Dual Targeting of Hypoxia and Homologous Recombination Repair Dysfunction in … · However, HAP sensitivity was enhanced by suppres-sion of HR (assessed by radiation-induced RAD51

Small Molecule Therapeutics

Dual Targeting of Hypoxia and Homologous RecombinationRepair Dysfunction in Triple-Negative Breast Cancer

Francis W. Hunter, Huai-Ling Hsu, Jiechuang Su, Susan M. Pullen, William R. Wilson, and Jingli Wang

AbstractTriple-negative breast cancer (TNBC) is an aggressive malignancy with poor clinical outcome and few

validated drug targets. Two prevalent features of TNBC, tumor hypoxia and derangement of homologous

recombination (HR) repair, are potentially exploitable for therapy. This study investigated whether

hypoxia-activated prodrugs (HAP) of DNA-damaging cytotoxins may inhibit growth of TNBC by simul-

taneously addressing these two targets. We measured in vitro activity of HAP of DNA breakers (tirapa-

zamine, SN30000) and alkylators (TH-302, PR-104, SN30548) in TNBC cell lines and isogenic models, and

related this to measures of HR repair and expression of prodrug-activating enzymes. Antitumor activity of

HAP was examined in isogenic BRCA2-knockout xenograft models and compared with platinum chemo-

therapy. All five HAP selectively inhibited growth of TNBC cell lines under hypoxia. Sensitivity to HAP

was not strongly associated with BRCA1 genotype. However, HAP sensitivity was enhanced by suppres-

sion of HR (assessed by radiation-induced RAD51 focus formation) when BRCA1 and PALB2were knocked

down in a common (MDA-MB-231) background. Furthermore, knockout of BRCA2 markedly sensitized

DLD-1 cells to the clinical nitrogen mustard prodrugs TH-302 and PR-104 and significantly augmented

sterilization of clonogens by these agents in xenografts, both as monotherapy and in combination with

radiotherapy, but had less effect on activity of the benzotriazine di-N-oxide SN30000. PR-104 monotherapy

was more effective than cisplatin at inhibiting growth of BRCA2-knockout tumors at equitoxic doses. This

study demonstrates the potential for HAP of nitrogen mustards to simultaneously exploit hypoxia and

HR defects in tumors, with translational implications for TNBC and other HR-deficient malignancies. Mol

Cancer Ther; 13(11); 2501–14. �2014 AACR.

IntroductionBreast cancer is a disease characterized by substantial

histologic and molecular heterogeneity, with wide dispa-rities in prognosis and response to therapy. Triple-nega-tive breast cancer (TNBC), defined by negative clinicalassays for expression of estrogen receptor (ER), proges-terone receptor and amplification of HER2, accounts for10% to 24%of invasive breast cancers (1) andencompassesan aggressive albeit heterogeneous subtype associatedwith young age at diagnosis (1), high histologic grade(2), visceral and central nervous system (CNS) metastasis(3), and worse prognosis than hormone receptor–positivetumors (4). Most patients relapse within 3 years of pri-

mary diagnosis with aggressive, chemoresistant metasta-ses and rapid progression to death (5, 6). TNBC is alsoclosely related to the poor prognosis basal-like breastcancer (BLBC) subtype defined by PAM50 gene expres-sion analysis. Although these classifications are not syn-onymous, approximately 80% of TNBC are also BLBC (7).Antagonists of ER and HER2 signaling, which have dras-tically improved outcomes for ER-positive and HER2-positive breast cancers, are not indicated for TNBC, andchemotherapy is the sole modality for systemic manage-ment of advanced disease.

There has been significant recent interest in exploitingthe link between TNBC and the BRCA1 pathway fortherapy. The vast majority of mammary carcinomas inwomen carrying germline BRCA1 mutations are triple-negative, and although BRCA1 is infrequently mutated insporadic TNBC (8, 9), suppression by miRNA (10), epi-genetic silencing (11), and other nongenetic causes of"BRCAness" phenotypes may implicate dysfunction ofgenes epistatic with BRCA1 more widely in TNBC.BRCA1 plays a critical function in resolution of DNAdouble-strand breaks (DSB) by homologous recombina-tion (HR) repair, particularly DSB associated with cross-links at DNA replication forks (12). As a result, TNBCshow deficiency in HR and may be more sensitive to

Auckland Cancer Society Research Centre, Faculty of Medical and HealthSciences, University of Auckland, Auckland, New Zealand.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: William R. Wilson, Auckland Cancer SocietyResearch Centre, Faculty of Medical and Health Sciences, University ofAuckland, 85Park Road, Grafton, Private Bag 92019, Auckland, 1142, NewZealand. Phone: 64-9-9236883; Fax: 64-9 3737571; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-14-0476

�2014 American Association for Cancer Research.

MolecularCancer

Therapeutics

www.aacrjournals.org 2501

on June 24, 2021. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst September 5, 2014; DOI: 10.1158/1535-7163.MCT-14-0476

http://mct.aacrjournals.org/

therapies that generate cross-links or DSB (13), includingplatinum drugs, alkylating agents, anthracyclines andPARP inhibitors, although the efficacy of this approachis yet to be validated in definitive clinical studies.

Hypoxia is an adverse pathologic feature of manytumors including breast cancer (14).Although earlydefin-itive characterization of tumor hypoxia preceded molec-ular classification of breast cancer (15), recent compellingevidence across multiple technology platforms links hyp-oxia specifically with TN/BL subtypes, where it maynegatively influence treatment outcome (16–24), raisingthe possibility that drugs targeted to tumor hypoxia maybe an effective strategy for TNBC. Several classes ofhypoxia-activated prodrugs (HAP) have been rationallydeveloped to exploit tumor hypoxia (14). These includethe clinical stage benzotriazine di-N-oxide HAP tirapa-zamine (25) and nitrogen mustard prodrugs TH-302 (26)and PR-104 (27), in addition to advanced preclinical com-pounds such as the tirapazamine analogue SN30000 (28)and a nitro-chloromethylbenzindoline (nitroCBI) that is aprodrug of a potent DNA minor groove alkylator (29).These agents are enzymatically reduced in hypoxic tumortissue to DNA-damaging metabolites that are selectivelytoxic to hypoxic cells.

