suppression of nuclear factor-kbby glucocorticoid receptor ...ment of aromatase...

Patient Focused Therapies in Breast Cancer

Suppression of Nuclear Factor-kB byGlucocorticoid Receptor BlocksEstrogen-Induced Apoptosis inEstrogen-Deprived Breast Cancer CellsPing Fan1, Doris R. Siwak2, Balkees Abderrahman1, Fadeke A. Agboke3,Smitha Yerrum1, and V. Craig Jordan1

Abstract

Our clinically relevant finding is that glucocorticoids blockestrogen (E2)-induced apoptosis in long-term E2-deprived(LTED) breast cancer cells. However, the mechanism remainsunclear. Here, we demonstrated that E2 widely activatedadipose inflammatory factors such as fatty acid desaturase1 (FADS1), IL6, and TNFa in LTED breast cancer cells.Activation of glucocorticoid receptor (GR) by the syntheticglucocorticoid dexamethasone upregulated FADS1 and IL6,but downregulated TNFa expression. Furthermore, dexa-methasone was synergistic or additive with E2 in upregulat-ing FADS1 and IL6 expression, whereas it selectively andconstantly suppressed TNFa expression induced by E2 inLTED breast cancer cells. Regarding regulation of endoplas-mic reticulum stress, dexamethasone effectively blockedactivation of protein kinase RNA-like endoplasmic reticulumkinase (PERK) by E2, but it had no inhibitory effects

on inositol-requiring protein 1 alpha (IRE1a) expressionincreased by E2. Consistently, results from reverse-phaseprotein array (RPPA) analysis demonstrated that dexameth-asone could not reverse IRE1a-mediated degradation ofPI3K/Akt-associated signal pathways activated by E2.Unexpectedly, activated GR preferentially repressed nuclearfactor-kB (NF-kB) DNA-binding activity and expression ofNF-kB–dependent gene TNFa induced by E2, leading to theblockade of E2-induced apoptosis. Together, these data sug-gest that trans-suppression of NF-kB by GR in the nucleusis a fundamental mechanism thereby blocking E2-inducedapoptosis in LTED breast cancer cells. This study providedan important rationale for restricting the clinical use ofglucocorticoids, which will undermine the beneficial effects ofE2-induced apoptosis in patients with aromatase inhibitor–resistant breast cancer.

IntroductionAntihormone therapy is a standard treatment of estrogen

receptor (ER)-positive breast cancer (1). However, resistance tothis therapy is an inevitable challenge in the clinic. Paradoxically,estrogen (E2) has the capacity to induce apoptosis in antihormonetherapy–resistant breast cancermodels in vitro and in vivo (2–5). Infact, E2-induced apoptosis has clinical significance for the treat-ment of aromatase inhibitor–resistant breast cancer (6) andreduction of breast cancer incidence in estrogen replacementtherapy (ERT) for postmenopausal women (7). Further clinicallyrelevant laboratory findings suggest that the anti-inflammatory

agent, dexamethasone, and the synthetic progestin medroxypro-gesterone acetate (MPA), which has glucocorticoid activity, canblock E2-induced apoptosis in long-term E2-deprived (LTED)breast cancer cells (8). However, antiapoptotic mechanisms ofglucocorticoids are unknown.

Long-termE2deprivation is a selective pressure onbreast cancercell lines (9), as well as for patients during antihormone thera-py (10), which results in stress responses for adaptation to the E2deficiency (10, 11). In addition to elevation of ERa expres-sion (4, 5), many signaling pathways, including metabolism,stress, and inflammatory responses, are modulated after E2 dep-rivation (10, 11). Notably, all of these alterations result inapoptosis in response to E2, instead of proliferation (4, 5). It isconfirmed that nuclear ERa is an initial site for E2 to induceapoptosis in LTED breast cancer cells, which can be completelyblocked by the tamoxifen (12). Our further observations dem-onstrate that accumulation of stress responses, including endo-plasmic reticulum stress, oxidative stress, and inflammatorystress, is a major mechanism by which E2 induces apopto-sis (12, 13). Particularly, endoplasmic reticulum is a criticalregulatory site for conveying signals between the nucleus andcytoplasm to decide the cell fate (12, 14, 15). The endoplasmicreticulum stress sensor, protein kinase RNA-like endoplasmicreticulumkinase (PERK) is responsible for homeostasis of unfold-ed proteins and is also a key driver of E2-induced apopto-sis (12, 14, 15). Specifically, PERK links endoplasmic reticulumstress with oxidative stress and increases transcription factor

1Department of Breast Medical Oncology, The University of Texas MD AndersonCancer Center, Houston, Texas. 2Department of Systems Biology, The Universityof Texas MD Anderson Cancer Center, Houston, Texas. 3Department of Oncol-ogy, Lombardi Comprehensive Cancer Center, Georgetown University,Washington, D.C.

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

Corresponding Author: V. Craig Jordan, The University of Texas MD AndersonCancer Center, 1515 Holcombe Blvd., Houston, TX 77030. Phone: 713-745-0600;Fax: 713-794-4985; E-mail: [email protected]

Mol Cancer Ther 2019;18:1684–95

doi: 10.1158/1535-7163.MCT-18-1363

�2019 American Association for Cancer Research.

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NF-kB DNA-binding activity to induce TNFa in E2-deprivedbreast cancer cells (12, 15, 16). Our recent findings havedemonstrated that the PERK/NF-kB/TNFa axis plays a criticalrole in E2-induced apoptosis (15, 16). In parallel, two otherendoplasmic reticulum stress sensors, inositol-requiring pro-tein 1 alpha (IRE1a) and ATF-6, mainly mediate endoplasmicreticulum-associated degradation of PI3K/Akt-associated signal-ing pathways (14). These different functions of endoplasmicreticulum stress sensors suggest that abnormal protein foldingand lipid metabolism occur after treatment with E2.

Furthermore, stress responses widely activate inflammatoryfactors, such as IL6, FADS1, and TNFa, in LTED breast cancercells after treatment with E2 (12, 13). Glucocorticoids haveclinical implications with potent anti-inflammatory action andthey control stress responses (17). Their binding receptor GR is amultitasking transcription factor that exerts its biological func-tions via trans-activation or trans-repression of various nucleartranscription factors depending on the cellular context (18, 19). Inaddition to interaction between ERa and GR (20), additionalstudies have demonstrated that GR trans-represses stress-responsive transcription factors, such as activator protein-1(AP-1) and NF-kB (21, 22). Our results confirmed that NF-kBis a critical transcription factor for linking stress response andapoptosis in MCF-7:5C cells after treatment with E2, which ismediated by another transcription factor, STAT3 (15). Neverthe-less, how GR interacts with these transcriptional factors to affectE2-induced apoptosis in LTEDbreast cancer cells remains unclear.

