Estrogen receptor-α directly regulates the hypoxia- …Estrogen receptor-α directly regulates the...

6
Estrogen receptor-α directly regulates the hypoxia- inducible factor 1 pathway associated with antiestrogen response in breast cancer Jun Yang a,b,1 , Alaa AlTahan b , Dylan T. Jones a , Francesca M. Buffa a , Esther Bridges a , Rodrigo B. Interiano b , Chunxu Qu c , Nathan Vogt b , Ji-Liang Li a , Dilair Baban d , Jiannis Ragoussis d,2 , Robert Nicholson e , Andrew M. Davidoff b , and Adrian L. Harris a a Growth Factor Group, Cancer Research UK, Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom; b Department of Surgery, St. Jude Childrens Research Hospital, Memphis, TN 38105; c Department of Bioinformatics, St. Jude Childrens Research Hospital, Memphis, TN 38105; d Genomics Group, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom; and e Tenovus Centre for Cancer Research, Welsh School of Pharmacy, Cardiff University, Cardiff CF10 3NB, United Kingdom Edited by Joan S. Brugge, Harvard Medical School, Boston, MA, and approved October 13, 2015 (received for review November 17, 2014) A majority of breast cancers are driven by estrogen via estrogen receptor-α (ERα). Our previous studies indicate that hypoxia-induc- ible factor 1α (HIF-1α) cooperates with ERα in breast cancer cells. However, whether ERα is implicated in the direct regulation of HIF-1α and the role of HIF-1α in endocrine therapy response are un- known. In this study we found that a subpopulation of HIF-1α targets, many of them bearing both hypoxia response elements and estrogen response elements, are regulated by ERα in normoxia and hypoxia. Interestingly, the HIF-1α gene itself also bears an estrogen response element, and its expression is directly regulated by ERα. Clinical data revealed that expression of the HIF-1α gene or a hypoxia metagene signature is associated with a poor outcome to endocrine treatment in ERα + breast cancer. HIF-1α was able to confer endocrine therapy resistance to ERα + breast cancer cells. Our findings define, for the first time to our knowledge, a direct regulatory pathway between ERα and HIF-1α, which might modulate hormone response in treatment. ERα | HIF-1α | tamoxifen E strogen receptor-α (ERα) is an estrogen-dependent nuclear transcription factor that is critical for mammary epithelial cell division and breast cancer progression (1, 2). ERα is expressed in 70% of breast tumors (3), the majority of which depend on estrogen signaling, thereby providing the rationale for using antiestrogens as adjuvant therapy to treat breast cancer (4). Tamoxifen is a first generation selective ER modulator and has been widely used in breast cancer prevention and treatment (4). Although now replaced by aromatase inhibitors as first-line treatment in postmenopausal women, it still remains important in premenopausal breast cancer and after failure of aromatase inhibitors. Tamoxifen acts as an antagonist in breast cancer cells by competing with estrogen for the ER. Tamoxifen-bound ER recruits the nuclear receptor corepressor and histone deacetylase (HDAC), as opposed to coactivators, leading to transcriptional repression (5). Although hundreds of thousands of patients have benefited from tamoxifen treatment, its efficacy is limited to an average time of 15 mo in patients with metastatic disease (6), as resistance often develops (7). Many mechanisms have been proposed to account for tamoxifen resistance (8), including loss of ERα ex- pression or expression of truncated ER isoforms, posttranslational modification of ERα, deregulation of ERα coactivators, and in- creased receptor tyrosine kinase signaling. Recent studies further indicate that somatic ERα mutation (9, 10), as well as genomic amplification of distant ER response elements (11), could con- tribute to hormone therapy resistance. Our clinical studies suggest that the in vivo tumor environment may play a role in tamoxifen resistance, as hypoxia-inducible factor 1α (HIF-1α) protein expression was associated with tamoxifen resistance in neoadjuvant, primary therapy of ERα + breast cancers (12), as well as resistance to chemoendocrine therapy (13). HIF-1α is a master regulator of oxygen homeostasis, which is rapidly degraded in normoxia by the tumor suppressor, von HippelLindau protein (VHL), but is stabilized in hypoxia (14). This pro- cess is mainly determined by the hydroxylation of HIF-1α catalyzed by prolyl hydroxylases. HIF-1α has been associated with an ag- gressive phenotype of breast cancer: that is, large tumor size, high grade, high proliferation, and lymph node metastasis (15). In- creased HIF-1α is also associated with ERα-positivity (15), whereas HIF-1β, the partner of HIF-1α, has been shown to function as a potent coactivator of ER-dependent transcription (16). Further studies revealed that in ER + T47D breast cancer cells, combined hypoxia and E2 treatment had additive effects on expression of some genes (17), although the mechanism is not clear. We have previously shown that HIF-1α and ERα can co- ordinate expression of genes, such as lysine-specific demethylase 4B/Jumonji domain-containing 2B (KDM4B/ JMJD2B), an H3K9me3/me2 histone demethylase, which is targeted by both ERα and HIF-1α and epigenetically regulates cell cycle progression Significance About 1.7 million new cases of breast cancer occur every year, 70% of which are estrogen receptor-α (ERα) positive. Antiestrogen therapy to block ERα function is the most important approach in treatment of ERα + patients. However, resistance eventually will develop for various reasons. Here we demonstrate that hypoxia- inducible factor 1α (HIF-1α) is a direct transcriptional target of ERα, which may compensate for ERα function loss because many other ERα targets are also HIF-1α targets. We further show that HIF-1α is able to confer cancer cell resistance to ERα antagonists tamox- ifen and fulvestrant, and the expression of HIF-1α is associated with poor survival to endocrine therapy in ERα + patients. Our findings thus have revealed a previously unidentified mechanism for antiestrogen resistance. Author contributions: J.Y., A.M.D., and A.L.H. designed research; J.Y., A.A., D.T.J., E.B., R.B.I., N.V., D.B., and J.R. performed research; J.Y., J.-L.L., and R.N. contributed new reagents/ analytic tools; J.Y., A.A., F.M.B., and C.Q. analyzed data; and J.Y., A.M.D., and A.L.H. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Data deposition: The data reported in this paper have been deposited in the Gene Ex- pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE61799). 1 To whom correspondence should be addressed. Email: [email protected]. 2 Present address: McGill University and Genome Quebec Innovation Centre, McGill Uni- versity, Montreal, QC, Canada. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1422015112/-/DCSupplemental. 1517215177 | PNAS | December 8, 2015 | vol. 112 | no. 49 www.pnas.org/cgi/doi/10.1073/pnas.1422015112 Downloaded by guest on April 13, 2020

Transcript of Estrogen receptor-α directly regulates the hypoxia- …Estrogen receptor-α directly regulates the...