By deploying in vitro isogenic models, we (30, 31) andothers (26, 32) have demonstrated that several HAP arecapable of exploiting HR defects analogous to those fre-quently observed in TNBC (8, 10). A comparison of chem-ical classes in Rad51d-knockout Chinese hamster cellssuggested that the DNA cross-linking HAP (TH-302 andPR-104) may have greater selectivity for HR dysfunctionthan benzotriazine-di-N-oxides or nitroCBI (30). HAPmay therefore be uniquely positioned to simultaneouslyexploit hypoxia and HR dysfunction in TNBC, anapproach that is further supported by observations thathypoxia itself downregulatesHR repair in tumors (33, 34).Here, we investigate the potential for HAP to inhibittumor growth by dual targeting of hypoxia andHR repairdefects in preclinical models.

Materials and MethodsCompounds

SN30000, tirapazamine, TH-302, mechlorethamine(HN2), PR-104, PR-104A, PR-104H, the nitroCBI SN30548,the corresponding aminoCBI SN30550, FSL-61, pimoni-dazole, cisplatin, and olaparib were either synthesized atthe Auckland Cancer Society Research Centre (Auckland,New Zealand) or purchased from suppliers as indicatedin Supplementary Table S1. Purity of batches synthesizedin-housewas confirmedbyhigh-performance liquid chro-matography (HPLC).Drug stock solutions (solvents listedin Supplementary Table S1) were stored at �80�C.

Cell linesTNBC lines with known BRCA1 genotype (35) were

obtained fromAsterand (SUM1315MO2, SUM149PT, andSUM159PT), ATCC (HCC1937 and MDA-MB-436), Cali-

per Life Sciences (MDA-MB-231-D3H2LN, a highly met-astatic subclone of MDA-MB-231; henceforth, D3H2LN),Dr. A. Patterson (University ofAuckland,Auckland,NewZealand; MDA-MB-468), and Dr. G. Krissansen (Univer-sity of Auckland; BT549). The TNBC lines were propa-gated in culture as described in Supplementary Table S2.HEK293 cells were obtained from Open Biosystems andcultured in RPMI with 10% FCS. DLD-1 cells with homo-zygous knockout of BRCA2 (line HD105-007, henceforth,DLD-1 BRCA2�/�) and isogenic BRCA2wild-typeDLD-1cells were licensed from Horizon Discovery. The DLD-1lines were maintained in McCoy’s 5A modified mediumwith 10% FCS and 2 mmol/L L-glutamine. All cell lineswere cultured in humidified CO2 incubators at 37

�C for�2months cumulative passage fromauthenticated frozenstocks confirmed to be Mycoplasma negative by PCR-ELISA (Roche). Cell lines that were not obtained from acommercial supplier were authenticated by short tandemrepeat profiling (CellBank Australia).

RNAi-mediated suppression of HR genesTRIPZ lentiviral plasmids carrying doxycycline-induc-

ible shRNA were purchased from Open Biosystems. Sev-en BRCA1-targeted shRNAs and four PALB2-targetedshRNAs were screened by comparing induction of theturboRFP reporter gene, using a fluorescence plate reader,and depletion of target mRNA, measured by quantitativereal-time PCR, in transiently transfected HEK293 cellsinduced with 0.5 mg/mL doxycycline for 48 hours. PCRprimer sequences for measuring BRCA1 and PALB2mRNA are given in Supplementary Table S3. The shRNAsequences selected on the basis of these screens,V2THS_254648 (henceforth, shBRCA1), V3THS_369350(henceforth shPALB2), together with a nonsilencingTRIPZ shRNA (TRIPZ control), were packaged into len-tiviruses and transduced into D3H2LN cells using amultiplicity of infection of 1. Stable transductants wereselected in puromycin and induced with 0.5 mg/mLdoxycycline for 48 hours before aseptically sorting thebrightest 30% of turboRFP-expressing cells (12–16 � 103cells in total) using a BD FACSAria II flow cytometer.Sorted pools were maintained without doxycycline untilexperimentation. Fluorescence and phase contrast micro-graphs were captured using a Nikon TE2000E invertedmicroscope with attached Nikon Digital Sight (DS-5Mc)cooled color camera.

Cytotoxicity assaysIn vitro antiproliferative activity of drugs was mea-

sured using well-established (30) IC50 assays with asulforhodamine B (SRB; Sigma-Aldrich) colorimetricendpoint. For each cell line, the linearity of SRB stainingand associated optimal seeding density (electronic par-ticle counter; Beckman Coulter; Supplementary TableS4) was determined by titrating cell number. To assaysensitivity to the PARP inhibitor olaparib, cells werecontinuously exposed to drug for 5 days under aerobicconditions before SRB staining. All other drug exposures

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Mol Cancer Ther; 13(11) November 2014 Molecular Cancer Therapeutics2502




were for 4 hours, with 4 to 5 days regrowth in drug-freemedium. To assay drug sensitivity in D3H2LN cells withshRNA-mediated knockdown of HR genes, log-phasecultures were induced with 2 mg/mL doxycycline for72 hours before drug treatment. Doxycycline was alsopresent in the regrowth medium after removal ofdrugs. Hypoxic incubations were performed in a H2/Pdcatalyst–scrubbed anaerobic chamber (Coy LaboratoryProducts)withmediumandconsumables preequilibratedfor >3 days to remove residual oxygen. Hypoxic cytotox-icity ratio (HCR) was defined as (IC50 oxic)/(IC50 hypoxic).Hypersensitivity factor (HF) for cell lines with shRNAknockdown or genetic deletion of HR genes was definedas (IC50 HR-proficient line)/(IC50 HR-defective line), where theHR-proficient line was D3N2LN-TRIPZ control or DLD-1 wild-type, respectively. All ratios (HCR and HF) areintraexperiment comparisons.