We sought here to further understand how glucocorticoidsmodulate E2-induced stress responses and affect E2-induced apo-ptosis via GR in two LTED breast cancer cells, MCF-7:5C andMCF-7:2A. These two cell lines have different levels of antioxidantsystems: MCF-7:2A cells are more resistant than MCF-7:5C cells,which leads to a delayed and moderate apoptosis in MCF-7:2Acells after E2 treatment (11). In this study, our results demonstratethat E2 and dexamethasone have similar regulatory effects onactivation of the adipose inflammatory factors FADS1 and IL6/IL6receptor (IL6R). Furthermore, they are synergistic or additive inincreasing FADS1 and IL6/IL6R expression in these cells aftercombination treatment. However, dexamethasone selectivelysuppresses induction of TNFa expression by E2 in LTED breastcancer cells. With respect to endoplasmic reticulum stress, dexa-methasone distinctively modulates two unfolded proteinresponse (UPR) sensors activated by E2. This difference demon-strates that dexamethasone effectively blocks activation of PERKby E2 but has no inhibitory effects on the lipid metabolism–

associated sensor IRE1a. Consistently, results fromRPPA analysisdemonstrate that dexamethasone cannot reverse degradation ofPI3K/Akt–associated pathwaysmediated by IRE1a in LTEDbreastcancer cells. A mechanistic finding is that dexamethasone pref-erentially suppresses NF-kB DNA-binding activity, which pre-vents activation of NF-kB–dependent TNFa and ultimatelyresults in complete blockade of E2-induced apoptosis. Together,these findings have important clinical implications for the con-servative therapeutic application of glucocorticoids in treatmentof advanced aromatase inhibitor–resistant breast cancer.

Materials and MethodsMaterials

Estradiol (E2) and dexamethasone were purchased fromSigma-Aldrich. The caspase-8 inhibitor Z-IETD-FMK was

bought from BioVision. Recombinant human TNFa wasobtained from Biomyx. For Western blotting, antibodiesagainst total eIF2a, phosphorylated eIF2a, IRE-1a, total Akt,phosphorylated Akt, total PDK1, phosphorylated PDK1, totalSTAT3, phosphorylated STAT3, NF-kB p65, NF-kB p105,cleaved PARP, and caspase-7 were obtained from Cell SignalingTechnology. ERa (sc-544) and GR (sc-8992) were purchasedfrom Santa Cruz Biotechnology.

Cell culture conditions and cell proliferation assaysWild-type MCF-7 cells and E2-deprived MCF-7:5C and

MCF-7:2A cells were cultured as described previously (12). TheDNA fingerprinting pattern of these cell lines is consistentwith that reported by the ATCC. All cell lines were validatedaccording to their short tandem repeat (STR) profiles at TheUniversity of Texas MD Anderson Cancer Center CharacterizedCell Line Core (CCLC). The STR patterns of all cell lines wereconsistent with those from the CCLC standard cells (Supple-mentary Table S1). The DNA content of the cells, a measure ofproliferation, was determined as described previously (12) usinga DNA fluorescence Quantitation kit (Bio-Rad Laboratories).

Annexin V binding assay to detect apoptosisA FITC Annexin V Apoptosis Detection Kit I (BD Pharmingen)

was used to quantify apoptosis of MCF-7:5C and MCF-7:2A cellsthrough flow cytometry according to the manufacturer's instruc-tions. In brief, MCF-7:5C and MCF-7:2A cells were seeded in10-cm dishes. The next day, the cells were treated with differentcompounds for different periods. Cells were suspended in 1�binding buffer, and 1 � 105 cells were stained simultaneouslywith FITC-labeled Annexin V and propidium iodide (PI) for15 minutes at room temperature. The cells were analyzed usinga BD Accuri C6 plus flow cytometer.

NF-kB (p65) transcription factor DNA-binding assayMCF-7:5C and MCF-7:2A cells were treated with a vehicle con-

trol (0.1% DMSO), E2 (1 nmol/L), dexamethasone (0.1 mmol/L),or a combination of them for different times. Nuclear proteinwas extracted from the cells according to the manufacturer'sinstructions (Cayman Chemical). NF-kB (p65) DNA-bindingactivity in the cells was detected using an NF-kB (p65)Transcription Factor Assay Kit (Cayman Chemical).

RPPAMCF-7:5C cells were seeded in 6-well plates. Twenty-four hours

later, cells were treated with a vehicle control (0.1% DMSO), E2(1 nmol/L), dexamethasone (0.1 mmol/L), MPA (1 mmol/L), or acombination of dexamethasone or MPA with E2 for differenttimes. Cells were then harvested in lysis buffer (1% Triton X-100,50 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl, 1.5 mmol/LMgCl2, 1 mmol/L EGTA, 100 mmol/L NaF, 10 mmol/L sodiumpyrophosphate, 1 mmol/L Na3VO4, 10% glycerol containingfreshly added protease and phosphatase inhibitors). Proteinsamples obtained from these cellswere analyzed in the FunctionalProteomics Reverse Phase Protein Array (RPPA) Core at UT MDAnderson Cancer Center (Houston, TX).

ImmunoblottingMCF-7:5CandMCF-7:2A cellswere harvested in cell lysis buffer

(Cell Signaling Technology) supplemented with Protease Inhib-itor Cocktail Set I and Phosphatase Inhibitor Cocktail Set II

Dexamethasone Blocks Estrogen-Induced Apoptosis

www.aacrjournals.org Mol Cancer Ther; 18(10) October 2019 1685




(Calbiochem). Immunoblotting of different proteins was per-formed as described previously (12).

qRT-PCRTotal RNA isolated from cells using an RNeasy Micro kit

(Qiagen) was converted to first-strand cDNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems).Quantitative real-time PCR assays were performed with SYBRGreenPCRMasterMix (AppliedBiosystems) andaQuantStudio 6Flex real-time PCR System (Applied Biosystems). All primers weresynthesized by Integrated DNA Technologies. All data were nor-malized using reference gene 36B4.

GR siRNA transfectionMCF-7:5C cells were seeded in 6-well plates. The next day, cells

were transfected with human GR SMARTpool siRNA (Dharma-con, L-003424-00-0005) or scrambled siRNA (D-001810-10-05)at 50 nmol/L according to the manufacturer's instructions. Tar-geting sequences used in the experiments were listed in Supple-mentary Table S2.

Statistical analysisAll reported values are means � SD. Statistical comparisons

were assessed using two-tailed Student t tests. Results were con-sidered statistically significant if the P value was less than 0.05.

ResultsCross-talk between ERa and GR regulates expression of theirrespective target genes in LTED breast cancer cells

Our earlier publications have demonstrated that E2 activatesnuclear ERa to induce stress-associated apoptosis (12) and thatdexamethasone blocks E2-induced apoptosis in MCF-7:5Ccells (8). The first question we raised in this study was howcross-talk between GR and ERa regulates respective receptor–target genes in LTED breast cancer cells after combination treat-ment with E2 and dexamethasone. We observed that treatmentwith dexamethasone increased GR-responsive element (GRE)activity in MCF-7:5C cells, which was completely blocked by theanti-glucocorticoid compoundRU486 (Supplementary Fig. S1A).It was expected that E2 greatly reduced ERa expression at both theprotein and mRNA level. Dexamethasone clearly decreased ERamRNA expression, but only slightly reduced ERa protein expres-sion. Combination treatment with E2 and dexamethasonedecreased ERa expression to a greater degree (Fig. 1A and B). Asfor GR, treatment with E2 markedly decreased GR mRNA expres-sion, but slightly reduced GR protein expression. In comparison,dexamethasone did not alter GRmRNA expression, but decreasedGR protein expression. Combination treatment with E2 anddexamethasone maintained GR protein expression similar tothose in cells treated with dexamethasone alone, despite theincrease in GR mRNA expression (Fig. 1A and C).

To further examine regulation of the ERa and GR target genes,we treated MCF-7:5C cells with E2, dexamethasone, or a combi-nation of them for different times. E2 substantially upregulatedexpression of the classical ERa target gene pS2, whereas dexa-methasone downregulated it and slightly decreased pS2 expres-sion induced by E2 after 24 hours of treatment (Fig. 1D). Weobserved a similar regulatory pattern for ERa-tethering c-Fosexpression (23). Treatment with E2 dramatically upregulatedc-Fos expression, and dexamethasone downregulated c-Fos

mRNA expression and slightly inhibited c-Fos expression inducedbyE2 after 24hours of treatment (Fig. 1E). The inhibitory effects ofdexamethasone on the expression of ERa-modulated genes weretime-dependent. Our results demonstrated that dexamethasonegradually inhibited induction of pS2 and c-Fos expression by E2when the treatment time was prolonged (Fig. 1F and G; Supple-mentary Fig. S1B and S1C).