Page 1: Estrogen receptor-α directly regulates the hypoxia- …Estrogen receptor-α directly regulates the hypoxia-inducible factor 1 pathway associated with antiestrogen response in breast

Estrogen receptor-α directly regulates the hypoxia-inducible factor 1 pathway associated withantiestrogen response in breast cancerJun Yanga,b,1, Alaa AlTahanb, Dylan T. Jonesa, Francesca M. Buffaa, Esther Bridgesa, Rodrigo B. Interianob, Chunxu Quc,Nathan Vogtb, Ji-Liang Lia, Dilair Baband, Jiannis Ragoussisd,2, Robert Nicholsone, Andrew M. Davidoffb,and Adrian L. Harrisa

aGrowth Factor Group, Cancer Research UK, Molecular Oncology Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford,John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom; bDepartment of Surgery, St. Jude Children’s Research Hospital, Memphis,TN 38105; cDepartment of Bioinformatics, St. Jude Children’s Research Hospital, Memphis, TN 38105; dGenomics Group, Wellcome Trust Centre for HumanGenetics, University of Oxford, Oxford OX3 7BN, United Kingdom; and eTenovus Centre for Cancer Research, Welsh School of Pharmacy, Cardiff University,Cardiff CF10 3NB, United Kingdom

Edited by Joan S. Brugge, Harvard Medical School, Boston, MA, and approved October 13, 2015 (received for review November 17, 2014)

A majority of breast cancers are driven by estrogen via estrogenreceptor-α (ERα). Our previous studies indicate that hypoxia-induc-ible factor 1α (HIF-1α) cooperates with ERα in breast cancer cells.However, whether ERα is implicated in the direct regulation ofHIF-1α and the role of HIF-1α in endocrine therapy response are un-known. In this studywe found that a subpopulation of HIF-1α targets,many of them bearing both hypoxia response elements and estrogenresponse elements, are regulated by ERα in normoxia and hypoxia.Interestingly, the HIF-1α gene itself also bears an estrogen responseelement, and its expression is directly regulated by ERα. Clinical datarevealed that expression of the HIF-1α gene or a hypoxia metagenesignature is associated with a poor outcome to endocrine treatmentin ERα+ breast cancer. HIF-1α was able to confer endocrine therapyresistance to ERα+ breast cancer cells. Our findings define, for the firsttime to our knowledge, a direct regulatory pathway between ERαand HIF-1α, which might modulate hormone response in treatment.

ERα | HIF-1α | tamoxifen

Estrogen receptor-α (ERα) is an estrogen-dependent nucleartranscription factor that is critical for mammary epithelial

cell division and breast cancer progression (1, 2). ERα isexpressed in ∼70% of breast tumors (3), the majority of whichdepend on estrogen signaling, thereby providing the rationale forusing antiestrogens as adjuvant therapy to treat breast cancer (4).Tamoxifen is a first generation selective ER modulator and hasbeen widely used in breast cancer prevention and treatment (4).Although now replaced by aromatase inhibitors as first-linetreatment in postmenopausal women, it still remains importantin premenopausal breast cancer and after failure of aromataseinhibitors. Tamoxifen acts as an antagonist in breast cancer cellsby competing with estrogen for the ER. Tamoxifen-bound ERrecruits the nuclear receptor corepressor and histone deacetylase(HDAC), as opposed to coactivators, leading to transcriptionalrepression (5). Although hundreds of thousands of patients havebenefited from tamoxifen treatment, its efficacy is limited to anaverage time of 15 mo in patients with metastatic disease (6), asresistance often develops (7). Manymechanisms have been proposed toaccount for tamoxifen resistance (8), including loss of ERα ex-pression or expression of truncated ER isoforms, posttranslationalmodification of ERα, deregulation of ERα coactivators, and in-creased receptor tyrosine kinase signaling. Recent studies furtherindicate that somatic ERα mutation (9, 10), as well as genomicamplification of distant ER response elements (11), could con-tribute to hormone therapy resistance.Our clinical studies suggest that the in vivo tumor environment

may play a role in tamoxifen resistance, as hypoxia-inducible factor1α (HIF-1α) protein expression was associated with tamoxifen

resistance in neoadjuvant, primary therapy of ERα+ breast cancers(12), as well as resistance to chemoendocrine therapy (13).HIF-1α is a master regulator of oxygen homeostasis, which is

rapidly degraded in normoxia by the tumor suppressor, von Hippel–Lindau protein (VHL), but is stabilized in hypoxia (14). This pro-cess is mainly determined by the hydroxylation of HIF-1α catalyzedby prolyl hydroxylases. HIF-1α has been associated with an ag-gressive phenotype of breast cancer: that is, large tumor size, highgrade, high proliferation, and lymph node metastasis (15). In-creased HIF-1α is also associated with ERα-positivity (15),whereas HIF-1β, the partner of HIF-1α, has been shown tofunction as a potent coactivator of ER-dependent transcription(16). Further studies revealed that in ER+ T47D breast cancercells, combined hypoxia and E2 treatment had additive effects onexpression of some genes (17), although the mechanism is notclear. We have previously shown that HIF-1α and ERα can co-ordinate expression of genes, such as lysine-specific demethylase4B/Jumonji domain-containing 2B (KDM4B/JMJD2B), anH3K9me3/me2 histone demethylase, which is targeted by bothERα and HIF-1α and epigenetically regulates cell cycle progression

Significance

About 1.7 million new cases of breast cancer occur every year, 70%of which are estrogen receptor-α (ERα) positive. Antiestrogentherapy to block ERα function is the most important approach intreatment of ERα+ patients. However, resistance eventually willdevelop for various reasons. Here we demonstrate that hypoxia-inducible factor 1α (HIF-1α) is a direct transcriptional target of ERα,which may compensate for ERα function loss because many otherERα targets are also HIF-1α targets. We further show that HIF-1αis able to confer cancer cell resistance to ERα antagonists tamox-ifen and fulvestrant, and the expression of HIF-1α is associatedwith poor survival to endocrine therapy in ERα+ patients. Ourfindings thus have revealed a previously unidentified mechanismfor antiestrogen resistance.