Liquid chromatography/tandem mass spectrometryanalysis of SN30000 metabolismMetabolic depletion of SN30000, and production of

the corresponding stable 1-oxide and nor-oxide reducedmetabolites, in hypoxic and aerobic TNBC cells wasquantified using a validated liquid chromatography/tan-dem triple-quad mass spectrometry assay (LC/MS-MS)as described previously (36).

Western immunoblottingLysates were harvested from log-phase cell cultures

using radioimmunoprecipitation assay buffer and totalprotein concentration measured by bicinchoninic acid(BCA) assay. Immunoblotting for expression of reduc-tases in cell lines used well-validated mouse monoclonalprimary antibodies for POR (sc25263; Santa Cruz Biotech-nology; ref. 37) andAKR1C3 (NP6.G6.A6; Sigma-Aldrich;ref. 38) as described previously. For RAD51 immunoblot-ting, 30 mg samples of 2-mercaptoethanol- and heat-dena-tured proteinwere resolved on 4% to 12%polyacrylamidegradient gels (Invitrogen), blocked, transferred to poly-vinylidenedifluoride (PVDF) membrane, and probedwith an anti-RAD51 primary antibody (rabbit polyclonalab63801; Abcam) that we have previously validatedfor immunofluorescent detection of radiation-inducedRAD51 foci in cell lines (30). Themembraneswere probedwith anti-rabbit secondary antibody (Invitrogen). Theseconditionsdetected abandat 37 kDa, corresponding to theexpected molecular weight of RAD51, with weaker non-specific bands at approximately 30, 50, 55, and 70 kDa insome cell lines (Supplementary Fig. S1). For all blots,chemiluminescent images were acquired using a LAS-4000 ImageQuant (GE Healthcare). Expression of POR,RAD51, and AKR1C3 in cell lines was quantified bycomparing band density normalized against ACTB(mouse monoclonal MAB1501R; Chemicon) or TUBA(mouse monoclonal B-5-1-2; Sigma-Aldrich) as loadingcontrols in ImageJ using unprocessed, unsaturatedimages. Values plotted are mean and SEM of the intraex-periment antigen/actin ratio for two independent experi-

ments. For clarity, blots in the main text have beencropped to retain six bandwidths above and below anti-gens. Full, uncropped replicate blots with molecular sizemarkers and quantitation are provided in SupplementaryData. Borders of cropped blots are indicated by blackmargins.

RAD51 immunofluorescenceFor analyzing induction of RAD51 foci in response to

ionizing radiation (IR) in vitro, cells were cultured onsterile poly-D-lysine–coated glass coverslips (BD Bios-ciences) and treated with 8 or 10 Gy IR (Eldorado modelG 60Co radiotherapymachine) or shamradiation. The cellswere fixed in 2% paraformaldehyde (Sigma-Aldrich)10 hours after irradiation, rehydrated in ice-cold PBS,permeabilized in 0.25% Triton X-100 (Sigma-Aldrich) inPBS for 20 minutes, and blocked using 5% goat serum(Invitrogen) in 0.1% PSB–Tween 20 (Global Science) for 30minutes at 20�C. The specimens were then probed withanti-RAD51 primary antibody (rabbit polyclonal ab63801;Abcam) diluted at 1:1,000 in blocking buffer for 1 hour at20�C. The coverslips were washed thoroughly in PBS andprobed with either Cy3- (TNBC wild-type and DLD-1cells) or Alexa Fluor 488–conjugated (shRNA-expressingD3H2LN cells) anti-rabbit secondary antibodies (bothfrom Invitrogen) diluted at 1:500 in blocking buffer for30minutes in darkness at 20�C. Cells were counterstainedwith 2.5 mg/mL 40,6-diamidino-2-phenylindole (DAPI;Sigma-Aldrich) for 1minute andmounted on glassmicro-scope slides using ProLongGold (Invitrogen). Slideswereair-dried before storing at 4�C. Images of random fieldswere captured using a LeicaDMRmicroscopewithNikonDigital Sight DS-U1 camera and 100� objective lens withstandardized exposure conditions. Nuclei presenting �2RAD51 foci were scored as positive by manual counting.Typically, >150 nuclei were scored per slide in eachindependent experiment. To assay induction of RAD51foci in D3H2LN cells with shRNA-mediated knockdownof BRCA1 and PALB2, cells were cultured on coverslips inthe presence of 2 mg/mL doxycycline for 72 hours beforeirradiation.

POR enzyme activityPOR enzymatic activity in cellular S-9 fractions was

determinedby spectrophotometric assay as cyanide-resis-tant, NADPH-dependent reduction of cytochrome c asreported elsewhere (39). Total protein in S-9 fractions wasmeasured by BCA assay.

FSL-61 fluorogenic assaysEnzymatic activation of the fluorogenic one-electron

reductase probe FSL-61 was measured as before (40).Briefly, 106 cells were seeded into non-tissue culture–treated 24-well plates in 0.5-mL preequilibrated PhenolRed–free MEMa with 5% FCS inside an anaerobic cham-ber, and incubated for 30minutes. The cells were exposedto 300 mmol/L FSL-61 for 3 hours and then stored indarkness on ice for

LSRII flow cytometer with BD FACSDiva software (Bec-tonDickson). The excitationwavelengthwas 355 nm,withemission at 425 to 475 nm.

Xenograft modelsAnimal studies were performed in accordance with the

New Zealand Animal Welfare Act 1999 and ResearchApproval 001190 from the Animal Ethics Committee ofthe University of Auckland. DLD-1 (1.5 � 106) or DLD-1BRCA2�/� (3 � 106) cells were inoculated, in 0.1 mL of30% Matrigel/MEMa (BD Biosciences), into the subcutisof anesthetized female, 18 to 21 gNIH-III nudemice (bredat the University of Auckland). Tumor growth was mon-itored by calipermeasurement using the formula: volume¼ 0.5 � length � width2.