Notably, both E2 and dexamethasone upregulated expressionof theGR target gene serum and glucocorticoid-regulated kinase 1(SGK1; ref. 24). Dexamethasone was more potent than E2 inincreasing SGK1 expression. Furthermore, they were additive inincreasing SGK1 expression after 24 hours of combination treat-ment (Fig. 1H). With extension of the treatment time, inductionof SGK1 expression was further increased (Fig. 1I; SupplementaryFig. S1D). Examination of another GR target gene, glucocorticoid-induced leucine zipper (GILZ), dexamethasone moderately upre-gulated its expression, whereas E2 significantly decreased it.Remarkably, combination treatment synergistically increased theexpression of GILZ after 24 hours (Supplementary Fig. S1E). Incontrast, combination treatment with E2 and dexamethasonedownregulated the expression of GILZ in wild-type MCF-7 cellsdespite that E2 and dexamethasone alone had similar regulatorytendency to that in LTED breast cancer cells (SupplementaryFig. S1F). Together, these results suggested that GR and ERacross-talk regulates the expression of their respective target genes,depending on the context of cells.

Dexamethasone selectively blocks induction of TNFaexpression after E2 treatment

E2 widely activates inflammatory genes, including TNFa, IL6,and FADS1, in LTEDbreast cancer cells (12, 13). To determine theanti-inflammatory effects of dexamethasone on LTED breastcancer cells after treatment with E2, we treated MCF-7:5C withE2, dexamethasone, or a combination of them for different times.Our results demonstrated that both E2 and dexamethasone sup-pressed TNFa mRNA expression after 24 hours of treatment.Dexamethasone was more potent than E2 in inhibiting TNFaexpression (Fig. 2A). After 48 hours of treatment, E2 began toincrease TNFa expression, whereas dexamethasone consistentlyinhibited it. Combination treatment with E2 and dexamethasonecompletely inhibited TNFa expression (Fig. 2B). With extensionof the treatment time to 72 hours, induction of TNFa expressionby E2 peaked. In contrast, dexamethasone remarkably inhibitedTNFa expression and blocked E2-induced TNFa expression(Fig. 2C). We observed a similar pattern in the regulation ofanother TNF family member, lymphotoxin beta (LTB), by E2 anddexamethasone in MCF-7:5C cells (Fig. 2D; SupplementaryFig. S2A and S2B). MPA has glucocorticoid activity (8), whichalso significantly blocked the increased expression of TNFainduced by E2 (Fig. 2E). Different from induction of TNFa andLTB expression, treatment with E2 quickly increased FADS1 andIL6R expression in MCF-7:5C cells after 2 hours of treatment(Supplementary Fig. S2C and S2D). Furthermore, E2 increasedFADS1 expression much greater in MCF-7:5C cells than in wild-type MCF-7 cells (Fig. 2F), indicating that fatty acid metabolismwasmore active in LTEDbreast cancer cells after E2 treatment thanin wild-type MCF-7 cells. Similar to E2, dexamethasone increasedFADS1, IL6, and IL6R expression after 24 hours of treatment. E2and dexamethasone were synergistic or additive in upregulatingFADS1, IL6, and IL6R expression after combination treatment(Fig. 2G–I). As anticipated, the effect ofMPAwasweaker than that

Fan et al.

Mol Cancer Ther; 18(10) October 2019 Molecular Cancer Therapeutics1686




of dexamethasone in increasing FADS1 expression at the sameconcentration (Supplementary Fig. S2E), and MPA further upre-gulated FADS1 and IL6 expression after combination treatmentwith E2 (Supplementary Fig. S2F andS2G). These results indicatedthat glucocorticoids regulate the expressionof these inflammatoryfactors with distinct mechanisms in LTED breast cancer cells.

Differential modulation of the endoplasmic reticulum stresssensors IRE-1a and PERK by dexamethasone

In addition to regulating inflammation, dexamethasone isimplicated to be a stress controller in the clinic (17). Accumula-tion of endoplasmic reticulum stress is a key event in induction ofapoptosis by E2 in LTED breast cancer cells (12, 14, 15). Todetermine how dexamethasone modulates endoplasmic reticu-lum stress, we examined alteration of three sensors (IRE1, PERK,and ATF6) in MCF-7:5C cells after treatment with E2, dexameth-

asone, or a combination of them. Our results demonstrated thatIRE1 mRNA expression levels were quickly elevated after 2 hoursof exposure to E2 (Fig. 3A). In comparison, we found no signif-icant changes in PERKmRNA expression after E2 treatment exceptfor transient upregulation of it by E2 during 4–6 hours of treat-ment (Fig. 3B). With respect to ATF6, treatment with E2 barelyaltered its mRNA expression (Supplementary Fig. S2H). Dexa-methasone slightly reduced basal levels of IRE1 but was unable toreduce IRE1 mRNA expression elevated by E2 (Fig. 3C). As forPERK, both E2 anddexamethasoneweakly reducedmRNAexpres-sion, but the combination of them increased it (Fig. 3D). Incontrast, both E2 and dexamethasone remarkably reduced PERKmRNA expression in wild-type MCF-7 cells. After combinationtreatment, PERKmRNA expression levels were similar to E2 alonetreatedMCF-7 cells (Supplementary Fig. S2I). ATF6mRNAexpres-sion levels did not change remarkably after the combination

Figure 1.

Regulation of respective target genes by interaction between ERa and GR. A, ERa and GR protein expression in MCF-7:5C cells. Cells were treated with E2(1 nmol/L), dexamethasone (Dex, 0.1 mmol/L), or a combination of them for 24 and 48 hours. Cell lysates were then harvested for Western blotting. B and C, ERaand GRmRNA expression in MCF-7:5C cells. Cells were treated with E2 (1 nmol/L), dexamethasone (0.1 mmol/L), or a combination of them for 24 hours. ERa (B)and GR (C) mRNA expression was quantitated by RT-PCR. � , P < 0.05; �� , P < 0.001. D–I, Regulation of the ERa and GR target genes. MCF-7:5C cells were treatedwith E2 (1 nmol/L), dexamethasone (0.1 mmol/L), or a combination of them for 24 or 72 hours as indicated. mRNA expression for pS2 after 24 hours of treatment(D), c-Fos after 24 hours of treatment (E), pS2 after 72 hours of treatment (F), c-Fos after 72 hours of treatment (G), SGK-1 after 24 hours of treatment (H), andSGK-1 after 72 hours of treatment (I) was quantitated by RT-PCR. � , P < 0.05; �� , P < 0.001.






treatment (Fig. 3E). Treatment with E2 increased mRNA expres-sion of GRP78, an important chaperone protein in the endoplas-mic reticulum, which could be blocked upon adding dexameth-asone to the treatment (Supplementary Fig. S2J). Consistent withregulation of mRNA expression, IRE1a protein expressionincreased continuously after 12 hours of treatment with E2(Fig. 3F). Dexamethasone neither altered IRE1a protein expres-sion nor affected the induction of IRE1a protein expression by E2after 24 hours of treatment (Fig. 3G). Attenuation of proteintranslation by PERK is mainly through activation of its down-stream signal eukaryotic initiation factor 2 alpha (eIF2a; ref. 14).We previously observed transient activation of eIF2a by E2 inMCF-7:5C cells after 6 hours of treatment (15). In this study, E2consistently increased phosphorylation of eIF2a, which was

effectively blocked by dexamethasone, even though dexametha-sone itself increased the phosphorylation of eIF2a after 24 hoursof treatment (Fig. 3H). All of these results suggested that IRE1a isactivated by E2 prior to PERK in LTED breast cancer cells. Also,dexamethasone differentially regulates these sensors in responseto lipid and protein metabolism in the endoplasmic reticulum.

