeutics Program, NCI.

hodamine B assays, seeded into 96-well plates, were treated with AF foritional 72 hours, and cell viability was assessed as pre-y described (29).

noblot analysisal protein lysates were obtained as previously de-d (30). Antibodies used are as follows: HIF-1α andD- Transduction Laboratories); HIF-1β, SULT1A1,
, and AhR (Novus Biologicals); actin (Chemicon Inter-al); and γH2AX (Cell Signaling, Inc.).
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ime PCRcular endothelial growth factor (VEGF), HIF-1α, p21,OX, actin, and CYP1A1 expression was measured bymePCR as previously described (30). Primers and probesre listed in Supplementary Table S1 and Table 1. VEGFrs and probe were previously described (30). 18S rRNAsed as an internal control.

α protein translational assayF-7 cells, treated for 12 hours with AF (0.25 μmol/L),abeled with 35S-methionine/cysteine (MP Biomedicals)viously described (31). Total 35S incorporation wasored by both size fractionation of total lysates andrecipitation.

rase activityF-7 cells were transfected using Effectene (Qiagen)GL2-TK-HRE, pGL3-control (30), NF-κB-Luc, activa-otein (AP-1)-luc (supplied by Dr. Nancy Colburn,Frederick, MD), and XRE-Luc (supplied by Dr. F.J.lez, NCI, NIH, Bethesda, MD). Efficiency of transfec-as assessed by cotransfection with pQTK-Renillaega). Results are expressed as fold increase of lucifer-vels relative to normoxic nontreated controls.

fection with small interfering RNA for HIF-1α,A1, and AhRll interfering RNA (siRNA) targeting SULT1A1 (s5′-CA-GAGTGTGCGAATCAA), AhR (5′-TTGGATTAAAT-TTGTGA), HIF-1α (5′-AGGACAAGTCACCACAGGA),egative control were purchased from Qiagen.

al studiesdies were conducted in a facility that is accredited bysociation of Assessment and Accreditation of Labora-nimal Care with an approved animal protocol. MCF-71 × 107) were injected s.c. into the flank of female
ic nude (NCr/nu) mice (Animal Production Area,rederick, MD). β-Estradiol cypionate (3 mg/kg) was
1. Primers used for real-ti

5′-ATT5′-TGC5′-ACG5′-AAG5′-GAG5′-AAC5′-TGC5′-AAT5′-TGC5′-TGG

5′-TTG

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CYP1A1
rd GGGCACATGCTGACG-3′ e TGGCTCATCCTTGACAG-3′
p21
rd CGACTGTGATGCGC-3′ e TTCCATCGCTCACGGG-3′
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rd GCCTGGCCGTGTTG-3′ e TGCTCATAGGCACTGTTTTCTT-3′
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HIF-1α (intron 5-exon 6)
rd TTTTTTTTTCCCTAGCATTGT-3′ e TTACTGTTGGTATCATATACGTGAA-3′
HIF-1α (exon5-exon 6)
rd CCGAGGAAGAACTATGAACATAA-3′ an Forwa 5′-TAGReverse 5′-TGAGGTTGGTTACTGTTGGTATCATATA-3′
CACTGCACAGGCCACATTCAC-3′

Cancer Research

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istered i.m. every 7 days. Tumor size was determinedlecting length and width measurements, and calculat-e tumor weight (mg) as [tumor length × (tumor2]/2, in which the tumor length is the longest dimen-mm) and the tumor width is the narrowest dimen-mm). AF (saline/0.05% Tween 80) was dosed i.p.ice per group were treated daily for 4 days with

0 mg/kg) or vehicle control. When mice were sacri-(day 4), tumors from each animal were harvestedsed to analyze mRNA and protein expression, as pre-y described (32).