For ex vivo clonogenic assays, tumors were grown totreatment size of 300 to 500 mm3 and stratified to cohortsthat were dosed with SN30000, TH-302, or PR-104, byintraperitoneal (i.p.) injection, at 155, 150, and 578mg/kg,respectively. These doses corresponded to 75% of empir-ically determined MTD in this mouse strain. In the drugand radiation combination cohorts, drugs were adminis-tered 5 minutes after 10 Gy single-dose, whole-bodyradiotherapy (Eldorado 78 60Co radiotherapy machine)or sham irradiation. Tumors were excised 18 hours laterand mechanically and enzymatically disaggregated tosingle cells, which were then plated in dilution series intriplicate for evaluation of clonogenic survival. Colonieswere scored 10 days thereafter by crystal violet staining.Sterilization of tumor clonogens by treatments is reportedas Log10 Cell Killing, defined as�log10(Surviving Fraction)by reference to plating efficiency of cells derived fromuntreated tumors. Cohort sizes were 3 for drug-onlygroups and 4 for combination therapy groups.

For tumor growth delay, xenografts were grown to 250to 400mm3 and stratified to cohorts thatwere treatedwith10 Gy local-tumor radiotherapy or single i.p. injection ofPR-104 or cisplatin at 578 and 5.1 mg/kg, respectively,which corresponded to 75% of MTD. Tumor growthkinetics was evaluated by caliper measurement asdescribed above. Survival analysis was performed usinglog-rank tests with the endpoint defined as tumor volume>3-fold higher than volume on the day of treatment.Cohort sizes were 5 for DLD-1 and 8 for DLD-1BRCA2�/�.

Pimonidazole immunohistochemistryMice bearing subcutaneousDLD-1 orDLD-1BRCA2�/�

xenografts with mean volume of 350 mm3 were dosedwith pimonidazole at 60 mg/kg or saline by i.p. injec-tion. The tumors were excised 2 hours thereafter andfixed in 4% paraformaldehyde for 24 hours, washedthree times in PBS, and then cryoprotected using 20%(w/v) sucrose-PBS followed by 30% sucrose-PBS. Thetissue was embedded in optimal cutting temperature(OCT) and frozen for cryosectioning. Eight-micrometersections were stained with anti-pimonidazole antibody(Hypoxyprobe 1-Mab1; HPI, Inc.), counterstained with

DAPI, and imaged using a Leica DMR microscope withNikon Digital Sight DS-U1 camera and 25� objectivelens with standardized exposure conditions.

Statistical analysisUnless otherwise indicated in figure legends, values are

mean and SEM of multiple independent experiments.Student t tests, ANOVA,Mann–WhitneyU tests, log-ranktests, and Spearman correlations were computed inSigmaPlot v12 (Systat Software). � P < 0.05; ��, P < 0.01;��, P < 0.001.

ResultsHypoxia-selective cytotoxicity of HAP in TNBC celllines

To compare the potential of HAP representing mul-tiple chemical classes to inhibit growth of TNBC cells,we examined in vitro sensitivity of eight TNBC cell linesof known BRCA1 genotype (Supplementary Table S2) tofive HAP (benzotriazine di-N-oxides tirapazamine andSN30000; alkylator prodrugs TH-302, PR-104A, andnitroCBI SN30548) under hypoxia (Fig. 1A). TH-302was the most potent hypoxic cytotoxin (mean IC50 for8 cell lines 0.071 mmol/L), followed by SN30548 (0.40mmol/L), tirapazamine (3.0 mmol/L), PR-104A (3.2mmol/L), and SN30000 (3.9 mmol/L). Cytotoxicity wasstrongly suppressed by oxygen in all cases (Fig. 1B) withHCR (Fig. 1B and Supplementary Fig. S2) greatest forTH-302 (range, 150–880) and least for PR-104A (range,7.9–73). There was no obvious relationship betweenBRCA1 mutational status and cytotoxic potency or hyp-oxia selectivity. Sensitivity to the active metabolites ofPR-104A (i.e., PR-104H) and SN30548 (i.e., SN30550)again showed no clear relationship with BRCA1 geno-type, as was also the case for HN2, which we used as amodel for the aliphatic mustard active metabolite fromTH-302, bromo-isophosphoramide mustard (41). Cis-platin showed a similar cell line dependence to HN2(r ¼ 0.83; P ¼ 0.01). The BRCA1-mutant MDA-MB-436was the most sensitive line to the cross-linking agents(Fig. 1C), and was also exquisitely sensitive to thePARP1/2 inhibitor olaparib (Fig. 1D). However, therewas no statistically significant relationship betweenBRCA1 genotype (wild-type vs. mutant) and eitheraerobic or hypoxic potency of any of the agents tested(Supplementary Table S5).

One-electron reductase activity in TNBC cell linesGiven that HAP activation in hypoxic cells requires

one-electron reduction (14), variation in reductase activ-ity could contribute to cell line differences in HAPsensitivity. Comparison of SN30000 reduction toits one-oxide and nor-oxide metabolites showed >97%inhibition by oxygen in all cell lines, with a difference ofonly approximately 2-fold in rates of anoxic metabolicreduction across the panel (Fig. 2A). The well-charac-terized one-electron reductase POR was expressed in

Hunter et al.





all cell lines, with significant variation (range, 0.3–1.2as the ratio of POR/ACTB; Fig. 2B; Supplementary S3and S4); protein expression correlated significantly withPOR enzymatic activity in the same cells (r ¼ 0.89; P ¼2 � 10�7; Fig. 2C). Activation of the one-electron reduc-tase flow cytometry probe FSL-61 in hypoxic cells(Fig. 2D) showed larger variation between lines, anddid not correlate with POR activity (r ¼ 0.0; P ¼ 1.0),

which is consistent with its reported activation by mul-tiple one-electron reductases (40). With the exceptionof SN30000, for which hypoxic activation correlatedwith POR expression and sensitivity correlated withPOR enzymatic activity (Supplementary Fig. S5), noneof these measures of one-electron reductase activitycorrelated with sensitivity of TNBC cells to other HAPin univariate analyses (Supplementary Table S6).