Activated GR does not affect degradation of PI3K-associatedpathways mediated by IRE-1a

To investigate the functional regulation of various proteinsrelated with lipid metabolism or stress responses by ERa and GR,we performed RPPA analysis of MCF-7:5C cells after treatmentwith E2, dexamethasone, or a combination of them for differenttimes. We screened more than four hundred proteins and

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Figure 2.

Regulation of inflammatory responses in MCF-7:5C cells by dexamethasone (Dex). A–C, Regulation of TNFa expression by dexamethasone. MCF-7:5C cells weretreated with E2 (1 nmol/L), dexamethasone (0.1 mmol/L), or a combination of them for 24 (A), 48 (B), and 72 (C) hours. TNFamRNA expression was quantitatedby RT-PCR. � , P < 0.05; �� , P < 0.001. D, Regulation of LTB expression by dexamethasone. MCF-7:5C cells were treated the same as in A–C for 72 hours. LTBmRNA expression was quantitated by RT-PCR. �, P < 0.05; �� , P < 0.001. E, Regulation of TNFa expression by MPA. MCF-7:5C cells were treated with E2(1 nmol/L), MPA (1 mmol/L), or a combination of them for 72 hours. TNFamRNA expression was quantitated by RT-PCR. � , P < 0.05; �� , P < 0.001. F, Expressionof FADS1 in MCF-7 and MCF-7:5C cells. MCF-7 cells were transferred to E2-free medium for 3 days. MCF-7 and MCF-7:5C cells were then seeded in 6-well platesand treated with a vehicle (0.1% EtOH) and E2 (1 nmol/L) for different times as indicated. FADS1 mRNA expression was quantitated by RT-PCR. Regulation ofFADS1, IL6, and IL6R by dexamethasone. MCF-7:5C cells were treated with E2 (1 nmol/L), dexamethasone (0.1 mmol/L), or a combination of them for 24 hours.FADS1 (G), IL6 (H), and IL6R (I) mRNA expression was quantitated by RT-PCR. �� , P < 0.001.

Fan et al.





demonstrated that E2 was more potent than dexamethasone inupregulating- and downregulating the expression of many pro-teins that widely localize in the nucleus, cytosol, and plasmamembrane (Fig. 4A). As expected, E2 decreased ERa proteinexpression, but increased the phosphorylationof ERa. In contrast,dexamethasone did not affect ERa protein expression or phos-phorylation (Fig. 4A). Expression of the ERa-dependent geneinsulin-like growth factor receptor-1 (IGF-1R; ref. 25) was quicklyupregulated by E2, but dexamethasone did not affect its expres-sion. Similarly, PI3K/Akt was rapidly activated by E2 andremained activated for 48 hours, but dexamethasone did notaffect the phosphorylation of Akt (Fig. 4A). Subsequently, weobserved clear degradation of Akt-associated proteins (such asmTOR, p70S6K, and4E-BP1, etc) inMCF-7:5C cells after 72hoursof E2 treatment. Treatment with dexamethasone maintained thephosphorylation of Akt at levels similar to those in control cells,

but did not prevent the degradation of these Akt-associatedproteins by E2. In addition, PDK1 and Akt are parallel signals,and both of them are downstream signals of PI3K (26).We foundthat E2 weakly increased the phosphorylation of PDK1 at an earlystage of treatment and decreased it at 72 hours. Dexamethasonehad almost no effect on phosphorylation of PDK1 (Fig. 4A). Theregulatory pattern for PDK1 was similar to that for Akt in that E2began to remarkably downregulate its expression after 72 hours.Dexamethasone could not block the degradation of PDK1. Inaddition, treatmentwith E2 activated the stress response pathwaysp38 and STAT3. Dexamethasone effectively blocked the phos-phorylation of p38 andmoderately blocked the phosphorylationof STAT3 (Fig. 4A). We validated these alterations detected byRPPA via Western blotting (Fig. 4B–D). MPA had a regulatorypattern similar to that for dexamethasone in signaling pathwaysdetected by RPPA (Supplementary Fig. S3). Together, these

Figure 3.

Regulation of endoplasmic reticulum stress in MCF-7:5C cells by dexamethasone (Dex). A and B, IRE-1 and PERK expression regulated by E2. MCF-7:5Ccells were treated with E2 (1 nmol/L) for different times as indicated. IRE-1 (A) and PERK (B) mRNA expression was quantitated by RT-PCR.� , P < 0.05; �� , P < 0.001. C–E, Regulation of three sensors mRNA expression by dexamethasone. MCF-7:5C cells were treated with E2 (1 nmol/L),dexamethasone (0.1 mmol/L), or a combination of them for 24 hours. IRE-1 (C), PERK (D), and ATF6 (E) mRNA expression was quantitated by RT-PCR.� , P < 0.05; �� , P < 0.001. F, IRE-1a protein expression. MCF-7:5C cells were treated with E2 (1 nmol/L) for different times as indicated. IRE-1a proteinexpression was measured by Western blot analysis. G, Regulation of IRE-1a protein expression by dexamethasone. MCF-7:5C cells were treated with E2(1 nmol/L), dexamethasone (0.1 mmol/L), or a combination of them for 24 and 48 hours. IRE-1a protein expression was measured by Western blotanalysis. H, Regulation of eIF2a phosphorylation by dexamethasone. MCF-7:5C cells were treated with E2 (1 nmol/L), dexamethasone (0.1 mmol/L), or acombination of them for 24 and 48 hours. Phosphorylated eIF2a was measured by Western blot analysis.






findings suggested that GR mainly exerts its biological functionson nuclear transcription factors but not in the cytosol of LTEDbreast cancer cells.

Dexamethasone suppresses NF-kB DNA-binding activity andassociated apoptosis induced by E2 in LTED breast cancer cells

Recently, we have found that induction of TNFa expression byE2 is strictly dependent on NF-kB DNA-binding activity in LTEDbreast cancer cells (15). Selective suppression of TNFa expression(Fig. 2A–C) implies that the function of NF-kB is repressed bydexamethasone. Treatment with E2 or dexamethasone reducedNF-kB mRNA expression after 24 hours, and combination treat-ment decreased it to a greater extent (Supplementary Fig. S4A–S4C).Wedid not see a reduction inNF-kBp65protein expression,but NF-kB p105 protein expression decreased after 72 hours ofcombination treatmentwith E2 anddexamethasone (Supplemen-tary Fig. S4D). Notably, NF-kB DNA–binding activity wasincreased by E2 after 48 hours of treatment in MCF-7:5C cells(Fig. 5A; Supplementary Fig. S4E) versus 6 days inMCF-7:2A cells(Supplementary Fig. S4F), whereas this activity was completelyblocked by dexamethasone (Fig. 5A; Supplementary Fig. S4F).Our recent study demonstrated that NF-kB is an oxidative stress

