receptor-α directly regulates the - inducible factor 1 pathway associated with antiestrogen response in breast cancer

Jun 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 Genetics, 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 estrogen resistance in neoadjuvant, primary therapy of ERα breast cancers receptor-α (ERα). Our previous studies indicate that hypoxia-induc- (12), as well as resistance to chemoendocrine therapy (13). ible factor 1α (HIF-1α) cooperates with ERα in breast cancer cells. HIF-1α is a master regulator of oxygen , which is However, whether ERα is implicated in the direct regulation of rapidly degraded in normoxia by the tumor suppressor, von Hippel– HIF-1α and the role of HIF-1α in endocrine therapy response are un- Lindau (VHL), but is stabilized in hypoxia (14). This pro- known. In this study we found that a subpopulation of HIF-1α targets, cess is mainly determined by the hydroxylation of HIF-1α catalyzed many of them bearing both hypoxia response elements and estrogen by prolyl hydroxylases. HIF-1α has been associated with an ag- response elements, are regulated by ERα in normoxia and hypoxia. gressive phenotype of breast cancer: that is, large tumor size, high Interestingly, the HIF-1α itself also bears an estrogen response grade, high proliferation, and lymph node metastasis (15). In- element, and its expression is directly regulated by ERα.Clinicaldata creased HIF-1α is also associated with ERα-positivity (15), revealed that expression of the HIF-1α gene or a hypoxia metagene whereas HIF-1β, the partner of HIF-1α, has been shown to signature is associated with a poor outcome to endocrine treatment function as a potent coactivator of ER-dependent transcription + + in ERα breast cancer. HIF-1α was able to confer endocrine therapy (16). Further studies revealed that in ER T47D breast cancer + resistance to ERα breast cancer cells. Our findings define, for the first cells, combined hypoxia and E2 treatment had additive effects on time to our knowledge, a direct regulatory pathway between ERα expression of some (17), although the mechanism is not and HIF-1α, which might modulate hormone response in treatment. clear. We have previously shown that HIF-1α and ERα can co- ordinate expression of genes, such as lysine-specific demethylase ERα | HIF-1α | tamoxifen 4B/Jumonji domain-containing 2B (KDM4B/JMJD2B), an H3K9me3/me2 histone demethylase, which is targeted by both strogen receptor-α (ERα) is an estrogen-dependent nuclear ERα and HIF-1α and epigenetically regulates cell cycle progression Etranscription factor that is critical for mammary epithelial cell division and breast cancer progression (1, 2). ERα is Significance expressed in ∼70% of breast tumors (3), the majority of which depend on estrogen signaling, thereby providing the rationale for About 1.7 million new cases of breast cancer occur every year, 70% using antiestrogens as adjuvant therapy to treat breast cancer (4). of which are estrogen receptor-α (ERα) positive. Antiestrogen Tamoxifen is a first generation selective ER modulator and has therapy to block ERα function is the most important approach in + been widely used in breast cancer prevention and treatment (4). treatmentofERα patients. However, resistance eventually will Although now replaced by aromatase inhibitors as first-line develop for various reasons. Here we demonstrate that hypoxia- treatment in postmenopausal women, it still remains important inducible factor 1α (HIF-1α) is a direct transcriptional target of ERα, α in premenopausal breast cancer and after failure of aromatase which may compensate for ER function loss because many other α α α inhibitors. Tamoxifen acts as an antagonist in breast cancer cells ER targets are also HIF-1 targets. We further show that HIF-1 α by competing with estrogen for the ER. Tamoxifen-bound ER is able to confer cancer cell resistance to ER antagonists tamox- ifen and fulvestrant, and the expression of HIF-1α is associated recruits the nuclear receptor corepressor and histone deacetylase + with poor survival to endocrine therapy in ERα patients. Our (HDAC), as opposed to coactivators, leading to transcriptional findings thus have revealed a previously unidentified mechanism repression (5). Although hundreds of thousands of patients have for antiestrogen resistance. benefited from tamoxifen treatment, its efficacy is limited to an average time of 15 mo in patients with metastatic disease (6), as 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., resistance often develops (7). Many mechanisms have been proposed to N.V., D.B., and J.R. performed research; J.Y., J.-L.L., and R.N. contributed new reagents/ account for tamoxifen resistance (8), including loss of ERα ex- 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. pression or expression of truncated ER isoforms, posttranslational The authors declare no conflict of interest. modification of ERα, deregulation of ERα coactivators, and in- creased receptor tyrosine kinase signaling. Recent studies further This article is a PNAS Direct Submission. α Data deposition: The data reported in this paper have been deposited in the Gene Ex- indicate that somatic ER mutation (9, 10), as well as genomic pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE61799). amplification of distant ER response elements (11), could con- 1To whom correspondence should be addressed. Email: [email protected]. tribute to hormone therapy resistance. 2Present address: McGill University and Genome Quebec Innovation Centre, McGill Uni- Our clinical studies suggest that the in vivo tumor environment versity, Montreal, QC, Canada. may play a role in tamoxifen resistance, as hypoxia-inducible factor This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1α (HIF-1α) protein expression was associated with tamoxifen 1073/pnas.1422015112/-/DCSupplemental.

