Identification of Novel Small Molecule Inhibitors of ... · Inducible Factor-1 That Differentially Block Hypoxia-Inducible Factor-1 Activity and Hypoxia-Inducible Factor-1A Induction

Identification of Novel Small Molecule Inhibitors of Hypoxia-

Inducible Factor-1 That Differentially Block Hypoxia-Inducible

Factor-1 Activity and Hypoxia-Inducible Factor-1A Induction

in Response to Hypoxic Stress and Growth Factors

Noan-Minh Chau,1Paul Rogers,

2Wynne Aherne,

2Veronica Carroll,

1Ian Collins,

3

Edward McDonald,3Paul Workman,

4and Margaret Ashcroft

1

1Cell Growth Regulation and Angiogenesis Team, 2Analytical Technology and Screening Team, 3Medicinal Chemistry Department, and4Signal Transduction and Molecular Pharmacology Team, Cancer Research UK Centre for Cancer Therapeutics,Institute of Cancer Research, Sutton, Surrey, United Kingdom

Abstract

Hypoxia-inducible factor-1 (HIF-1) is a transcriptional com-plex that is activated in response to hypoxia and growthfactors. HIF-1 plays a central role in tumor progression, inva-sion, and metastasis. Overexpression of the HIF-1A subunithas been observed in many human cancers and is associatedwith a poor prognostic outcome with conventional treatments.Targeting HIF-1 using novel small molecule inhibitors is,therefore, an attractive strategy for therapeutic development.We have generated U2OS human osteosarcoma cells stablyexpressing a luciferase reporter construct under the control ofa hypoxia response element (U2OS-HRE-luc). The U2OS-HRE-luc cells were robustly and reproducibly sensitive to hypoxicstress in a HIF-1–dependent manner. We developed anautomated U2OS-HRE-luc cell-based assay that was used in ahigh-throughput screen to identify compounds that inhibitedHIF-1 activity induced by treatment with the hypoxia mimetic,deferoxamine mesylate. We performed a pilot screen of theNational Cancer Institute Diversity Set of 2,000 compounds. Weidentified eight hit compounds, six of these were also identifiedby Rapisarda et al. in an independent hypoxia screen. However,there were two novel hit compounds, NSC-134754 and NSC-643735, that did not significantly inhibit constitutive luciferaseactivity in U2OS cells (U2OS-luc). We showed that both NSC-134754 and NSC-643735 significantly inhibited HIF-1 activityand HIF-1A protein induced by deferoxamine mesylate.Interestingly, NSC-134754 but not NCS-643735 inhibited HIF-1 activity and HIF-1A protein induced by hypoxia andsignificantly inhibited Glut-1 expression. Finally, we showedthat both NCS-134754 and NCS-643735 inhibited HIF-1Aprotein induced by insulin-like growth factor-1. Our cell-basedassay approach has successfully identified novel compoundsthat differentially target hypoxia and/or growth factor–mediated induction of HIF-1A. (Cancer Res 2005; 65(11): 4918-28)

Introduction

Solid tumors characteristically contain areas of hypoxia (lowoxygen tension), which is a powerful stimulus for the expression of

genes involved in proliferation, glycolysis, and angiogenesis.Adaptation of tumor cells to a hypoxic environment results in anaggressive and metastatic cancer phenotype that is associatedwith resistance to radiation therapy, chemotherapy, and a poortreatment outcome (1–5).The transcription factor hypoxia-inducible factor-1 (HIF-1) is

central to the regulation of a growing number of hypoxia-activatedgenes and consists of HIF-1a and HIF-1h subunits. The mRNAs ofHIF-1a and HIF-1h, also known as the aryl hydrocarbon receptornuclear translocator (ARNT), are constitutively expressed in cells.However, HIF-1a protein expression is tightly regulated by changesin cellular oxygen and growth factors. Both subunits contain abasic-helix-loop-helix and PER-ARNT-SIM (PAS) domain importantfor heterodimerzation and DNA binding. Two other a-subunitshave been identified in addition to HIF-1a and are called HIF-2aand HIF-3a. Interestingly, HIF-3a has been shown to exist as anumber of splice variants (6) and is thought to negatively regulatethe HIF pathway (7, 8).The HIF-1a subunit is primarily regulated at the level of protein

stability. In normoxia, HIF-1a is rapidly degraded via targetedubiquitination and subsequent degradation by the proteasome.This negative regulation is mediated by direct binding of the vonHippel-Lindau (VHL) tumor suppressor protein, a component of anE3 ubiquitin ligase, and is dependent on prolyl hydroxylation ofHIF-1a at residues 402 and 564 (9, 10). In response to physiologicalhypoxia, HIF-1a becomes rapidly stabilized and is localized to thenucleus, where it binds to HIF-1h to form the HIF-1 complex. HIF-1specifically binds to a short DNA sequence, 5V-ACGTG-3V, known asthe hypoxia responsive element (HRE) within target genes. Uponbinding, HIF-1 recruits the coactivator cAMP-responsive elementbinding protein (CREB)-binding protein/p300 and various otherproteins to activate transcription (11, 12).HIF-1 plays a central role in tumor progression and angiogen-

esis in vivo . Oncogenic activation (e.g., Ha-ras , myc , or src) or lossof tumor suppressor function (e.g., p53, PTEN, or VHL) isassociated with HIF-1–mediated tumor progression (13, 14). Inaddition, exposure to a variety of growth factors [insulin, insulin-like growth factor-1 (IGF-1), IGF-2, and angiotensin II] has alsobeen shown to increase HIF-1 activity in normoxic and hypoxicconditions. HIF-1a is overexpressed in many human cancers (15).The resistance of hypoxic cells to killing and the aggressiveness ofhighly hypoxic tumors are, in part, due to the overexpression ofHIF-1a. The recent finding showing that radiation itself activatesHIF-1 adds to the therapeutic challenge of a vascular hypoxictumor (2, 16). Elegant studies have shown that targeting theinteraction between HIF-1a and the p300/CREB-binding protein

Note: P. Workman is a Cancer Research UK Life Fellow.Requests for reprints: Margaret Ashcroft, Cell Growth Regulation and

Angiogenesis Team, Cancer Research UK Centre for Cancer Therapeutics, Instituteof Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, United Kingdom.Phone: 44-208-722-4035; Fax: 44-208-722-4205; E-mail: [email protected].