HIF-1α assayue lysates were prepared as previously described (32)ed for the quantitative determination of HIF-1α usingchemiluminescence assay (Meso-Scale). HIF-1α con-tion (pg/mL) was determined using a recombinantn (R&D Systems) as standard.

tical analysisults are either representative or average of at least threendent experiments done. Statistical analysis was per-d using ANOVA test and t test (Prism, GraphPad).

lts

hibits HIF-1α transcriptional activity in MCF-7t cancer cellstest whether AF inhibited HIF-1 activity, MCF-7 cellsransiently transfected either with pGL2-TK-HRE, con-g the luciferase reporter gene under control of threeof a hypoxia response element (HRE) or with a control(pGL3 control). Hypoxia increased HRE-dependentase expression by 49-fold, relative to cells cultured un-rmoxic conditions (Fig. 1A, left). AF inhibited lucifer-pression in a dose-dependent manner, but did notconstitutive luciferase expression (Fig. 1A, right), sug-g that inhibition of luciferase by AF was HIF-1 depen-AF also inhibited endogenous HIF-1 transcriptionaly, as indicated by inhibition of hypoxic induction of(Fig. 1B, left), CA9, and LOX mRNA expression (Fig.ht), similar to the effects of siRNA targeting HIF-1α.trast, AF caused up to 40-fold increase of p21 mRNAsion, under both normoxic and hypoxic conditionsC, left), indicating that AF differentially affects genesion in MCF-7 cells.ably, neither NF-κB–dependent (Fig. 1D, left) nor AP-endent transcriptional activities (right) were inhibited, further showing that AF specifically inhibits HIF-1.

hibits HIF-1α and HIF-2α protein accumulationell type–dependent fashionaddress whether AF inhibited HIF-1α and HIF-2α pro-cumulation, we tested six cell lines from the NCI60 pan-were reported to be sensitive to AF. Hypoxia increasedα protein expression in all the cell lines examined,
as HIF-2α was induced to detectable levels in three cellT47D, CAKI, and UACC257). AF (0.5 μmol/L) inhibited
HIF-1AhR p

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protein accumulation, albeit to a different extent, in7, T47D, CAKI, and OVCAR3. In contrast, HIF-2α wasy decreased by AF in T47D and CAKI cells but not in257 (Fig. 2A), suggesting that AF inhibits HIF-1α andprotein accumulation in a cell type–specific fashion,

gh HIF-2α seems to be slightly less susceptible to AFtion, relative to HIF-1α.ther experiments showed that AF inhibited HIF-1αn accumulation in MCF-7 cells in a dose-dependentn, with ∼80% decrease at 0.25 μmol/L (Fig. 2B). By con-IF-1β was only inhibited by 15%, and actin levels wereanged in the presence of up to 1 μmol/L of AF. Kineticments showed that AF (0.25 μmol/L) caused little or notion of HIF-1α protein at 8 hours, but completely abro-its accumulation after 12 to 24 hours of treatmentC). AF also inhibited HIF-1α protein accumulation in-by the iron chelator desferrioxamine (100 μmol/L;), suggesting that its effects are not restricted to hyp-

ignaling. By contrast, AF induced p21 protein accumu-in MCF-7 cells cultured under either normoxia (dataown) or hypoxia (Fig. 1C, right), consistent with theion of p21 mRNA expression and showing a differentialon distinct target proteins.

ctional AhR pathway is not required for theition of HIF-1α expression by AFvious studies have indicated that AF is a ligand of18). Indeed, AF caused a 7- to 8-fold increase inependent luciferase expression (Fig. 3A) and induced200-fold higher levels of CYP1A1 mRNA expression incells (Fig. 3B), showing that AF was able to induce

ependent transcriptional activity. TCDD, used as pos-ontrol, induced a 15-fold increase in XRE-dependentase expression (Fig. 3A) and up to 1,160-fold higherof CYP1A1 mRNA expression (Fig. 3B), relative toated cells.address whether inhibition of HIF-1α by AF required aonal AhR pathway, we took advantage of AhR100 cells,–derived cells that express low levels of AhR and arent to the cytotoxic effects of AF (18). Indeed, AF causedxicity in parental MCF-7 cells but not in AhR100 cells,t concentrations as high as 2 μmol/L (Fig. 3C). Consis-ith a functional impairment of the AhR pathway, TCDDtion of XRE-dependent luciferase expression was de-d by 75% in AhR100 cells, relative to parental MCF-7and induction of CYP1A1 mRNA expression was de-d by 95% (Fig. 3A and B). More importantly, AF faileduce XRE-dependent luciferase expression in AhR100 cellsnly modestly induced CYP1A1 mRNA expression (35%levels induced in wild-type MCF-7 cells; Fig. 3A and B).ver, AF inhibited hypoxic induction of HIF-1 transcrip-activity irrespective of a functional AhR, as shown byhibition of HRE-dependent luciferase expression inand AhR100 cells (Fig. 3D). Accordingly, AF also com-inhibited hypoxic induction of HIF-1α protein accu-

ion in AhR100 cells (Fig. 3E), showing that inhibition of
α expression by AF is independent from a functionalathway.
Cancer Res; 70(17) September 1, 2010 6839