15172–15177 | PNAS | December 8, 2015 | vol. 112 | no. 49 www.pnas.org/cgi/doi/10.1073/pnas.1422015112 Downloaded by guest on September 23, 2021 (18). The genomic locus of KDM4B bears both HIF-1α and ERα directly regulate their expression. The top hits were ERα and binding elements (18, 19). These data collectively suggest that HIF-1α (Fig. 1C). Thus, these data indicate that a subgroup of HIF-1α and ERα are functionally associated. However, how these genes that are targeted by hypoxia/HIF-1α is also regulated by two important oncogenic pathways interact has not yet been de- ERα signaling, which is dual responsive to hormone and oxygen. fined. In addition, whether HIF-1α plays an autonomous role in Interestingly, some of these genes can be bound by p53 in murine modulating endocrine therapy efficacy, such as tamoxifen re- embryonic stem cells although the biological function is unclear sistance, is unknown. In this study, we investigated the role of ERα (20). Cancer genomic sequencing reveals that p53 is more com- + in the regulation of HIF-1 signaling and how HIF-1 signaling is monly mutated in triple-negative breast cancers than ERα involved in endocrine drug response. patients (21), whereas MCF7 is p53 wild-type. ZNF263 is a tran- scription factor that regulates FoxA1 expression (22). FoxA1 is a Results pioneer factor that facilitates ERα for genomic binding (23), ERα Signaling Regulates Hypoxia/HIF-1α Pathway. We have previously which further suggests that the estrogen-ER signaling pathway is shown that knockdown of ERα significantly down-regulated his- involved in hypoxia/HIF response. tone demethylase KDM4B expression (18), a HIF-1α transcrip- To further confirm that ERα and HIF-1α directly bind their tional target, suggesting that HIF-1α function is compromised by response elements in a subgroup of genes, we reanalyzed pub- loss of ERα even in hypoxia. To study whether ERα signaling is lished ChIP sequencing data (24, 25). We found that among the involved in the regulation of the hypoxia/HIF pathway, we used a 356 genes bound by HIF-1α, 202 (57%) of them were identified + chemical genetics approach in which the ERα breast cancer cell as the common genes bound by ERα as well (Fig. 1D and Table line MCF7 was treated with ICI182780 (fulvestrant) in normoxia S1). KDM4B was one of the targets of both ERα and HIF-1α and hypoxia to perform a global gene-expression profile analysis (Table S1), consistent with our previous studies (18, 26). Pathway (Fig. 1A). ICI182780 is an ER antagonist with no agonist effects, analysis reveals that these common genes are involved in me- which works by downregulating the ERα expression. Clinically, tabolism, cancer, and important signaling pathways, including ICI182780 has been used in hormone receptor-positive metastatic Notch, MAPK, and pathways (Fig. 1E). breast cancer in postmenopausal women with disease progression following antiestrogen therapy. The gene-expression profiling re- ERα Signaling Regulates HIF-1α Expression. Some genes, such as STC2, sults showed that a cluster of genes, such as stanniocalcin 2 VEGFA,andKDM4B, bear both ERα and HIF-1α binding elements (STC2), stanniocalcin 1 (STC1), solute carrier family 2 (Facilitated (18, 19, 27–30), and thus we initially hypothesized that blockade of transporter), Member 1 (SLC2A1) (also known as Glut-1), the ER pathway might disrupt HIF-1α binding to its target genes. and lysyl oxidase (LOX) that were induced in hypoxia were down- Surprisingly, we found that ERα signaling actually directly regulates MEDICAL SCIENCES regulated by ICI182780 in both normoxia and hypoxia (Fig. 1A). HIF-1α expression. When MCF7 cells were grown without estrogen We then queried the iLINCS (integrative LINCS) genomics data for 4 d and then placed in hypoxia or treated with the hypoxia mi- portal to search compounds that regulate a similar gene pattern as metic deferoximine, 17-β-estradiol (E2) greatly enhanced HIF-1α ICI182780 induced in MCF7 cells. Among the top 15 hits, most