inducer after E2 treatment in MCF-7:5C cells (27). Therefore,dexamethasone effectively inhibited expression of the oxidativestress indicatorHMOX1 thatwas increasedbyE2 after suppressionof NF-kB activity (Supplementary Fig. S4G). In addition, E2increased expression of the stress-responsive transcription factorsnuclear factor erythroid-derived 2-like 2 (Nrf2) and hypoxia-inducible factor 1-alpha (HIF-1a), whereas dexamethasonereduced the basal levels of their expression. Furthermore, dexa-methasone effectively blocked the induction of their expressionby E2 (Supplementary Fig. S4H and S4I). With respect to apo-ptotic biomarkers, E2 increased cleaved PARP and caspase-7 after72 hours of treatment in MCF-7:5C cells, but not at 24 hours.Dexamethasone alone did not change the expression of thesetwo biomarkers but completely blocked the cleavage of PARPand caspase-7 induced by E2 (Fig. 5B). We observed similareffects on another LTED breast cancer cell line, MCF-7:2A cells(Supplementary Fig. S5A), which exhibit delayed apoptosisafter E2 treatment (11). Importantly, treatment with dexa-methasone completely blocked E2-induced apoptosis in twoLTED breast cancer cells (Fig. 5C and D; SupplementaryFig. S5B and S5C). The GR antagonist RU486 alone could notblock E2-induced apoptosis, but it remarkably reversed inhibitory

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Figure 4.

Regulation of signaling pathways by E2 and dexamethasone (Dex) in MCF-7:5C cells. A, Regulation signaling pathways detected by RPPA. MCF-7:5Ccells were treated with E2 (1 nmol/L), dexamethasone (0.1 mmol/L), or a combination of them for different times as indicated. Next, cell lysates wereharvested for RPPA analysis. B–E, Validation of signaling pathways regulated by E2 and dexamethasone. MCF-7:5C cells were treated with E2(1 nmol/L), dexamethasone (0.1 mmol/L), or a combination of them for 24 and 72 hours. Cell lysates were then harvested. Expression of p-Akt andtotal Akt (B), p-PDK1 and total PDK1 (C), p-p38 and total p38 (D), and p-STAT3 and total STAT3 (E) was measured by Western blotting.

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effects of dexamethasone on the blockade of E2-induced apopto-sis (Fig. 5C and D). Furthermore, low dose of TNFa remarkablyinduced apoptosis after 48 hours of treatment in MCF-7:5C cells,which could be effectively blocked by dexamethasone (Fig. 5E;Supplementary Fig. S5D). Consistently, the inhibitor of caspase-8, a downstream signal of TNFa, completely blocked E2-inducedapoptosis in LTED breast cancer cells (Supplementary Fig. S5Eand S5F). All of these results suggested that NF-kB/TNFa axisis crucial for E2 to induce apoptosis and activated GR is potentto suppress its induction by E2, thereby blocking E2-inducedapoptosis.

Dexamethasone modulates E2-induced inflammation andapoptosis via GR

To confirm that dexamethasonemodulates E2-induced inflam-mation and apoptosis via GR, we effectively knocked down GRexpression in MCF-7:5C cells using a specific siRNA (Fig. 6A). Wethen treated GR siRNA-transfected cells with E2, dexamethasone,and a combination of them for 72 hours, using scrambled siRNA–transfected cells as controls. The results demonstrated that deple-tion ofGR alone increased the percentage of Annexin V binding inthe cells, indicating thatGRhas the potential to regulate apoptosisinMCF-7:5C cells (Fig. 6B).Downregulation ofGR expressiondid

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Figure 5.

Suppression of NF-kB DNA-binding activity and associated apoptosis in LTED breast cancer cells by dexamethasone (Dex). A, Suppression of NF-kB DNA-binding activity by dexamethasone. MCF-7:5C cells were treated with E2 (1 nmol/L), dexamethasone (0.1 mmol/L), or a combination of them for 72 hours. Cellswere then harvested for extraction of nuclear protein. The NF-kB DNA-binding activity in the cells was measured using an NF-kB (p65) transcription factor assaykit. � , P < 0.05. B, Inhibition of apoptotic biomarkers by dexamethasone. MCF-7:5C cells were treated as described inA for 24 hours and 72 hours. Cleaved PARPand caspase-7 were measured byWestern blot analysis. C and D, Blockade of E2-induced apoptosis by dexamethasone. MCF-7:5C cells were treated with E2(1 nmol/L), dexamethasone (0.1 mmol/L), RU486 (1 mmol/L), or in different combinations as indicated for 72 hours. Apoptosis was measured by an annexin Vbinding assay through flow cytometry. �, P < 0.05; �� , P < 0.001. E, Inhibition of TNFa-induced apoptosis by dexamethasone. MCF-7:5C cells were treated withTNFa (2.5 ng/mL), dexamethasone (0.1 mmol/L), or in a combination of them for 48 hours. Apoptosis was measured by an annexin V binding assay through flowcytometry. �� , P < 0.001.






not affect the ability of E2 to induce apoptosis, but dexamethasonecould not prevent E2-induced apoptosis under these conditions(Fig. 6B). Consistently, expression of the E2-induced apoptosis-associated inflammatory factors TNFa, LTB, HMOX1, and Bimwas not affected after knockdown of GR, whereas dexamethasone

could not effectively block their expression induced by E2 afterdepletion of GR (Fig. 6C–E; Supplementary Fig. S6A). As forregulation of FADS1 and IL6 expression, dexamethasone couldbarely upregulate their expression after knockdownofGR. It couldnot further increase IL6 or FADS1 expression after combination

Figure 6.

Dexamethasone (Dex) modulates E2-induced inflammation and apoptosis via GR. A, Knockdown of GR expression in MCF-7:5C cells. Cells were transfected withscrambled siRNA or GR siRNA for 72 hours. Cell lysates were then harvested. GR expression was measured byWestern blot analysis. B, Regulation of E2-inducedapoptosis after knockdown of GR. MCF-7:5C cells were transfected with scrambled siRNA or GR siRNA for 72 hours. Then, scrambled siRNA-transfected cellswere treated with a vehicle (0.1% EtOH) or E2 (1 nmol/L). GR siRNA-transfected cells were treated with a vehicle (0.1% EtOH), E2 (1 nmol/L), dexamethasone(0.1 mmol/L), or a combination of them for 72 hours. Cells were then harvested for annexin V binding assay. �� , P < 0.001. C–F, Regulation of inflammation andapoptosis-associated factors after depletion of GR. MCF-7:5C cells were knocked down of GR and treated with E2 or dexamethasone as described in B for72 hours. TNFa (C), LTB (D), HMOX1 (E), and FADS1 (F) mRNA expression was quantitated by RT-PCR. � , P < 0.05; �� , P < 0.001.

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with E2 when GR expression was downregulated (Fig. 6F; Sup-plementary Fig. S6B). Our results suggested that dexamethasoneprimarily targets GR to modulate E2-induced inflammatoryresponses and apoptosis. Both E2 and dexamethasone commonlyupregulate FADS1 and IL6/IL6R expression via respective recep-tors. However, they exerted distinct effects on NF-kB DNA-binding activity, which was increased by E2 but suppressed bydexamethasone. This is a fundamental mechanism underlyingdexamethasone blockade of E2-induced apoptosis (Fig. 7).