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1. AF inhibits HIF-1αiptional activity. A, MCF-7ere transfected with pGL2-E (left) or pGL3-Controland cultured underia or hypoxia in thee or presence of increasingtrations of AF. B, MCF-7ere cultured under normoxiaxia for 16 h in thee or presence of increasingtrations of AF, and VEGFlevels were measured (left).CF-7 cells were culturedormoxic or hypoxicns for 16 h either in thee or presence of AFmol/L) or after transfectionsiRNA targeting HIF-1αIF-1α downregulationg siRNA trasfection). Levelsand CA9 mRNA areed as fold increase relativereated normoxic controls.-7 cells were culturedormoxic or hypoxicns for 16 h in the absenceence of AF (0.25 μmol/L).of p21 mRNA and proteinsessed. D, MCF-7 cells,cted with NF-κB-luc (left) orc (right), were treated forder normoxic or hypoxicns in the absence or

ce of AF (0.25 μmol/L),ecrosis factor α (30 ng/mL),-tetradecanoylphorbol-13-

Cancer Research

tion for Cancer


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has bactivaA potesion oabilitythat mliferatparenMDA/activitdepenby eithWe

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hibits HIF-1α in MDA/SULT1A1, but not inMB-231 parental breast cancer cellsrestricted spectrum of AF activity in the NCI60 cells

een attributed to a requirement for its intracellulartion (18) by pathway(s) yet to be completely elucidated.ntial correlation between sensitivity to AF and expres-f SULT1A1 has also been suggested, consistent with theof SULT1A1 to induce the formation of AF metabolitesediate DNA damage (33). Indeed, AF exerted antipro-ive effects in MDA/SULT1A1, but not in MDA-MB-231tal cells (Fig. 3C). However, both MDA-MB-231 andSULT1A1 cells express little or no AhR transcriptionaly, as indicated by minimal if any induction of XRE-dent luciferase (Fig. 3A) or CYP1A1 mRNA expressioner AF or TCDD (Fig. 3B).then tested whether AF inhibited HIF-1 transcriptionaly in MDA-MB-231 and MDA-SULT1A1 cells. AF com-inhibited HIF-1–dependent luciferase expression in

SULT1A1, but did not affect its expression in MDA-
1 parental cells, showing that exogenous expression ofA1was sufficient tomediate AF-de
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ere measured.

acrjournals.org


activity (Fig. 3D). Accordingly, AF almost completely in-d hypoxic induction of HIF-1α protein inMDA/SULT1A1ut not in MDA-MB-231 (Supplementary Fig. S1; Fig. 3E).t, we evaluated expression of HIF-1α in MCF-7 cellsected with either negative control siRNA or siRNA tar-SULT1A1 or AhR (Supplementary Fig. S2A-B). AF in-

d hypoxic induction of HIF-1α protein by 75% in cellsected with negative control siRNA and by 50% in cellsected with AhR siRNA, relative to hypoxia-treatedSupplementary Fig. S2B). In contrast, downregulationT1A1 almost completely prevented the inhibition ofby AF (15% inhibition, compared with hypoxia; Sup-

ntary Fig. S2B), showing that SULT1A1 expression wasated in HIF-1α inhibition by AF and further supportingnclusion that inhibition of HIF-1α by AF is indepen-rom a functional AhR pathway.

ition of HIF-1α by AF is independent fromdamage
uction of DNA damage by AF, measured by phosphoryla-
pendent inhibition of tion of H2AX, paralleled results obtained in cytotoxicity assay.

2. AF inhibits HIF-1αaccumulation in humancell lines. A, cancer cellre incubated for 16 h in thee or absence of AFol/L) under normoxic orconditions. Protein levels

-1α, HIF-2α, and actin wereed. B, MCF-7 cells wereed for 16 h under normoxicxic conditions in thee or presence of increasingtrations of AF, as indicated.levels of HIF-1α, HIF-1β,tin were assessed byn blot. C and D, MCF-7 cellsltured under normoxicns in the absence ore of AF (0.25 μmol/L,) or DFX (100 μmol/L, D) forrotein levels of HIF-1α and




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Terzuoli et al.

