NF-kBRegulatesRadioresistanceMediatedByb1-Integrin inThree-Dimensional Culture of Breast Cancer Cells
Kazi Mokim Ahmed, Hui Zhang, and Catherine C. Park
Abstractb1-integrin induction enhances breast cancer cell survival after exposure to ionizing radiation (IR), but the
mechanisms of this effect remain unclear. Although NF-kB initiates prosurvival signaling pathways post-IR,the molecular function of NF-kB with other key elements in radioresistance, particularly with respectto extracellular matrix-induced signaling, is not known. We discovered a typical NF-kB–binding site in theb1-integrin promoter region, indicating a possible regulatory role for NF-kB. Using three-dimensionallaminin-rich extracellular matrix (3D lrECM) culture, we show that NF-kB is required for b1-integrintransactivation in T4-2 breast cancer cells post-IR. Inhibition of NF-kB reduced clonogenic survival andinduced apoptosis and cytostasis in formed tumor colonies. In addition, T4-2 tumors with inhibition of NF-kBactivity exhibit decreased growth in athymic mice, which was further reduced by IR with downregulatedb1-integrin expression. Direct interactions between b1-integrin and NF-kB p65 were induced in nonmalig-nant breast epithelial cells, but not in malignant cells, indicating context-specific regulation. As b1-integrinalso activates NF-kB, our findings reveal a novel forward feedback pathway that could be targeted to enhancetherapy. Cancer Res; 73(12); 3737–48. �2013 AACR.
IntroductionAside from well-known genetic and epigenetic alterations,
increasing evidence suggests that microenvironmental factorssubstantially contribute to acquired or developed cancer ther-apy resistance (1, 2). Integrins, a family of transmembrane cellsurface receptors, are composed of 18 a and 8 b subunits, thatcritically mediate cell–extracellular matrix (ECM) interactions(3). b1-integrins are aberrantly expressed in human breastcarcinomas and play multifaceted roles in determining cellfate, effecting cell survival, proliferation, apoptosis, invasion,metastasis, and tissue organization (4, 5). Similar to otherintegrin receptors, b1-integrins are overexpressed in variouscancers, including breast cancers, and have been shown tomediate tumor cell resistance to chemotherapy (6, 7) andradiation therapy (8, 9). Therefore, understanding b1-integ-rin-mediated signaling is crucial for the optimization anddevelopment of innovative chemo- and radiotherapeuticapproaches.
Several experimental models have shown the efficacy of b1-integrin inhibitors to inhibit metastasis in colon and breastmodels, refractory tumors, and advanced metastatic disease(10–12). Targeting of b1-integrin has also shown strong poten-tial to sensitize cancer cells to conventional radiotherapies andchemotherapies (13–15). In addition to preclinical studies,clinical trials evaluating b1-integrin antagonists are still ongo-ing. To date, 3 b1-integrin inhibitors have been or are beingevaluated in clinical trials: ATN-161, volociximab (M200), andJSM6427. In contrast to these anti-integrinmonotherapies thattarget specific a/b integrin heterodimers and have only beentested as monotherapy so far and with limited efficacy, AIIB2(b1-integrin function–blocking antibody), used in this study,may be more effective as it targets multiple integrins simul-taneously. However, the underlying molecular mechanismsof b1-integrin-mediated resistance to radiotherapy remainlargely unclear.
NF-kB, a stress-sensitive heterodimeric transcription factorin the regulation of the stress-responsive genes, is activatedupon phosphorylation and proteolysis of IkB or by an IkB-independent pathway (16). Tumor cells express high levels ofconstitutive NF-kB activity (17), leading to increased cellsurvival via antagonism of apoptotic pathways (18). NF-kBhas been directly implicated in the cellular resistance toradiation and chemotherapy (19). IR-induced NF-kB activityis associated with enhanced survival in human leukemic K562cells (20) and papillomavirus-transformed human keratino-cytes (21). In addition, the link between constitutive NF-kBactivity, basal apoptosis, and radiosensitivity has beenreported in breast carcinoma cell lines (22, 23). Thus, theelevated NF-kB activity identified in human tumors may leadnot only to apoptosis suppression but also to radiotherapy
Authors' Affiliation: Department of Cancer and DNADamage Responses,Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley;and Department of Radiation Oncology, University of California San Fran-cisco, San Francisco, California
Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).
Corresponding Author: Catherine C. Park, Department of RadiationOncology, Comprehensive Cancer Center, University of California SanFrancisco, 1600 Divisadero Street H1031, San Francisco, CA 94143.Phone: 415-353-7175; Fax: 415-353-9883; E-mail:[email protected]
resistance. However, the cooperative function of NF-kB withother key stress elements in radioresistance remains to beelucidated.
Recently, we discovered that a typical binding site for NF-kB was located in human b1-integrin promoter. In ourprevious studies, we have shown that downmodulation ofb1-integrin, via inhibitory monoclonal antibody AIIB2, effec-tively synergizes with IR to modify Akt-mediated radiore-sistance in breast cancer cell lines in three-dimensionallaminin-rich extracellular matrix (3D lrECM) cell culturemodel (8, 24). In addition, we showed that AIIB2 dramati-cally enhanced radiotherapy efficacy in human breast cancerxenografts (13). Thus, we hypothesized that b1-integrin-mediated resistance to radiation may be functionally regu-lated by NF-kB. The aim of the present study was toinvestigate the relationship between NF-kB and b1-integrinpathways in radiation-induced cell death in malignantbreast cells in an in vitro 3D culture and tumor growth invivo. Here, we show that upon b1-integrin inhibition, radio-sensitization is regulated by NF-kB via increased transcrip-tional activity, and a loop-like b1-integrin–NF-kB–b1-integ-rin pathway is activated post-IR. Our results suggest apromising approach to radiosensitize malignant breast can-cers by targeting NF-kB/b1-integrin pathways.