I2005 American Association for Cancer Research.

Cancer Res 2005; 65: (11). June 1, 2005 4918 www.aacrjournals.org

Research Article

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significantly reduces tumor growth in vivo and support HIF-1 as apotential therapeutic target (17, 18). Consequently, an increase inthe development of strategies to target HIF-1 activity has inten-sified in recent years.A variety of small molecule inhibitors of HIF-1 activity have been

shown to have antitumor and antiangiogenic activity in vivo . Theseinclude the microtubule depolymerizing agent 2-methoxyestradiol(19); inhibitors of the redox protein, thioredoxin-1 (20, 21); YC-1, anagent developed for circulatory disorders (22); PX-478, a smallmolecule inhibitor that targets both constitutive and hypoxia-induced HIF-1a levels (23); topotecan, a Topo-I inhibitor identifiedin a cell-based high-throughput hypoxia screen (24, 25); the Hsp90inhibitors, geldanamycin and its analogue 17-allylamino,17-demethoxygeldanamycin (26); and the small molecule inhibitor,chemotin, which blocks the interaction between HIF-1a andp300/CREB (18).In this study, we have developed a robust cell-based assay that we

have used in a high-throughput screen to identify small moleculeinhibitors of the HIF pathway. We performed a pilot screen of theNational Cancer Institute (NCI) Diversity Set of 2,000 compounds,enabling us to compare our hits with a previous hypoxia screen (24).In addition to identifying six hit compounds, which were previouslydescribed (24), we discovered two novel hit compounds, NSC-134754 and NSC-643735, which we evaluated further. We show herethat NSC-134754 and NSC-643735 differentially block HIF-1 activityand HIF-1a expression in response to hypoxic stress. Importantly,we show that NCS-134754 blocks both the hypoxia and growthfactor–mediated pathways regulating HIF-1a expression.

Materials and Methods

Plasmid constructs and antibodies. A full-length human HIF-1a

expression construct (pCMVh-HA-HIFa) was provided by Dr. Andrew Kung

(Dana-Farber Cancer Institute, Boston, MA). The pGL-HRE luciferasereporter construct contains a triple repeat of the iNOS HRE binding sequence

and was kindly provided by Dr. Giovanni Melillo (NCI, Bethesda, MD). The

pGL3-basic and control vectors were obtained from Promega (Southampton,

United Kingdom). The HIF-1a–specific monoclonal antibody was obtainedfrom BD Biosciences (Oxford, United Kingdom). The h-actin–specificmonoclonal antibody was obtained from Abcam (Cambridge, United

Kingdom). The Glut-1 antibody polyclonal antibody was obtained fromAlpha Diagnostic International (Wiltshire, United Kingdom).

Cell culture. Stable transfection was used to generate the U2OS-HRE-luc,

U2OS-basic, and U2OS-luc control cells using the constructs pGL-HRE, pGL3-

basic, and pGL3-control, respectively. The pGL3-HRE construct has threetandem copies of the iNOS HRE fused to the luciferase reporter gene. The

pGL3-control vector also contains the luciferase reporter gene and expression

is driven by SV40 promoter and enhancer sequences resulting in constitutive

luciferase activity. The pGL3-basic vector contains the luciferase reportergene only. pSV2-(hygromycin) was cotransfected and selection was carried

out using hygromycin (Boehringer Mannheim, Berkshire, United Kingdom).

Stable clones were isolated and expanded for further evaluation. All cell lines

were maintained in DMEM (Life Technologies, Paisley, United Kingdom)containing 10% FCS and cultured at 37jC in 5% CO2. Transient transfections

were carried out using the calcium phosphate precipitation method. Cells

were harvested either in passive lysis buffer (Promega, Southampton, UnitedKingdom) or in 2� sample buffer [125 mmol/L Tris (pH 6.8), 4% SDS, 0.01%

bromophenol-blue, 10% h-mercaptoethanol, 10% glycerol] to assess lucifer-

ase activity or protein expression respectively. For assessment of protein

expression samples were separated by SDS-PAGE and Western blot analysiswas done as previously described (27).

HIF-1A induction. Cells were exposed to hypoxic conditions (1% O2,

5% CO2, and 94% N2) in a LEEC dual gas incubator at 37jC. HIF-1 was

induced in normoxia using the hypoxia mimetic agent deferoxamine

mesylate (DFX, Sigma, Poole, United Kingdom) at a concentration of 500Amol/L. A stock of DFX (500 mmol/L) was freshly prepared in sterile H2O

immediately before each experiment. For experiments using the phosphoi-

nositide 3 kinase inhibitor, cells were treated with 10 or 80 Amol/L

LY294002 (Calbiochem, Nottingham, United Kingdom) for f30 minutesbefore exposure to hypoxia (1% O2) or treatment with DFX (500 Amol/L)

for up to 16 hours. For experiments using NSC-134754, NSC-643735, and

NSC-607097, cells were treated with 20 Amol/L of each compound (unless

otherwise indicated) for f30 minutes before exposure to hypoxia (1% O2)or treatment with DFX (500 Amol/L) for up to 16 hours. For treatment with

IGF-1, HCT116 cells were starved for 36 to 48 hours then stimulated

with 50 ng/mL recombinant IGF-1 (Sigma) for 6 to 8 hours as described

previously (27). Compounds were added to the cells 30 minutes beforeIGF-1 stimulation.