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, AF induced significantly higher levels of γH2AX in sen-CF-7 and MDA/SULT1A1 cells than in resistant AhR100

DA-MB-231 cells (Fig. 3E). However, AF was equally ableibit hypoxic induction of HIF-1α protein in AhR100 and

MDA-MB-231, and MDA/SULT1A1 were cultured under normoxia or undeevels of HIF-1α, actin, and γH2AX were measured.

SULT1A1 cells, showing a complete dissociation betweenion of DNA damage and HIF-1α inhibition (Fig. 3E).

(35), Acells t



have previously shown that agents that inhibit topoi-ase I or II may affect HIF-1α protein translation). Consistent with the finding that induction of DNAge by AF does not seem to involve topoisomerases

xic conditions in the absence or presence of AF (0.25 μmol/L) for

3. AF inhibits HIF-1α in an AhR-independent fashion. A, MCF-7, AhR100, MDA-MB-231, and MDA/SULT1A1 were transfected with XRE-luc andated with TCDD (1 μmol/L) or AF (0.25 μmol/L) for 16 h. ***, P < 0.001, relative to control. B, MCF-7, AhR100, MDA-MB-231, and MDA/SULT1A1 wereunder normoxia for 16 h in the absence or presence of AF (0.25 μmol/L) or TCDD (1 μmol/L). CYP1A1 mRNA expression was analyzed.0.001, relative to control. C, MCF-7, AhR100, MDA-MB-231, and MDA/SULT1A1 were treated with increasing concentrations of AF as indicated. Cell viability was assessed. D, MCF-7, AhR100, MDA-MB-231, and MDA/SULT1A1 were transfected with pGL2-TK-HRE and then treated forder normoxic or hypoxic conditions in the absence or presence of AF (0.25 μmol/L). ***, P < 0.001; **, P < 0.005, relative to hypoxia. E, MCF-7,

F was able to inhibit HIF-1α protein expression inransfected with siRNA targeting topo I or topo IIα

Cancer Research



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lementary Fig. S3B). Furthermore, AF inhibited theic induction of HIF-1α protein accumulation in thece of aphidicolin, which blocks DNA polymeraserevents DNA damage (Supplementary Fig. S4), furtherng that AF inhibited HIF-1α by a DNA damage–ndent pathway.

es not affect HIF-1α degradation, but decreaseste of HIF-1α translation-1α steady-state is the result of a balance between pro-anslation and its degradation. To investigate whetherected HIF-1α degradation, we performed experimentspresence of inhibitors of either proteasome activity32) or prolyl hydroxylase enzymes (DMOG, a global in-r of 2-oxoglutarate–dependent dioxygenase enzymes),cause an accumulation of HIF-1α under normoxicions. As shown in Fig. 4A, AF inhibited HIF-1α proteinulation regardless of inhibition of the proteasome or
hydroxylases, suggesting that AF did not affect degra- relativ
and HIF-1β levels (left) were assessed as described in Materials and Methods. Totaasured as an indication of total protein synthesis.

acrjournals.org


hibited normoxic accumulation of HIF-2α in a sensi-on Hippel-Lindau (VHL)–deficient renal cancer cell line, which does not express HIF-1α), indicating that aonal VHL pathway, essential for normoxic degradationHIF-α subunit, is not required for the inhibition ofby AF (Fig. 4B). Finally, experiments conducted inesence of cycloheximide failed to show significant dif-es in HIF-1α protein half-life between cells cultured insence or presence of AF (half-life ∼60 min; Fig. 4C).together, these results show that AF does not affectprotein degradation.

then assessed whether AF inhibited HIF-1α translationluating the rate of HIF-1α protein synthesis in nor-MCF-7 cells in the absence or presence of AF. Cells

pulse labeled with [35S] methionine for 60 minutes atpoint HIF-1α was immunoprecipitated and analyzedGE and autoradiography. As shown in Fig. 4D (left),nificantly decreased 35S-labeled HIF-1α accumulation
e to nontreated cells, suggesting that it may affect its
of HIF-1α protein. Consistent with these results, AF rate of translation. Importantly, under the same experimental