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Figure 1. Hypoxia-selectivecytotoxicity of HAP in TNBC celllines in vitro. Antiproliferativeactivity of the prodrugstirapazamine (TPZ), SN30000, PR-104A, TH-302, and nitroCBI(SN30548) in a panel of eight TNBCcell lines, four carrying BRCA1mutations and four with wild-typeBRCA1, exposed for 4 hours underhypoxic (A) or aerobic (B)conditions. The values shown aremean þ SEM of three to sixindependent determinations ofhalf-maximal inhibitoryconcentration (IC50). The range ofHCR observed for each compoundis indicated numerically above thebars in B. C, antiproliferativeactivity of PR-104H (an activemetabolite of PR-104A),mechlorethamine (HN2; ananalogue of the active metaboliteof TH-302), aminoCBI (SN30550;the active metabolite of nitroCBI),and cisplatin in TNBC cell linesexposed to compounds underaerobic conditions for 4 hours. Thevalues plotted are mean þ SEM ofthree to six independent IC50determinations. D, antiproliferativeactivity of the clinical PARPinhibitor olaparib in TNBC cellsexposed continuously to drug for120 hours. The values are mean þSEM of three to ten independentdeterminations.

Targeting Hypoxia and HR Repair Defects in TNBC

www.aacrjournals.org Mol Cancer Ther; 13(11) November 2014 2505




HR repair and its relationship to HAP sensitivity inTNBC cells

Although BRCA1 genotype did not show an obviousrelationship with HAP sensitivity above, the mutationsinvestigated may have significant phenotypic differencesand HR status may also be influenced by other mutationsand epigenetic changes in these cells. We therefore eval-uated HR function by quantifying radiation-inducedRAD51 focus formation, which showed marked differ-

ences between cell lines (Fig. 3A). MDA-MB-436 showedthe lowest HR activity with no detectable induction ofRAD51 foci, consistent with its marked sensitivity toolaparib (Fig. 1D). Overall, lines with BRCA1 mutationsshowed a reduced proportion of nuclei with RAD51 foci(mean 18% vs. 56% of irradiated cells; Fig. 3B) but thisdifference was not statistically significant in our smallpanel (P ¼ 0.06, Mann–Whitney U test). We also testedRAD51 protein expression (Fig. 3C and Supplementary

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Figure 2. One-electron reductase activity in TNBC cells. A, metabolic activation of SN30000 by TNBC cells under hypoxic and aerobic conditions. The rate ofsummed production of stable 1-oxide and nor-oxide metabolites was normalized for cell density and actual (i.e., measured) SN30000 concentration. Valuesare mean þ SEM from three independent experiments, each measuring three separate cultures. B, evaluation of POR protein expression in TNBC cells byWestern blot analysis. POR:ACTIN band densitometry ratios, normalized against MDA-MB-468 cells, are shown numerically below the image, where bluecoloring corresponds to BRCA1 wild-type lines and red to BRCA1-mutant lines, and are the mean and SEM determination of two experiments. C,POR enzymatic activity in TNBC cell lines measured as cyanide-resistant, NADPH-dependent reduction of cytochrome c by spectrophotometric assay.Values are mean þ SEM of determinations from two biologic replicates, each with three technical replicates. D, reductive activation of the fluorogenic agentFSL-61 measured by flow cytometry in TNBC cells. Values are mean þ SEM of geometric mean of fluorescence in three independent experiments.

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Figs. S1 and S6) given that increased RAD51 can partiallycompensate for HR dysfunction (42). Although RAD51expression trended higher in BRCA1-mutant lines, thisdifference was not significant (Supplementary Table S5).At least one surrogate marker of HR—RAD51 foci, ola-parib or cisplatin sensitivity—was strongly correlated toTH-302, PR-104A, HN2, and PR-104H sensitivity underhypoxia and to tirapazamine and SN30000 sensitivityunder aerobic conditions (Supplementary Table S6). Col-lectively, these data suggested that HR repair competencemay influence sensitivity of TNBC cell lines to someclasses of HAP, although other determinants are likely tocontribute across a panel of genetically diverse cell lines.

RNAi-mediated suppression of HR repair sensitizesTNBC cells to HAP in vitroTo further investigate HR repair as a determinant of

sensitivity toHAP,we turned to isogenicmodels inwhichthis variable could be isolated.Wegenerateddoxycycline-inducible lentiviral shRNA vectors to suppress the HRgenesBRCA1 andPALB2 inHR-competentD3H2LNcells.Hairpins that efficiently suppressed BRCA1 or PALB2upon induction with doxycycline were identified byscreening transiently transfectedHEK293 cells for expres-sion of the turboRFP reporter gene, using a fluorescenceplate reader (Supplementary Fig. S7), and depletion oftarget mRNA, measured by quantitative real-time PCR(Supplementary Fig. S8). The most effective shRNA

against each target, in addition to a nonsilencing TRIPZshRNA, were stably transduced into D3H2LN cells, andpoolswith high expression of the bicistronic cassette wereisolated by fluorescence-activated cell sorting of thebrightest 30% of turboRFP-expressing cells (Supplemen-tary Fig. S9). Exposure to doxycycline for 72 hours gaveoptimal turboRFP induction without cytotoxicity at 2 mgdoxycyline/mL (Supplementary Fig. S10). These condi-tions efficiently elicited expression of the linked turboRFPreporter gene (Fig. 4A) and resulted in partial suppressionof BRCA1 (47% of noninduced) and PALB2 transcripts(42% of noninduced) with no effect of the control vector(Fig. 4B). Suppression of BRCA1 and PALB2 resulted inreduction ofHRactivity asdemonstratedby the radiation-induced RAD51 focus assay, although this did not reachstatistical significance for PALB2 (Fig. 4C). This loss of HRwas associated with a 2- to 5-fold increase in sensitivity toHN2, chlorambucil, cisplatin, and PR-104Hunder aerobicconditions and 2- to 3-fold increased sensitivity to TH-302,PR-104A, SN30000, and cisplatin under hypoxic condi-tions (Fig. 4D).