High-throughput screening assay. U2OS-HRE-luc cells were grown in

DMEM complete medium with 10% fetal bovine serum and their use in a

high-throughput screen initially evaluated using 96-well plates. The assay

was subsequently satisfactorily transferred to a 384-well plate format for

compound screening. U2OS-HRE-luc cells were plated into 384-well plates at

4,000 cells/well. The following day, compounds were added 30minutes before

DFX treatment (500 Amol/L). The final concentration of compounds was

18.18 Amol/L. Total activity controls included DMSO ( final concentration

0.2%) plus DFX (n = 32), and the positive control was LY294002 ( final

concentration 80 Amol/L, n = 8). Other controls were DMSO only (n = 16) and

DFX only (n = 8). The plates were incubated overnight at 37jC in a humidified

incubator. Luciferase reporter activity wasmeasured using the ReporterLight

Plus assay reagents (Cambrex, Berkshire, United Kingdom) according to the

manufacturer’s instructions and after 10 minutes the plates were read on a

Topcount plate reader (Perkin-Elmer Life Sciences, Buckinghamshire, United

Kingdom). The mean ZVfactor, calculated using the DMSO plus DFX versus

the DMSO only controls, was 0.52 F 0.09. It is worth noting that a thorough

comparison of the SteadyGlo (Promega) and Lumitech ReportaLight Plus

luciferase reagents was made. Both reagents were comparable in terms of

their sensitivity and duration of effectiveness. However, the latter was used in

the high-throughput screen because it was more economical.Hits were identified as those compounds that inhibited the readout (DFX

plus DMSO) by greater than three standard deviations of the mean of the

compounds on each plate. These compounds were selected and their

activity confirmed (n = 4) in the same assay. To exclude the possibility thatthe inhibition was due to loss of cells, compounds were assayed at the same

concentration in an ATP viability assay (Cambrex, Berkshire, United

Kingdom) according to the manufacturer’s instructions. In addition, the

effect of the hits on luciferase activity was determined using the Vialightreagents in a cell-free assay using ATP (1 Amol/L) as a substrate. All

compounds were prepared and stored in DMSO (2%). To control for any

effect that might have been due to the DMSO, cells were either untreated orDMSO was added to untreated cells at the same final concentration and

volume as used for compound treated cells.

Luciferase reporter assays. HIF-1 activity was determined in the U2OS-

HRE-luc cells, U2OS-basic-luc cells, or U2OS-luc control cells in six-wellformat. Cells were washed twice with ice-cold PBS and lysed with 200 AL of

Passive Lysis buffer (Promega) and incubated at room temperature for 10

minutes. Lysates were centrifuged and supernatants were stored at �80jCuntil assayed. Luciferase assays were done by pipetting 20 AL of cell lysateinto each well of a 96-well plate and analyzed immediately after addition

of luciferase reagent (Promega) in a luminometer (Dynex Technologies,

Worthing, United Kingdom).Sulforhodamine-B assay. Analysis of cell growth inhibition was done by

sulforhodamine-B assay. Cells were plated into clear flat-bottomed 96-well

plates and left to attach overnight. They were treated with 5 AL/well ofserially diluted compounds in quadruplicate and incubated for 72 hours.The cells were cultured in a final volume of 200 AL. At the end of the

incubation period, the medium was removed and the cells were fixed

immediately with ice-cold 10% trichloroacetic acid (VWR International,

Poole, United Kingdom) for 30 minutes. The cells were then washed fivetimes with H2O and allowed to dry before staining with 0.4% (w/v)

sulforhodamine B (Sigma) dissolved in 1% acetic acid for 10 minutes.

Novel Small Molecule Inhibitors of HIF-1

www.aacrjournals.org 4919 Cancer Res 2005; 65: (11). June 1, 2005



Unbound dye was removed by five washes with 1% acetic acid, and protein-bound dye was extracted with 10 mmol/L unbuffered Tris base for 5

minutes for determination of absorbance in a microplate reader. The

absorbance value was read at a wavelength of 540 to 570 nm.

Results

Effect of hypoxic stress on luciferase activity and HIF-1Ainduction in U2OS-HRE-luc cells. We have generated a humanosteosarcoma cell line (U2OS) stably expressing a triple tandemrepeat of a HRE fused to a luciferase reporter gene (U2OS-HRE-luc).To assess whether the U2OS-HRE-luc cells could be developed into ahigh-throughout cell-based assay to identify HIF-1 inhibitors, weinitially characterized the responsiveness of the U2OS-HRE-luc cellsto hypoxic stress. To do this, U2OS-HRE-luc cells were exposed toeither hypoxia (1% O2) or treated with the hypoxia mimetic agent,

DFX at 500 Amol/L over a time course of 24 hours. In response tohypoxia, the highest increase in luciferase activity was achieved at16 hours of incubation (Fig. 1A). Interestingly, there was a decreasein HIF-1a protein levels at 24 hours of hypoxic treatment. We haveobserved this decrease in many other cell lines including MRC-5,MCF-7, U87MG, RPE-1, and HCT116 (data not shown) and theresponse is consistent with observations described elsewhere (28).This was in contrast to the response of DFX-treated U2OS-HRE-luccells in which HIF-1a protein levels continued to increase withincreasing luciferase activity being exhibited over a 24-hour period(Fig. 1B). The results suggest that increases in luciferase activity inthe U2OS-HRE-luc cells were due to HIF-1 activation as aconsequence of HIF-1a induction. To support this, the cells weretransiently transfected with a HIF-1a expression construct orcontrol vector. As seen in Fig. 1C , the presence of exogenous HIF-1a

Figure 1. Effect of hypoxic stress on luciferase activity and HIF-1a induction in U2OS-HRE-luc, U2OS-basic, and U2OS-luc control cells. U2OS-HRE-luc cells wereexposed to normoxia, hypoxia (1% O2; A) or (B ) DFX (500 Amol/L) for the times indicated. Samples were assayed for luciferase activity. Graph shows luciferasereporter activity as a fold induction relative to normoxia (norm ). Experiments were done in duplicate. C, U2OS-HRE-luc cells were transiently transfected with 10 AgHIF-1a or the vector control (�), expression constructs. Twenty-four hours after transfection, cells were harvested for luciferase assay and Western blot analysis.Graph shows luciferase reporter activity as a fold induction relative to normoxia. Experiments were done in duplicate. Western blot, analysis (bottom ) shows HIF-1aprotein expression. Actin was used as a load control. D, U2OS-HRE-luc cells or U2OS-basic-luc cells were exposed to normoxia (norm), hypoxia (Hyp, 1% O2) orDFX (500 Amol/L) for 8 hours. Graph shows relative light units (RLU ). Experiments were done in duplicate. E, U2OS-luc control cells were exposed to normoxia (norm),hypoxia (Hyp, 1% O2) or DFX (500 Amol/L) for 8 hours. Graph shows relative light units. Experiments were done in duplicate.