4. AF does not affect HIF-1α protein half-life but decreases its translation. A, MCF-7 cells were cultured, in the absence or presence of AF (0.25 μmol/L),nder hypoxia or in the presence of MG132 (20 μmol/L) or DMOG (100 μmol/L) for 16 h. HIF-1α and actin levels were analyzed. B, A498 cells werefor 16 h with AF (0.25 μmol/L) under normoxic or hypoxic conditions, and HIF-2α and actin levels were assessed. C, MCF-7 cells were culturedypoxia in the absence or presence of AF (0.25 μmol/L) for 12 h (time 0), when CHX (40 μg/mL) was added for the indicated times. HIF-1α levels wereed by densitometry (bottom). Values were normalized to the expression of actin and expressed as percentage relative to time 0 (arbitrarily considered100%). D, MCF-7 cells were treated under normoxic conditions in the absence or presence of AF (0.25 μmol/L) for 12 h. Newly synthesized

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Terzuoli et al.

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ions AF did not significantly affect the synthesis ofor that of total proteins (Fig. 4D, right).

transcription is required for the inhibition ofα mRNA expression by AF

to medium. C and D, MCF-7 cells were cultured under normoxia or hypoycin D (5 μg/mL). HIF-1α mRNA (C) and HIF-1α pre-mRNA (D) expression

ulation of HIF-1α translation under hypoxic conditionspoorly understood, and recent evidence suggests that

measufound



of HIF-1α mRNA may be a crucial factor affecting theHIF-1α translation (36). AF, under hypoxic but not nor-conditions, caused a 50% decrease of HIF-1αmRNA ex-on, relative to normoxic cells (P = 0.005; Fig. 5A). Next, toss whether AF inhibited HIF-1αmRNA transcription, we

16 h in the absence or presence of AF (0.25 μmol/L) and/orssessed. ***, P < 0.001; **, P < 0.005, relative to medium.

5. Active transcription is required for the inhibition of HIF-1αmRNA expression by AF. A and B, MCF-7 cells were cultured under normoxia or hypoxiain the absence or presence of AF (0.25 μmol/L). HIF-1α mRNA (A) and HIF-1α pre-mRNA (B) expression was analyzed. **, P < 0.005; *, P < 0.01,

red the levels of HIF-1α pre-mRNA. Unexpectedly, wethat hypoxia induced up to 5.8-fold higher levels of

Cancer Research



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pre-mRNA, relative to normoxic cells (Fig. 5B). Nota-spite the increase of HIF-1α pre-mRNA under hypoxicions, which was confirmed using a different set of pri-data not shown), hypoxia only marginally affected theof HIF-1α mRNA, suggesting possible abnormalities inmRNA maturation and/or processing. AF did not af-

IF-1α pre-mRNA expression under normoxic conditions,duced the hypoxic induction to levels 2-3-fold higherhose present in normoxic cells.further address the mechanism by which AF affectedmRNA levels, we performed experiments in the pres-f actinomycin-D, an inhibitor of transcription. Wethat hypoxic induction of HIF-1α pre-mRNA, both insence and presence of AF, was completely abrogatedition of actinomycin-D (Fig. 5D), showing that it wasdent on active transcription. Surprisingly, we alsothat inhibition of mature HIF-1α mRNA expression, observed in hypoxic cells, was completely reversedition of actinomycin-D, showing that induction rathernhibition of transcription is required for HIF-1α mRNAegulation in the presence of AF (Fig. 5C). Consistenthese results, addition of actinomycin-D also partiallyed the inhibitory effect of AF on HIF-1α protein accu-ion (Supplementary Fig. S5), suggesting that the effects-1α mRNA expression were causally related to the in-n of HIF-1α protein.en together, these results show that active transcrip-n hypoxic cells treated with AF, is required for the in-n of HIF-1α mRNA expression.

hibits HIF-1α expression in MCF-7 xenograftstest whether AF was able to inhibit HIF-1α expressionor xenografts, MCF-7 cells were implanted s.c. in fe-athymic nude mice. When tumors reached ∼200 mg,n = 5/group) were randomized to receive either vehiclel or AF (60 mg/kg, i.p.) daily for 4 days. As shown inA, AF exerted a cytostatic effect on tumor growth01), relative to vehicle-treated mice. Notably, AF inhib-IF-1α protein accumulation in tumor lysates by 90%.005), relative to vehicle-treated mice (Fig. 6B) andexpression of the HIF-1 target genes VEGF, CA9, andby ∼ 70% (P < 0.05; Fig. 6C), showing that AF inhibitsexpression and activity in MCF-7 xenografts.

