Regulation of docetaxel chemosensitivity by NR2F6 in breast cancer

in Endocrine-Related Cancer
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  • 1 Department of Thyroid, Breast and Vascular Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province, People’s Republic of China
  • | 2 State Key Laboratory of Cancer Biology, Biotechnology Center, School of Pharmacy, Fourth Military Medical University, Xi’an, Shaanxi Province, People’s Republic of China

Correspondence should be addressed to J Zhang or R Ling: vascularzhang@163.com or lingruiaoxue@126.com

*(J Zhang, H Meng and M Zhang contributed equally to this work)

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Docetaxel (DTX)-based chemotherapy significantly eliminates rest cancerous cells and decreases the risk of death, thus remaining the mainstay of treatment for operable breast cancer (BCa). However, resistance or incomplete response to DTX occurs frequently, resulting in disease recurrence and poor prognosis. There is an urgent need to identify and understand the key factors and corresponding molecular bases driving this complicated pathogenesis. Herein, both data mining and profiling analysis using clinical BCa biopsies showed that expression levels of the nuclear receptor subfamily 2, group F, member 6 (NR2F6), a recently characterized central transcription factor for cancer immune surveillance, were significantly downregulated in DTX-resistant BCa. This downregulation, possibly regulated by leptin signaling, predicted a poor postoperative chemotherapy survival in DTX-resistant BCa. In both genetically engineered cell models and patient-derived xenograft models, we provided evidence that BCa cells with insufficient NR2F6 expression were less responsive to DTX treatment. Mechanistically, NR2F6 functioned as a potent corepressor of platelet-derived growth factor B receptor gene (PDGFRB) transcription by recruiting HDAC2 onto the PDGFRB promoter. Stable PDGFRB inhibition ameliorated NR2F6 deficiency-impaired response to DTX in BCa cells, indicating that NR2F6’s effect on DTX response is mediated, at least in part, through transcriptional repression of PDGFRB. Collectively, our findings define NR2F6 as an negative regulator of cell survival and DTX resistance, probably by serving as a convergent point linking leptin signaling and PDGF-B/PDGFRβ axis, in BCa cells.

Abstract

Docetaxel (DTX)-based chemotherapy significantly eliminates rest cancerous cells and decreases the risk of death, thus remaining the mainstay of treatment for operable breast cancer (BCa). However, resistance or incomplete response to DTX occurs frequently, resulting in disease recurrence and poor prognosis. There is an urgent need to identify and understand the key factors and corresponding molecular bases driving this complicated pathogenesis. Herein, both data mining and profiling analysis using clinical BCa biopsies showed that expression levels of the nuclear receptor subfamily 2, group F, member 6 (NR2F6), a recently characterized central transcription factor for cancer immune surveillance, were significantly downregulated in DTX-resistant BCa. This downregulation, possibly regulated by leptin signaling, predicted a poor postoperative chemotherapy survival in DTX-resistant BCa. In both genetically engineered cell models and patient-derived xenograft models, we provided evidence that BCa cells with insufficient NR2F6 expression were less responsive to DTX treatment. Mechanistically, NR2F6 functioned as a potent corepressor of platelet-derived growth factor B receptor gene (PDGFRB) transcription by recruiting HDAC2 onto the PDGFRB promoter. Stable PDGFRB inhibition ameliorated NR2F6 deficiency-impaired response to DTX in BCa cells, indicating that NR2F6’s effect on DTX response is mediated, at least in part, through transcriptional repression of PDGFRB. Collectively, our findings define NR2F6 as an negative regulator of cell survival and DTX resistance, probably by serving as a convergent point linking leptin signaling and PDGF-B/PDGFRβ axis, in BCa cells.

Introduction

Worldwide, breast cancer (BCa) is so far the most frequently diagnosed female cancer. In China, BCa is now the most common cancer among women and the sixth leading cause of cancer-related women death (Fan et al. 2014). Optimal postoperative chemotherapy (adjuvant therapy) can significantly eliminate rest tumor cells and reduce mortality, thus remaining the mainstay of treatment for patients with either advanced BCa or resistance to endocrine therapy (Chang et al. 2005). The taxanes (docetaxel (DTX) and paclitaxel) are a group of antimicrotubule therapeutic agents that cause apoptosis by inhibiting the depolymerisation of the β-tubulin subunit of microtubules. Compared to paclitaxel, DTX displays less side-effects (Kastl et al. 2012). However, acquired resistance to DTX, a multifactorial process governed by an array of signals acting at different levels, occurs frequently and severely impedes therapeutic efficacy of DTX. Thus, there is an urgent need to identify and understand the key factors and signaling pathways driving this complicated pathogenesis. Recent advance in this field suggests that platelet-derived growth factor BB and its receptors (PDGF-B/PDGFRβ) play important regulatory roles in both normal and malignant mammary gland. PDGF-B/PDGFRβ axis promotes cell survival, regulates tumor-microenvironment dynamics and exhibits potent pro-lymphangiogenic effects, thus representing an attractive therapeutic target for BCa treatment (Jitariu et al. 2018). However, directly targeting PDGF-B/PDGFRβ axis is challenging because of its vast functional complexity and its crucial role in embryologic development (all PDGF-B or PDGFRβ knockout are embryonic lethal) (Andrae et al. 2008). To this end, identification of key regulators that circumvent PDGF-B/PDGFRβ function is an alternative approach to the direct targeting of PDGF-B/PDGFRβ axis.

To identify the key factors that are potentially involved in DTX resistance in BCa, we previously carried out data mining on established expression profiles (GSE6434) from the Gene Expression Omnibus (GEO) database. One differentially expressed transcript, namely the human NR2F6 gene, was found to be significantly upregulated in DTX-resistant BCa samples. This gene, mapped to human chromosome 19p13.11, encodes the nuclear receptor subfamily 2, group F, member 6 (NR2F6, also called EAR-2), a recently characterized central transcription factor for cancer immune surveillance, with uncharacterized role in BCa progression. NR2F6 expression is induced in acute myeloid leukemia (AML) and suppression of NR2F6 expression causes terminal differentiation and apoptosis in AML cells (Ichim et al. 2011). NR2F6 inhibits hematopoietic cell differentiation and induces myeloid dysplasia (Ichim et al. 2018). Interestingly, genetic ablation of NR2F6 significantly improves survival in the murine transgenic TRAMP prostate cancer model by developing host-protective immunological memory. The latter observation defines NR2F6 as an essential immune checkpoint in effector T cells, governing the amplitude of anti-cancer immunity (Hermann-Kleiter et al. 2015). Nevertheless, regardless of various reports on the expression patterns of NR2F6 beyond hematopoietic system (Ichim et al. 2018), its functional details and corresponding molecular bases in multiple malignancies remain largely obscure.

Data mining, and the fact that dysregulation of immune surveillance contributes essentially to impaired response to chemotherapy (Ndibe et al. 2015, Nicolini et al. 2018), prompted us to systematically investigate the expression and clinicopathologic relevance of NR2F6 signaling in BCa patients with poor response to DTX, along with its biological significance in vitro and in vivo.