expression (Fig. 2A). However, ICI182780 significantly reduced are HIF-1α inducers or ERα modulators, including tamoxifen and HIF-1α and ERα expression in hypoxia (Fig. 2B), consistent with ICI182780 (Fig. 1B). We analyzed these genes through ChIP en- its biological function in suppressing HIF-1α targets (Fig. 1). In richment analysis of transcription factors (ChEA) through the addition, ICI1820780 treatment did not affect HIF-1α expression − LINCS canvas browser II to examine what transcriptional factors in 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. Extracted RNA from duplicated biological samples was subject to microarray analysis. Heatmap shows a subgroup of genes that are dual responsive to hormone and oxygen. (B) The dually responsive genes were queried with the LINCS program to search Connectivity MAP (CMAP) for compounds that induced a similar pattern to ICI182780. (C) ChEA of transcription factor binding to the dual responsive genes. (D) Venn diagram shows the common gene bound by both ERα and 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 Downloaded by guest on September 23, 2021 ERα binding element because FOXA1 is a pioneer factor that facilitates ERα recruitment (23). In fact, all ER-chromatin in- teractions and gene-expression changes depend on the presence of FOXA1 (23). To test whether ERα is able to bind the ERE of the HIF-1α gene, we treated MCF7 cells with E2 or tamoxifen and then performed ChIP and real-time PCR assays. The results clearly showed that ERα bound to this ERE compared with the surrounding negative regions, and E2 and tamoxifen treatment further enhanced ERα binding (Fig. 3D). Next, we cloned the ERE sequences (Fig. S2 A and B) into a luciferase reporter and performed a luciferase assay to assess the regulatory function of ERα. Indeed, the luciferase activity was significantly high, but the ERE mutant abrogated the activity in MCF7 cells (Fig. 3E). We also tested the binding of ERα in hypoxia condition. The results showed that ERα still bound at the ERE of HIF-1α under hypoxia (Fig. 3F). Thus, these data demonstrate that we have identified HIF-1α as a direct target of ERα, potentially explaining why HIF-1α is associated with ER-positivity in breast cancer samples. Interestingly, we did not see positive correlation between Fig. 2. ERα signaling regulates HIF-1α expression. (A) After 4-d hormonal HIF-1α and ERα at transcript levels from tumor cohorts; in- starvation, MCF7 cells were treated with 500 μM of DFO or 1% O2 for 6 h in the presence or absence of 100 nM of E2. The whole-cell lysates were subject stead, we found a negative correlation between them (Fig. S3 A − to Western blotting with indicated antibodies. V, Vehicle. (B) MCF7 and and B). This may be explained by the hypothesis that ER breast μ MDA-MB-231 cells were treated with 1 M of ICI182780 in hypoxia (1% O2) cancer has been epigenetically remodeled to express high for 24 h. The whole-cell lysates were subject to Western blotting with in- α α HIF-1 transcript. This finding led us to further speculate that dicated antibodies. (C and D)ER in MCF7 cells was knockdown with two α α different siRNAs (C) or SMARTpool (D), which were treated with 200 μMof ER loss need to be compensated by high HIF-1 activity. DMOG (C) or hypoxia (D). The whole-cell lysates were subject to Western blotting with indicated antibodies. (E) MCF7 cells were treated with siRNA SMARTpool against ERα, treated with 10 μM G132 for 16 h. The whole-cell lysates were subject to Western blotting with indicated antibodies.

ERα with two separate siRNA oligos or a SMARTpool of siRNA greatly reduced HIF-1α induction under hypoxia or hypoxic mimetic dimethyloxalylglycine (DMOG) treatment (Fig. 2 C and D). The proteasome inhibitor MG132, which stabilizes HIF-1α protein ex- pression by preventing protein degradation, failed to rescue ERα knockdown-mediated down-regulation of HIF-1α (Fig. 2E). These data clearly show that the ERα signaling pathway directly regulates HIF-1α expression.