ause of the potentially confounding effects of tumor sizeF-1α expression, we also conducted experiments inMCF-7 tumor–bearing animals were only treated forwith either vehicle control or AF (120 mg/kg, i.p.). Asin Supplementary Fig. S6, AF did not affect tumor

h under these conditions, yet it significantly inhibitedα protein accumulation and VEGF mRNA expressionlementary Fig. S6B and C, respectively), suggesting thation of HIF-1α and HIF-1 target genes by AF is indepen-rom its cytostatic/cytotoxic activity.

ssion

-1 has emerged over the last several years as an attrac-rget for the development of novel cancer therapeutics.

levelsthe de

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d others have identified small-molecule inhibitors ofthat act by distinct mechanisms of action, including,t limited to, inhibition of HIF-1α translation (31, 34,), inhibition of HIF-1 DNA binding (39) or transcrip-activity (40–42), inhibition of protein-protein interac-3), increased protein degradation (44, 45), or inhibitionochondrial respiration (46). Several HIF-1 inhibitorsfied at the NCI share the property of inducing DNAge, including topotecan (30) and NSC 644221 (34),gh DNA damage and HIF-1 inhibition seem to be con-ant but independent events.induces DNA damage and acts as a ligand of AhR. AFantiproliferative activity in a fairly limited number ofn cancer cell lines, and activation of AhR seems to beed for its conversion to DNA-damaging species, atin part, by transcriptional activation of CYP1A1 andA1, two XRE target genes (18). In this article, we pro-vidence that AF inhibits HIF-1α expression in MCF-7n an AhR-independent fashion, as indicated by the fol-results: (a) AF inhibited HIF-1α protein accumula-

in MCF-7 and AhR100 cells, in which the AhRay is functionally impaired; (b) AF did not inhibitα in the resistant MDA-MB-231 breast cancer cells,did in MDA/SULT1A1, MDA-MB-231 cells transfectedULT1A1; and (c) siRNA targeting SULT1A1 signifi-blocked the ability of AF to inhibit HIF-1α incells (Supplementary Fig. S2). Along with the ability

to exert antiproliferative activity in MCF-7 and MDA-A1, but not in AhR100 or MDA-MB-231, these resultst that (a) inhibition of HIF-1α by AF is independenta functional AhR pathway, and (b) there is no corre-between cytotoxicity and HIF-1α inhibition. Indeed,s completely ineffective in inducing antiproliferativein AhR100 cells, yet it was able to inhibit HIF-1α ex-

on. In addition, AF induced significantly lower levels2AX, a marker of DNA damage, in AhR100 and MDA-1 cells, which did not correlate with its ability tot HIF-1α. These results not only show a dissociationen cytotoxicity and HIF-1 inhibition, but they alsohe possibility that cancer cells found to be “resistant”cytotoxic effects of AF may be sensitive to HIF-1α

tion.ignificant number of small-molecule inhibitors of HIF-ntified thus far seems to affect HIF-1 translation (31,38), yet regulation of HIF-1α translation under hypoxicions is still poorly understood. We were then intriguedfact that AF also seemed to inhibit HIF-1α synthesis.r experiments showed that AF inhibited HIF-1αmRNAssion by ∼50%, under hypoxic, but not normoxicions. The magnitude of HIF-1α mRNA inhibition wasly to account for the almost complete inhibition ofprotein accumulation, raising the possibility that ad-

al mechanisms were implicated. Using primers thatically detect HIF-1α pre-mRNA, we discovered thatia (in the absence or presence or AF) induced higherof transcript, relative to normoxic cells. The increased
of HIF-1α pre-mRNA did not seem to correlate withcreased expression of HIF-1α mRNA in the presence



of AF.tinomfor thThe diand depotentsing apresenand a(a) aof HIFwhichpressiwith (proteilationinhibit