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led to an increase in luciferase activity compared with vectorcontrol, confirming that luciferase activity is HIF-1–dependent,consistent with our previous observations (26). In addition togenerating the U2OS-HRE-luc cell line, U2OS cells stably expressinga basic luciferase construct, which did not contain any HREsequences, were also generated (U2OS-basic). As expected, nosignificant increase in luciferase activity was observed in responseto hypoxia or DFX treatment in the U2OS cells stably expressing thebasic luciferase vector (Fig. 1E). To address whether hypoxia orDFX treatment itself affected luciferase activity, we also generateda U2OS cell line stably expressing a control luciferase reporter,which contains an SV40 site and enhancer sequences (U2OS-luc).Figure 1E shows that these cells have constitutively high basalluciferase activity in normoxia as expected. Interestingly, exposureof the U2OS-luc cells to hypoxia (1% O2) resulted in a significantdecrease in the luciferase activity, suggesting that hypoxia affectedluciferase reporter gene expression. DFX treatment of these cellshad no significant effect on luciferase activity (Fig. 1E).Development of the U2OS-HRE-luc cell-based assay. We next

wanted to assess the robustness of the hypoxia-mediated responsein the U2OS-HRE-luc cells. A recent study using U251-HRE cells in ahigh-throughput hypoxia screen described a reoxygenation of cellsfor 1.5 hours immediately after hypoxia and before lysis (24). Wewere particularly interested in assessing both HIF-1 activity andHIF-1a induction in response to hypoxia and upon reoxygenation ofthe U2OS-HRE-luc cells. U2OS-HRE-luc cells were exposed tonormoxia or hypoxia for 6 hours. Cells were either lysed immediatelyor reoxygenated by exposure to normoxia before lysis. Consistentwith previous studies (29), HIF-1a protein levels were significantlyreduced by at least 50% upon reoxygenation of the cells, presumablyvia rapid degradation by the proteasome (Fig. 2A). Consequently,luciferase reporter activity was also significantly reduced (Fig. 2B).This was observed even when cells were exposed to hypoxia forlonger periods ( for up to 16 hours; data not shown). Our datasuggested that performing a high-throughput hypoxia screen aspreviously described (24) would not be feasible using the U2OS-HRE-luc cells, because we wanted to ensure that luciferase reporteractivity was a direct measure of HIF-1a protein induction. Wetherefore used DFX as an alternative method for HIF-1a stabiliza-tion. By way of validating the U2OS-HRE-luc cells in an automatedhigh-throughput cell-based assay in response to DFX, the effects ofDFX versus normoxia were assessed in 96-well format. We havepreviously shown that the phosphoinositide 3-kinase inhibitorLY294002 completely blocks HIF-1a protein expression and HIF-1activity in the U2OS-HRE-luc cells (27). We, therefore, assessed HIF-1activity in response to DFX in the presence and absence of LY294002.Cells were untreated or treated with either DMSO or LY294002 30minutes before DFX treatment and incubated for 16 hours.Luciferase activity was assessed and the coefficient of variationwas expressed as a percentage. A robust and reproducible increasein HIF-1 activity by DFX was observed and was inhibited byLY294002 (Fig. 2C). The mean ZVfactor for the screen was found tobe satisfactory at 0.52 F 0.09.High-throughput screen of 2,000 compounds. Following the

validation and automation of the U2OS-HRE-luc cell-based assay, apilot screen of f2,000 compounds from the NCI Diversity Set wasdone in 384-well format. This enabled us to make a directcomparison of our data with those obtained from an independentcell-based screen done in hypoxic conditions (24). Eight hitcompounds were identified from the NCI Diversity Set of 2,000compounds. This represented a hit rate of 0.4%. The criteria for a

hit in the screen were defined as any compound that caused areduction in DFX-induced luciferase activity by more than threestandard deviations from the control, and did not result in morethan 50% growth inhibition in a 24-hour viability assay norinterfere with the luciferase reagent alone to cause a change inreadout by >50%. We identified six hit compounds (NSC-675865,NSC-607097, NSC-259968, NSC-259969, NCS-25485, and NCS-131547) that were also identified in an independent hypoxia screen(Table 1; ref. 24). Four of these compounds (NSC-259968, NSC-259969, NSC-25485, and NSC-131547) were excluded because ofnonspecific DNA binding activity (Table 1). In addition, weidentified two novel compounds, NSC-134754 and NSC-643735,which we evaluated further (Table 1). Interestingly, topotecan(NSC-609699), camptothecin 20-ester(S) (NSC-606985), and thecamptothecin analogue, 9-glycineamido-20(S)-camptothecin (NSC-639174), which were described as hits by Rapisarda et al. (24)also inhibited luciferase activity in our screen by 43.4%, 79.8%,and 23.9%, respectively. However, these compounds did not satisfyour criteria for a hit and were, therefore, not evaluated further.

Figure 2. HIF-1a protein and HIF-1 activity induced by hypoxia are rapidlydecreased upon exposure to normal oxygen tension. U2OS-HRE-luc cellswere exposed to hypoxia (1% O2) or normoxia for 6 hours. Cells were then eitherharvested immediately or reoxygenated for 5 minutes before lysis. Sampleswere assayed for Western blot analysis and luciferase activity. A, Western blotanalysis shows HIF-1a protein. Actin was used as a load control. B, graphshows luciferase reporter activity for normoxia (norm), hypoxia (hyp, 1% O2), andreoxygenation (reoxy) as a fold induction relative to normoxia (norm). Eachexperiment was done in duplicate. C, U2OS-HRE-luc cells were plated into96-well plates. LY294002 (80 Amol/L) was added 30 minutes before treatmentwith or without DFX (500 Amol/L) for 16 hours in normoxia. Graph showsluciferase reporter activity as relative light units. n = 8 for each treatmentcondition. Coefficients of variation (%) are shown above each column.