Materials and methods

Data mining

To provide the preliminary data on the clinical relevance of NR2F6 transcripts expression, we performed the data mining on the GSE6434 datasheet from GEO database, using the GEO2R platform. This datasheet consists of 11 DTX-sensitive and 13 DTX-resistant surgical specimens from BCa patients (Chang et al. 2005).

Human samples

The procedures involved in the human study were strictly conformed to the ethical standards of Helsinki Declaration and were approved by the Ethics Committee of Fourth Military Medical University (Reference Number XJYYLL-2011562-A). All participants provided written informed consent before enrolment in this study. A total of 79 patients with locally advanced BCa, who had received docetaxel monotherapy (60 to 100 mg/m2 i.v. over 1 h every 3 weeks) or received neoadjuvant docetaxel therapy (75 mg/m2 of DTX was administered every 3 weeks for a total of six cycles), were enrolled from Department of Thyroid, Breast and Vascular Surgery in Xijing Hospital during October 2011 to July 2018 (Supplementary Table 1, see section on supplementary materials given at the end of this article). Patients were subdivided into DTX sensitive (n = 44, tumors with 25% residual disease or less after four cycles of DTX treatment) and DTX resistant (n = 35, tumors with greater than 25% residual disease after four cycles of DTX treatment), according to the reported guideline (Chang et al. 2005). The patients with neoadjuvant chemotherapy were defined as ‘DTX sensitive’ as the absence of any residual invasive carcinoma at both the primary site or in axillary lymph nodes. Three core biopsy specimens were snap frozen in liquid nitrogen and immediately stored at −80°C, and another two core biopsy specimens were formalin-fixed, paraffin-embedded and subjected to histological examination.

Immunohistochemistry, immunofluorescence and TUNEL staining

Five-micrometer-thick parraffin sections were routinely deparaffinized and rehydrated and were subjected to antigen retrieval at 95°C for 30 min using retrieval buffer (Abcam). Sections were then incubated at 4°C overnight with different primary antibodies as listed in the Supplementary Table 2. After a thorough rinse, sections were incubated with biotinylated secondary antibodies or Texas Red/FITC-conjugated second antibodies (Thermo Fisher Scientific) at room temperature for 1–2 h. Final immunohistochecal reaction was developed using the Vectastain Elite ABC kit (Vector Labs). Immunofluorescent staining was observed under an inverted microscope (Axio Imager M1 microscope, Zeiss). To detected the in situ apoptosis, 5-μm-thick parraffin sections were stained using a TUNEL kit (Roche), as instructed by the manufacturer.

Cell treatment

Human BCa cell lines (MCF-7 and MDA-MB-231) were obtained from American Type Culture Collection (Manassas, VA, USA). MCF-7 cells were non-invasive and hormone receptor positive but MDA-MB-231 cells were invasive and triple negative (Nestal de Moraes et al. 2015). Both cell lines were authenticated in December 2017 using STR DNA profiling analysis (Promega) and used less than 15 passages. BCa cells were cultured in DMEM (Thermo Fisher Scientific) supplemented with 10% heat-inactivated FBS and 100 U/mL penicillin/streptomycin (GIBCO) in a humidified incubator (37°C, 5% CO2). The DTX-resistant sublines (MCF-7/LR and MDA-MB-231/LR) were generated by gradually exposing the parental BCa cells to different doses of DTX and were maintained in a 10 nM final concentration of docetaxel (Zhang et al. 2015). To study the potential regulation of NR2F6 mRNA expression by leptin signaling, BCa cells were incubated for different durations with 1.2 nM of recombinant human leptin (R&D Systems), or treated with different doses of leptin for 12 h, followed by other assays. To confirm the leptin regulation of NR2F6 expression, MCF-7 cells were stimulated with different doses of leptin as indicated, in the presence or absence of PEG-LPrA2 (a leptin receptor antagonist, 1.2 nM), for 12 h, followed by RT-qPCR analysis.

To knockdown the endogenous expression of the leptin receptor OB-R, we incubated MCF-7 cells with LEPR siRNA or scramble Ctrl siRNA (Ambion-Thermo Fisher Scientific) for 48 h using Lipofectamine 3000 (Thermo Fisher Scientific) (Liu et al. 2018). To manipulate the NR2F6 expression, BCa cells were transfected either with NR2F6 shRNA (Zhang and Dufau 2000) or with pCMV6-DDK-NR2F6, along with their corresponding negative controls, for 48 h using Lipofectamine 3000. To generate the MCF-7 cells stably deprived of PDGFRB expression, MCF-7 cells were transfected with PDGFRB shRNA or Ctrl shRNA from OriGene (Beijing, CHINA) (Esparza-Lopez et al. 2016) for 48 h using TurboFectin Transfection Reagent (OriGene), followed by selection with 0.5 μg/mL puromycin (Sigma-Aldrich).

DTX-resistant BCa patient-derived xenograft (PDX) model

To establish the expression vector of NR2F6, NR2F6 cDNA was released from pCMV6-DDK-NR2F6 and was then cloned into Retro-EGFP-Puro vector (Addgene, Watertown, MA, USA), as per the manufacturer’s instructions. The Retro-EGFP-Puro vector was a gift from Georgios Stathopoulos (Giannou et al. 2015). High-titer virus was prepared according a previously validated protocol (Ichim and Wells 2011). PDX model was established as described elsewhere (DeRose et al. 2011). Briefly, local recurrent BCa tumors were minced into ~20 mm3 slices and were implanted into the cleared inguinal fat pad of female nude mice concurrently with a s.c. estradiol pellet (Innovative Research of America, Sarasota, FL, USA). Five days after implantation, mice received i.p. injection of DTX (10 mg/kg, twice per week). Following growth to ~200 mm3 of the tumor in passage 1 (PDX1), tumor fragments was prepared again for sequential passage of the tumor (designated as PDX2, PDX3, and et al.). At the end of 5 and 17 days after PDX3 implantation, mice received intratumoral (I.T.) injection of 2 μg of Retro-EGFP-NR2F6 (or empty vector) complexed with 2 μL of Lipofectamine 3000 dissolved in 50 μL of DMEM. Mice received a concurrent DTX treatment as described previously. The xenografts were allowed to grow for about 1 month and tumor volumes were recorded and calculated according to the published formula every week (Ning et al. 2016). At the end of 28 days after PDX3 implantation, mice were killed and xenografts were harvested and subjected to other assays.

Measurement of in vitro cytotoxicity

Forty-eight hours after transfections, BCa cells were seeded onto a 96-well plate at the density of 1 × 104 cells/well. Cells were then treated with different doses of DTX or paclitaxel (PTX) as indicated for 48 h. Subsequent cell viability and apoptosis were analyzed using a MTT Assay Kit (Sigma-Aldrich) and an Apoptosis ELISA kit (Roche Diagnostics), respectively.