DiscussionThe discovery of E2-induced apoptosis not only has clinical

relevance to treat aromatase inhibitor–resistant breast cancer andreduce breast cancer incidence in postmenopausal women (6, 7),butalsoageneralprincipalhasemerged tounderstandsex steroid–induced apoptosis in long-term androgen-deprived prostate can-cer (28). Our further findings demonstrate that dexamethasoneandMPA with glucocorticoid activity block E2-induced apoptosisin LTED breast cancer cells (8). This observation has clinical

significance for the interpretation of breast cancer incidence inpostmenopausalwomenreceivingdifferenthormonereplacementtherapy (HRT) in theWomen'sHealth Initiative (7, 29).However,the mechanisms remain unclear. It is known that E2 initiallyoveractivates nuclear ERa that leads to the accumulation of stressresponses includingendoplasmic reticulumstress,oxidative stress,and inflammatory stress in LTEDbreast cancer cells (12, 13). Theseunresolved stress responses cause E2-induced apoptosis in thesecells (12–16).Among thesestress-associated factors, thePERK/NF-kB/TNFa axis plays a critical role in mediating E2-induced apo-ptosis (15, 16). Herein, we demonstrate that dexamethasonepreferentially and consistently represses NF-kB DNA binding,which results in selective inhibitory effects on the induction ofTNFa expression. Consequently, dexamethasone completelyblocks E2-induced apoptosis in LTED breast cancer cells.

NF-kB is a key transcription factor in mediating inflammationand stress responses (30, 31). Furthermore, its transcriptionalactivity is suppressed by the lipogenesis transcription factorsCCAAT/enhancer binding protein b (C/EBPb) and PPARg inLTED breast cancer cells (15, 27), indicating that NF-kB has aclose relationship with lipid metabolism. In this study, NF-kB isregulated by both E2 and dexamethasone but in an oppositedirection in LTED breast cancer cells. We have observed that E2increases NF-kBDNA-binding activity via activation of PERK, butnot through the canonical pathway in MCF-7:5C cells (15, 16).Unlike E2, dexamethasone decreases NF-kBDNA-binding activityand consistently represses the expression of the NF-kB–dependent gene TNFa. These data suggest that dexamethasonepreferentially inhibits NF-kB DNA binding in LTED breast cancercells. Our result is consistent with a report that activated GRpotently prevents NF-kB from chromatin occupancy (32). Inaddition, the GR target gene GILZ can bind to NF-kB directlyand inhibit its DNA-binding activity (33). Therefore, suppressionof the NF-kB/TNFa axis by dexamethasone is one of the majormechanisms that blocks E2-induced apoptosis.

Glucocorticoids are prescribed as anti-inflammatory and anti-stress agents in the clinic (34, 35), and they exhibit proapoptoticor antiapoptotic effects in a tissue- or cell type–dependent man-ner (36). In MCF-7–derived cell lines, treatment with dexameth-asone inhibits cell proliferationbutdoesnot induceapoptosis (8).As for the regulation of inflammatory responses, dexamethasoneis not a complete inhibitor of them in LTED breast cancer cells,which increases the expression of adipose inflammatory factorsFADS1 and IL6/IL6R and upregulates more upon adding dexa-methasone to E2. This may be related to the activation of thelipogenesis transcription factor C/EBPd by treatment with dexa-methasone (37). Unexpectedly, dexamethasone differentiallyregulates the function of endoplasmic reticulum stress sensors,which prevents E2 from activation of PERK, but it has no inhib-itory effects on IRE1a expression increased by E2. This distinctiveregulation of PERK and IRE1a also indicates that dexamethasonedifferentially regulates protein and lipidmetabolism in the endo-plasmic reticulum (12, 14–16). Although how dexamethasonedifferentially regulates these two sensors remains unclear, treat-mentwithdexamethasone alone increasesphosphorylationof thePERK downstream signal eIF2a (Fig. 3H), implying that dexa-methasone does not directly inhibit PERK function. Most likely,E2 increases the expression of some short half-life nuclear pro-teins, leading to accumulation of unfolded proteins in the endo-plasmic reticulum. In contrast, dexamethasone has the potentialto inhibit expression of these nuclear proteins, thereby reducing

Figure 7.

Modulation of E2-induced apoptosis in LTED breast cells by glucocorticoids.Both E2 and dexamethasone bind to respective nuclear receptors and exertdistinct biological effects on NF-kB DNA binding. Specifically, E2 increasesNF-kB DNA-binding activity via the endoplasmic reticulum stress sensorPERK (15) and activates the NF-kB/TNFa axis to induce apoptosis. Incontrast, dexamethasone directly suppresses NF-kB DNA-binding activity,which results in selective blockade of E2-induced NF-kB–dependent TNFaexpression and associated apoptosis. As for the regulation of otherinflammatory factors, both E2 and dexamethasone activate IL6 and FADS1,and they are synergistic or additive in increasing IL6 and FADS1 expressionafter combination treatment.






the unfolded protein burden in the endoplasmic reticulum. TheAP-1 familymember c-Fos is one of the candidates as a short-livedtranscription factor, which is activated by E2 (Fig. 1G; ref. 38). Insupport of our view, other studies have demonstrated that acti-vated GR preferentially suppresses the function of AP-1 familymembers (39, 40). In addition, synergistic upregulation of SGK1expression by E2 and dexamethasone facilitates maintenance ofthe homeostasis in the endoplasmic reticulum to antiapopto-sis (41, 42). All of these findings suggest that activated GR is apotent regulator of multiple stress-responsive transcription fac-tors, thereby modulating stress and inflammatory responses.

Finally, it is important to note that our results mainly focusedon MCF-7–derived cells due to the limitation of cell lines (43).MCF-7 and T47D are two representative ERa- and GR-positivebreast cancer cell lines. After long-term E2 deprivation, there is nochange in GR expression in MCF-7 and T47D. As for the expres-sion of ERa, MCF-7–derived cell lines continue to express ERa.However, T47D-derived cell line loses ERa, thereby having noresponse to E2 (44). This is also the reason why E2-inducedapoptosis is observed indifferent sources ofMCF-7–derived LTEDcell lines (4, 5).

In summary, E2 and dexamethasone regulate E2-induced stressand apoptosis through their respective receptors. Cross-talkbetween ERa and GR exists (45, 46), but ERa is more potentthan GR in widely activating signaling pathways in LTED breastcancer cells. In this study, activation of GR by dexamethasone orMPA blocks E2-induced apoptosis via trans-suppression of NF-kBDNA-binding activity.We found a similarmechanism underlyingblockageof E2-inducedapoptosis by the agonistof PPARg inMCF-7:5C cells (27). These results demonstrate that different ligandsactivate respective transcription factors to commonly suppress theDNA-binding activity of NF-kB, a molecule critical to mediatestress, inflammation, and apoptosis in LTED breast cancercells (15, 16, 27). They also imply that the biological functionof ERa is affected by several other transcription factors dependingon the ligands present in the nucleus, thereby dynamically remo-deling the chromatin occupancy (47–49). Transcriptional activa-tor FoxA1 has been identified as a pioneer factor to be involved inthe dynamic chromatin transitions after activation of GR and ERain breast cancer cells (50). These findings have built up a strongbasis for us to further investigate how GR and ERa interact witheach other on the chromatin occupancy in the endocrine-resistantbreast cancer cells, compared with the wild-type breast cancercells. Our data further provide a novel understanding of E2-induced apoptosis through extensive interactions of ERa with

inflammation- and stress-associated transcription factors, such asNF-kB and PPARg (15, 16, 27), which facilitates apoptosis-associated inflammation and stress after long-term E2 deficiency.Importantly, these transcription factors are also therapeutic targetsfor different diseases (51–53). Together, this study not onlysupports the deployment of antiglucocorticoids to enhance sexsteroid–induced apoptosis in cancer therapy, but also provides aninsight into themechanismofMPAwith glucocorticoid activity toincrease breast cancer incidence in the WHI. Ongoing strategicstudies in our laboratory are addressing the wide application ofglucocorticoid action in regulating sex steroid–induced apoptosisin a range of models of normal and cancer cells.