Furthermore, we found that NSC-359449 and NSC-254681 hadminimal effects on luciferase activity and although Rapisarda et al.(24) described these compounds as hits, they did not inhibitvascular endothelial growth factor mRNA expression.Nonspecific effects of NSC-134754, NSC-607097, and NSC-

643735. Cell-based assays may potentially identify compounds thatact on multiple pathways. Therefore, evaluation of the hits in avariety of secondary assays is required for assessment of theirmode of action against the intended target. Our primary interestwas to address whether the hit compounds inhibited HIF-1 activityand HIF-1a protein levels in response to hypoxia. In addition, thenonspecific effects of the compounds on reporter genes wereinvestigated. Of the hit compounds identified, three were studiedin secondary assays. Two of these, NSC-134754 and NSC-643735,were discovered as novel hits in our screen. NSC-134754 is asemisynthetic analogue of the natural alkaloid, emetine (30), andNSC-643735 is a structural analogue of actinomycin D aglycone(31). The third, NSC-607097, a quinocarmycin analogue (32), wasalso identified by Rapisarda et al. (24) and was included forcomparison. The chemical structures of the three compounds areshown in Fig. 3. To rule out any nonspecific effects on luciferaseactivity, the compounds were initially assessed on U2OS-luc controlcells, which were the U2OS cells stably expressing a constitutiveluciferase reporter construct (Fig. 4). Our results indicated thatboth NSC-134754 and NSC-607097 inhibited the luciferase activitynonspecifically to some degree. To determine whether a compoundwas more inhibitory of luciferase activity increased by HIF-1a and,therefore, could be considered as a selective HIF-1 inhibitor, a ratioof the percentage inhibition of HIF-1 activity in the U2OS-HRE-luccells over the percentage inhibition under the same treatmentcondition in the U2OS-luc control cells was calculated. A compoundwas considered to have a greater effect on inhibiting HIF-1 activitycompared with a nonspecific effect if an arbitrary ratio of 2 orabove was obtained. The experiments using U2OS-HRE-luc and

U2OS-luc control cells were performed in parallel so that a directcomparison could be made. Analysis of the results showed thatNSC-134754 inhibited hypoxia and DFX-induced activity overnonspecific activity by a ratio of >2. The same was observed forNSC-643735 following DFX treatment. However, NSC-607097was borderline in terms of specificity as the ratio of inhibition inU2OS-HRE-luc cells versus U2OS-luc control cells was 1.4 in hypoxia

Table 1. Comparison of compounds identified in the screen done by Rapisarda et al. (24) with those identified in our high-throughput screen

Hit compounds %Inhibition

primary

screen

%Inhibition

reconfirmation

(n = 4)

%Growth

inhibition

at 24 h

%Inhibition

luciferase

reagent

Hit compounds

(24)

Compounds excluded

from both screens

NSC-134754 76.1 73.0 0.07 3.4 � �NSC-643735 93.2 94.6 0.0 6.2 � �NSC-675865 90.2 91.9 0.05 8.7 + �NSC-607097 105.8 106.9 27.8 7.3 + �NSC-259968 101.3 101.4 0.2 6.2 + {NSC-259969 96.8 98.0 0.1 8.4 + {NSC-25485 102.8 103.1 11.5 7.6 + {NSC-131547 103.8 103.5 0.1 8.5 + {

NSC-609699*NSC-606985*

NSC-639174*

NSC-359449

NSC-254681

NOTE: Hit compounds identified by both screens (+). Two novel compounds (NSC-134754 and NSC-643735) were identified in our screen. NSC-259968,

NSC-259969, NSC-25485, and NSC-131547 were excluded from both screens because of their nonspecific DNA binding activity ({). NSC-609699, NSC-606985, and NSC-639174 are camptothecin and its analogues (*). They were inhibitory in our screen, but they did not meet the criteria used for

identification of a hit in our assay.

Figure 3. Chemical structures of NSC-134754, NSC-607097, and NSC-643735.A, NSC-134754: 3-Ethyl-9,10-dimethoxy-2-(1,2,3,4-tetrahydro-isoquinolin-1-ylmethyl)-1,6,7,11b-tetrahydro-4H -pyrido[2,1-a ]isoquinoline. B, NSC-607097:7-Cyano-5,7,8,9,10,11,11A,12-octahydro-5-(hydroxymethyl)-4-methoxy-13-methyl-8,11-iminoazepino[1,2-b ]isoquinoline-10-carboxylic acid. C,NSC-643735: 2-Amino-3-(5-oxo-5H -benzo[a ]phenoxazin-10-yl)-propionic acid.

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although it was >2 for DFX. All three compounds were, therefore,found to have HIF-1–specific effects and were evaluated further.Effects of NSC-134754, NSC-607097, and NSC-643735 on HIF-

1 activity and HIF-1A protein levels. To determine whether thecompounds decreased hypoxia-induced HIF-1 activity, 20 Amol/L ofeach compound was added to U2OS-HRE-luc cells 30 minutesbefore treatment with DFX (500 Amol/L) or exposure to hypoxia(1% O2) for 16 hours. NSC-134754 (compound A) and NSC-607097(compound B) both completely inhibited the increases in luciferaseactivity induced by hypoxia, but NSC-643735 (compound C) did not(Fig. 5A and B). Changes in HIF-1a protein levels stabilized by DFXand hypoxia in the presence and absence of the compounds wereexamined by Western blotting (Fig. 5C). Our data showed that NSC-134754 (compound A) decreased HIF-1a protein levels stabilized byboth DFX and hypoxia. NSC-607097 (compound B) decreasedhypoxia-stabilized HIF-1a to a greater extent than DFX-stabilizedHIF-1a; and NSC-643735 (compound C) did not have any effect onhypoxia-stabilized HIF-1a protein level, which corresponded to itsinability to inhibit HIF-1 activity under hypoxic conditions (Fig. 5B).These observations were reflected in the levels of Glut-1

protein, a HIF-1 target gene product (Fig. 5D). In normoxia, therewas no detectable Glut-1 protein. Following treatment of U2OS-