In vivo chemosensitivity assay

Forty-eight hours after transfections, BCa cells (5 × 106) were injected subcutaneously into flanks of 6-week-old female nude mice. Twelve days later, mice received i.p. injection of DTX (10 mg/kg, twice per week) or (20 mg/kg, twice per week). Following growth for another 23 days, mice were killed and tumors were harvested for other assays. Tumor volumes were recorded and calculated on a weekly basis, as described previously. All procedures were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines and were approved by the Ethics Committee of Fourth Military Medical University (Reference Number XJYYLL-2011562-B).

In vitro tubulin polymerization assay

Effects of NR2F6 overexpression on the polymerization of microtubule proteins isolated from different BCa cells were evaluated using a fluorescence-based microtubule polymerization assay kit (Sigma-Aldrich), as per the manufacturer’s instructions. Purified tubulin-containing proteins (>99% purity), suspended in the reaction buffer containing 80 mM PIPES, 2 mM MgCl2, 0.5 mM EGTA, 1 mM GTP (pH 6.9) and 15% glycerol were transferred to a pre-warmed 96-well plate, and the reaction was then initiated by addition of tubulin. Final spectrophotometry was carried out at 340 nm at 37 °C on a fluorescence microplate reader (Thermo Fisher Scientific).

RT-qPCR

Total RNA was prepared from BCa cells and tissues using RNeasy Mini Kit (Qiagen). After removal of the contaminated DNA using DNA-free™ KitDNase (Thermo Fisher Scientific), RNA samples were subjected to RT using the SuperScript III RT system (Thermo Fisher Scientific), according to the manufacturer’s instructions. Subsequent qPCR was carried out as described previously (Dong et al. 2016), using GAPDH as the internal control. Relative expression levels of target genes were then quantified by SYBR Green intercalation using the 2−ΔΔCt method on a MiniOpticon™ system (Bio-Rad) (Zhang et al. 2014). The primers used were: NR2F6 (accession#: NM_005234.3), 5′-TCTCCCAGCTGTTCTTCATGC-3′ and 5′-CCAGTTGAAGGTACTCCCCG-3′ (Ichim et al. 2011); LEPR (accession#: NM_002303.5), 5′-GGAAGATGTTCCGAACCCCA-3′ and 5′-ACCACATGTCACTGATGCTG-3′ (Ma et al. 2019); PDGFRB (accession#: NM_002609.4), 5′-CAACTTCGAGTGGACATACCC-3′ and 5′-AGCGGATGTGGTAAGGCATA-3′ (Fujino et al. 2018); and GAPDH (accession#: NM_002046.6), 5′-GGCCTCCAAGGAGTAAGACC-3′ and 5′-AGGGGTCTACATGGCAACTG-3′ (Ichim et al. 2011).

Immunoblotting

Total protein samples were prepared from BCa cells and tissues using a Protein Isolation and Purification Kit (GenScript, Nanjing, China). Approximately, 30 μg of protein samples were separated by SDS-PAGE and transferred to a PVDF membrane (Thermo Fisher Scientific) for standard Western blotting/immunoblotting, as described elsewhere (Li et al. 2011a). The antibodies used for the current study were listed in Supplementary Table 2.

Luciferase reporter assay

The promoter fragments of human PDGFRB harboring the putative NR2F6-binding site (~1.9 kb upstream of the translation start site) were amplified by PCR and cloned into the pGL3-Luc reporter vector (Promega) using the CloneEZ ® PCR Cloning Kit (GenScript). The functional binding of NR2F6 onto the PDGFRB promoter was further validated by mutation of the putative NR2F6-binding site by replacing TGACCT with TtcaCT in using the QuikChange Site-Directed Mutagenesis Kit (Agilent). The mutated promoter fragments of human PDGFRB gene were also cloned into the pGL3-Luc reporter vector as described previously. For reporter assay, 0.5 μg of reporter plasmid (WT or Mu pGL3-Luc reporter plasmids), pCMV6-DDK-NR2F6 and pRL-TK Renilla reporter plasmid were co-transfected into HeLa cells using Lipofectamine 3000. Forty-eight hours later, cells were stimulated for 6 h with 1.2 nM of DTX, in the presence or absence of co-treatment with 100 ng/mL of trichostatin A (TSA, a potent histone deacetylase (HDAC) inhibitor, Sigma-Aldrich), followed by measurement of the relative luciferase activity using the dual luciferase reporter assay system (Promega).

Chromatin immunoprecipitation (ChIP) and double ChIP

MCF-7 cells were transfected with NR2F6 shRNA or Scramble shRNA as described previously. Forty-eight hours later, cells were challenged for 12 h with 1.2 nM of DTX, in the presence or absence of co-treatment with 100 ng/mL of trichostatin A. Subsequently, cells were harvested, crosslinked with 1% formaldehyde and sonicated. Soluble chromatin was immunoprecipitated and subjected to ChIP and double ChIP assays, as described in previous publications (Zhang et al. 2012, 2014). Primers used for ChIP and double ChIP were: 5′-ACTATCCAACAGTTACTATGC-3′ and 5′-TGGTGTCTCATCTGTAACATG-3′ (Accession#: NC_000005.10).

Statistical analysis

Data were analyzed using GraphPad Prism 5 from at least three independent experiments and were expressed as mean ± s.d. Statistical comparisons were determined by either Student’s t-test or one-way ANOVA, followed by Tukey post-hoc analyses wherever appropriate. The association between PDGFRB and NR2F6 mRNA levels was assessed using the Pearson Chi-Square test and the survival difference was analyzed using Kaplan–Meier method. P < 0.05 was considered statistically significant.

Results

Downregulation of NR2F6 predicts a poor postoperative chemotherapy survival in DTX-resistant BCa

To explore the expression profile of NR2F6 in BCa, we firstly analyzed the transcriptomic profiles of 11 DTX-sensitive and 13 DTX-resistant surgical BCa specimens deposited in the GEO database using the GEO2R platform. Expression of NR2F6 transcripts was found to be significantly decreased in DTX-resistant samples compared to DTX-sensitive samples (Fig. 1A). To verify this finding, we examined the expression levels of NR2F6 mRNA in our 79-patient cohort. An approximate 56.4% reduction in NR2F6 mRNA levels in DTX-resistant BCa (n = 35) compared with DTX-sensitive (n = 44) was observed based on RT-qPCR analysis (Fig. 1B). This downregulation of NR2F6 expression was further validated at the protein level by immunohistochemistry (Fig. 1C). Interestingly, levels of NR2F6 mRNA were significantly correlated with postoperative chemotherapy survival and Kaplan–Meier plots demonstrated that low NR2F6 mRNA levels predicted a poor postoperative docetaxel chemotherapy survival in our 79-patient BCa cohort (P < 0.0001, Fig. 1D). To further confirm the association between NR2F6 downregulation and the pathogenesis of DTX resistance, we generated DTX-resistant BCa sublines according to the reported protocol. The IC50 value was estimated by MTT assay from dose-response curves obtained following 48 h exposure to DTX. The results showed that IC50 values of DTX in MCF-7/DTX and MDA-MB-231/DTX were 11.3-fold and 8.5-fold high compared to their parental cells, respectively (Fig. 1E). Subsequent RT-qPCR (Fig. 1F) and immunoblotting (Fig. 1G) analyses revealed a unanimous reduction in NR2F6 expression at both transcriptional and translational levels. Interestingly, when naive MCF-7 and MDA-MB-231 cells were stimulated with 5 nM of DTX for different durations, NR2F6 expression was observed to be gradually induced and this DTX-elicited stimulatory effect appeared to be exhibited in a dose-dependent manner (Fig. 1H). Taken together, the results point to a close link between NR2F6 downregulation and acquired resistance to DTX in BCa.