Author contributions: J.Y., A.M.D., and A.L.H. designed research; J.Y., A.A., D.T.J., E.B., R.B.I.,N.V., D.B., and J.R. performed research; J.Y., J.-L.L., and R.N. contributed new reagents/analytic tools; J.Y., A.A., F.M.B., and C.Q. analyzed data; and J.Y., A.M.D., and A.L.H.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The data reported in this paper have been deposited in the Gene Ex-pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE61799).1To whom correspondence should be addressed. Email: [email protected] address: McGill University and Genome Quebec Innovation Centre, McGill Uni-versity, Montreal, QC, Canada.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1422015112/-/DCSupplemental.

15172–15177 | PNAS | December 8, 2015 | vol. 112 | no. 49 www.pnas.org/cgi/doi/10.1073/pnas.1422015112

Dow

nloa

ded

by g

uest

on

Apr

il 13

, 202

0

Page 2: Estrogen receptor-α directly regulates the hypoxia- …Estrogen receptor-α directly regulates the hypoxia-inducible factor 1 pathway associated with antiestrogen response in breast

(18). The genomic locus of KDM4B bears both HIF-1α and ERαbinding elements (18, 19). These data collectively suggest thatHIF-1α and ERα are functionally associated. However, how thesetwo important oncogenic pathways interact has not yet been de-fined. In addition, whether HIF-1α plays an autonomous role inmodulating endocrine therapy efficacy, such as tamoxifen re-sistance, is unknown. In this study, we investigated the role of ERαin the regulation of HIF-1 signaling and how HIF-1 signaling isinvolved in endocrine drug response.

ResultsERα Signaling Regulates Hypoxia/HIF-1α Pathway.We have previouslyshown that knockdown of ERα significantly down-regulated his-tone demethylase KDM4B expression (18), a HIF-1α transcrip-tional target, suggesting that HIF-1α function is compromised byloss of ERα even in hypoxia. To study whether ERα signaling isinvolved in the regulation of the hypoxia/HIF pathway, we used achemical genetics approach in which the ERα+ breast cancer cellline MCF7 was treated with ICI182780 (fulvestrant) in normoxiaand hypoxia to perform a global gene-expression profile analysis(Fig. 1A). ICI182780 is an ER antagonist with no agonist effects,which works by downregulating the ERα expression. Clinically,ICI182780 has been used in hormone receptor-positive metastaticbreast cancer in postmenopausal women with disease progressionfollowing antiestrogen therapy. The gene-expression profiling re-sults showed that a cluster of genes, such as stanniocalcin 2(STC2), stanniocalcin 1 (STC1), solute carrier family 2 (Facilitatedglucose transporter), Member 1 (SLC2A1) (also known asGlut-1),and lysyl oxidase (LOX) that were induced in hypoxia were down-regulated by ICI182780 in both normoxia and hypoxia (Fig. 1A).We then queried the iLINCS (integrative LINCS) genomics dataportal to search compounds that regulate a similar gene pattern asICI182780 induced in MCF7 cells. Among the top 15 hits, mostare HIF-1α inducers or ERα modulators, including tamoxifen andICI182780 (Fig. 1B). We analyzed these genes through ChIP en-richment analysis of transcription factors (ChEA) through theLINCS canvas browser II to examine what transcriptional factors

directly regulate their expression. The top hits were ERα andHIF-1α (Fig. 1C). Thus, these data indicate that a subgroup ofgenes that are targeted by hypoxia/HIF-1α is also regulated byERα signaling, which is dual responsive to hormone and oxygen.Interestingly, some of these genes can be bound by p53 in murineembryonic stem cells although the biological function is unclear(20). Cancer genomic sequencing reveals that p53 is more com-monly mutated in triple-negative breast cancers than ERα+patients (21), whereas MCF7 is p53 wild-type. ZNF263 is a tran-scription factor that regulates FoxA1 expression (22). FoxA1 is apioneer factor that facilitates ERα for genomic binding (23),which further suggests that the estrogen-ER signaling pathway isinvolved in hypoxia/HIF response.To further confirm that ERα and HIF-1α directly bind their

response elements in a subgroup of genes, we reanalyzed pub-lished ChIP sequencing data (24, 25). We found that among the356 genes bound by HIF-1α, 202 (57%) of them were identifiedas the common genes bound by ERα as well (Fig. 1D and TableS1). KDM4B was one of the targets of both ERα and HIF-1α(Table S1), consistent with our previous studies (18, 26). Pathwayanalysis reveals that these common genes are involved in me-tabolism, cancer, and important signaling pathways, includingNotch, MAPK, and insulin pathways (Fig. 1E).

ERα Signaling Regulates HIF-1α Expression. Some genes, such as STC2,VEGFA, and KDM4B, bear both ERα and HIF-1α binding elements(18, 19, 27–30), and thus we initially hypothesized that blockade ofthe ER pathway might disrupt HIF-1α binding to its target genes.Surprisingly, we found that ERα signaling actually directly regulatesHIF-1α expression. When MCF7 cells were grown without estrogenfor 4 d and then placed in hypoxia or treated with the hypoxia mi-metic deferoximine, 17-β-estradiol (E2) greatly enhanced HIF-1αexpression (Fig. 2A). However, ICI182780 significantly reducedHIF-1α and ERα expression in hypoxia (Fig. 2B), consistent withits biological function in suppressing HIF-1α targets (Fig. 1). Inaddition, ICI1820780 treatment did not affect HIF-1α expressionin MDA-MD-231 cells (Fig. 2B), an ERα− cell line. Knockdown of

Fig. 1. ERα signaling regulates hypoxia/HIF pathway. (A) MCF7 cells were treated with 1 μM of ICI182780 in normoxia and hypoxia (1% O2) for 24 h. ExtractedRNA from duplicated biological samples was subject to microarray analysis. Heatmap shows a subgroup of genes that are dual responsive to hormone andoxygen. (B) The dually responsive genes were queried with the LINCS program to search Connectivity MAP (CMAP) for compounds that induced a similarpattern to ICI182780. (C) ChEA of transcription factor binding to the dual responsive genes. (D) Venn diagram shows the common gene bound by both ERαand HIF-1α. (E) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation of the common genes from D.