HRE-luc cells with DFX (500 Amol/L) or hypoxia (1% O2), Glut-1expression increased. In the presence of NSC-134754 (compoundA) and NSC-607097 (compound B), there was a decrease in bothhypoxia and DFX-induced Glut-1. In the presence of NSC-643735(compound C), Glut-1 levels were reduced in the DFX-treatedcells, but not in the hypoxia-treated cells, thus confirming theresult of the luciferase activity in U2OS-HRE-luc cells (Fig. 5Aand B). These effects were more apparent when a range ofconcentrations (1–10 Amol/L) was used. In the DFX-treated cells,all three compounds inhibited HIF-1 activity. NSC-134754(compound A) and NSC-643735 (compound C) decreased HIF-1aprotein levels, but NSC-607097 (compound B) was much lesseffective (Fig. 6A). In hypoxia-treated cells, NSC-643735 (com-pound B) had no effect on HIF-1a and HIF-1 activity, confirmingour results described above. The other two compounds, NSC-134754 (compound A) and NSC-607097 (compound B), signifi-cantly inhibited both HIF-1 activity and HIF-1a protein levels inhypoxia (Fig. 7). Similar data were observed in U2OS cells stablyexpressing an HRE repeat (6�) fused to h-galactosidase (U2OS-HRE-hgal; data not shown), demonstrating that the effects werereproducible in a different reporter system.Effects of NSC-134754, NSC-607097, and NSC-643735 on cell

growth.We found that NSC-134754 (compound A) and NSC-643735(compound C) did not significantly inhibit cell growth at 18 Amol/L(the concentration used in high-throughput screening) whenassessed in a 24-hour viability assay, and compound NSC-607097(compound B) inhibited growth by <30%. We also determined theIC50 for growth inhibition for each compound using a sulforhod-amine B assay over a 72-hour period. The IC50 values for growthinhibition for NSC-134754, NSC-607097, and NSC-643735 weref0.5, 0.1, and 100 Amol/L, respectively (Table 2).Effects of NSC-134754, NSC-607097, and NSC-643735 on HIF-

1A induced in response to insulin-like growth factor-1. It isclear that HIF-1 is regulated by hypoxia and growth factors by twoindependent mechanisms (13). We next assessed whether NSC-134754 (compound A), NSC-607097 (compound B), and NSC-643735(compound C) could block HIF-1a induced by IGF-1. To do this,HCT116 human colon carcinoma cells were used as previouslydescribed (27). HIF-1a protein was induced following exposure ofserum-starved HCT116 cells to IGF-1 (50 ng/mL) for 7 hours innormoxia. NSC-134754 (compound A) and NCS-607097 (com-pound B) completely blocked HIF-1a induced by IGF-1 (Fig. 8),which mirrored their effects in hypoxia (Fig. 7). Interestingly, NCS-643735 (compound C) also significantly inhibited IGF-1–inducedHIF-1a expression despite having no effect on hypoxia-inducedHIF-1a or HIF-1 activity (Figs. 7 and 8). Our data suggest that NCS-134754 could prove to be the most promising therapeutically fortargeting both hypoxia and growth factor–mediated HIF-1aexpression and that NCS-643735 (compound C) could be used tospecifically target growth factor–mediated HIF-1a expression. Table2 summarizes the effects of NSC-134754, NSC-607097, and NSC-643735 on HIF-1 activity and HIF-1a protein levels in U2OS cells.

Discussion

Before embarking on any drug discovery project, one of thefundamental questions to be answered is why a particular target isvalid for therapeutic intervention. To satisfactorily answer thisquestion, it is necessary to show that the target has a greaterrelevance in disease than in health. There are several lines ofevidence to indicate that elevated levels of HIF-1a and its resultant

Figure 4. Effect of NSC-134754, NSC-607097, and NSC-643735 onluciferase activity of U2OS-luc control cells. U2OS-luc control cells were treatedwith (A) 20 Amol/L NSC-134754 (lane A), NSC-607097 (lane B ), NSC-643735(lane C ), or vehicle control (DMSO) 30 minutes before exposure to normoxia(norm) or DFX (500 Amol/L) for 16 hours. Graph shows luciferase reporteractivity as relative light units. Each experiment was done in duplicate.(B) Western blot analysis shows HIF-1a protein expression. Actin was used as aload control. Graph shows luciferase reporter activity as relative light units.Experiments were done in duplicate.





activity promotes tumor progression and the development oftreatment resistance (33–35). In addition, it has also been shownthat inhibiting HIF-1a can reduce tumor growth in vivo (17, 18,36, 37). Accordingly, much interest has been generated in thesearch for HIF-1 inhibitors in recent years.The aim of this study was to develop and implement a

strategy to therapeutically target the HIF pathway. In theabsence of any known direct enzymatic regulators of HIF thatwould be feasible to inhibit in a biochemical assay, we developeda cell-based assay for a high-throughput screen to identify smallmolecule inhibitors of HIF-1. Here, we describe the generation,characterization, and validation of the U2OS-HRE-luc cell-basedassay and the outcome of a high-throughput pilot screen usingthis system. We also generated secondary cell-based assays(U2OS-basic-luc and U2OS-luc control cells) that were suitablefor evaluating the hit compounds in initial deconvolutionstudies, with the aim of establishing their specificity for theHIF-1 pathway.