Figure 1
Figure 1

Downregulation of NR2F6 expression predicts a poor postoperative chemotherapy survival in docetaxel (DTX)-resistant breast cancer (BCa). (A) Data analysis from GSE6434 (Gene Expression Omnibus dataset) revealed that the NR2F6 transcripts were significantly decreased (P < 0.001) in DTX-resistant BCa biopsies. (B) RT-qPCR analysis validated that expression levels of NR2F6 mRNA were significantly downregulated in DTX-resistant BCa biopsies from our 79-patient cohort (P < 0.001). (C) Localization of NR2F6 protein in BCa tissues was assessed by immunohistochemistry. Bar = 15 μm (D) Kaplan–Meier analysis of the association of NR2F6 mRNA expression and overall survival of patients receiving DTX-based chemotherapy in our 79-patient cohort. (E) BCa cells were seeded onto a 96-well plate at the density of 1 × 104 cells/well. Cells were then treated with different doses of DTX for 48 h, followed by MTT assay. The IC50 (half maximal inhibitory concentration) values of DTX in different cells were calculated accordingly. (F) Relative expression levels of NR2F6 mRNA in different BCa cells were assayed using RT-qPCR analysis. (G) Relative expression levels of NR2F6 protein in different BCa cells were assayed using immunoblotting analysis. (H) MCF-7 and MDA-MB-231 cells were challenged with 5 nM of DTX for different durations as indicated, followed by immunoblotting analysis. A full color version of this figure is available at https://doi.org/10.1530/ERC-19-0229.

Citation: Endocrine-Related Cancer 27, 5; 10.1530/ERC-19-0229

Leptin signaling negatively regulates NR2F6 expression in BCa

Accumulating data evidence a causal link between obesity (characterized by overweight condition and high serum level of leptin secreted by adipose tissue) and cancer incidence, progression and metastasis and chemoresistance in various types of cancer (Candelaria et al. 2017). Indeed, in our study, profiling analysis using three pairs of DTX-sensitive and DTX-resistant surgical BCa specimens revealed that LEPR mRNA or OB-R expression were both noticeably induced in DTX-resistant specimens (Fig. 2A and B). To gain mechanistic insight into the potential regulation of NR2F6 expression by leptin, we studied the effects of leptin treatment on NR2F6 expression in naïve MCF-7 and MDA-MB-231 cells. As shown by RT-qPCR, leptin substantially inhibited the expression levels of NR2F6 mRNA in a time- (Fig. 2C) and dose-dependent (Fig. 2D) manner. In line with these results, co-incubation with PEG-LPrA2, a leptin receptor antagonist, effectively abolished the inhibitory effect on NR2F6 mRNA expression in leptin-challenged MCF-7 cells (Fig. 2E). To further confirm the potential regulation of NR2F6 expression by leptin, we knocked down LEPR expression using siRNA treatment in MCF-7 cells (Fig. 2F). Expectedly, OB-R depletion totally neutralized leptin-repressed NR2F6 expression in MCF-7 cells (Fig. 2G). We thus identified a negative regulation of NR2F6 expression by leptin signaling in BCa cells.

Figure 2
Figure 2

Regulation of NR2F6 expression by leptin signaling. (A) Relative expression levels of NR2F6 mRNA in three pairs of DTX-sensitive and DTX-resistant BCa biopsies were evaluated using RT-qPCR. (B) Relative expression levels of NR2F6 protein in three pairs of DTX-sensitive and DTX-resistant BCa biopsies were evaluated using immunoblotting. (C) BCa cells were incubated for different durations with 1.2 nM of recombinant human leptin, followed by RT-qPCR. Different superscript letters denote groups that are statistically different (P < 0.05). (D) BCa cells were incubated with different doses of leptin for 12 h, followed by RT-qPCR. Different superscript letters denote groups that are statistically different (P < 0.05). (E) MCF-7 cells were challenged with different doses of leptin, in the presence or absence of co-treatment with 1.2 nM of PEG-LPrA2, for 12 h, followed by RT-qPCR. Different superscript letters denote groups that are statistically different (P < 0.05). (F) Transient knockdown of OB-R in MCF-7 cells was verified using immunoblotting. (G) 48 h after siRNA treatment, MCF-7 cells were stimulated with different doses of leptin for 12 h, followed by measurement of NR2F6 mRNA expression using RT-qPCR. Different superscript letters denote groups that are statistically different (P < 0.05). A full color version of this figure is available at https://doi.org/10.1530/ERC-19-0229.

Citation: Endocrine-Related Cancer 27, 5; 10.1530/ERC-19-0229

NR2F6 augmentation potentiates sensitivity to DTX in a patient-derived xenograft model

To ask whether NR2F6 deficiency affected DTX sensitivity in BCa, we employed a patient-derived xenograft model (Fig. 3A) based on a previously validated protocol (DeRose et al. 2011). Reintroduction of the exogenous NR2F6 expression in PDX tumors via intratumoral injection of Retro-EGFP-NR2F6 significantly improved DTX sensitivity, as reflected by the reduced tumor formation when comparing DTX + Retro-EGFP-NR2F6 group to DTX + Mock infection group at 21 and 28 days after tissue implantation (Fig. 3B). Consistently, Retro-EGFP-NR2F6-injected tumors displayed a lower tumor weight and a smaller tumor size relative to Mock infection group at the end of the PDX study (Fig. 3C). This differential DTX response was even more evident by evaluation of qualitative endpoints using morphological approaches, including TUNEL staining and CD133 immunostaining. Overexpression of NR2F6 (evidenced by high levels of EGFP expression in Retro-EGFP-NR2F6-injected tumors) dramatically promoted apoptosis and decreased CD133 expression in DTX-challenged PDX tumors (Fig. 3D). Because CD133 is a membrane-bound pentaspan glycoprotein that is highly expressed in tumor initiating cells and its expression status is positively associated with tumor progression (Schmohl & Vallera 2016), the available data suggest that NR2F6 augmentation bears a potential therapeutic effect in BCa. To be noted, differential response to DTX was unlikely due to differences in uptake of the drug, because Retro-EGFP-NR2F6-injected tumors and Mock infection tumors exhibited similar expression levels of γ-H2AX (Fig. 3E), a marker of DNA damage (Ivashkevich et al. 2012). Collectively, these results suggest that NR2F6 status in BCa has a profound influence on DTX sensitivity.