Yang et al. PNAS | December 8, 2015 | vol. 112 | no. 49 | 15173

MED

ICALSC

IENCE

S

Dow

nloa

ded

by g

uest

on

Apr

il 13

, 202

0

Page 3: Estrogen receptor-α directly regulates the hypoxia- …Estrogen receptor-α directly regulates the hypoxia-inducible factor 1 pathway associated with antiestrogen response in breast

ERα with two separate siRNA oligos or a SMARTpool of siRNAgreatly reduced HIF-1α induction under hypoxia or hypoxic mimeticdimethyloxalylglycine (DMOG) treatment (Fig. 2 C and D). Theproteasome inhibitor MG132, which stabilizes HIF-1α protein ex-pression by preventing protein degradation, failed to rescue ERαknockdown-mediated down-regulation of HIF-1α (Fig. 2E). Thesedata clearly show that the ERα signaling pathway directly regulatesHIF-1α expression.

HIF-1α Gene Bears Estrogen Response Element and ERα EnhancesHIF-1α Transcription. HIF-1α is labile and subject to rapid protea-somal degradation. Most studies focus on the regulation of proteinstability of HIF-1α but how HIF-1α is transcriptionally regulated isnot well understood. The down-regulation of HIF-1α by knock-down of ERα was not rescued by the proteasome inhibitor MG132,indicating that ERα regulates HIF-1α expression in a proteinstability-independent manner (Fig. 2E). Supporting this hy-pothesis, the ERα antagonist tamoxifen treatment resulted indown-regulation of HIF-1α expression in MCF7 but not MDA-MB-231 cells, although tamoxifen caused ERα protein up-regulation (Fig. S1A), suggesting impaired ERα transcriptionalfunction is involved. To determine whether ERα signaling di-rectly regulates HIF-1α gene expression, we treated hormone-starved MCF7 and MDA-MB-231 cells with estrogen. RT-PCRdata revealed that HIF-1α, KDM4B, and STC2 mRNA wassignificantly up-regulated in MCF7 but not MDA-MB-231cells (Fig. 3A and Fig. S1 B and C). RT-PCR results furthershowed that HIF-1α mRNA level was significantly reducedafter ICI182780 treatment, indicating an ER-mediated tran-scriptional mechanism regulates HIF-1α transcription.By analyzing the HIF-1α genomic sequence that bears 15 exons

and 14 introns (Fig. 3C), we found that there is a canonical estrogenresponse element (ERE) located in the first intron. Interestingly,there is also a FOXA1 binding site that is 64 nucleotides down-stream of the ERE (Fig. 3C), further supporting it as a bona fide

ERα binding element because FOXA1 is a pioneer factor thatfacilitates ERα recruitment (23). In fact, all ER-chromatin in-teractions and gene-expression changes depend on the presenceof FOXA1 (23). To test whether ERα is able to bind the ERE oftheHIF-1α gene, we treated MCF7 cells with E2 or tamoxifen andthen performed ChIP and real-time PCR assays. The resultsclearly showed that ERα bound to this ERE compared with thesurrounding negative regions, and E2 and tamoxifen treatmentfurther enhanced ERα binding (Fig. 3D). Next, we cloned theERE sequences (Fig. S2 A and B) into a luciferase reporter andperformed a luciferase assay to assess the regulatory function ofERα. Indeed, the luciferase activity was significantly high, but theERE mutant abrogated the activity in MCF7 cells (Fig. 3E). Wealso tested the binding of ERα in hypoxia condition. The resultsshowed that ERα still bound at the ERE of HIF-1α under hypoxia(Fig. 3F). Thus, these data demonstrate that we have identifiedHIF-1α as a direct target of ERα, potentially explaining whyHIF-1α is associated with ER-positivity in breast cancer samples.Interestingly, we did not see positive correlation between

HIF-1α and ERα at transcript levels from tumor cohorts; in-stead, we found a negative correlation between them (Fig. S3 Aand B). This may be explained by the hypothesis that ER− breastcancer has been epigenetically remodeled to express highHIF-1α transcript. This finding led us to further speculate thatERα loss need to be compensated by high HIF-1α activity.

Fig. 2. ERα signaling regulates HIF-1α expression. (A) After 4-d hormonalstarvation, MCF7 cells were treated with 500 μM of DFO or 1% O2 for 6 h inthe presence or absence of 100 nM of E2. The whole-cell lysates were subjectto Western blotting with indicated antibodies. V, Vehicle. (B) MCF7 andMDA-MB-231 cells were treated with 1 μM of ICI182780 in hypoxia (1% O2)for 24 h. The whole-cell lysates were subject to Western blotting with in-dicated antibodies. (C and D) ERα in MCF7 cells was knockdown with twodifferent siRNAs (C) or SMARTpool (D), which were treated with 200 μM ofDMOG (C) or hypoxia (D). The whole-cell lysates were subject to Westernblotting with indicated antibodies. (E) MCF7 cells were treated with siRNASMARTpool against ERα, treated with 10 μM G132 for 16 h. The whole-celllysates were subject to Western blotting with indicated antibodies.

Fig. 3. ERα directly binds EREs on the HIF-1α gene to enhance HIF-1α tran-scription. (A) After 3-d hormonal starvation, MCF7 cells were treated with100 nM of E2 for 24 h. Real-time PCR was performed to assess HIF-1α geneexpression. (B) MCF7 cells were treated with 1 μM of ICI182780 for 24 h. Real-time PCR was performed to assess HIF-1α gene expression. (C) The genestructure of HIF-1α and the sequence that bears ERα binding sites (ERE) andFOXA1 binding sites, as indicated. The black bar represents each exon and theblack line represents each intron of HIF-1α. NB1 and NB2 represent the primerlocation for ChIP PCR to assess ERα binding as negative controls, as shown in D.(D) After 3-d hormonal starvation, MCF7 cells were treated with 100 nM of E2or tamoxifen for 24 h. ChIP PCR was performed to assess ERα binding amongthe regions as indicated in C. (E) The ERE region and the mutant were clonedinto pGL3 luciferase reporter. The plasmids and the pRL Renilla Luciferase controlvector (to normalize the ERE reporter) were transfected into MCF7 cells. Dualluciferase assay was performed. (F) MCF7 cells were incubated in hypoxia for 8 h.ChIP PCR was performed to assess ERα binding at ERE of HIF-1α. **P < 0.01.