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

Authors' ContributionsConception and design: P. Fan, B. Abderrahman, V.C. JordanDevelopment of methodology: P. FanAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): P. Fan, D.R. Siwak, F.A. AgbokeAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): P. FanWriting, review, and/or revision of the manuscript: P. Fan, D.R. Siwak,B. Abderrahman, V.C. JordanAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): P. Fan, D.R. Siwak, S. YerrumStudy supervision: P. Fan, B. Abderrahman, V.C. Jordan

AcknowledgmentsThis work was supported by aDepartment of Defense Breast ProgramCenter

of Excellence AwardW81XWH-06-1-0590 (to V.C. Jordan), the NIH/NCI underaward number P30-CA016672 (to P.W. Pisters), Susan G. Komen for the CureFoundation under award number SAC100009 (to V.C. Jordan), and CancerPrevention Research Institute of Texas (CPRIT) for the STARs and STARs plusAwards (to V.C. Jordan). V.C. Jordan thanks the George and Barbara BushFoundation for Innovative Cancer Research and the benefactors of the Dallas/Fort Worth Living Legend Chair of Cancer Research for their generous support.The authors thankDonNorwood in theDepartment of Scientific Publications atMD Anderson Cancer Center for editing the manuscript. We also thank the MDAnderson's Characterized Cell Line Core for validating cell lines, which isfunded by NCI #CA16672.

The costs of publicationof this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 12, 2018; revised April 26, 2019; accepted July 1, 2019;published first September 11, 2019.

References1. Jordan VC, Brodie AM.Development and evolution of therapies targeted to

the estrogen receptor for the treatment and prevention of breast cancer.Steroids 2007;72:7–25.

2. Yao K, Lee ES, Bentrem DJ, England G, Schafer JI, O'Regan RM, et al.Antitumor action of physiological estradiol on tamoxifen-stimulatedbreast tumors grown in athymic mice. Clin Cancer Res 2000;6:2028–36.

3. Liu H, Lee ES, Gajdos C, Pearce ST, Chen B, Osipo C, et al. Apoptoticaction of 17beta-estradiol in raloxifene-resistant MCF-7 cells in vitroand in vivo. J Natl Cancer Inst 2003;95:1586–97.

4. Song RX, Mor G, Naftolin F, McPherson RA, Song J, Zhang Z, et al. Effect oflong-term estrogen deprivation on apoptotic responses of breast cancercells to 17 beta-estradiol. J Natl Cancer Inst 2001;93:1714–23.

5. Lewis JS, Meeke K, Osipo C, Ross EA, Kidawi N, Li T, et al. Intrinsicmechanism of estradiol-induced apoptosis in breast cancer cells resistantto estrogen deprivation. J Natl Cancer Inst 2005;97:1746–59.

6. Ellis MJ, Gao F, Dehdashti F, Jeffe DB, Marcom PK, Carey LA, et al. Lower-dose vs. high-dose oral estradiol therapy of hormone receptor-positive,aromatase inhibitor-resistant advanced breast cancer: a phase 2 random-ized study. JAMA 2009;302:774–80.

7. Anderson GL, Chlebowski RT, Aragaki AK, Kuller LH, Manson JE, Gass M,et al. Conjugated equine oestrogen and breast cancer incidence and mor-tality inpostmenopausalhysterectomy:extendedfollow-upoftheWomen'sHealth Initiative Randomised Trial. Lancet Oncol 2012;13:476–86.

8. Sweeney EE, Fan P, Jordan VC. Molecular modulation of estrogen-inducedapoptosis by synthetic progestins in hormone replacement therapy: aninsight into the Women's Health Initiative Study. Cancer Res 2014;74:7060–8.

9. Fan P, Jordan VC. Acquired resistance to selective estrogen receptor mod-ulators (SERMs) in clinical practice (tamoxifen & raloxifene) by selectionpressure in breast cancer cell populations. Steroids 2014;90:44–52.

Fan et al.





10. MillerWR, Larionov A.Molecular effects of oestrogen deprivation in breastcancer. Mol Cell Endocrinol 2011;340:127–36.

11. Sweeney EE, Fan P, Jordan VC. Mechanisms underlying differentialresponse to estrogen-induced apoptosis in long-term estrogen-deprivedbreast cancer cells. Int J Oncol 2014;44:1529–38.

12. Fan P, Griffith OL, Agboke FA, Anur P, Zou X, McDaniel RE, et al. c-Srcmodulates estrogen-induced stress and apoptosis in estrogen-deprivedbreast cancer cells. Cancer Res 2013;73:4510–20.

13. Ariazi EA, Cunliffe HE, Lewis-Wambi JS, Slifker MJ, Willis AL, Ramos P,et al. Estrogen induces apoptosis in estrogen deprivation-resistant breastcancer through stress responses as identified by global gene expressionacross time. Proc Natl Acad Sci U S A 2011;108:18879–86.

14. Fan P, Cunliffe HE, Maximov PY, Agboke FA, McDaniel RE, Sweeney EE,et al. Integration of downstream signals of insulin-like growth factor-1receptor by endoplasmic reticulum stress for estrogen-induced growth orapoptosis in breast cancer cells. Mol Cancer Res 2015;13:1367–76.

15. Fan P, Tyagi AK, Agboke FA,Mathur R, Pokharel N, Jordan VC.Modulationof nuclear factor-kappa B activation by the endoplasmic reticulum stresssensor PERK to mediate estrogen-induced apoptosis in breast cancer cells.Cell Death Discov 2018;4:15.

16. Fan P, Jordan VC. How PERK kinase conveys stress signals to nuclearfactor-kB to mediate estrogen-induced apoptosis in breast cancer cells?Cell Death Dis 2018;9:842.

17. Rhen T, Cidlowski JA. Antiinflammatory action of glucocorticoids—newmechanisms for old drugs. N Engl J Med 2005;353:1711–23.

18. Desmet SJ, De Bosscher K. Glucocorticoid receptors: finding the middleground. J Clin Invest 2017;127:1136–45.

19. RatmanD, VandenBergheW,Dejager L, Libert C, Tavernier J, Beck IM, et al.How glucocorticoid receptors modulate the activity of other transcriptionfactors: a scope beyond tethering. Mol Cell Endocrinol 2013;380:41–54.

20. Karmakar S, Jin Y, Nagaich AK. Interaction of glucocorticoid receptor(GR) with estrogen receptor (ER) a and activator protein 1 (AP1) indexamethasone-mediated interference of ERa activity. J Biol Chem2013;288:24020–34.

21. Jonat C, Rahmsdorf HJ, Park KK, Cato AC, Gebel S, Ponta H, et al.Antitumor promotion and antiinflammation: down-modulation of AP-1 (Fos/Jun) activity by glucocorticoid hormone. Cell 1990;62:1189–204.

22. Scheinman RI, Gualberto A, Jewell CM, Cidlowski JA, Baldwin AS. Char-acterization of mechanisms involved in transrepression of NF-kB byactivated glucocorticoid receptors. Mol Cell Biol 1995;15:943–53.

23. HeldringN, IsaacsGD,DiehlAG,SunM,CheungE,Ranish JA,etal.Multiplesequence-specific DNA-binding proteins mediate estrogen receptor signal-ing through a tethering pathway. Mol Endocrinol 2011;25:564–74.