Genetic deletion of BRCA2 markedly augmentscytotoxicity and antitumor activity of the nitrogenmustard prodrugs TH-302 and PR-104

As demonstrated above, shRNA knockdown only par-tially suppressed BRCA1 and PALB2 expression and HRrepair activity in D3H2LN cells, resulting in modest

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Figure 4. RNAi-mediated suppression of HR repair sensitizes TNBC cells to HAP in vitro. A, phase-contrast (PC) and fluorescence micrographs illustratinginduction of shRNA expression, with concomitant induction of turboRFP reporter expression, in D3H2LN cells stably transduced with shRNA to BRCA1exposed to doxycycline for 72 hours. Analogous images were obtained for shPALB2 and TRIPZ control lines but have been excluded for simplicity. B,doxycycline-induced, shRNA-mediated suppression of target mRNA in stably transduced D3H2LN cells. Changes in abundance of BRCA1 and PALB2transcriptsweremeasured by quantitative real-timePCR in reference toACTB using the relative quantificationmethod, and are plotted asmeanþSEMof foldchanges relative towild-typeD3H2LNcells assayed in parallel. Statistical significance of changes in transcript abundancewas evaluated byone-wayANOVA.C, quantitation of RAD51 foci in doxycycline-induced and noninduced TRIPZ control, shBRCA1, and shPALB2 D3H2LN cells 10 hours after treatment with8 Gy IR. The values plotted are mean þ SEM of two independent cultures. Statistical significance was assessed using two-way ANOVA. D, increasedsensitivity of D3H2LN cells to mechlorethamine (HN2), chlorambucil (CHL), cisplatin (CisPt), and PR-104H under aerobic conditions following doxycycline-induced knockdownofBRCA1 andPALB2 (left); increased sensitivity of D3H2LN cells to TH-302, PR-104A, SN30000, and cisplatin under hypoxic conditionsfollowingdoxycycline-induced knockdownofBRCA1 andPALB2 (right). D,HFwasdefinedas the intraexperiment quotient (IC50 Noninduced/IC50 Induced) and themeanþ SEM from four to seven independent experiments is plotted. Statistical significance of effects of BRCA1 and PALB2 knockdown on drug sensitivitywas established by comparing HF distributions for each compound in shBRCA1/shPALB2 to TRIPZ control cells using Student two-tailed t tests. �, P < 0.05;��, P < 0.01; ��, P < 0.001.

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hypersensitivity to cross-linking agents. As a furtherisogenic model of HR deficiency, we investigated aDLD-1 colorectal adenocarcinoma cell line with homozy-gous deletion of exon 11 of BRCA2 (43). These cellsdemonstrated complete loss of radiation-induced RAD51foci compared with parental DLD-1 cells (Fig. 5A), with ahighly significant difference between the two lines(Fig. 5B). Both lines were in the upper range of one-electron reductase activity as compared with the TNBCpanel for reductive activation of FSL-61 (SupplementaryFig. S11), and showed very weak expression of the aldo-keto reductase AKR1C3 (Supplementary Figs. S12 andS13) that has been shown to mediate oxygen-insensitivetwo-electron activation of PR-104A (38). BRCA2�/� cellswere 18- to 28-fold more sensitive to HN2, PR-104H,chlorambucil, and cisplatin than their isogenic counter-part under aerobic conditions (Fig. 5C), with 10- to 13-foldincreased sensitivity to TH-302, PR-104A, and cisplatinunder hypoxic conditions (Fig. 5C) without compromis-ing hypoxia selectivity of the HAP (Fig. 5D). BRCA2�/�

cells were only modestly (2-fold) more sensitive toSN30000 under normoxia and were not significantlymore sensitive under hypoxic conditions (two-wayANOVA, P ¼ 0.01 and 0.9, respectively). The absoluteIC50 values for the DLD-1 line and its BRCA2-null deriv-ativewere in the range for theTNBCcell lines investigatedabove, and for other cell lines studied in our laboratory(Supplementary Figs. S14 and S15), suggesting thatthe model recapitulates variability in HAP sensitivityobserved in wild-type cancer cell lines.To examine effects of HR derangement on antitumor

activity of HAP, we grew DLD-1 BRCA2�/� and wild-type xenografts subcutaneously in female NIH-III nudemice and used IR as a tool to distinguish the radiation-resistant hypoxic tumor fraction. IHC analysis demon-strated that both DLD-1 and DLD-1 BRCA2�/� tumorscontainpimonidazole-bindinghypoxic cell fractions char-acteristic ofmany xenograft models (Fig. 6A). To compareradiosensitivity of the two xenograft models, and toaddress this potentially confounding variable, we com-pared tumor growth following administration of a single10 Gy dose of localized external beam radiotherapy orsham irradiation (Supplementary Fig. S16). Radiotherapysignificantly delayed growth of both DLD-1 and DLD-1BRCA2�/� xenografts (log-rank tests,P¼ 0.006 and 0.003,respectively). The median time to endpoint ratio (IR/sham) was 2.8 for both models, indicating equivalentsensitivity to radiotherapy. Next, we measured steriliza-tion of clonogens in DLD-1 and DLD-1 BRCA2�/� xeno-grafts by ex vivo culturing of single cells recovered fromtumors 18 hours after treatment with a single i.p. dose ofSN30000, TH-302, or PR-104 (thewater-soluble phosphatepre-prodrug of PR-104A) at equivalent toxicity (75% ofMTD), either as monotherapy or 5 minutes after admin-istering 10 Gy whole-body radiotherapy (Fig. 6B).SN30000, TH-302, and PR-104 were all inactive as singleagents in HR-competent DLD-1 tumors (two-wayANOVA, P > 0.5). Consistent with our in vitro cytotoxicity