Figure 3
Figure 3

Ectopic expression of NR2F6 by intratumoral injection potentiates sensitivity to DTX in a patient-derived xenograft model. (A) Protocols employed for the intratumoral (I.T.) injection in a patient-derived xenograft model. (B) Following the I.T. injection of Retro-EGFP-NR2F6 or empty vector complexed with Lipofectamine 3000, mice received a concurrent DTX treatment as described in Materials and methods. The xenografts were allowed to grow for about 1 month, and tumor volumes were recorded and calculated on a weekly basis (*P < 0.05 and **P < 0.01 when comparing DTX + Mock infection to DTX + Retro-EGFP-NR2F6). (C) Summary of tumor weights for the indicated groups (n = 10/group). (D) Representative images showing validation of EGFP-NR2F6 overexpression by FACS, TUNEL staining or immunostaining with the indicated markers. (E) DTX-induced DNA damage in xenografts was evaluated by immunoblotting analysis ofγ-H2AX expression. A full color version of this figure is available at https://doi.org/10.1530/ERC-19-0229.

Citation: Endocrine-Related Cancer 27, 5; 10.1530/ERC-19-0229

Manipulation of NR2F6 expression in BCa cells affects response to DTX

Next, we tried to provide the direct evidence to demonstrate the functional relevance between NR2F6 dysregulation and DTX sensitivity in BCa cells. Toward this end, we transiently knocked down the expression levels of the endogenous NR2F6 using shRNA treatment (Fig. 4A). Cell viability assay showed a more than two-fold increase in cell numbers after NR2F6 depletion for MCF-7 and MDA-MB-231 cells at the end of 48 h following DTX treatment (Fig. 4B). In accordance with this finding, induction of apoptosis by different doses of DTX was substantially abolished in NR2F6-delepted MCF-7 and MDA-MB-231 cells (Fig. 4C and D). We next investigated whether NR2F6 also affected in vivo response to DTX using the BCa cells-derived xenograft model. The NR2F6-delepted BCa cells were implanted into flanks of host mice, followed by i.p. injection of DTX or saline. Apparently, NR2F6-delepted BCa cells developed tumors with lower latency, as reflected by the measurement of tumor formation following treatment with DTX or saline (Fig. 4E). In another experimental setting, we expressed exogenous NR2F6 in MCF-7/DTX and MDA-MB-231/DTX cells (Fig. 4F). Ectopic expression of NR2F6 attenuated cell viability (Fig. 4G) and potentiated apoptosis (Fig. 4H and I) in DTX-challenged MCF-7/DTX and MDA-MB-231/DTX cells. Likewise, NR2F6 overexpression in both BCa cells significantly ameliorated in vivo response to DTX, which was evident from quantitative endpoint (i.e. tumor formation rate, Fig. 4I). These findings indicate that the endogenous NR2F6 may function as an important factor governing the DTX response in BCa cells.

Figure 4
Figure 4

Manipulation of NR2F6 expression affects response to DTX in BCa cells. (A) 48 h after shRNA transfection, transient knockdown of NR2F6 expression was validated using immunoblotting. BCa cells were seeded onto a 96-well plate at the density of 1 × 104 cells/well. Cells were then treated with different doses of DTX for 48 h, followed by MTT assay (B) (*P < 0.05 and **P < 0.01 when comparing Scramble to NR2F6 shRNA) and apoptosis ELISA (C and D). Different superscript letters denote groups that are statistically different (P < 0.05). (E) Effects of NR2F6 depletion on in vivo DTX sensitivity were evaluated using a BCa cell-derived xenograft model, as described in Materials and methods. Tumor volumes were recorded accordingly on a weekly basis (*P < 0.05 and **P < 0.01 when comparing DTX + Scramble to DTX + NR2F6 shRNA). (F) Ectopic of exogenous NR2F6 in BCa cells was validated using immunoblotting. BCa cells with different transfections were seeded onto a 96-well plate at the density of 1 × 104 cells/well. Cells were then treated with different doses of DTX for 48 h, followed by MTT assay (G) (*P < 0.05 and **P < 0.01 when comparing Scramble to NR2F6 shRNA) and apoptosis ELISA (H-I). Different superscript letters denote groups that are statistically different (P < 0.05). (J) Effects of NR2F6 overexpression on in vivo DTX sensitivity were evaluated using a BCa cell-derived xenograft model, as described in Materials and methods. Tumor volumes were recorded accordingly on a weekly basis (*P < 0.05 and **P < 0.01 when comparing DTX + Vector to DTX + DDK-NR2F6). A full color version of this figure is available at https://doi.org/10.1530/ERC-19-0229.

Citation: Endocrine-Related Cancer 27, 5; 10.1530/ERC-19-0229

NR2F6 expression potentiates resistance to paclitaxel in BCa cells

To further address whether the NR2F6-potentiated chemosensitivity applies to other taxols, we treated the NR2F6-depleted cells with different doses of paclitaxel (PTX) for 48 h. Interestingly, ablation of endogenous NR2F6 significantly increased cell resistance to PTX, as determined by cell viability (Fig. 5A) and apoptosis (Fig. 5B) assays. Consistently, NR2F6-delepted BCa cells developed tumors with lower latency, as reflected by the measurement of tumor formation following treatment with PTX or saline for a 35-day duration (Fig. 5C). Thus, the chemosensitivity-promoting effect is not specific to DTX. As docetaxel mainly targets microtubules to inhibit BCa progression, we further examined the effects of NR2F6 overexpression on tubulin polymerization in DTX-challenged MCF-7/DTX and MDA-MB-231/DTX cells. In line with the previous report (Duran et al. 2017), MCF-7/DTX and MDA-MB-231/DTX cells demonstrated an impaired tubulin polymerization than their parental cells. This impairment could be effectively reversed by NR2F6 overexpression (Fig. 5D and E). This observation raised an interesting possibility that NR2F6 may confer chemosensitivity to DTX also by targeting tubulin polymerization.

Figure 5
Figure 5

Ablation of endogenous NR2F6 confers resistance to paclitaxel (PTX) treatment in BCa cells. BCa cells were seeded onto a 96-well plate at the density of 1 × 104 cells/well. Cells were then treated with different doses of PTX for 48 h, followed by MTT assay (A) (*P < 0.05 and **P < 0.01 when comparing Scramble to NR2F6 shRNA) and apoptosis ELISA (B). Different superscript letters denote groups that are statistically different (P < 0.05). (C) Effects of NR2F6 depletion on in vivo PTX sensitivity (20 mg/kg, twice per week) were evaluated using a BCa cell-derived xenograft model, as described in Materials and methods. Tumor volumes were recorded accordingly on a weekly basis (*P < 0.05 and **P < 0.01 when comparing PTX + Scramble to PTX + NR2F6 shRNA). (D and E) Inhibition of tubulin polymerization by NR2F6 overexpression using an in vitro tubulin polymerization assay kit (Sigma-Aldrich). A full color version of this figure is available at https://doi.org/10.1530/ERC-19-0229.