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Notably, experiments conducted in the presence of ac-ycin D showed that active transcription was requirede downregulation of HIF-1α mRNA expression by AF.screpancy between accumulation of HIF-1α pre-mRNAcrease of HIF-1α mRNA levels might have suggested aial effect of AF plus hypoxia on HIF-1α mRNA proces-nd/or maturation. However, results obtained in thece of actinomycin-D argue against this conclusionre consistent with transcriptional induction of eitherrepressor, which in turn is responsible for inhibition-1α mRNA expression, or (b) noncoding RNA species,may account for both inhibition of HIF-1α mRNA ex-on and translation. The latter possibility is consistenta) the mechanism of action of AF, which implicatesn nucleic acid complexes (33, 35), (b) the lack of corre-

enes (VEGF, CA9, and PDK-1) in tumor lysates from mice treated with vehto levels detected in mRNA harvested from normoxic MCF-7.

between magnitude of HIF-1α mRNA inhibition andion of HIF-1α translation, and (c) the ability of topo-

pathwscripti



, a DNA-damaging agent that also inhibits HIF-1αation, to increase the levels of antisense transcripts ofIF-1α genomic sequence (47). Several miRs have beenly identified that indeed target the 3′-untranslated re-f HIF-1α mRNA, leading to the inhibition of HIF-1αn levels (48–50). It is then plausible that the effects ofamaging agents on HIF-1 protein translation may atin part implicate noncoding RNA species that targetmRNA expression and/or HIF-1α mRNA translation.

r experiments are required to characterize the spec-of miR induced by agents that concomitantly inhibitand induce DNA damage to formally show the exis-

of a mechanistic link.proposed mechanism of HIF-1α inhibition by AF isivably associated with effects on other genes and

AF was assessed (P < 0.05). Values are expressed as fold change

6. AF inhibits HIF-1α expression and transcriptional activity in MCF-7 xenografts. A, MCF-7 cells were implanted into nude mice (n = 5/group)wed to grow up to ∼200 mg, when treatment with AF (60 mg/kg daily × 4 d i.p.) was started. Tumor weight was measured as described in Materialsthods, (Mann-Whitney test; *, P < 0.01). B, quantitative determination of HIF-1α protein levels (**, P < 0.05). C, mRNA expression of HIF-1

icle or

ays. However, AF did not inhibit NF-κB or AP-1 tran-onal activities and potently induced p21 mRNA and

Cancer Research



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n expression, ruling out a global effect on mRNA expres-nd/or protein translation. Moreover, gene array experi-(using the Affymetrix human 133 2.0 plus chip)ted that 1.78% of genes were induced >2-fold, andof genes were inhibited >2-fold in MCF-7 cells treatedF (0.25 μmol/L) for 16 hours, showing that AF does nota global inhibition of gene expression (data not shown).ence of inhibition of HIF-1α expression in xenograft(Fig. 6) further corroborates potential translational im-ons of the findings described in this article. Inhibition-1α in AhR100 cells also raises the possibility that AFmodulate HIF-1–dependent pathways even in cellsre not sensitive to its cytotoxic effects, a feature thatbe emphasized by more protracted schedules of ad-ration, as opposed to the ones generally used for cyto-agents. Finally, modulation of both mRNA expressionRNA translation induced by AF may potentially con-
to its anticancer activity and unveils novel properties
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ns of differential activity of drugs against human tumor cell lines:velopment of mean graph and COMPARE algorithm. J Natl Cancert 1989;81:1088–92.

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osure of Potential Conflicts of Interest

otential conflicts of interest were disclosed.

owledgments

hank members of the Melillo's laboratory, Yves Pommier, and Robertmaker for the helpful discussion.

Support

ral funds from the NCI, NIH, under contract no. N01-CO-12400. Theof this publication does not necessarily reflect the views or policiesepartment of Health and Human Services, nor does mention of tradecommercial products, or organizations imply endorsement by the U.S.ent. This research was supported by the Developmental Therapeutics, DCTD, of the NCI, NIH.costs of publication of this article were defrayed in part by the paymentcharges. This article must therefore be hereby marked advertisement innce with 18 U.S.C. Section 1734 solely to indicate this fact.

ived 03/26/2010; revised 06/16/2010; accepted 06/30/2010; publishedirst 08/24/2010.
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Cancer Research



2010;70:6837-6848. Published OnlineFirst August 24, 2010.Cancer Res Erika Terzuoli, Maura Puppo, Annamaria Rapisarda, et al.

Expression in an AhR-Independent FashionαInhibits HIF-1Aminoflavone, a Ligand of the Aryl Hydrocarbon Receptor,

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Aminoflavone, a Ligand of the Aryl Hydrocarbon - Cancer Research

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