Materials and MethodsCell culture
The isogenic cell lines, nonmalignant S1 and malignantT4-2 cells, from the HMT3522 human breast cancer pro-gression series were maintained as described previously(25). The cell series was established in an attempt torecapitulate the stochastic and prolonged nature of breastcancer progression by continuously culturing S1 cells,derived from reduction mammoplasty, in the absence ofserum followed by EGF removal and injection into mice, togive rise to T4-2 cells (26, 27). The S1 cells were propagatedas monolayers on plastic in the presence of 10 mg/mL EGF(BD Biosciences); the T4-2 cells were grown as monolayerson dishes coated with collagen type I (Vitrogen 100, CeltrixLaboratories) in the absence of EGF. Three dimensional(3D) cultures were prepared by growing S1 and T4-2 cells toconfluence as monolayers, followed by trypsinization andseeded as single cells (8.5 � 105 cells/mL) at the density of2.5 � 104 cells/mL and 1.8 � 104 cells/mL, respectively, intoEHS matrices (Trevigen). Human breast cancer cell linesMDA-MB-231 and MCF-7 were obtained from the AmericanType Culture Collection, and maintained in Dulbecco'sModified Eagle Medium (DMEM/F12; UCSF Cell CultureFacility, San Francisco, CA) with 5% FBS and penicillin/streptomycin.
Apoptosis and proliferation assaysApoptosis and proliferation were detected using terminal
deoxynucleotidyl transferase-mediated dUTP nick end label-ing (TUNEL) and indirect immunofluorescence of Ki-67 nucle-ar antigen, respectively, in samples taken from 3D lrECM atday 6 (MCF-7) and 7 (T4-2 and MDA-MB-231; Figs. 2A and 4A)as described previously (28).
Confocal microscopyConfocal images were acquired by using a Zeiss LSM 710
inverted laser scanning confocal microscope equipped with anexternal argon laser. Using a Zeiss Fluor 40�W (1.3 numericalaperture) objective, images were captured at the colony mid-section. Relative immunofluorescence intensity of images wasstandardized by comparing only cultures that were processedidentically and stained in the same experiment.
Other materials and methodsRadiosensitivity assay, Western blotting, immunofluores-
cence, NF-kB DNA-binding assays, real-time PCR analysis,immunoprecipitation, in vivo caspase-3/7 activity assay, tumorinhibition assay in nude mice, immunohistochemistry stain-ings, and statistics are described in the Supplementary Infor-mation section.
ResultsNF-kB inhibition sensitizes human malignant breastcancer T4-2 cells to ionizing radiation
We have previously shown that malignant T4-2 colonies aresignificantly more resistant to radiation-induced death com-pared with nonmalignant counterpart S1 acinar structures in3D lrECM (13). We used clonogenic survival assays to verifythat malignant T4-2 cells indeed had increased reproductivecapacity compared with S1 cells post-IR (Fig. 1A). In addition,b1-integrin inhibition byAIIB2monoclonal antibody increasedradiosensitivity in T4-2 cells (Fig. 1B). It is well documentedthat NF-kB is activated by IR and plays a central role inradiation resistance (23, 29). The activity of NF-kB is largelyregulated by its subcellular localization. Using immunoflour-escence for p65 (a major subunit of NF-kB), we measured theeffect of IR on the activation of NF-kB in T4-2 and S1 cells in 2-dimensional (2D) monolayer culture. In S1 cells, 4-Gy IR rarelyinduced nuclear translocation of p65. In contrast, nuclear p65was observed in 8.6% of Sham-irradiated T4-2 cells and 59% ofT4-2 cells exposed to 4-Gy at 4 hours (Figs. 1C and D). Using amicrowell colorimetric assay, we also found that NF-kB DNA-binding activity was significantly increased in a time-depen-dent manner in T4-2, but not S1, cells after exposure to 4-Gy X-ray. To determine the binding specificity, oligos with intactbinding site for NF-kB (wild-type) or the mutated site (mutat-ed) as a competitor for p65 binding to the NF-kB consensussequence were used. The addition of a 10-fold molar excess ofthe wild-type oligo significantly competed for binding of p65 tothe NF-kB consensus sequence; however, no competition wasobserved with a 10-fold molar excess of the mutated oligo (Fig.1E). In addition, using TFBIND software, we discovered that anNF-kB–binding site (gggaggcccc; -96 to -87) was located in thehuman b1-integrin promoter region (Fig. 1F). These resultsindicated that b1-integrin–mediated radioresistance of T4-2cells is regulated by NF-kB. If NF-kB positively regulates b1-integrin, inhibition of NF-kB activity should increase radio-sensitivity. To test this, NF-kB activity was inhibited by 4-Methyl-N-(3-phenylpropyl)benzene-1,2-diamine (JSH-23) andthen colony formation assay was conducted. As shown in Fig.1B, JSH-23 resulted in a significant decrease in surviving clones.Overall, these findings show that b1-integrin is a novel target of
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NF-kB in radioresistance of breast cancer cells, but not non-malignant human mammary epithelial cells.