Citation: Endocrine-Related Cancer 27, 5; 10.1530/ERC-19-0229

NR2F6 regulates PDGFRB transcription in BCa cells

To understand the molecular basis of NR2F6 function, we carried out in silico analysis using two different algorithms (UCSC Genome and JASPAR CORE), and we found that PDGFRB might be a potential down-stream target of NR2F6. In favor of this, NR2F6 mRNA expression was observed to be negatively correlated to PDGFRB mRNA expression in our 79-patient BCa cohort (P = 0.0078, Fig. 6A). This negative correlation was further verified by immunofluorescence in three pairs of DTX-sensitive and DTX-resistant surgical BCa specimens (Fig. 6B). Further bioinformatic analysis showed that the promoter of PDGFRB gene contains a consensus binding sequence of NR2F6 (TGACCT, Fig. 6C). We therefore anticipated that PDGFRB transcription may be directly repressed by NR2F6 in BCa cells. Indeed, ablation of NR2F6 expression by shRNA induced a significant increase in PDGFRB transcription, whereas ectopic expression of exogenous NR2F6 caused a noticeable reduction in PDGFRB mRNA levels (Fig. 6D and E), lending support to our hypothesis. To ask whether PDGFRB chromatin acts as the direct downstream target of NR2F6, we performed the luciferase reporter assay. Cotransfection with the pGL3-Luc-PDGFRB reporter plasmid and pCMV6-DDK-NR2F6 resulted in a ~45.9% reduction in the luciferase reporter activity, and this inhibitory effect was further enhanced by co-treatment with 1.2 nM of DTX. In contrast, co-treatment with 100 ng/mL of TSA (a potent HDAC inhibitor) or mutation of the putative NR2F6-binding site in the PDGFRB chromatin by replacing TGACCT with TtcaCT both totally countervailed the NR2F6-suppressed PDGFRB transcription, regardless of the DTX co-treatment (Fig. 6F and G). To further elucidate the in vivo occupancy of NR2F6 at the PDGFRB promoter, we performed chromatin immunoprecipitation (ChIP) in NR2F6 shRNA or Scramble shRNA transfected MCF-7 cells. qPCR analysis using the primers flanking the putative NR2F6-binding site in the PDGFRB chromatin revealed a robust recruitment of NR2F6 onto the PDGFRB promoter. This recruitment was further enhanced by co-treatment with 1.2 nM of DTX, but not by co-treatment with 100 ng/mL of TSA (Fig. 6H). To further verify the physical interaction of the NR2F6-HDAC complex within the PDGFRB chromatin, we carried out a double ChIP in MCF-7 cells. A simultaneous co-association of NR2F6 and HDAC2 within the PDGFRB chromatin was observed when DTX co-treatment was present, and the recruitment of HDAC2 was totally abolished upon TSA stimulation (Fig. 6I). Notably, the recruitment of NR2F6 and corresponding co-association of HDAC2 within the PDGFRB chromatin were both vanished in NR2F6-depleted MCF-7 cells (Fig. 6H and I). The observed preferential binding of NR2F6-HDAC2 to the PDGFRB promoter sequences containing the NR2F6 consensus element suggests a direct regulatory relationship between NR2F6-HDAC complex and PDGFRB in BCa.

Figure 6
Figure 6

NR2F6 regulates PDGFRB transcription in BCa cells. (A) Association between PDGFRB and NR2F6 mRNA levels was assessed in 79 cases of BCa using RT-qPCR, followed by Pearson Chi-Square test. (B) Co-localization of PDGFRB and NR2F6 in DTX-sensitive and DTX-resistant BCa biopsies were evaluated using double immunofluorescence staining plus confocal microscopy analysis. Bar = 10 μm (C) Prediction of a putative NR2F6-binding site in the promoter of PDGFRB gene. (D) 48 h after shRNA transfection, expression levels of PDGFRB mRNA were assayed using RT-qPCR in different BCa cells. (E) 48 h after pCMV6-DDK-NR2F6 transfection, expression levels of PDGFRB mRNA were assayed using RT-qPCR in different BCa cells. (F) Schematic of WT or mutated pGL3-PDGFRB-Luc reporter plasmids. (G) 0.5 μg reporter plasmids (WT or Mu pGL3-Luc reporter plasmids), pCMV6-DDK-NR2F6 and pRL-TK Renilla reporter plasmid were co-transfected into HeLa cells using Lipofectamine 3000. Forty-eight hours later, cells were stimulated for 6 h with 1.2 nM of DTX, in the presence or absence of co-treatment with 100 ng/mL of trichostatin A (TSA), followed by measurement of the relative luciferase activity using the dual luciferase reporter assay system. Different superscript letters denote groups that are statistically different (P < 0.05). (H) ChIP-qPCR analysis showing recruitment of NR2F6 onto a specific region of the PDGFRB promoter. This recruitment was further enhanced by treatment with DTX. The direct binding of NR2F6 onto the PDGFRB chromatin was totally abolished in the presence of TSA co-treatment or NR2F6 shRNA. Different superscript letters denote groups that are statistically different (P < 0.05). (I) Double ChIP-qPCR analysis of NR2F6-HDAC2 complex onto the PDGFRB promoter in MCF-7 cells upon challenge for 12 h with 1.2 nM of DTX. The direct binding of NR2F6-HDAC2 complex onto the PDGFRB chromatin was totally abolished in the presence of TSA co-treatment or NR2F6 shRNA. Different superscript letters denote groups that are statistically different (P < 0.05). A full color version of this figure is available at https://doi.org/10.1530/ERC-19-0229.

Citation: Endocrine-Related Cancer 27, 5; 10.1530/ERC-19-0229

Stable PDGFRB inhibition ameliorates NR2F6 deficiency-impaired response to DTX

Final efforts were initiated toward evaluating whether deregulation of PDGFRB alone could explain for the NR2F6 deficiency-impaired response to DTX. At the in vitro level, we generated MCF-7 cells stably depleted of endogenous PDGFRB (MCF-7/PDGFRB sh) and then transiently transfected MCF-7/PDGFRB sh cells with NR2F6 shRNA or Scramble shRNA. As expected, NR2F6 silence exerted no effects on PDGFRB expression level in MCF-7/PDGFRB sh cells (Fig. 7A). Consequently, stable PDGFRB ablation effectively abolished NR2F6 knockdown-induced cell proliferation (Fig. 7B) and promoted NR2F6 deficiency-impaired apoptosis upon DTX challenge (Fig. 7C). We solidified these in vitro data by assessing the effects of stable PDGFRB inhibition on tumor formation using a BCa cells-derived xenograft model. NR2F6 deficiency, caused by transfection with NR2F6 shRNA, resulted in a significant increase in the DTX-challenged tumor growth of MCF-7 xenograft tumors. This stimulatory effect was partially but effectively reversed by co-inhibition of PDGFRB expression using shRNA in MCF-7 cells, especially during the late stage of tumor development (Fig. 7D). Thus, PDGFRB antagonism might be an effective therapy modality for DTX-resistant BCa, in which NR2F6 deficiency usually expedites a poor response to DTX.