15174 | www.pnas.org/cgi/doi/10.1073/pnas.1422015112 Yang et al.

Dow

nloa

ded

by g

uest

on

Apr

il 13

, 202

0

Page 4: Estrogen receptor-α directly regulates the hypoxia- …Estrogen receptor-α directly regulates the hypoxia-inducible factor 1 pathway associated with antiestrogen response in breast

Tamoxifen-Bound ERα Inhibits HIF-1α Expression. Tamoxifen is an an-tagonist of ERα, competing with estrogen for the ER binding.Tamoxifen-bound ERα recruits the corepressors nuclear receptorcorepressor and HDAC to silence gene transcription of ERα tar-gets (5). The discovery of enhancing HIF-1α by E2-bound ERαprompted us to examine the effect of tamoxifen-bound ERα.Tamoxifen treatment of MCF7 cells greatly increased HDAC1binding on the ERE of HIF-1α. Interestingly, ICI182780 did notaffect HDAC1 binding, but remarkably reduced the ERα binding(Fig. 4A). These data indicate that the two compounds inhibit ERαfunction through different mechanisms.We then used an in vitro established tamoxifen-resistant MCF7

(TamR-MCF7) cell line and BT474 cells, which are intrinsicallyresistant to tamoxifen (Fig. 4 B and C). These cells were cultured inmedia containing tamoxifen for propagating. When ERα was de-pleted in these cells, HIF-1α expression was up-regulated (Fig. 4 Band C). Overexpression of ERα in TamR-MCF7 cells significantlyreduced HIF-1α expression, but not for the parental MCF7 cellsin normoxia (Fig. 4D). Longer exposure of the film showed thatoverexpression of ERα enhanced HIF-1α in parental cells (Fig.4D). Interestingly, we noticed that the basal levels of HIF-1α ex-pression in Tam-MCF7 cells is higher than in parental cells, in-dicating epigenetic effects are involved after cells acquire tamoxifenresistance. These data indicate that E2-bound ERα induces—buttamoxifen-bound ERα suppresses—HIF-1α expression (Fig. 4E).

HIF-1α Confers Tamoxifen Resistance on ERα+ Breast Cancer Cells. Be-cause HIF-1α was a downstream target of ERα and enhanced ERαtarget expression, we hypothesized that HIF-1α may modulateendocrine efficacy in ER+ breast cancers. We therefore assessedthe role of HIF in regulation of breast cancer cell survival orproliferation in response to inhibitors of ERα. We first used ret-roviral vector-mediated transduction to stably introduce HIF-1α

and HIF-2α into T47D and MCF7 cells (Fig. 5A and Fig. S4).HIF-2α expression significantly suppressed proliferation of bothcell lines, whereas expression of HIF-1α did not. Thus, we wereunable to generate stable cell lines with HIF-2α. Although bothparental MCF7 and T47D and their derivative HIF-1α–expressingcells responded to tamoxifen treatment, the HIF-1α–expressingcells were at least twofold more resistant in normoxia (Fig. S4) andlong-term treatment showed more remarkable effect (Fig. 5B),demonstrating that HIF-1α is able to confer tamoxifen resistance.We also treated the cells with ICI182780. Although the parentaland HIF-1α expressing cells responded to ICI182780 similarly after1 wk of treatment (Fig. S4B), long-term treatment (18 d or 4 wk)with 10 nM or 100 nM led to development of more resistant col-onies (Fig. 5 B and C). Although 1,000 nM of ICI182780 efficientlysuppressed both cell lines, HIF-1α–expressing cells gave rise tomore large resistant colonies (Fig. 5C).We further tested the capacity for tumorsphere formation of the

HIF-1α–expressing and parental cells using a mammosphere 3Dculture system. HIF-1α did not affect tumorsphere formation;however, HIF-1α conferred significant resistance to tamoxifen andICI182780 compared with the parental control (Fig. 5 D and E)(P < 0.01). We also tested another drug, RITA, which inhibits

Fig. 4. Tamoxifen-bound ERα inhibits HIF-1α expression. (A) MCF7 cells werecultured in standard DMEM and treated with 1,000 nM of tamoxifen orICI182780 for 48 h. ChIP PCR was performed to assess HDAC1 and ERα bindingthe ERE of HIF-1α. (B) MCF7 and TamR-MCF7 cells were transfected with siRNASMARTpool against ERα. After 36-h transfection, cells were incubated in nor-moxia and hypoxia for 16 h. Western blotting was used to assess the indicatedproteins. (C) BT474 cells were treated as in B. (D) MCF7 and TamR-MCF7 cellswere transfected with ERα expression plasmids. MCF7 cells were cultured innormoxia in standard DMEM while Tam-MCF7 cells were cultured in phenol-free DMEM with 100 nM of tamoxifen. After 48-h transfection, cells wereharvested for Western blotting. The two HIF-1α blots were for short and longexposure, respectively. (E) The cartoon shows the E2-bound and tamoxifen-bound ERα exert opposite effect on HIF-1α transcription.