NF-kB activation is required for radiation-induced b1-integrin overexpressionIn a 3D laminin-rich ECM (lr-ECM)–based cell culturemodel
(Fig. 2A; experimental schema), which better mimics physio-logic growth conditions than 2D cultures (8, 30–32), malignantbreast cancer T4-2 cells showed higher levels of total andphospho (T788/789) b1-integrins compared with its counter-part nonmalignant S1 cells (Fig. 2B). These protein levels werefurther induced in T4-2 cells post-IR (4-Gy X-ray; Fig. 2C).Interestingly, treatment of T4-2 colonies, formed at day 4, with
NF-kB inhibitors JSH-23 or IMD-0354, resulted in a decrease inphospho and total b1-integrins, which was further reduced byIR (Fig. 2C and Supplementary Fig. S1A). At 30 mmol/L con-centration of JSH-23, which selectively blocks nuclear trans-location of NF-kB p65 and its transcription activity, bothphospho and total b1-integrins were significantly reducedindependently of radiation exposure. (Fig. 2C). IMD-0354, anIKK-b inhibitor that blocks IkB-a phosphorylation and NF-kBavailability, also significantly reduced phospho and total b1-integrins by 5 mmol/L and 10–20 mmol/L, respectively, with orwithout IR (Supplementary Fig. S1A). Similarly, IR-inducedincreases in b1-integrin mRNA expression were significantlyreduced by JSH-23 in a dose-dependent manner (Fig. 2D). To
Figure 1. Inhibition of NF-kB or b1-integrin increased radiosensitivity inmalignant breast cancer T4-2 cells. Aand B, T4-2 malignant breast cellsand its counterpart nonmalignant S1breast epithelial cells were leftuntreated (A) or T4-2 cells weretreated with NF-kB activationinhibitor JSH-23 (5 mmol/L) or b1-integrin inhibitory antibody AIIB2 (0.1mg/mL; B) before exposure to 1-, 2-,4- or 8-Gy X-ray. Clonogenic survivalwas measured 14 days after IR.Colonies consisting of more than 50cells were scored as survivingcolonies and normalized againstnonirradiated clones (n ¼ 3, mean �SD). C and D, immunofluorescenceanalysis of NF-kBp65 in S1 and T4-2cells exposed to sham or 4-Gy IR.Arrows indicate nuclear p65. Scalebar, 200 mm (C). Graphicalrepresentation of the p65-positivenuclei. A representative experimentfrom n ¼ 3 is shown (D). E, NF-kBDNA-binding assay using nuclearextracts of S1 and T4-2 cellsexposed to shamor 4-Gy IR. Data aremean � SD of pooled resultsfrom 3 independent experiments;��,P < 0.01. F, an NF-kB–binding siteis identified in the promoter region ofb1-integrin gene.
confirm inhibition of b1-integrin activity, phosphorylation ofFAK397, a critical indicator of downstream b1-integrin signal-ing, was measured. As shown in Supplementary Fig. S1B,inhibition of b1-integrin by NF-kB inhibitor JSH-23 signifi-cantly reduced the levels of total and phosphorylated FAK.These results clearly show that IR-induced b1-integrin expres-sion and its downstream signaling are mediated by NF-kB.Similar to b1-integrin, protein levels of p65/p50, the majorinducible NF-kB dimer, were also higher in T4-2 than S1 cells
(Fig. 2B).Wepreparedwhole-cell lysates for immunoblotting ofp65 and p50 to determine whether NF-kB transactivationfollowing IR was associated with an increase in p65/p50 levels.Compared with Sham (0 Gy) IR control cells, a clear increase inp65/p50 was observed post-IR; in addition, these levels weredecreased by JSH-23 in a dose-dependent manner (Fig. 2E).Together with Fig. 2C, these results indicate that NF-kB andb1-integrin are components of a coordinated response toradiation.
Figure 2. Radiation-induced b1-integrin expression in 3D lrECM inhuman breast cancer T4-2 cellswas mediated by NF-kB throughincreased transcriptional activity.A, experimental schema. T4-2colonies andS1acini formed at day4 and 6, respectively, were treatedwith NF-kB activation inhibitorJSH-23 or vehicle (DMSO), andthen exposed to sham or 4-GyX-ray at day 5 (T4-2) and 7 (S1).Whole-cell lysates were prepared48 hours post-IR for Western andNF-kB DNA-binding assays. B,Western blot analyses on theexpression of phospho and totalb1-integrins and p65/p50 (majorheterodimer of NF-kB) using wholelysates prepared from malignantbreast cancer T4-2 andnonmalignant S1 breast epithelialcells at day 7 and 9, respectively, in3D lrECM. b-actin serves as aninternal loading control. C and E,Western blot analyses of phosphoand total b1-integrins (C), and p65and p50 (E) in T4-2 cells treatedwith different concentrations ofJSH-23 and exposed to sham or4-Gy X-ray. The lysates were alsoblotted for b-actin as an internalloading control. Right, relativeexpression levels of phospho-b1-integrin (C) and NF-kB p65/p50 (E)normalized to the expression levelsof b-actin. D, quantitative RT-PCRanalysis of b1-integrin mRNAexpression in T4-2 cells treated asabove. Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) serves as an internalcontrol. F, NF-kB DNA-bindingassay using whole-cell lysates ofT4-2 and S1 cells exposed to shamor 4-Gy X-ray as shown in theschematic diagram in A. The 50 bpb1-integrin promoter regioncontaining wild-type or mutatedNF-kB–binding sequence wassynthesized and used as a DNAprobe to assess the binding. Dataare mean � SD of pooled resultsfrom 3 independent experiments;��, P < 0.01.