data, BRCA2 deletion did not significantly affect antitu-mor activity of SN30000 as a single agent (P ¼ 0.9);however, TH-302 and PR-104 had marked monotherapyactivity inHR-deficient tumors (P < 0.001 for both agents),with surviving fractions of 6� 10�3 and 1� 10�3, respec-tively. PR-104 was modestly active in DLD-1 wild-typetumors in combination with radiotherapy (one additionallog of cell killing; P > 0.001), whereas TH-302 (P¼ 0.2) andSN30000 (P¼ 0.7)were inactive in this context. Deletion ofBRCA2 dramatically increased sterilization of radiother-apy-resistant tumor cells bybothTH-302 andPR-104,withcell killing beyond the dynamic range of the assay (sur-viving fraction

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Figure 5. Genetic deletion ofBRCA2 sensitizes tumor cells toHAP in vitro. A, fluorescencemicrographs of DLD-1 andDLD-1BRCA2�/� cells fixed and stainedfor induction of nuclear RAD51 foci 8 hours after exposure to either 8 Gy IR or mock radiation. B, proportion of irradiated and unirradiated DLD-1 and DLD-1BRCA2�/� nuclei presenting with �2 RAD51 foci. Values are mean þ SEM of two independent experiments. Significance was assessed using two-wayANOVA. ��, P < 0.001. C, enhanced sensitivity of DLD-1 BRCA2�/� cells to cytotoxins under aerobic conditions (left) and to HAP under aerobic and hypoxicconditions (right). HFwas definedas the intraexperiment quotient (IC50 DLD-1/IC50 DLD-1 BRCA2�/�) and themeanþSEM from three to six independent assays isshown. D, HCR of TH-302, PR-104A, SN30000, and cisplatin in DLD-1 and DLD-1 BRCA2�/� cells in vitro. HCR was defined as the intraexperimentquotient (IC50 aerobic/IC50 hypoxic) and the mean þ SEM from three independent assays is shown.

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Figure 6. Genetic deletion ofBRCA2 markedly augmentsantitumor activity of the nitrogenmustard prodrugs TH-302 and PR-104. A, fluorescence micrographsof thin sections from DLD-1 andDLD-1 BRCA2�/� tumorsadministered pimonidazole by i.p.injection at 60 mg/kg andimmunostained 2 hours thereafterfor hypoxia. Representativeimages are shown. B, sterilizationof clonogens in DLD-1 and DLD-1BRCA2�/� tumors administeredSN30000 (155 mg/kg), TH-302(150 mg/kg), or PR-104 (578mg/kg) by single i.p. injection eitheras monotherapy (left) or 5 minutesfollowing 10 Gy IR (right). Thesedrug doses corresponded to 75%of murine MTD determinedempirically in the current study. Thesurviving fraction (SF) for eachtreatment was determined byindexing plating efficiency againstunirradiated tumors treatedwith nodrug. Log10 Cell Kill was defined as�log10(SF), and the mean þ SEMfor three to four (monotherapy) orfour to five (combination therapy)tumors is shown. Statisticalsignificance was evaluated usingtwo-way ANOVA. In thecombination setting, cell killing inthree of four tumors treated with IRþ TH-302 and four of four tumorstreated with IR þ PR-104 wasbeyond the assay limit (SF < 10�5).��, P < 0.01; ��, P < 0.001. Tumorgrowth delay (C) and Kaplan–Meiersurvival analysis (D) ofmice bearing DLD-1 or DLD-1BRCA2�/� tumors andadministered cisplatin (5.1 mg/kg)or PR-104 (578 mg/kg)monotherapy, which was 75% ofMTD in this mouse strain, by singlei.p. injection. Cohort sizes were 5(DLD-1) and 8 (DLD-1 BRCA2�/�),and mean þ SEM of tumor volumeis shown. Growth delay curveswere truncated when the firstanimal in each cohort wassacrificed for humane reasons. Thestudy endpoint for survival analysiswas defined as tumor volume �3-fold tumor volume on the day oftreatment. Statistical significanceof differences in survival wasevaluated using log-rank tests.






chronic hypoxia downregulates expression of key com-ponents of the HR machinery, offsetting chemo- andradioresistance (33, 34). Our present finding that DNAcross-linking HAP are able to exploit HR dysfunctionsimilarly to the widely used clinical cross-linkers cis-platin and chlorambucil in human tumor cell cultures(Figs. 4D and 5C), and are more active than nitroCBI orbenzotriazine di-N-oxides in this context, is consistentwith earlier studies in Chinese hamster ovary (CHO)models (30). The correlation between sensitivity to eachof the DNA cross-linking agents across cell lines sug-gests that cellular sensitivity is dominated by DNA-damage responses that are generic across these diverseagents. SN30000 and tirapazamine also show similarcell line dependence under aerobic conditions, consis-tent with the idea that replication fork arrest is a com-mon lesion across both the benzotriazine di-N-oxides(32) and cross-linking agents (27). We note that our datado not prove that compromised cross-link repair issolely responsible for the observed hypersensitivity ofHR-defective cells; higher endogenous levels of DNAlesions and a correspondingly lower threshold to exog-enous agents might also contribute.