Figure 7
Figure 7

Effects of stable PDGFRB depletion on cell viability and DTX sensitivity in NR2F6-depleted cells. (A) MCF-7 cells stably deprived of endogenous PDGFRB expression were established as described in Materials and methods. These cells were then transiently transfected with NR2F6 shRNA or Scramble shRNA for 48 h, followed by immunoblotting analysis. MCF-7 cells with different transfections were seeded onto a 96-well plate at the density of 1 × 104 cells/well. Cells were then treated with different doses of DTX for 48 h, followed by MTT assay (B) (*P < 0.05 and **P < 0.01 when comparing NR2F6 shRNA + Ctrl shRNA to NR2F6 shRNA + PDGFRB shRNA) and apoptosis ELISA (C). Different superscript letters denote groups that are statistically different (P < 0.05). (D) Effects of PDGFRβ manipulation on in vivo DTX sensitivity were evaluated using a NR2F6-depleted MCF-7 cell-derived xenograft model, as described in Materials and methods. Tumor volumes were recorded accordingly on a weekly basis (*P < 0.05 and **P < 0.01 when comparing DTX + NR2F6 shRNA + Ctrl shRNA to DTX + NR2F6 shRNA + PDGFRB shRNA). (E) Proposed working model for the current study. A full color version of this figure is available at https://doi.org/10.1530/ERC-19-0229.

Citation: Endocrine-Related Cancer 27, 5; 10.1530/ERC-19-0229

Discussion

Unveiling mechanisms that dominate transcriptional control of chemoresistance is of high clinical relevance to understand the oncological origin and to further develop compelling targets for transcriptional targeting therapy (Javan & Shahbazi 2017). In the present communication, we described a critical role of NR2F6 in maintaining docetaxel chemosensitivity in BCa. First, data mining and profiling analysis using a 79-patient cohort both showed that NR2F6 expression was significantly downregulated in DTX-resistant BCa biopsies, and this downregulation predicted a poor postoperative docetaxel chemotherapy survival. Second, repression of NR2F6 expression in BCa cells appeared to be elaborately controlled by leptin signaling. Third, we highlighted that the observed chemosensitivity promoting effects depend on NR2F6 abundance in BCa cells, and we validated the therapeutic potential of using the replenishment of NR2F6 against DTX resistance in a patient-derived xenograft model. Last, our mechanistic study revealed that the chemosensitivity promoting effects of NR2F6 were, at least in part, mediated through transcriptional repression of PDGFRB signaling. Overall, these findings define NR2F6 as an intracellular transcriptional checkpoint in BCa cells, governing proper cellular response to DTX chemotherapy (Fig. 7E).

NR2F6 plays differential roles in specific subsets of cancer. For example, NR2F6 expression is found to be significantly induced in leukemia (Ichim et al. 2011), cervical cancer (Niu et al. 2016) and colorectal cancer (Li et al. 2011b). Of particular interest, in ovarian cancer cells, NR2F6 has been shown to confer cisplatin resistance by activating the Notch3 signaling pathway (Li et al. 2019). These studies highlight that NR2F6 acts as a potent driver of cell proliferation/survival and chemotherapy resistance in these malignancies. By contrast, NR2F6 has been shown to be a central checkpoint for cancer immune surveillance, and NR2F6 depletion remarkably promotes tumor outgrowth and progression in the autochthonous prostate cancer model TRAMP (Hermann-Kleiter et al. 2015). NR2F6 inhibits cell growth in thyroid via transcriptional repression of thyroid hormone nuclear receptor subtype beta1 (TRbeta1) signaling (Zhu et al. 2000). Likewise, in our study, we observed that NR2F6 downregulation is tightly associated with a poor postoperative docetaxel chemotherapy survival in BCa (Fig. 1). These results together suggest that NR2F6 may pay a dual role during tumor development and progression. The paradoxical role of NR2F6 may be attributed to cell context, tumor types and recruitment of different coregulators (Klepsch et al. 2016).

Despite much efforts focused on the identification of the downstream effectors of NR2F6, very little is known about the molecular bases that regulate NR2F6 expression. In our experiments, treatment with increasing doses of leptin resulted in a clear-cut biphasic inhibitory effect upon NR2F6 mRNA expression, suggestive of the involvement of leptin signaling in local control of NR2F6 expression (Fig. 2). In this sense, increased leptin levels have been associated with tumor progression and poor prognosis in BCa (Cleary 2013, Battle et al. 2014), and leptin is able to promote chemoresistance in BCa by mediating microenvironment effects and establishing a self-reinforcing signaling circuit in BCa stem cells (Candelaria et al. 2017), yet the mechanistic relevance of this system in the pathogenesis of DTX resistance remains unexplored. Of particular interest, we observed that leptin suppressed NR2F6 mRNA expression in a time- and dose-dependent manner. Such a response curve is similar to that reported for the inhibitory effects of Snail on NR2F6 mRNA expression during adipocyte differentiation (Pelaez-Garcia et al. 2015). Because leptin regulation requires central signaling through STAT3 pathway (Buettner et al. 2008; Buettner et al. 2006), and because our bioinformatics analysis has revealed a putative STAT3-binding site in the NR2F6 promoter (Supplementary Fig. 1), the available data may reflect the complex interaction of distinct transcription factors in the control of NR2F6 expression in different physiopathological systems. Of note, specificity of the inhibitory effects of leptin upon NR2F6 expression further confirmed the observation that the inhibition of NR2F6 expression by leptin was substantially abolished by co-treatment with a leptin receptor antagonist PEG-LPrA2 or by knockdown of leptin receptor expression in MCF-7 cells (Fig. 2). This intriguing leptin/STAT3/NR2F6 axis is currently under investigation in our lab.

Further evidence for a functional role of NR2F6 in the control of DTX response is strengthened by our gain- and loss-of-function approaches. In both genetically engineered cell models and murine xenograft models, we provided evidence that BCa cells with deficient NR2F6 expression were less responsive to DTX treatment (Fig. 3). Estrogen production through aromatization of the adrenal gland-derived androgens is well known to negatively affect chemosensitivity, and DTX treatment can significantly suppress aromatase in BCa cells (Miyoshi et al. 2004). Interestingly, in normal breast tissues, transcriptional levels of aromatase expression is specifically inhibited by NR2F6, in concert with other corepressors. Contrarily, in advanced BCa tissues, the levels of NR2F6 and other corepressors decrease, resulting in a deregulated activation of aromatase expression (Chen et al. 2005). It is therefore a logical hypothesis that proper control of aromatase activity through transcriptional repression of aromatase mRNAs by NR2F6 is crucial in combating malignant behavior of BCa cells. It will be of future interest to deconvolute how NR2F6 harmoniously regulates aromatase activation and DTX response at the transcriptional level. Importantly, by employing a patient-derived xenograft model, we have shown that intratumoral overexpression of NR2F6 could significantly promote apoptosis and decrease activation of tumor initiating cells, finally enhancing efficacy of DTX chemotherapy (Fig. 3). The latter observation provides a rational mechanistic basis envisioning targeted manipulation of NR2F6 in BCa cells as a tempting intracellular checkpoint-targeting strategy to improve the DTX therapy.