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Next, we asked whether transcriptional activity directedthrough NF-kBwas involved in the upregulation of b1-integrinpost-IR. To determine this, we conducted an ELISA-basedDNAbinding assay in 3D lrECM to quantify the binding of NF-kB toits binding site identified in the b1-integrin promoter (Fig. 1F).A 50 bp oligo containing the kB binding site on the b1-integrinpromoter region was synthesized and then used as a DNAprobe to assess binding of IR-activated NF-kB in T4-2 and S1cells. As shown in Fig. 2F, NF-kB DNA-binding activity wasmuch higher (�4-fold) in T4-2 comparedwith S1 cells and 4-GyX-ray significantly increased binding activity. However, thebinding activities were not induced with or without radiationwhen a 50 bp DNA probe containing a mutation in the kB sitewas used. To test the generalizability of these results, theinduction of DNA binding activity of NF-kB observed in T4-2 cells post–4-Gy X-ray was confirmed in another cell line(Supplementary Fig. S1B). Similar to T4-2 cells, binding activ-ities were significantly increased (�3.8-fold) post-IR in humanmalignant breast cancer MDA-MB-231 cells. As expected, thisinduction of DNA binding of NF-kB was abolished when themutated probe was used. Together, these data identified b1-integrin as a downstream transcriptional target of NF-kB in 3DlrECM T4-2 cell cultures.
Inhibition of NF-kB activity reduces colony size viaincreasing apoptosis and decreasing proliferation inmalignant breast cancer colonies in 3D lrECMWe showed previously that inhibition of b1-integrin by
monoclonal antibody AIIB2 significantly increased TUNEL-positive nuclei and decreased Ki-67–positive nuclei in a panelof malignant breast cancer colonies (28). Figure 2 shows thatNF-kB positively regulates b1-integrin, which suggests thatNF-kB inhibition may reduce the colony size via increasingapoptosis and decreasing proliferation in malignant breastcells. To test this hypothesis, malignant T4-2 colonies andnonmalignant S1 acini on top of 3D lrECM gels with 5%Matrigel formed at day 4 and 6, respectively, were treated withNF-kB inhibitor JSH-23 and then exposed to sham or a singledose of 4-Gy X-ray 24 hours after treatment. Upon NF-kBinhibition, T4-2 colony size was significantly reduced at 48hours. IR further reduced the size in a dose-dependent manner(data for 20 and 30mmol/Lwere not shown; Figs. 3A andB). Themaximum size difference was reached in colonies treated with40 mmol/L JSH-23 and exposed to IR, associated with a sub-stantial approximately 40% decrease in the average colonydiameter. In contrast, the average diameter of S1 acini was onlymarginally reduced by 20–40 mmol/L JSH alone, and no furtherreductionwas observed by the addition of IR (data for 10 and 30mmol/L were not shown; Supplementary Figs. S3A and S3B).Next, to correlate the decrease in colony size by JSH-23 with
apoptosis and proliferation in malignant breast cells, immu-nofluorescence (IF) staining for TUNEL and caspase-3/7 activ-ity assay, and IF staining for Ki-67 nuclear antigen, respectively,were conducted. Consistent with the decrease in size of T4-2colonies by JSH-23 as described above, TUNEL-positive nucleiwere significantly increased in sham-treated colonies and afurther induction was observed after IR exposure (Figs. 3A andC). At the concentration of 40 mmol/L JSH, a 5.8 times increase
in TUNEL-positive nuclei was observed comparedwith controlcells post-IR (8% versus 49%; Fig. 3C). To verify IR- and JSH-23–induced apoptosis, we conducted a caspase-3/7 activity assay(Fig. 3D). A 9-fold induction of caspase activity was observed byJSH-23 (40 mmol/L) alone, which was further increased with IR.The combined treatment of JSH-23 or b1-integrin inhibitoryAIIB2 monoclonal antibody with IR was associated with thehighest caspase activity compared with either single agentused alone. In addition, we analyzed the effect of caspaseinhibition by applying the pan-caspase inhibitor Z-VAD-FMKand found that Z-VAD-FMK reversed apoptosis mediated byNF-kB inhibition with or without IR, further supporting thefinding that NF-kB inhibition induces apoptosis of T4-2 cells.In addition, the induction of apoptosis by JSH-23�IR wasassociated with a decrease in proliferation. A significantdecrease in the percentage of Ki-67–positive cells with JSH-23 treatment was observed in sham IR cells, which was furtherreduced by IR in a clearly dose-dependent manner (data for 20and 30 mmol/L were not shown; Figs. 3A and E). Themaximumreduction of proliferation was observed in T4-2 colonies trea-ted with 40mmol/L JSH-23 compared with control cells post-IR(33% versus 11%; Fig. 3E). Together with Fig. 2, these resultsprovide strong evidence that b1-integrin–induced resistanceto IR is regulated by NF-kB.