HIF-1α Gene Bears Estrogen Response Element and ERα Enhances HIF-1α Transcription. HIF-1α is labile and subject to rapid protea- somal degradation. Most studies focus on the regulation of protein stability of HIF-1α but how HIF-1α is transcriptionally regulated is not 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 protein stability-independent manner (Fig. 2E). Supporting this hy- pothesis, the ERα antagonist tamoxifen treatment resulted in down-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 transcriptional function is involved. To determine whether ERα signaling di- Fig. 3. ERα directly binds EREs on the HIF-1α gene to enhance HIF-1α tran- rectly regulates HIF-1α , we treated hormone- scription. (A) After 3-d hormonal starvation, MCF7 cells were treated with 100 nM of E2 for 24 h. Real-time PCR was performed to assess HIF-1α gene starved MCF7 and MDA-MB-231 cells with estrogen. RT-PCR μ HIF-1α KDM4B STC2 expression. (B) MCF7 cells were treated with 1 MofICI182780for24h.Real- data revealed that , ,and mRNA was time PCR was performed to assess HIF-1α gene expression. (C) The gene significantly up-regulated in MCF7 but not MDA-MB-231 structure of HIF-1α and the sequence that bears ERα binding sites (ERE) and cells (Fig. 3A and Fig. S1 B and C). RT-PCR results further FOXA1 binding sites, as indicated. The black bar represents each exon and the showed that HIF-1α mRNA level was significantly reduced black line represents each intron of HIF-1α. NB1 and NB2 represent the primer after ICI182780 treatment, indicating an ER-mediated tran- location for ChIP PCR to assess ERα binding as negative controls, as shown in D. scriptional mechanism regulates HIF-1α transcription. (D) After 3-d hormonal starvation, MCF7 cells were treated with 100 nM of E2 α By analyzing the HIF-1α genomic sequence that bears 15 exons or tamoxifen for 24 h. ChIP PCR was performed to assess ER binding among C the regions as indicated in C.(E)TheEREregionandthemutantwerecloned and 14 introns (Fig. 3 ), we found that there is a canonical estrogen into pGL3 luciferase reporter. The plasmids and the pRL Renilla Luciferase control response element (ERE) located in the first intron. Interestingly, vector (to normalize the ERE reporter) were transfected into MCF7 cells. Dual there is also a FOXA1 binding site that is 64 nucleotides down- luciferase assay was performed. (F) MCF7 cells were incubated in hypoxia for 8 h. stream of the ERE (Fig. 3C), further supporting it as a bona fide 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. Downloaded by guest on September 23, 2021 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 receptor corepressor and HDAC to silence gene transcription of ERα tar- gets (5). The discovery of enhancing HIF-1α by E2-bound ERα prompted us to examine the effect of tamoxifen-bound ERα. Tamoxifen treatment of MCF7 cells greatly increased HDAC1 binding on the ERE of HIF-1α. Interestingly, ICI182780 did not affect HDAC1 binding, but remarkably reduced the ERα binding (Fig. 4A). These data indicate that the two compounds inhibit ERα function through different mechanisms. We then used an in vitro established tamoxifen-resistant MCF7 (TamR-MCF7) cell line and BT474 cells, which are intrinsically resistant to tamoxifen (Fig. 4 B and C). These cells were cultured in media containing tamoxifen for propagating. When ERα was de- pleted in these cells, HIF-1α expression was up-regulated (Fig. 4 B and C). Overexpression of ERα in TamR-MCF7 cells significantly reduced HIF-1α expression, but not for the parental MCF7 cells in normoxia (Fig. 4D). Longer exposure of the film showed that overexpression 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 tamoxifen resistance. These data indicate that E2-bound ERα induces—but tamoxifen-bound ERα suppresses—HIF-1α expression (Fig. 4E). Fig. 5. HIF-1α confers tamoxifen resistance to ER+ breast cancer cells. + HIF-1α ConfersTamoxifenResistanceonERα Breast Cancer Cells. Be- (A) Western blotting to assess MCF7 cells expressing HIF-1α.(B)TheMCF7 α α α – α