24. Itani OA, Liu KZ, Cornish KL, Campbell JR, Thomas CP. Glucocorticoidsstimulate human sgk1 gene expression by activation of a GRE in its 5'-flanking region. Am J Physiol Endocrinol Metab 2002;283:E971–9.

25. Fan P, Agboke FA, McDaniel RE, Sweeney EE, Zou X, Creswell K, et al.Inhibition of c-Src blocks oestrogen-induced apoptosis and restores oes-trogen-stimulated growth in long-term oestrogen-deprived breast cancercells. Eur J Cancer 2014;50:457–68.

26. Biondi RM, Kieloch A, Currie RA, Deak M, Alessi DR. The PIF-bindingpocket in PDK1 is essential for activation of S6K and SGK, but not PKB.EMBO J 2001;20:4380–90.

27. FanP,AbderrahmanB,ChaiTS, YerrumS, JordanVC.Targetingperoxisomeproliferator-activated receptor g to increase estrogen-induced apoptosis inestrogen-deprived breast cancer cells. Mol Cancer Ther 2018;17:2732–45.

28. Maximov PY, Abderrahman B, Curpan RF, Hawsawi YM, Fan P, Jordan VC.A unifying biology of sex steroid-induced apoptosis in prostate and breastcancers. Endocr Relat Cancer 2018;25:R83–R113.

29. Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefa-nick ML, et al. Risks and benefits of estrogen plus progestin in healthypostmenopausal women: principal results from the Women's HealthInitiative randomized controlled trial. JAMA 2002;288:321–33.

30. Zhang K, Kaufman RJ. From endoplasmic-reticulum stress to the inflam-matory response. Nature 2008;454:455–62.

31. Rius J, GumaM, SchachtrupC, Akassoglou K, Zinkernagel AS,Nizet V, et al.NF-kappaB links innate immunity to the hypoxic response through tran-scriptional regulation of HIF-1alpha. Nature 2008;453:807–11.

32. Oh KS, Patel H, Gottschalk RA, Lee WS, Baek S, Fraser IDC, et al.Anti-inflammatory chromatinscape suggests alternative mechan-

isms of glucocorticoid receptor action. Immunity 2017;47:298–309.

33. Cheng Q, Fan H, Ngo D, Beaulieu E, Leung P, Lo CY, et al. GILZ over-expression inhibits endothelial cell adhesive function through regulationof NF-kB and MAPK activity. J Immunol 2013;191:424–33.

34. De Bosscher K, Haegeman G, Elewaut D. Targeting inflammation usingselective glucocorticoid receptor modulators. Curr Opin Pharmacol 2010;10:497–504.

35. Kadmiel M, Cidlowski JA. Glucocorticoid receptor signaling in health anddisease. Trends Pharmacol Sci 2013;34:518–30.

36. Gruver-Yates AL, Cidlowski JA. Tissue-specific actions of glucocorticoids onapoptosis: a double-edged sword. Cells 2013;2:202–23.

37. Mitani T, Takaya T, Harada N, Katayama S, Yamaji R, Nakamura S, et al.Theophylline suppresses interleukin-6 expression by inhibiting glucocor-ticoid receptor signaling in pre-adipocytes. Arch Biochem Biophys 2018;646:98–106.

38. Ferrara P, Andermarcher E, Bossis G, Acquaviva C, Brockly F, Jariel-Encontre I, et al. The structural determinants responsible for c-Fos proteinproteasomal degradation differ according to the conditions of expression.Oncogene 2003;22:1461–74.

39. Yang-Yen HF, Chambard JC, Sun YL, Smeal T, Schmidt TJ, Drouin J, et al.Transcriptional interference between c-Jun and the glucocorticoid receptor:mutual inhibition of DNA binding due to direct protein-protein interac-tion. Cell 1990;62:1205–15.

40. Weikum ER, de Vera IMS, Nwachukwu JC, Hudson WH, Nettles KW,Kojetin DJ, et al. Tethering not required: the glucocorticoid receptor bindsdirectly to activator protein-1 recognition motifs to repress inflammatorygenes. Nucleic Acids Res 2017;45:8596–608.

41. Anacker C, CattaneoA,MusaelyanK, Zunszain PA,HorowitzM,Molteni R,et al. Role for the kinase SGK1 in stress, depression, and glucocorticoideffects on hippocampal neurogenesis. Proc Natl Acad Sci U S A 2013;110:8708–13.

42. Wang Y, Zhou D, Phung S, Warden C, Rashid R, Chan N, et al. SGK3sustains ERa signaling and drives acquired aromatase inhibitor resistancethroughmaintaining endoplasmic reticulum homeostasis. Proc Natl AcadSci U S A 2017;114:E1500–8.

43. Sweeney EE, McDaniel RE, Maximov PY, Fan P, Jordan VC. Models andmechanisms of acquired antihormone resistance in breast cancer: signif-icant clinical progress despite limitations. Horm Mol Biol Clin Investig2012;9:143–63.

44. Pink JJ, Bilimoria MM, Assikis J, Jordan VC. Irreversible loss of the oestro-gen receptor in T47D breast cancer cells following prolonged oestrogendeprivation. Br J Cancer 1996;74:1227–36.

45. De Bosscher K, Vanden Berghe W, Haegeman G. Cross-talk betweennuclear receptors and nuclear factor kappaB. Oncogene 2006;25:6868–86.

46. Cvoro A, Tzagarakis-Foster C, Tatomer D, Paruthiyil S, Fox MS, LeitmanDC. Distinct roles of unliganded and liganded estrogen receptors intranscriptional repression. Mol Cell 2006;21:555–64.

47. Carroll JS, Meyer CA, Song J, Li W, Geistlinger TR, Eeckhoute J, et al.Genome-wide analysis of estrogen receptor binding sites. Nat Genet 2006;38:1289–97.

48. Fullwood MJ, Liu MH, Pan YF, Liu J, Xu H, Mohamed YB, et al. Anoestrogen-receptor-alpha-bound human chromatin interactome. Nature2009;462:58–64.

49. Miranda TB, Voss TC, Sung MH, Baek S, John S, Hawkins M, et al.Reprogramming the chromatin landscape: interplay of the estrogen andglucocorticoid receptors at the genomic level. Cancer Res 2013;73:5130–9.

50. Swinstead EE, Miranda TB, Paakinaho V, Baek S, Goldstein I, Hawkins M,et al. Steroid receptors reprogram FoxA1 occupancy through dynamicchromatin transitions. Cell 2016;165:593–605.

51. StrausDS,GlassCK.Anti-inflammatoryactionsofPPARligands:newinsightson cellular and molecular mechanisms. Trends Immunol 2007;28:551–8.

52. Vandevyver S, Dejager L, Van Bogaert T, Kleyman A, Liu Y, Tuckermann J,et al. Glucocorticoid receptor dimerization induces MKP1 to protectagainst TNF-induced inflammation. J Clin Invest 2012;122:2130–40.

53. Donovan JM, Zimmer M, Offman E, Grant T, Jirousek M. A novel NF-kBinhibitor, edasalonexent (CAT-1004), in development as a disease-modifying treatment for patients with Duchenne muscular dystrophy:phase 1 safety, pharmacokinetics, and pharmacodynamics in adultsubjects. J Clin Pharmacol 2017;57:627–39.






2019;18:1684-1695. Published OnlineFirst September 11, 2019.Mol Cancer Ther Ping Fan, Doris R. Siwak, Balkees Abderrahman, et al. Cancer CellsBlocks Estrogen-Induced Apoptosis in Estrogen-Deprived Breast

B by Glucocorticoid ReceptorκSuppression of Nuclear Factor-

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