To test whether the increase in radiosensitivity by NF-kBinhibition is a commonmechanism for other malignant breastcancer cell lines, MDA-MB-231 andMCF-7 cells were seeded in3D lrECM (experimental schema is shown in Fig. 4A) toexamine the percentage of TUNEL- and Ki-67–positive cellswith JSH-23 treatment with and without IR. As shown in Figs.4B and D, the percentage of TUNEL-positive cells was signif-icantly increased in both MDA-MB-231 and MCF-7 cells andfurther induced post-IR.We did find a differential sensitivity toJSH-23 among the cell lines. Notably, the level of apoptosis waslower inMCF-7 cells thanT4-2 (34% versus 49%) andMDA-MB-231 (34% vs. 44%) cells after treatment with 40 mmol/L JSH-23post-IR, indicating less relative sensitivity in this cell line. Toreach the percentage of apoptosis in MCF-7 cells that wassimilar to T4-2 or MDA-MB-231, a higher concentration ofJSH-23 was required (Figs. 3C and 4B and D). Similar dose-dependent findings were reflected in the degree of cytostaticresponse, measured by the percentage of Ki-67–positive cells(Figs. 4C and E). Although we treated MCF-7 cells with higherconcentrations of JSH-23 (60 and 80mmol/L), the percentage ofKi-67–positive cells remained higher than T4-2 and MDA-MB-231 cells post-IR (Figs. 3E and 4C and E). These results indicatethat b1-integrin may be differentially regulated by NF-kBamong different malignant breast lines.
Interactions of b1-integrin with NF-kB p65 anda5-integrin are oppositely regulated in S1 and T4-2cells post-IR
NF-kB physically and/or functionally interacts with manyproteins involved in cell proliferation and survival (23). Inlight of our current results that IR-induced b1-integrin isregulated by NF-kB, we used immunoprecipitation to testthe hypothesis that NF-kB physically interacts with b1-integrin. This showed that NF-kB p65 was able to interact
with b1-integrin in sham-irradiated S1 and T4-2 cells in 3DlrECM (Supplementary Figs. S4A and S4B). Because theprotein levels of b1-integrin and p65 were high in T4-2 andfurther induced post-IR compared with S1 cells (Figs. 2B, C,and E), we expected that IR would increase b1-integrin/p65interaction in T4-2 than S1 cells. However, we found that theinteraction was strikingly absent post-IR in relatively resis-tant T4-2 cells and was enhanced in nontumorigenic S1 cells(Supplementary Figs. S4A and S4B; marked with a box).
Together with Figs. 1A and B, these results indicate thatNF-kB may inhibit b1-integrin function via physical inter-action to increase radiosensitivity in nontransformed cells,but not in malignant cells. As we have shown previously (8),the interaction of b1-integrin with one of its heterodimericpartners a5-integrin was almost absent in S1, but very highin T4-2, cells without radiation. Although IR slightly inducesa5b1-integrin complex in both S1 and T4-2 cells, the level ofinteraction was much higher in T4-2 than S1 cells post-IR
Figure 3. Inhibition of NF-kBactivityin T4-2 cells resulted in decreasedcolony size and cell proliferation,and increased apoptosis in 3DlrECM culture. A, colony size,apoptosis (TUNEL), andproliferation (detected by indirectimmunofluorescence of Ki-67nuclear antigen) of T4-2 cellstreated with JSH-23 (40 mmol/L),and then exposed to sham or 4-GyX-ray as shown in the schematicdiagram in Fig. 2A. Phase-contrastphotographs of the colonies weretaken to measure the diameter of100 colonies with or without IRusing Spot Advanced software;Scale bar, 100 mm (top). T4-2 cellsmears fixedontoglass slideswereprepared from the colonies at day 7for apoptosis (middle) andproliferation (bottom) assays.Arrows indicate positive nuclei forTUNEL (red) and Ki-67 (green),respectively. Scale bar, 50 mm. B,graphical representation of therelative diameter of T4-2 colonies;P < 0.01 (��). C and E, graphicalrepresentation of the TUNEL (C)and Ki-67–positive cells (E).Columns, mean (n¼ 3); bars, SD; P< 0.001 (���); P < 0.01 (��). D, theactivation of caspase-3/7,considered a reliable marker forcells undergoing apoptosis, washighly induced by NF-kB inhibitionpost-IR. T4-2 cells treated withJSH-23, AIIB2, or JSH-23þAIIB2with or without pan-caspaseinhibitor Z-VAD-FMK wereexposed to sham or 4-Gy X-ray(schematic diagram is shownin Fig. 2A), single cells weredissociated from lrECM andcaspase-3/7 activity wasmeasured by Caspase-Glo 3/7Assay Kit. Asterisks indicatesstatistically significant difference(��, P < 0.01) compared withDMSO-treated cells; columns,mean (n ¼ 3); bars, SD.
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(Supplementary Fig. S4C; marked with arrows). Togetherwith Supplementary Figs. S4A and S4B, these results suggestthat the induction of physical interaction of b1-integrin witha5-integrin, but not with p65, promotes radioresistance inmalignant breast cancer cells in 3D lrECM. The exact mech-anism underlying the interaction of b1-integrin with p65 anda5-integrin in radiosensitivity is the subject of ongoinginvestigations.