The validation and optimization of the U2OS-HRE-luc cell-based assay showed that this was a robust and reproduciblesystem that could be used to identify HIF-1 inhibitors. A high-throughput pilot screen of the NCI Diversity Set of f2,000compounds was done. Importantly, we also did a viability assay inparallel with the high-throughput screen to enable us to establishthe toxicity of the compounds in the U2OS-HRE-luc cell-basedassay. Interestingly, a comparison of our hit compounds withthose identified from a hypoxia screen done at the NCI recently(24) showed that many of the hit compounds were identical,despite a difference in cell line, the concentration at which thetest compounds were screened (1 versus 18 Amol/L), and mostnotably a difference in the mode by which HIF-1 activity wasinduced in both screens. Our screen was performed using thehypoxia mimetic DFX. Rapisarda et al. (24) identified topotecanand several camptothecin analogues as primary hits. Indeed, wealso found that topotecan and camptothecin inhibited HIF-1activity in our own screen. However, they did not meet our

Figure 5. Effect of NSC-134754, NSC-607097,and NSC-643735 on HIF-1 activity and HIF-1aprotein expression. U2OS-HRE-luc cells weretreated with 20 Amol/L NSC-134754 (lane A),NSC-607097 (lane B ), NSC-643735 (lane C ),or vehicle control (DMSO) in the presence orabsence of (A ) DFX (500 Amol/L) or (B) hypoxia(1% O2) for 16 hours. Samples were assayedfor luciferase activity and protein expressionby Western blot analysis. Graphs show luciferasereporter activity represented as relative light units.Experiments were done in duplicate. Westernblot analysis shows (C ) HIF-1a protein expressionand (D ) Glut-1 protein expression, respectively,in response to hypoxia and DFX. Actin was usedas a load control.

Cancer Research




criteria for progression as a HIF-1–selective hit. Although thevalues were very close to be considered a hit by our criteria, theydid not reproducibly cause a reduction in DFX-induced luciferaseactivity by more than three standard deviations from the controland, therefore, were not evaluated further. This does not mean,however, that the effects of these compounds are not interestingor therapeutically relevant. Topotecan is currently in use fortreatment of ovarian cancer and non–small cell lung carcinoma(24, 38) and recent studies have shown that topotecan reducesHIF-1a expression, vessel density, and HIF-1 targets in U251-HRExenografts (39).Characterization of the U2OS-HRE-luc cells showed that HIF-1

activity was highly sensitive to reoxygenation. Although not

surprising, this was in contrast to the observations obtained with

a U251-HRE cell-based assay described previously, where a

reoxygenation step was used before lysis (24). There is a

possibility that the loss of PTEN in the glioma U251 cells

(PTEN-null) may contribute to this effect. However, it is more

likely that the difference between the U2OS-HRE-luc and U251-

HRE systems is due to the different HRE-luciferase constructs

used. Rapisarda et al. (24) used a construct where the SV40

promoter of pGL2 was replaced with a herpes simplex virus TK

promoter fragment. Although this may have enabled the ability to

assess HIF-1–mediated luciferase activity in the U251-HRE system

even after significant reoxygenation of the cells, the resulting levels

of HIF-1a protein were not assessed. It is most likely that HIF-1a

protein expression would be significantly depleted by the

reoxygenation of the U251-HRE cells. In contrast to our cell-

based screen, the ability to assess and correlate the inhibition of

HIF-1 activity with effects on HIF-1a protein expression would,

therefore, not be possible within the screening parametersdescribed by Rapisarda et al. (24).Nevertheless, despite the differences in these independent

screens, the observation that several identical hit compounds

were identified suggests that the molecular targets involved in

the induction of HIF-1 activity by the iron chelator DFX clearly

overlap with those responsible for inducing HIF-1 activity in

response to hypoxia. Indeed, it is clear that the prolyl

hydroxylase enzymes, which target HIF-1a for hydroxylation

and subsequent ubiquitination by VHL and degradation by the

proteasome, require oxygen, ascorbate, 2-oxygluterate, and,

importantly, ferrous iron (40). The inactivation of the prolyl

hydroxylase enzymes, which results in the stabilization of HIF-1a

and subsequent activation of HIF-1, is therefore common to both

hypoxia and DFX treatments.We identified two novel compounds, NSC-134754 and NSC-

643735, which were evaluated further. It was important to

initially determine whether the compounds had any nonspecific

activity and to establish whether they had an inhibitory effect on

HIF-1 activity mediated by physiologic hypoxia, because our

screen used DFX. This analysis was also carried out on NSC-

607097, which was a hit identified in our screen and also by

Rapisarda et al. (24). NSC-134754 and NSC-643735 did not

significantly affect constitutive luciferase activity compared with

HIF-1–mediated luciferase activity. Although NSC-607097 was

found to have significant nonspecific activity, it was evaluated

further because we concluded that it was a borderline compound

in terms of its overall nonspecific activity.We correlated the effects of the compounds on HIF-1 activity

and HIF-1a expression in response to DFX, hypoxia, and IGF-1

Figure 6. NSC-607097 inhibits HIF-1 activity but does not inhibitHIF-1a protein induction in response to DFX. U2OS-HRE-luccells were treated with NSC-134754 (lane A ), NSC-607097(lane B ), or NSC-643735 (lane C ) over a range of concentrationsor with vehicle control (DMSO) in the presence or absence of DFX(500 Amol/L) for 16 hours and then harvested. Samples wereassayed for protein expression by Western blot analysis andluciferase activity. (A) Western blot analysis shows HIF-1a proteinexpression. Actin was used as a load control. (B) Graph showsluciferase reporter activity as a fold induction relative to theuntreated vehicle control. Experiments were done in duplicate.





treatment. We found that NSC-607097 inhibited HIF-1 activity in

DFX and hypoxia, and although NSC-607097 blocked theinduction of HIF-1a in hypoxia, it did not significantly affect

DFX-induced HIF-1a protein levels. Interestingly, while we found

that NSC-643735 blocked DFX-induced HIF-1 activity and HIF-1a

protein levels, NSC-643735 had no effect on HIF-1 activity or HIF-1ainduction in response to hypoxic conditions, highlighting a

mechanistic difference between the action of ‘‘chemical’’ hypoxia

and physiologic hypoxia on the cells. Consistent with ourobservations, both NCS-134754 and NSC-607097 blocked the

expression of Glut-1 protein in response to both DFX and hypoxia,

whereas NCS-643735 had no effect on Glut-1 expression in hypoxia.