Fig. 5. HIF-1α confers tamoxifen resistance to ER+ breast cancer cells.(A) Western blotting to assess MCF7 cells expressing HIF-1α. (B) The MCF7control and MCF7–HIF-1α cells were treated with tamoxifen or ICI182780 withindicated concentration for 18 d. Cell colonies were stained with Crystal violet.(C) The MCF7 control and MCF7–HIF-1α cells were treated with ICI182780 for4 wk. Cell colonies were stained with Crystal violet. (D) Tumorspheres gener-ated from the parental (Upper) and HIF-1α expressing (Lower) MCF7 cells weretreated for 5 d with 1 μM of tamoxifen, 1 μM of ICI182780, and 0.5 μM of RITA.Photos were taken under microscope (10×). (E) After counting 10 different 10×fields, the number of tumorsphere counts were averaged and compared withStudent’s t test (two tailed, **P < 0.01). (F) Kaplan–Meier analyses for relapsefree survival of the cohort of patients with ER-positivity, receiving tamoxifentreatment only without chemotherapy. Affymetrix ID for HIF-1α used was200989_at. The cut-off value used in analysis was 3,043 and the expressionrange of the probe was 439–17,198. Patient number for low HIF-1α (black) andhigh HIF-1α (red) is presented under the following months. (G) Kaplan-Meieranalyses for overall survival of the cohort of patients with ER-positivity, re-ceiving tamoxifen treatment with chemotherapy. The cutoff value was 3035and the expression range was 456–11726. Patient number for low HIF-1α(black) and high HIF-1α (red) are presented under the following months.

Yang et al. PNAS | December 8, 2015 | vol. 112 | no. 49 | 15175

MED

ICALSC

IENCE

S

Dow

nloa

ded

by g

uest

on

Apr

il 13

, 202

0

Page 5: Estrogen receptor-α directly regulates the hypoxia- …Estrogen receptor-α directly regulates the hypoxia-inducible factor 1 pathway associated with antiestrogen response in breast

HIF-1α expression and induces apoptosis of MCF7 cells (31). Theresults showed that RITA equally reduced the tumorsphere forma-tion in both HIF-1α–expressing and parental cells (Fig. 5 D and E).

Hypoxia Metagene Signature and High HIF-1α Gene Expression Showa Poor Response to Tamoxifen Treatment in ERα+ Breast Cancer. Todetermine whether hypoxia/HIF-1α is associated with tamoxifeneffectiveness in patients with breast cancer, we first examinedthe hypoxia status of two groups of breast cancer patients by ahypoxia/HIF-1α metagene signature (32). We then comparedrelapse-free survival of ERα+ breast cancers classified as hypoxicor normoxic by their gene-expression profiles with this hypoxia/HIF-1αmetagene signature. In those without adjuvant tamoxifentherapy, hypoxic cases had a significantly worse outcome thanthe normoxic breast cancers (P = 0.001) (Fig. S5A). A significantdifference in outcome for those treated with tamoxifen remained(P = 0.03) (Fig. S5A). Then we analyzed whether the HIF-1αgene expression itself correlated with tamoxifen response in alarge cohort of ER+ patients from public data (33). Kaplan–Meier analysis results showed that patients with high level ofHIF-1α gene expression had a poorer relapse-free survival toendocrine therapy or tamoxifen treatment alone (P = 0.0093)(Fig. 5G and Fig. S5B), although overall survival was not sig-nificantly different (Fig. S5C). When chemotherapy was includedfor those patients who received tamoxifen, HIF-1α is also asso-ciated with poor overall survival (P = 0.017) (Fig. 5H). Thesedata further indicate that HIF-1α may be directly involved inmodulating tamoxifen response in ER+ patients. Interestingly,high HIF-2α is associated with better survival in ER+ breastcancer patients who received endocrine therapy (Fig. S5D). Al-though statistically not significant, HIF-2α tends to be associatedwith better survival in tamoxifen-treated patients (Fig. S5E).These results were consistent with our findings that HIF-2αoverexpression was harmful for ERα+ cancer cells and the clin-ical data that HIF-2α is significantly lower in breast cancer tissuesthan the normal breast tissue (Fig. S6).

HIF-1α Overexpression Confers Advantage of Tumor Growth andResistance to Tamoxifen Treatment. To further investigate the ef-fect of HIF-1α on tamoxifen treatment in vivo, orthotopic xenografttumor was established using MCF7 cells overexpressing HIF-1α.Compared with the control, HIF-1α overexpression resulted inrapid tumor growth in NOD SCID-γ (NSG) mice that had to bekilled before the control reached comparable sizes of tumors (Fig.6A). Tamoxifen treatment only modestly delayed tumor growthwith HIF-1α overexpression (P > 0.05), similar to the in vitro data(Fig. 5), indicating that HIF-1α confers tamoxifen resistance.

DiscussionTo develop novel therapeutics to treat breast cancer, a deeperunderstanding of the molecular mechanism of ER-driven cancer isimportant, as the most common type of metastatic breast cancer isendocrine receptor-positive. The association of ERα-positivity andHIF-1α from clinical studies (12, 15, 34) supports our findings thatthese two pathways may act in cooperation to promote breastcancer progression. However, the basis of these previous clinicalobservations was unclear because ER− tumors have a greaterproliferation rate and are more hypoxic than ER+ tumors. Hence,it was unclear why HIF-1α should be more highly expressed inER+ tumors (15, 34). Our data, which demonstrate that ERαregulates HIF-1α expression, provide a mechanism for theseclinical observations. HIF-1α is a labile protein that is rapidly de-graded by VHL-mediated proteasomal degradation in normoxia butstabilized in hypoxia. However, the transcriptional regulation ofHIF-1α is not well studied, except in one report showing that theNF-κB pathway regulates HIF-1α gene expression (35). Here weshow that the HIF-1α gene bears a canonical ER-binding elementthat responds to estrogen signaling, demonstrating a direct

regulatory link between the ERα and HIF-1α pathways in breastcancer (Fig. 6B).Interestingly, we found a subgroup of genes that are dually

responsive to hormone and oxygen (Fig. 1). These genes wereup-regulated by hypoxia but the ERα antagonist ICI182780 sig-nificantly reduced their expression. Previous studies also show thatsome genes, such as KDM4B, STC2, and VEGFA, bear both ahypoxia response element and ERE (18, 19, 26–30). This findingmay indicate that the ER signaling pathway can be enhanced byHIF-1α induction and vice versa (Fig. 6B). The physiological sig-nificance of the cross-talk between these two pathways in breast orovary development warrants further study. Nevertheless, we en-visage that both pathways form a positive feedback loop to en-hance the common downstream target gene expression. But isHIF-1 required for ERα activity? We previously showed that de-pletion of HIF-1α only partially affected KDM4B expression inhypoxia, whereas depletion of ERα nearly abrogated KDM4Bexpression (18), indicating that HIF-1 might not be required forERα activity but synergizes with ERα. Here we further tested thisby knocking down HIF-1α in tamoxifen-resistant MCF7 andBT474 cells and obtained a similar result that KDM4B expressionwas only partially reduced (Fig. S7), further suggesting that ERαfunction may not rely on HIF-1α. However, we cannot exclude thepossibility that ERα may require HIF-1α function for activation ofcertain specific genes or under specific conditions.Another important conclusion of our findings is that overactive