Inhibition of NF-kB activity results in tumor growthinhibition in vivo associated with downregulated b1-integrin expressionTo test whether the in vitro observations in 3D lrECM
culture could be validated in vivo, we examined tumorgrowth from mice injected with vehicle or NF-kB–inhibitedT4-2 cells exposed to sham or 4-Gy X-ray. As shown in thetumor growth curves (Figs. 5A and B), NF-kB inhibition by
20 mmol/L of JSH-23 in T4-2 cells (T4þJSH) significantlydelayed the growth of tumors relative to control in athymicmice. We also found that tumors exposed to IR post-JSH-23treatment (T4þJSHþIR) grew dramatically slower than con-trol. The significant suppression of tumor formation wasobserved at day 13 after a subcutaneous injection of T4þJSHor T4þJSHþIR cells and continued till the day of sacrifice(day 28).
We next tested the expression levels of b1-integrin and NF-kB p65 by immunohistochemical staining (Fig. 5C). As seen inthe representative tissue sections, b1-integrin was reducedin T4þJSH tumors and a severe reduction was observed intumors formed by T4þJSHþIR. Similarly, total and nuclear NF-kB p65 were also reduced in T4þJSH and a further reductionwas observed in T4þJSHþIR tumors. Then, Western blotanalysis of tumor sections was conducted to further determinethe expression levels of b1-integrin and NF-kB. The Western
Figure 4. Inhibition of NF-kBactivityresulted in increased apoptosis anddecreased proliferation in MDA-MB-231 and MCF-7 cells in 3D lrECM. A,experimental schema. B–E,apoptosis (B and D) and proliferation(C and E) levels of MDA-MB-231 andMCF-7 colonies in 3D lrECM usingTUNEL and Ki-67 staining. MDA-MB-231 andMCF-7 colonies formedat day 3 and 4, respectively, weretreated with NF-kB activationinhibitor JSH-23 or vehicle (DMSO),and then exposed to shamor 4-Gy X-ray at day 4 (MDA-MB-231) and 5(MCF-7). Smears were fixed ontoglass slides 48 hours post-IR.Columns, mean (n ¼ 3); bars, SD;�,P < 0.05; ��,P < 0.01; ���,P < 0.001.
data also revealed a decrease in phospho and total b1-integrinstogether with p65/p50 in NF-kB–inhibited T4-2 tumors, whichwas further reduced by IR (Fig. 5D). The results of theseexperiments show that NF-kB inhibition in T4-2 cells with orwithout IR decreases the tumor growth via inhibition of b1-integrin expression in athymic mice. Together with Figs. 2C–F,the data in Fig. 5 strongly suggest that b1-integrin expression isregulated by NF-kB.
Inhibition of b1-integrin reversed IR-induced NF-kBprotein levels and DNA binding activity in 3D lrECM
The functional role of integrin-induced NF-kB in cell sur-vival was first showed by Scatena M and colleagues in a5b3-integrin-mediated endothelial cells (33). To determinewhetherb1-integrin function blocking monoclonal antibody AIIB2 wasable to inhibit IR-inducedNF-kBp65/p50 expression andDNA-binding activity, immunoblotting for p65 and p50, and anELISA-based DNA-binding assay were conducted in T4-2 cellsin 3D lrECM. As shown in Fig. 6A, IR-induced p65/p50 proteinlevels were significantly reduced by 0.2mg/mLAIIB2, suggesting
that NF-kB is a downstream target of b1-integrin. Inhibition ofb1-integrin also significantly reduced IR-induced NF-kB DNA-binding activity (Fig. 6B). Together with Figs. 1B and 2C and F,these results suggest a loop-like b1-integrin–NF-kB–b1-integ-rin pathway is activated post-IR to induce radioresistancein T4-2 cells in 3D lrECM (schematic presentation is shownin Fig. 6C).
DiscussionCancer cells dynamically interact with their microenvi-
ronment to remodel the surrounding stroma and facilitategrowth and invasion. Tumor ECM has been shown toincrease resistance to cytotoxic cancer therapy includingradiation (6, 7) and has also been associated with pooroutcomes in subgroups of patients with breast cancer(34). However, the underlying basis for ECM-mediated resis-tance to therapy, particularly after radiation, has not beenwell studied. We have previously shown that targeting b1-integrin leads to selective apoptosis and cytostasis in breastcancer cells in vivo without toxicity. In addition, a number of
Figure 5. NF-kB inhibition by 20mmol/L JSH-23 in T4-2 cellsdelayed tumor growth in nudemicevia inhibition of b1-integrinexpression.A, tumor growthcurvesobtained following subcutaneousinjection of T4-2 cells treated withvehicle (DMSO) or JSH-23 withsham (T4þJSH) or 4-Gy X-ray(T4þJSHþIR) in female NCR nudemice (nu/nu). Eight animals wereused per group and the datarepresent the mean � SE;��P < 0.01; ���, P < 0.001.B, whole tumors excised frommice injected with T4þJSH,T4þJSHþIR, or control cells. C,immunohistochemical staining ofthe tumor sections from T4þJSH,T4þJSHþIR, or control mice usingNF-kB p65 or b1-integrin antibody.After the immunoreaction, sectionswere counterstained withhematoxylin. Nuclear p65 and b1-integrin are indicated by yellow andwhite arrows, respectively. Imageswere captured at �1,000magnifications; scale bar, 50 mm.D, phospho and total b1-integrinsand NF-kB p65/50 expressions incontrol, T4þJSH and T4þJSHþIRmouse tumors detected byWestern blot analysis. Lamin A/Cserves as an internal control.