cause HIF-1 was a downstream target of ER and enhanced ER control and MCF7 HIF-1 cells were treated with tamoxifen or ICI182780 with MEDICAL SCIENCES target expression, we hypothesized that HIF-1α may modulate indicated concentration for 18 d. Cell colonies were stained with Crystal violet. + endocrine efficacy in ER breast cancers. We therefore assessed (C) The MCF7 control and MCF7–HIF-1α cells were treated with ICI182780 for the role of HIF in regulation of breast cancer cell survival or 4 wk. Cell colonies were stained with Crystal violet. (D) Tumorspheres gener- α ated from the parental (Upper) and HIF-1α expressing (Lower)MCF7cellswere proliferation in response to inhibitors of ER .Wefirstusedret- μ μ μ α treated for 5 d with 1 Moftamoxifen,1 M of ICI182780, and 0.5 MofRITA. roviral vector-mediated transduction to stably introduce HIF-1 Photos were taken under microscope (10×). (E) After counting 10 different 10× fields, the number of tumorsphere counts were averaged and compared with Student’s t test (two tailed, **P < 0.01). (F) Kaplan–Meier analyses for relapse free survival of the cohort of patients with ER-positivity, receiving tamoxifen treatment only without chemotherapy. Affymetrix ID for HIF-1α used was 200989_at. The cut-off value used in analysis was 3,043 and the expression range of the probe was 439–17,198. Patient number for low HIF-1α (black) and high HIF-1α (red) is presented under the following months. (G)Kaplan-Meier analyses for overall survival of the cohort of patients with ER-positivity, re- ceiving tamoxifen treatment with chemotherapy. The cutoff value was 3035 and the expression range was 456–11726. Patient number for low HIF-1α (black) and high HIF-1α (red) are presented under the following months.

and HIF-2α into T47D and MCF7 cells (Fig. 5A and Fig. S4). HIF-2α expression significantly suppressed proliferation of both cell lines, whereas expression of HIF-1α did not. Thus, we were unable to generate stable cell lines with HIF-2α. Although both parental MCF7 and T47D and their derivative HIF-1α–expressing cells responded to tamoxifen treatment, the HIF-1α–expressing cells were at least twofold more resistant in normoxia (Fig. S4)and long-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 parental and HIF-1α expressing cells responded to ICI182780 similarly after Fig. 4. Tamoxifen-bound ERα inhibits HIF-1α expression. (A) MCF7 cells were B cultured in standard DMEM and treated with 1,000 nM of tamoxifen or 1wkoftreatment(Fig. S4 ), long-term treatment (18 d or 4 wk) ICI182780 for 48 h. ChIP PCR was performed to assess HDAC1 and ERα binding with 10 nM or 100 nM led to development of more resistant col- the ERE of HIF-1α.(B) MCF7 and TamR-MCF7 cells were transfected with siRNA onies (Fig. 5 B and C). Although 1,000 nM of ICI182780 efficiently SMARTpool against ERα. After 36-h transfection, cells were incubated in nor- suppressed both cell lines, HIF-1α–expressing cells gave rise to moxia and hypoxia for 16 h. Western blotting was used to assess the indicated more large resistant colonies (Fig. 5C). . (C) BT474 cells were treated as in B.(D) MCF7 and TamR-MCF7 cells We further tested the capacity for tumorsphere formation of the α were transfected with ER expression plasmids. MCF7 cells were cultured in HIF-1α–expressing and parental cells using a mammosphere 3D normoxia in standard DMEM while Tam-MCF7 cells were cultured in phenol- culture system. HIF-1α did not affect tumorsphere formation; free DMEM with 100 nM of tamoxifen. After 48-h transfection, cells were α harvested for Western blotting. The two HIF-1α blots were for short and long however, HIF-1 conferred significant resistance to tamoxifen and exposure, respectively. (E) The cartoon shows the E2-bound and tamoxifen- ICI182780 compared with the parental control (Fig. 5 D and E) bound ERα exert opposite effect on HIF-1α transcription. (P < 0.01). We also tested another drug, RITA, which inhibits

Yang et al. PNAS | December 8, 2015 | vol. 112 | no. 49 | 15175 Downloaded by guest on September 23, 2021 HIF-1α expression and induces apoptosis of MCF7 cells (31). The results 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 Show + a Poor Response to Tamoxifen Treatment in ERα Breast Cancer. To determine whether hypoxia/HIF-1α is associated with tamoxifen effectiveness in patients with breast cancer, we first examined the hypoxia status of two groups of breast cancer patients by a hypoxia/HIF-1α metagene signature (32). We then compared + relapse-free survival of ERα breast cancers classified as hypoxic or normoxic by their gene-expression