HIF-1α function may partially compensate for estrogen signalingwhen ERα function is compromised, such as under the circum-stances of hormone therapy. When ERα+ breast cancer cells weretransduced with HIF-1α, the cancer cells became much more re-sistant to tamoxifen and ICI182780 treatment (Fig. 5). This find-ing is consistent with the fact that the HIF-1α and hypoxia genesignature were correlated with poorer survival in response tohormone therapy. Although the molecular mechanism by whichHIF-1α confers tamoxifen resistance needs to be further defined,our recent findings indicate that the histone demethylase KDM4Bis important in coordinating HIF-1α and ERα. KDM4B is a directtarget of both HIF-1α and ERα and regulates expression of manygenes in normoxia and hypoxia and cell cycle progression (18).Thus, HIF-1α may drive gene expression of KDM4B and othergenes to compensate for tamoxifen inhibition of ERα signaling.This may be more important in vivo as the common factors aresecreted extracellular signaling molecules. These results comple-ment recent data showing the importance of HIF-1α in triple-negative breast cancer that is driven by XBP1 in response to

Fig. 6. The working model between ERα and HIF-1 pathway. (A) Mammaryfat pad tumor growth assessment of HIF-1α–overexpressing MCF7 cellscompared with parental MCF7 cells implanted with tamoxifen citrate (5 mg;TAM) or placebo pellet (CTL) when HIF-1α tumors reached 150 mm3 (in-dicated by red arrow) in NSG female mice with estrogen pellets implanted(0.72 mg; n = 5 per group). NS, nonsignificant; ***P < 0.001. (B) HIF-1α genebears ERα binding site whose transcription is directly regulated by ERα sig-naling pathway. Under hypoxia, produced HIF-1α protein is stabilized. Dualresponsive genes bear both ERα and HIF-1α binding sites, which can beregulated by two signaling pathways. ERα directly regulates the HIF-1αpathway associated with antiestrogen response in breast cancer.

15176 | www.pnas.org/cgi/doi/10.1073/pnas.1422015112 Yang et al.

Dow

nloa

ded

by g

uest

on

Apr

il 13

, 202

0

Page 6: Estrogen receptor-α directly regulates the hypoxia- …Estrogen receptor-α directly regulates the hypoxia-inducible factor 1 pathway associated with antiestrogen response in breast

unfolded protein or endoplasmic reticulum stress (36). In-terestingly, XBP1 has been shown to confer both estrogenindependence and antiestrogen resistance in breast cancer celllines (37). Thus, it is also possible that cellular stress-inducedXBP1 might interact with HIF-1α to confer antiestrogen re-sistance. Therefore, our data suggest that targeting the HIF-1αsignaling pathway might increase efficacy of endocrine therapyin breast cancers.

Materials and MethodsCells were maintained in DMEM supplemented with 10% (vol/vol) FCS, 1%glutamine, and 1% penicillin-streptomycin. Western blot analysis, quanti-tative RT-PCR, tumorsphere formation, ChIP, retroviruses and plasmids,

siRNA transfection, cell viability and colony formation assays, luciferase re-porter assay, gene-expression profiling, data mining, patient details, andgene-expression profiling for patients sample, and in vivo xenograft ex-periment are described in SI Materials and Methods. Statistical analyses weretwo-tailed t tests, with P ≤ 0.05 considered statistically significant. All animalprocedures were approved by University of Oxford ethical review and workwas conducted in accordance with the UK Home Office guidelines, under theproject licence PPL 30/2771.

ACKNOWLEDGMENTS. We thank the Oxford and Royal Marsden NHSBiomedical Research Centres and Experimental Cancer Medicine Centre.This study was supported in part by Cancer Research UK, Breast CancerResearch Foundation (A.L.H., J.Y., and J.-L.L.), and Advancing Clinico-Genomic Trials European Union Project FP6-IST-026996 (to F.M.B.).

1. Carroll JS, Brown M (2006) Estrogen receptor target gene: An evolving concept. MolEndocrinol 20(8):1707–1714.

2. McDonnell DP, Norris JD (2002) Connections and regulation of the human estrogenreceptor. Science 296(5573):1642–1644.

3. Harvey JM, Clark GM, Osborne CK, Allred DC (1999) Estrogen receptor status by im-munohistochemistry is superior to the ligand-binding assay for predicting response toadjuvant endocrine therapy in breast cancer. J Clin Oncol 17(5):1474–1481.

4. Jordan VC (1995) Third annual William L. McGuire Memorial Lecture. “Studies on theestrogen receptor in breast cancer”—20 years as a target for the treatment andprevention of cancer. Breast Cancer Res Treat 36(3):267–285.

5. Huang HJ, Norris JD, McDonnell DP (2002) Identification of a negative regulatory surfacewithin estrogen receptor alpha provides evidence in support of a role for corepressorsin regulating cellular responses to agonists and antagonists. Mol Endocrinol 16(8):1778–1792.

6. Howell A, DeFriend D, Robertson J, Blamey R, Walton P (1995) Response to a specificantioestrogen (ICI 182780) in tamoxifen-resistant breast cancer. Lancet 345(8941):29–30.

7. Clarke R, Leonessa F, Welch JN, Skaar TC (2001) Cellular and molecular pharmacologyof antiestrogen action and resistance. Pharmacol Rev 53(1):25–71.

8. Musgrove EA, Sutherland RL (2009) Biological determinants of endocrine resistance inbreast cancer. Nat Rev Cancer 9(9):631–643.

9. Toy W, et al. (2013) ESR1 ligand-binding domain mutations in hormone-resistantbreast cancer. Nat Genet 45(12):1439–1445.

10. Robinson DR, et al. (2013) Activating ESR1 mutations in hormone-resistant metastaticbreast cancer. Nat Genet 45(12):1446–1451.