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studies have shown that b1-integrins regulate radiation-induced prosurvival signaling, leading to increased surviv-ability and reproductive capacity of human cancer cellsexposed to ionizing radiation (8, 9, 13, 14, 28). Inthe present study, we wished to further dissect the possiblemolecular mechanisms associated with b1-integrin regula-tion of survival in irradiated cancer cells. We discovered thata typical NF-kB–binding site was located in the promoterregion of the b1-integrin gene (Fig. 1F). Given the known roleof NF-kB in mediating an acute phase stress response to IR,we sought to further investigate a potential novel relation-ship between ECM-directed signaling and b1-integrin-medi-ated survival. Here, we show that NF-kB directly modifiesb1-integrin expression, in part through transcriptional acti-vation (Figs. 2D and F). This is associated with increasedsurvival post-IR, and sensitization of cells to IR-induced
death upon inhibition of NF-kB (Figs. 1B and 3C and D).This pathway is functional in several breast cancer cell lines,and notably oppositely regulated in normal human mam-mary epithelial cells (Supplementary Fig. S3). Finally, inhi-bition of b1-integrin reverses NF-kB transcriptional activity(Fig. 6B), suggesting a loop-like b1-integrin–NF-kB–b1-integrin regulatory pathway in malignant, but not normalcells.
b1-integrin signaling has been shown to mediate diverseroles in cancer progression including invasion, migration, andmetastasis (35, 36). In addition, b1-integrins mediate resis-tance to cytotoxic chemotherapy and radiation in severalhuman cancers (6–9). In addition, we have previously shownthat the combination of b1-integrin inhibitory antibodies withIR significantly reduced the dose that was necessary to achievethe same growth inhibitory effect in breast cancer xenografts
Figure 6. Inhibition of b1-integrinreversed IR-induced NF-kBexpression and DNA-binding activityin 3D lrECM. A, Western blotanalyses on the expression of NF-kBp65/p50 and b1-integrin usingwhole-cell lysates prepared frommalignant breast cancer T4-2 cells atday 7 in 3D lrECM as shownin Fig. 2A. b-actin serves as aninternal loading control. Right,relative expression levels of NF-kBp65/p50 normalized to theexpression levels of b-actin. B,NF-kB DNA-binding assay usingwhole-cell lysates prepared fromT4-2 cells as above. Data aremean�SD of pooled results from 3independent experiments (n ¼ 3);��, P < 0.01. C, schematicrepresentation of radiation-inducedloop-like b1-integrin–NF-kB–b1-integrin pathway in radioresistanceofmalignant breast cells in 3D lrECM.
in vivo, related to regulation of Akt signaling downstreamofb1-integrin (8, 13). Cordes and colleagues showed a FAK/cortac-tin-dependent regulation directly linked of b1-integrin-medi-ated survival post-IR in squamous cell carcinoma of thehead and neck (9). Thus, increasing evidence indicates thatinhibiting b1-integrins enhance the therapeutic efficacy ofradiotherapy through critical signaling pathways. However,modulation of gene expression is one of the most importantevents because it directly controls cellular adaptation togenotoxic conditions; yet little is known regarding the poten-tial transcriptional mechanisms involved in the b1-integrin-dependent survival pathway of cancer cells after IR treatment.Here, we show that IR induces p65 translocation into thenucleus (Figs. 1C and D) and concomitantly increases NF-kBbinding to the b1-integrin promoter region (86 bp upstream oftranscription initiation site; Fig. 1E), suggesting that IR-induced NF-kB activity transcriptionally upregulates b1-integrin.
To elucidate the functional consequences of this interac-tion, we measured therapeutic endpoints after inhibition ofNF-kB using JSH-23. We used a clonogenic assay to showthat reproductive capacity was significantly reduced in JSH-23-treated malignant breast cells after IR (Fig. 1B). Inaddition, as we have previously shown that using 3D lrECMaccurately predicted response between normal and malig-nant cells in vivo, we tested JSH-23 in this context. As shown,colony size and proliferation were dramatically reducedwhile apoptosis was increased in T4-2, but not S1, cells,consistent with the role of NF-kB in positively regulating b1-integrin post-IR in malignant, but not nonmalignant breastcells (Fig. 3 and Supplementary Fig. S3). To determine thebroader applicability of our findings, we tested luminal-like(MCF7) and basal-like (MDA-MB-231) breast cancer celllines for response to JSH-23 and IR. Interestingly, JSH-23highly induced apoptosis and reduced proliferation in basal-like breast cancers T4-2 and MDA-MB-231 compared withluminal-like breast cancer MCF-7 cells (Figs. 3C–E and 4),indicating that basal-like breast cancers may be moreresponsive to b1-integrin/NF-kB–targeted therapies. Yama-guchi N and colleagues reported that basal-like breastcancer cell lines that exhibited higher activation of NF-kBthan luminal subtypes are preferentially involved in prolif-eration (37). Together, these results are consistent with ourprevious findings that b1-integrin levels are much higher inbasal compared with luminal cell lines, consistent with ahigher dependence on integrin-related prosurvival signaling(8, 13, 28).