profiles with this hypoxia/ Fig. 6. The working model between ERα and HIF-1 pathway. (A) Mammary HIF-1α metagene signature. In those without adjuvant tamoxifen pad tumor growth assessment of HIF-1α–overexpressing MCF7 cells therapy, hypoxic cases had a significantly worse outcome than compared with parental MCF7 cells implanted with tamoxifen citrate (5 mg; α 3 the normoxic breast cancers (P = 0.001) (Fig. S5A). A significant TAM) or placebo pellet (CTL) when HIF-1 tumors reached 150 mm (in- dicated by red arrow) in NSG female mice with estrogen pellets implanted difference in outcome for those treated with tamoxifen remained = < α P = A HIF-1α (0.72 mg; n 5 per group). NS, nonsignificant; ***P 0.001. (B) HIF-1 gene ( 0.03) (Fig. S5 ). Then we analyzed whether the bears ERα binding site whose transcription is directly regulated by ERα sig- gene expression itself correlated with tamoxifen response in a α + naling pathway. Under hypoxia, produced HIF-1 protein is stabilized. Dual large cohort of ER patients from public data (33). Kaplan– responsive genes bear both ERα and HIF-1α binding sites, which can be Meier analysis results showed that patients with high level of regulated by two signaling pathways. ERα directly regulates the HIF-1α HIF-1α gene expression had a poorer relapse-free survival to pathway associated with antiestrogen response in breast cancer. endocrine therapy or tamoxifen treatment alone (P = 0.0093) (Fig. 5G and Fig. S5B), although overall survival was not sig- α α nificantly different (Fig. S5C). When chemotherapy was included regulatory link between the ER and HIF-1 pathways in breast B for those patients who received tamoxifen, HIF-1α is also asso- cancer (Fig. 6 ). ciated with poor overall survival (P = 0.017) (Fig. 5H). These Interestingly, we found a subgroup of genes that are dually data further indicate that HIF-1α may be directly involved in responsive to hormone and oxygen (Fig. 1). These genes were + α modulating tamoxifen response in ER patients. Interestingly, up-regulated by hypoxia but the ER antagonist ICI182780 sig- + high HIF-2α is associated with better survival in ER breast nificantly reduced their expression. Previous studies also show that KDM4B STC2 VEGFA cancer patients who received endocrine therapy (Fig. S5D). Al- some genes, such as , ,and , bear both a – though statistically not significant, HIF-2α tends to be associated hypoxia response element and ERE (18, 19, 26 30). This finding with better survival in tamoxifen-treated patients (Fig. S5E). may indicate that the ER signaling pathway can be enhanced by α B These results were consistent with our findings that HIF-2α HIF-1 induction and vice versa (Fig. 6 ). The physiological sig- + overexpression was harmful for ERα cancer cells and the clin- nificance of the cross-talk between these two pathways in breast or ical data that HIF-2α is significantly lower in breast cancer tissues ovary development warrants further study. Nevertheless, we en- than the normal breast tissue (Fig. S6). visage that both pathways form a positive feedback loop to en- hance the common downstream target gene expression. But is HIF-1α Overexpression Confers Advantage of Tumor Growth and HIF-1 required for ERα activity? We previously showed that de- Resistance to Tamoxifen Treatment. To further investigate the ef- pletion of HIF-1α only partially affected KDM4B expression in fect of HIF-1α on tamoxifen treatment in vivo, orthotopic xenograft hypoxia, whereas depletion of ERα nearly abrogated KDM4B tumor was established using MCF7 cells overexpressing HIF-1α. expression (18), indicating that HIF-1 might not be required for Compared with the control, HIF-1α overexpression resulted in ERα activity but synergizes with ERα. Here we further tested this rapid tumor growth in NOD SCID-γ (NSG) mice that had to be by knocking down HIF-1α in tamoxifen-resistant MCF7 and killed before the control reached comparable sizes of tumors (Fig. BT474 cells and obtained a similar result that KDM4B expression 6A). Tamoxifen treatment only modestly delayed tumor growth was only partially reduced (Fig. S7), further suggesting that ERα with HIF-1α overexpression (P > 0.05), similar to the in vitro data function may not rely on HIF-1α. However, we cannot exclude the (Fig. 5), indicating that HIF-1α confers tamoxifen