Importantly, all three compounds significantly inhibited IGF-1–

induced HIF-1a expression. Taken together, our studies show thatHIF-1a expression and HIF-1 activity can be differentially targeted

and we have identified novel inhibitors with selective action for

either blocking HIF-1a protein expression in response to hypoxia

(NSC-134754) and/or IGF-1 (NSC-134754 and NSC-643735). Theobservation that NSC-134754 blocked both hypoxia and IGF-1–

mediated HIF-1a induction, and also had limited nonspecific

effects, suggests that this compound has particularly interestingproperties that are HIF specific.This study has shown that the U2OS-HRE-luc cell-based assay

provides not only a robust method for identifying compounds

Table 2. Summary of the effects of NSC-134754, NSC-607097, and NSC-643735

Compound DFX Hypoxia IGF-1 IC50 of growth

inhibition (Amol/L)

HIF-1a protein HIF-1 activity HIF-1a protein HIF-1 activity HIF-1a protein HIF-1 activity

(A) NSC-134754 +++ +++ +++ +++ +++ n.d. 0.5

(B) NSC-607097 +/� +++ ++ +++ +++ n.d. 0.1

(C) NSC-643735 ++ ++ � � +++ n.d. 100

NOTE: The effect of NSC-134754 (A), NSC-607097 (B), and NSC-643735 (C) at 10 Amol/L on HIF-1a and HIF-1 activity in response to DFX, hypoxia, and

IGF-1 in U2OS-HRE-luc cells. +++, a compound that completely inhibits HIF-1a protein induction such that no protein is detected by Western analysis

or inhibits HIF-1 activity by >80%. ++, a compound that inhibits HIF-1a protein induction or HIF-1 activity by >50%. +/�, a compound that inhibits HIF-1a induction or HIF-1 activity by <5%. �, a compound that has no significant inhibitory effect on HIF-1a induction or HIF-1 activity.

Abbreviation: n.d., not determined.

Figure 7. Hypoxia-induced HIF-1a and HIF-1 activity arenot decreased by NSC-643735. U2OS-HRE-luc cells weretreated with NSC-134754 (lane A ), NSC-607097 (lane B), orNSC-643735 (lane C ) over a range of concentrations or withvehicle control (�) in normoxia (norm) or hypoxia (1% O2)for 16 hours. Samples were assayed for Western blot analysisand luciferase activity. (A) Western blot analysis shows HIF-1aprotein expression. Actin was used as a load control.(B ) Graph shows luciferase reporter activity as a fold inductionrelative to normoxia. Experiments were done in duplicate.

Cancer Research




that inhibit DFX-induced HIF-1 activity, but also selects forcompounds that are drug-like. The three hit compounds havemolecular weights within the range of 334 to 418, Clog P valuesin the range �1.6 to 5.0, and have one to three hydrogen bonddonors and four to seven hydrogen bond acceptors. Theseproperties are all within the range considered to be preferablefor drug development (41). Both NSC-134754 and NSC-643735have interesting chemical properties; NCS-134754 is related tothe benzoisoquinoline alkaloids known to be inhibitors ofprotein synthesis (30, 42), whereas NCS-643735 is a planartetracyclic compound with potential for redox chemistry,originally prepared as a structural analogue of actinomycin Daglycone (31). Interestingly, NSC-607097, also known as DX-52-1(32), has been investigated in a phase I clinical trial but thestudy was not continued due to toxicity (43). Because a cell-based assay approach was used to identify hits, the challenge isto establish how they inhibit HIF-1 activity. A thoroughevaluation of the activity of the two novel compounds in anumber of secondary assays is currently under way with the aimof defining their mechanism of action. Importantly, these studieswill impact on their potential optimization for preclinicaldevelopment and assessment in vivo . In view of the encouragingresults from the screen described here and to generate initialhits that might provide additional or alternative options for alead optimization program, we have also performed a largescreen of f70,000 compounds using this system. Although ourstudy focuses primarily on the effects of small molecule

inhibition of HIF-1 activity, our system also has the potentialfor assessing inhibitors of HIF-2.In conclusion, a cell-based assay approach has successfully

identified novel compounds that differentially target hypoxiaand/or growth factor–mediated induction of HIF-1a. Thecompounds described here, together with those emerging froma subsequent larger screen, will be evaluated further for use asmechanistic tools and as candidates for potential chemicaloptimization. In addition, the differential effects of thesecompounds on the hypoxia versus the growth factor–mediatedregulation of HIF will be investigated in vivo . These studies mayallow us to evaluate the contribution of converging signalingpathways on HIF and their respective relevance in HIF-dependent tumor progression and angiogenesis.

Acknowledgments

Received 12/13/2004; revised 2/8/2005; accepted 2/24/2005.Grant support: Cancer Research UK (CRC C309/A2984), Institute of Cancer

Research, and Association for International Cancer Research grant Ref:03-150(V. Carroll).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Giovanni Melillo (Developmental Therapeutic Program, TumorHypoxia Laboratory, NCI, Frederick, MD) for generously providing the pGL-HREluciferase reporter construct, Dr. Colin Porter and Nicola Ingram (Cancer Research UKCentre for Cell and Molecular Biology, ICR, London, United Kingdom) for kindlysupplying the HRE-h-galactosidase reporter constructs, Dr. Julia Bardos for criticalreview of the manuscript, Kati Rasanen for technical support, and all members of theAshcroft laboratory for their input and support.

Figure 8. Effects of NSC-134754, NCS-607097, andNCS-643735 on HIF-1a induced by IGF-1. (A ) HCT116cells were either treated with NSC-134754, NCS-607097,or NCS-643735 at the concentrations indicated 30minutes before (A) DFX (500 Amol/L) for 8 hours, or (B)serum-starved HCT116 cells were treated withNSC-134754, NCS-607097, and NCS-643735 for30 minutes before stimulation with IGF-1 (50 ng/mL) for8 hours. Samples were assessed by Western blot analysisfor HIF-1a protein levels. Actin was used as a load control.

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Cancer Research




2005;65:4918-4928. Cancer Res Noan-Minh Chau, Paul Rogers, Wynne Aherne, et al. Growth Factors

Induction in Response to Hypoxic Stress andαFactor-1Hypoxia-Inducible Factor-1 Activity and Hypoxia-InducibleHypoxia-Inducible Factor-1 That Differentially Block Identification of Novel Small Molecule Inhibitors of

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Identification of Novel Small Molecule Inhibitors of ... · Inducible Factor-1 That Differentially Block Hypoxia-Inducible Factor-1 Activity and Hypoxia-Inducible Factor-1A Induction