Cross-linking agents, such as cisplatin, are increasinglyadministered as part of first-line therapy for TNBC andother HR-deficient tumors (44); however, toxicity pre-cludes dosing cisplatin above 100 mg/m2 on a conven-tional 3-weekly schedule.We showhere, for the first time,that dysfunction of HR repair analogous to that observedin BRCA-related breast and ovarian cancer drasticallyenhances antitumor activity of DNA cross-linking HAPin xenografts (Fig. 6B). This observation raises the possi-bility that HAP may provide an alternative to platinumchemotherapy, with potential to address a clinically chal-lenging subpopulation of hypoxic cells and to amelioratetoxicity by limiting exposure of well-oxygenated normaltissue to the active agent. Accordingly, we demonstratedthat PR-104 is more effective than cisplatin at inhibitinggrowth of BRCA2-null xenografts when administered atequivalent levels of toxicity to mice (Fig. 6C and D). Thisresult must be qualified by the observation that micetolerate PR-104 doses that provide higher plasma phar-macokinetics than achieved in solid tumor oncologypatients (46). Interspecies scaling of TH-302 toxicokineticsappears to be more favorable (47). Thus, our finding thatTH-302 has similar selective activity to PR-104 in BRCA2-null xenografts (Fig. 6B) suggests that it may be a bettercandidate for exploiting HR dysfunction in humancancers that, such as DLD-1, do not highly express thePR-104A–activating reductase AKR1C3.

We reasoned that the striking single-agent activity ofPR-104 and TH-302 in BRCA2-null tumors despite acti-vation being restricted to the minority hypoxic fraction(Fig. 6B) must reflect significant bystander cell killingcaused by diffusion of active metabolites into better-oxy-genated zones. This interpretation aligns with spatiallyresolved pharmacokinetic/pharmacodynamic modelingundertaken in our laboratory, which estimated such

bystander effects to contribute 30% and 50% of PR-104monotherapy activity in SiHa and HCT116 tumors,respectively (48). Interestingly, an efficient bystandereffect places central importance onHR status in normoxiccells, suggesting that dysfunction of HR through muta-tions in genes, such as BRCA1 and BRCA2, rather thansuppression of HR by hypoxia, to be the more relevanttherapeutic target. However, macroregional heterogene-ity will place some cells beyond the reach of bystandereffects, implying that HAP may be expected to offeradvantages over cisplatin only in settings where hypoxialimits therapeutic outcome, an issue that is not yet wellunderstood in breast cancer.

The finding that SN30000 has limited capacity to exploitHR dysfunction in tumors, both as a single-agent and incombination with radiation (Fig. 6B), agrees with cellculture data in the present and previous studies (30, 32)and suggests that the benzotriazine di-N-oxide class is lesssuited than cross-linkers to exploiting this target. Thelatter may reflect the lesser dependence on HR for reso-lution of lesions induced by SN30000 under hypoxicconditions, and cell entrapment of the cytotoxic-free rad-icalmetabolites of SN30000precluding efficient bystandereffects. Interestingly, SN30000 showedno activity inwild-typeDLD-1 tumors in combinationwith radiation despitesignificant antitumor activity in HT29, SiHa, H1299, andHCT116 xenografts in previous studies (28, 36, 49). Thelikely explanation for this difference is thatDLD-1 cells areintrinsically resistant to SN30000 in culture (18th mostsensitive of 21 cell lines tested; Supplementary Fig. S14).

This study has translational implications beyondTNBC. Indeed high-grade serous ovarian carcinoma mayprovide earlier opportunities to clinically evaluate theactivity of HAP in HR-deficient tumors. Platinum-taxanechemotherapy is well established as standard-of-care inthe latter indication and many patients already undergoroutine BRCAmutation testing to determine eligibility forolaparib maintenance therapy in current phase III trials(NCT01874353 and NCT01844986). We also note withinterest that a subset of pancreatic adenocarcinomas har-bor mutations in BRCA2 (50), an indication in which TH-302 is currently undergoing phase III evaluation (trialNCT01746979). Our study provides a strong rationale forexplicitly evaluating a nitrogen mustard HAP in humancancers with HR dysfunction.

Disclosure of Potential Conflicts of InterestW.R Wilson has ownership interest (including patents) in and is a

consultant/advisory boardmember for Proacta, Inc. No potential conflictsof interest were disclosed by the other authors.

Authors' ContributionsConception and design: F.W. Hunter, W.R. Wilson, J. WangDevelopment of methodology: F.W. Hunter, H.-L. Hsu, J. WangAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F.W. Hunter, H.-L. Hsu, J. Su, S.M. Pullen,J. WangAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): F.W. Hunter, H.-L. Hsu, J. WangWriting, review, and/or revision of the manuscript: F.W. Hunter, J. Su,W.R. Wilson, J. Wang

Hunter et al.





Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): F.W.Hunter, H.-L. Hsu, S.M. PullenStudy supervision: W.R. Wilson, J. Wang

AcknowledgmentsThe authors thank Dr. Michael Hay for synthesis of SN30000, tirapa-

zamine, TH-302, and FSL-61, Dr. Moana Tercel for synthesis of SN30548and SN30550, and Mr. Stephen Edgar for assistance with FACS.

Grant SupportF.W. Hunter was supported by Postgraduate Scholarship, Genesis

Oncology Trust (grant 3627392); and Health Research Doctoral Schol-arship, University of Auckland; H.-L. Hsu was supported by Project

Grant, Health Research Council of New Zealand (grant 10/459); J. Su byDoctoral Scholarship from University of Auckland; S.M. Pullen byCancer Society, Auckland; W.R. Wilson by Project Grant, HealthResearch Council of New Zealand (grant 10/459); and J. Wang byProject Grant, Health Research Council of New Zealand (grant 10/459). This research was also supported by a grant 11-1103 from theHealth Research Council of New Zealand.

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 June 4, 2014; revised August 12, 2014; accepted August 25,2014; published OnlineFirst September 5, 2014.

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Hunter et al.




2014;13:2501-2514. Published OnlineFirst September 5, 2014.Mol Cancer Ther Francis W. Hunter, Huai-Ling Hsu, Jiechuang Su, et al. Dysfunction in Triple-Negative Breast CancerDual Targeting of Hypoxia and Homologous Recombination Repair

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Dual Targeting of Hypoxia and Homologous Recombination Repair Dysfunction in … · However, HAP sensitivity was enhanced by suppres-sion of HR (assessed by radiation-induced RAD51