11. Hsu PY, et al. (2013) Amplification of distant estrogen response elements deregulatestarget genes associated with tamoxifen resistance in breast cancer. Cancer Cell 24(2):197–212.

12. Generali D, et al. (2009) Phosphorylated ERalpha, HIF-1alpha, and MAPK signaling aspredictors of primary endocrine treatment response and resistance in patients withbreast cancer. J Clin Oncol 27(2):227–234.

13. Generali D, et al. (2006) Hypoxia-inducible factor-1alpha expression predicts a poorresponse to primary chemoendocrine therapy and disease-free survival in primaryhuman breast cancer. Clin Cancer Res 12(15):4562–4568.

14. Semenza GL (1998) Hypoxia-inducible factor 1: Master regulator of O2 homeostasis.Curr Opin Genet Dev 8(5):588–594.

15. Bos R, et al. (2001) Levels of hypoxia-inducible factor-1 alpha during breast carcino-genesis. J Natl Cancer Inst 93(4):309–314.

16. Brunnberg S, et al. (2003) The basic helix-loop-helix-PAS protein ARNT functions as apotent coactivator of estrogen receptor-dependent transcription. Proc Natl Acad SciUSA 100(11):6517–6522.

17. Seifeddine R, et al. (2007) Hypoxia and estrogen co-operate to regulate gene expressionin T-47D human breast cancer cells. J Steroid Biochem Mol Biol 104(3-5):169–179.

18. Yang J, et al. (2010) The histone demethylase JMJD2B is regulated by estrogen re-ceptor alpha and hypoxia, and is a key mediator of estrogen induced growth. CancerRes 70(16):6456–6466.

19. Beyer S, Kristensen MM, Jensen KS, Johansen JV, Staller P (2008) The histone deme-thylases JMJD1A and JMJD2B are transcriptional targets of hypoxia-inducible factorHIF. J Biol Chem 283(52):36542–36552.

20. Sharma SV, et al. (2010) A chromatin-mediated reversible drug-tolerant state incancer cell subpopulations. Cell 141(1):69–80.

21. Cancer Genome Atlas Network (2012) Comprehensive molecular portraits of humanbreast tumours. Nature 490(7418):61–70.

22. Frietze S, Lan X, Jin VX, Farnham PJ (2010) Genomic targets of the KRAB and SCANdomain-containing zinc finger protein 263. J Biol Chem 285(2):1393–1403.

23. Hurtado A, Holmes KA, Ross-Innes CS, Schmidt D, Carroll JS (2011) FOXA1 is a keydeterminant of estrogen receptor function and endocrine response. Nat Genet 43(1):27–33.

24. Schmidt D, et al. (2010) A CTCF-independent role for cohesin in tissue-specific tran-scription. Genome Res 20(5):578–588.

25. Schödel J, et al. (2011) High-resolution genome-wide mapping of HIF-binding sites byChIP-seq. Blood 117(23):e207–e217.

26. Yang J, et al. (2009) Role of hypoxia-inducible factors in epigenetic regulation viahistone demethylases. Ann N Y Acad Sci 1177:185–197.

27. Bouras T, et al. (2002) Stanniocalcin 2 is an estrogen-responsive gene coexpressedwith the estrogen receptor in human breast cancer. Cancer Res 62(5):1289–1295.

28. Law AY, Wong CK (2010) Stanniocalcin-2 is a HIF-1 target gene that promotes cellproliferation in hypoxia. Exp Cell Res 316(3):466–476.

29. Forsythe JA, et al. (1996) Activation of vascular endothelial growth factor genetranscription by hypoxia-inducible factor 1. Mol Cell Biol 16(9):4604–4613.

30. Mueller MD, et al. (2000) Regulation of vascular endothelial growth factor (VEGF)gene transcription by estrogen receptors alpha and beta. Proc Natl Acad Sci USA97(20):10972–10977.

31. Yang J, et al. (2009) Small-molecule activation of p53 blocks hypoxia-inducible factor1alpha and vascular endothelial growth factor expression in vivo and leads to tumorcell apoptosis in normoxia and hypoxia. Mol Cell Biol 29(8):2243–2253.

32. Buffa FM, Harris AL, West CM, Miller CJ (2010) Large meta-analysis of multiple cancersreveals a common, compact and highly prognostic hypoxia metagene. Br J Cancer102(2):428–435.

33. Györffy B, et al. (2010) An online survival analysis tool to rapidly assess the effect of22,277 genes on breast cancer prognosis using microarray data of 1,809 patients.Breast Cancer Res Treat 123(3):725–731.

34. Bos R, et al. (2004) Expression of hypoxia-inducible factor-1alpha and cell cycle pro-teins in invasive breast cancer are estrogen receptor related. Breast Cancer Res 6(4):R450–R459.

35. Rius J, et al. (2008) NF-kappaB links innate immunity to the hypoxic response throughtranscriptional regulation of HIF-1alpha. Nature 453(7196):807–811.

36. Chen X, et al. (2014) XBP1 promotes triple-negative breast cancer by controlling theHIF1α pathway. Nature 508(7494):103–107.

37. Gomez BP, et al. (2007) Human X-box binding protein-1 confers both estrogen in-dependence and antiestrogen resistance in breast cancer cell lines. FASEB J 21(14):4013–4027.

38. de Hoon MJ, Imoto S, Nolan J, Miyano S (2004) Open source clustering software.Bioinformatics 20(9):1453–1454.

39. Gyorffy B, Lánczky A, Szállási Z (2012) Implementing an online tool for genome-widevalidation of survival-associated biomarkers in ovarian-cancer using microarray datafrom 1287 patients. Endocr Relat Cancer 19(2):197–208.

40. Loi S, et al. (2007) Definition of clinically distinct molecular subtypes in estrogen receptor-positive breast carcinomas through genomic grade. J Clin Oncol 25(10):1239–1246.

41. Higgins GS, et al. (2010) Overexpression of POLQ confers a poor prognosis in earlybreast cancer patients. Oncotarget 1(3):175–184.

Yang et al. PNAS | December 8, 2015 | vol. 112 | no. 49 | 15177

MED

ICALSC

IENCE

S

Dow

nloa

ded

by g

uest

on

Apr

il 13

, 202

0