resistance. possibility that ERα may require HIF-1α function for activation of certain specific genes or under specific conditions. Discussion Another important conclusion of our findings is that overactive To develop novel therapeutics to treat breast cancer, a deeper HIF-1α function may partially compensate for estrogen signaling understanding of the molecular mechanism of ER-driven cancer is when ERα function is compromised, such as under the circum- + important, as the most common type of metastatic breast cancer is stances of hormone therapy. When ERα breast cancer cells were endocrine receptor-positive. The association of ERα-positivity and transduced with HIF-1α, the cancer cells became much more re- HIF-1α fromclinicalstudies(12,15,34)supportsourfindingsthat sistant to tamoxifen and ICI182780 treatment (Fig. 5). This find- these two pathways may act in cooperation to promote breast ing is consistent with the fact that the HIF-1α and hypoxia gene cancer progression. However, the basis of these previous clinical signature were correlated with poorer survival in response to − observations was unclear because ER tumors have a greater hormone therapy. Although the molecular mechanism by which + proliferation rate and are more hypoxic than ER tumors. Hence, HIF-1α confers tamoxifen resistance needs to be further defined, it was unclear why HIF-1α should be more highly expressed in our recent findings indicate that the histone demethylase KDM4B + ER tumors (15, 34). Our data, which demonstrate that ERα is important in coordinating HIF-1α and ERα. KDM4B is a direct regulates HIF-1α expression, provide a mechanism for these target of both HIF-1α and ERα and regulates expression of many clinical observations. HIF-1α is a labile protein that is rapidly de- genes in normoxia and hypoxia and cell cycle progression (18). graded by VHL-mediated proteasomal degradation in normoxia but Thus, HIF-1α may drive gene expression of KDM4B and other stabilized in hypoxia. However, the transcriptional regulation of genes to compensate for tamoxifen inhibition of ERα signaling. HIF-1α is not well studied, except in one report showing that the This may be more important in vivo as the common factors are NF-κB pathway regulates HIF-1α gene expression (35). Here we secreted extracellular signaling molecules. These results comple- show that the HIF-1α gene bears a canonical ER-binding element ment recent data showing the importance of HIF-1α in triple- that responds to estrogen signaling, demonstrating a direct negative breast cancer that is driven by XBP1 in response to

15176 | www.pnas.org/cgi/doi/10.1073/pnas.1422015112 Yang et al. Downloaded by guest on September 23, 2021 unfolded protein or endoplasmic reticulum (36). In- siRNA transfection, cell viability and colony formation assays, luciferase re- terestingly, XBP1 has been shown to confer both estrogen porter assay, gene-expression profiling, data mining, patient details, and independence and antiestrogen resistance in breast cancer cell gene-expression profiling for patients sample, and in vivo xenograft ex- lines (37). Thus, it is also possible that cellular stress-induced periment are described in SI Materials and Methods. Statistical analyses were XBP1 might interact with HIF-1α to confer antiestrogen re- two-tailed t tests, with P ≤ 0.05 considered statistically significant. All animal sistance. Therefore, our data suggest that targeting the HIF-1α procedures were approved by University of Oxford ethical review and work signaling pathway might increase efficacy of endocrine therapy was conducted in accordance with the UK Home Office guidelines, under the in breast cancers. project licence PPL 30/2771. Materials and Methods ACKNOWLEDGMENTS. We thank the Oxford and Royal Marsden NHS Biomedical Research Centres and Experimental Cancer Medicine Centre. Cells were maintained in DMEM supplemented with 10% (vol/vol) FCS, 1% This study was supported in part by Cancer Research UK, Breast Cancer glutamine, and 1% penicillin-streptomycin. Western blot analysis, quanti- Research Foundation (A.L.H., J.Y., and J.-L.L.), and Advancing Clinico- tative RT-PCR, tumorsphere formation, ChIP, retroviruses and plasmids, Genomic Trials European Union Project FP6-IST-026996 (to F.M.B.).

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