It has been well documented that the stress conditionsinduced by radiation can activate a cellular defense system,including NF-kB, the acute phase transcription factor thataffects the decision of cell fate after IR. NF-kB mediates thesurvival response of many signals by inhibiting p53-depen-dent apoptosis and upregulating antiapoptotic members ofthe Bcl-2 family, and caspase inhibitors such as XIAP andFLIP (29, 38). In addition to antiapoptotic responses, NF-kBregulates expression of the stress-responsive genes associ-ated with a prosurvival network (39, 40). Inhibition of NF-kBactivity increases the intrinsic radiosensitivity in several
(41, 42), but not all, human cancer cell lines (43). Thisapparent paradox indicates that IR-induced NF-kB subunitsmay cross-talk with other signaling elements and/or mayhave distinct tissue-specificity. To further investigate therelationship between b1-integrins and NF-kB, we provideevidence for components of the regulatory network showingthat phosphorylation of T788/789 of the b1-integrin cyto-plasmic tail were decreased by NF-kB inhibitors (JSH-23 andIMD-0354), along with total b1-integrins and the activecomponents of the NF-kB p65/p50 subunits (Figs. 2C andE and Supplementary Fig. S1A).
This study also shows interactions ofb1-integrinwithNF-kBp65 and one of its heterodimeric partnersa5-integrin. Protein–protein interactions can have positive implications for cancerprevention and therapy. The studies of Vassilev and colleagues(44, 45) showed in vivo proof-of-principle that inhibitors ofprotein–protein interactions can be efficacious anticancerdrugs. NF-kB physically and/or functionally interacts withmany proteins, including MEK, E2F1 transcription factor, andPML tumor suppressor, involved in the controlling of cellproliferation and survival (23, 46, 47). In the present study, wehave shown that the interactions ofb1-integrinwithNF-kBp65and a5-integrin were oppositely regulated in malignant breastcancer T4-2 and its counterpart radiosensitive nonmalignantbreast epithelial S1 cells. As shown in Supplementary Fig. S4,b1-integrin/p65 protein complex significantly increased post-IR in S1, but not T4-2, cells, indicating that NF-kB may protectS1 cells against radiation damage via physical interaction withb1-integrin. Furthermore, IR-induced a5b1-integrin complexwas much higher in T4-2 than S1 cells (Supplementary Fig.S4C), suggesting that the induction of b1-integrin interactionwitha5-integrin promotes radioresistance ofmalignant breastcancer T4-2 cells.
Another important implication of the current results isthe loop-like activation pathway of NF-kB/b1-integrin sig-naling in breast cancer radioresistance. It is well documen-ted that NF-kB is activated by radiation (23, 29), and NF-kBis now shown to bind directly to the b1-integrin promoter,resulting in b1-integrin overexpression and tumor radio-resistance. In addition, several previous studies have corre-lated NF-kB activation with integrin ligation. The functionalrole of integrin-induced NF-kB in cell survival was firstshown by Scatena and colleagues (33). In Figs. 6A and B,we showed that IR-induced NF-kB p65/p50 expression andDNA binding activity were inhibited by b1-integrin functionblocking monoclonal antibody AIIB2. This feed-forwardloop-like b1-integrin–NF-kB–b1-integrin pathway activatedpost-IR may cause tumor resistance. A schematic presenta-tion of the b1-integrin–NF-kB–b1-integrin loop in radio-resistance is proposed in Fig. 6C. We speculate that activa-tion of this pathway results in the failure of DNA-damaginganticancer modalities in breast cancers.
In summary, we report here a novel finding that b1-integrin is induced by exposure to radiation through NF-kB–mediated gene activation in 3D lrECM breast cancer cellculture and breast cancer xenografts. NF-kB–mediated b1-integrin overexpression is tightly associated with enhancedclonogenic survival and tumor repopulation. Our results
Ahmed et al.
Cancer Res; 73(12) June 15, 2013 Cancer Research3746
suggest that breast cancer therapy may be enhanced bytargeting the NF-kB/b1-integrin pathway of radiation-resis-tant tumors.
Disclosure of Potential Conflicts of InterestC.C. Park has ownership interest (including patents) in Oncosynergy. No
potential conflicts of interest were disclosed by the other authors.
Authors' ContributionsConception and design: K.M. Ahmed, C.C. ParkDevelopment of methodology: K.M. Ahmed, H. Zhang, C.C. ParkAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K.M. AhmedAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): K.M. AhmedWriting, review, and/or revision of the manuscript: K.M. Ahmed, C.C. Park
Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K.M. Ahmed, H. ZhangStudy supervision: K.M. Ahmed, C.C. Park
AcknowledgmentsThe authors thank Christopher Pham for technical assistance.
Grant SupportThis work was supported by NIH grant 1R01CA124891 to C.C. Park.The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.
Received September 11, 2012; revised March 20, 2013; accepted April 2, 2013;published OnlineFirst April 10, 2013.
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2013;73:3737-3748. Published OnlineFirst April 10, 2013.Cancer Res Kazi Mokim Ahmed, Hui Zhang and Catherine C. Park Three-Dimensional Culture of Breast Cancer Cells
1-Integrin inβB Regulates Radioresistance Mediated By κNF-
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