Accumulated data evidence that PDGF-B and PDGFRβ play important tumor-promoting roles in BCa. Expression levels of PDGF-B/PDGFRB axis are elevated in malignant lesions of the mammary gland such as ductal infiltrating carcinomas (Park et al. 2016). PDGFRβ is not only expressed in ductal carcinoma in situ but also enriched in the development of the architecture of the tumor vasculature, suggesting that PDGF-B/PDGFRβ axis may affect both tumor cells and tumor microenvironment (Plantamura et al. 2014). In favor of the latter hypothesis, it has been shown that PDGF-B/PDGFRβ promotes tumor angiogenesis and progression in triple negative breast cancers (TNBCs) through regulation of the HIF-1α/VEGFA signaling pathway (Jitariu et al. 2017). In keeping with the idea that suppressing PDGFRβ function in BCa could be of potential benefit to certain BCa patients, a series of inhibitors of PDGFRβ are currently at the level of clinical and/or preclinical trials (e.g. Imatinib (Kadivar et al. 2017), Pazopanib (Gril et al. 2013) and DNA vaccine (Kaplan et al. 2006)). As such, identification and replenishment of critical PDGFRB transcriptional suppressors may inhibit PDGFRβ function and affect both tumor cells and tumor microenvironment, thus enhancing the efficacy of tumor treatment. Our results indicate that NR2F6 represents as such a striking negative regulator of PDGFRB transcription. Additionally, recruitment of HDACs to target gene promoters has emerged as a general mechanism of transcription repression (Zhang et al. 2012). In light of our observation that NR2F6 recruits HDAC2 onto the PDGFRB promoter and this co-recruitment could repress the PDGFRB transcription to the maximum extent (Fig. 6), we propose that a joint application of ectopic expression of NR2F6, along with enhanced HDAC2 activity, may improve DTX response more effectively in advanced BCa.

In sum, the orphan nuclear receptor NR2F6 appears to be an important negative regulator of cell survival and DTX resistance in BCa cells, directly repressing transcription of PDGFRB, an essential tumor-promoting signaling and a potential therapeutic target for BCa progression. Thus, in the presence of synergistic network between NR2F6 and HDAC2, the activity of PDGF-B/PDGFRβ axis is maintained at the proper level within both tumor cells and tumor microenvironment. The exact molecular pathways by which leptin compromises NR2F6 expression and NR2F6 impairs the transcriptional amplitude of PDGFRB, along with other potential target genes of NR2F6 in BCa, warrant further investigation.

Supplementary materials

This is linked to the online version of the paper at https://doi.org/10.1530/ERC-19-0229.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by the International Cooperation Research Program (2016KW-010).

Author contribution statement

Z J and L R designed and supervised the experiments and wrote the manuscript. M H, Z M, Z C, H M and Y C performed the experiments and analyzed the data. W Z, H L and Y L helped to analyze the data. Z J contributed reagents and helped to analyze the data. L R contributed partial reagents. Z J supervised the manuscript submission.

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    Downregulation of NR2F6 expression predicts a poor postoperative chemotherapy survival in docetaxel (DTX)-resistant breast cancer (BCa). (A) Data analysis from GSE6434 (Gene Expression Omnibus dataset) revealed that the NR2F6 transcripts were significantly decreased (P < 0.001) in DTX-resistant BCa biopsies. (B) RT-qPCR analysis validated that expression levels of NR2F6 mRNA were significantly downregulated in DTX-resistant BCa biopsies from our 79-patient cohort (P < 0.001). (C) Localization of NR2F6 protein in BCa tissues was assessed by immunohistochemistry. Bar = 15 μm (D) Kaplan–Meier analysis of the association of NR2F6 mRNA expression and overall survival of patients receiving DTX-based chemotherapy in our 79-patient cohort. (E) BCa cells were seeded onto a 96-well plate at the density of 1 × 104 cells/well. Cells were then treated with different doses of DTX for 48 h, followed by MTT assay. The IC50 (half maximal inhibitory concentration) values of DTX in different cells were calculated accordingly. (F) Relative expression levels of NR2F6 mRNA in different BCa cells were assayed using RT-qPCR analysis. (G) Relative expression levels of NR2F6 protein in different BCa cells were assayed using immunoblotting analysis. (H) MCF-7 and MDA-MB-231 cells were challenged with 5 nM of DTX for different durations as indicated, followed by immunoblotting analysis. A full color version of this figure is available at https://doi.org/10.1530/ERC-19-0229.

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    Regulation of NR2F6 expression by leptin signaling. (A) Relative expression levels of NR2F6 mRNA in three pairs of DTX-sensitive and DTX-resistant BCa biopsies were evaluated using RT-qPCR. (B) Relative expression levels of NR2F6 protein in three pairs of DTX-sensitive and DTX-resistant BCa biopsies were evaluated using immunoblotting. (C) BCa cells were incubated for different durations with 1.2 nM of recombinant human leptin, followed by RT-qPCR. Different superscript letters denote groups that are statistically different (P < 0.05). (D) BCa cells were incubated with different doses of leptin for 12 h, followed by RT-qPCR. Different superscript letters denote groups that are statistically different (P < 0.05). (E) MCF-7 cells were challenged with different doses of leptin, in the presence or absence of co-treatment with 1.2 nM of PEG-LPrA2, for 12 h, followed by RT-qPCR. Different superscript letters denote groups that are statistically different (P < 0.05). (F) Transient knockdown of OB-R in MCF-7 cells was verified using immunoblotting. (G) 48 h after siRNA treatment, MCF-7 cells were stimulated with different doses of leptin for 12 h, followed by measurement of NR2F6 mRNA expression using RT-qPCR. Different superscript letters denote groups that are statistically different (P < 0.05). A full color version of this figure is available at https://doi.org/10.1530/ERC-19-0229.

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    Ectopic expression of NR2F6 by intratumoral injection potentiates sensitivity to DTX in a patient-derived xenograft model. (A) Protocols employed for the intratumoral (I.T.) injection in a patient-derived xenograft model. (B) Following the I.T. injection of Retro-EGFP-NR2F6 or empty vector complexed with Lipofectamine 3000, mice received a concurrent DTX treatment as described in Materials and methods. The xenografts were allowed to grow for about 1 month, and tumor volumes were recorded and calculated on a weekly basis (*P < 0.05 and **P < 0.01 when comparing DTX + Mock infection to DTX + Retro-EGFP-NR2F6). (C) Summary of tumor weights for the indicated groups (n = 10/group). (D) Representative images showing validation of EGFP-NR2F6 overexpression by FACS, TUNEL staining or immunostaining with the indicated markers. (E) DTX-induced DNA damage in xenografts was evaluated by immunoblotting analysis ofγ-H2AX expression. A full color version of this figure is available at https://doi.org/10.1530/ERC-19-0229.

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    Manipulation of NR2F6 expression affects response to DTX in BCa cells. (A) 48 h after shRNA transfection, transient knockdown of NR2F6 expression was validated using immunoblotting. BCa cells were seeded onto a 96-well plate at the density of 1 × 104 cells/well. Cells were then treated with different doses of DTX for 48 h, followed by MTT assay (B) (*P < 0.05 and **P < 0.01 when comparing Scramble to NR2F6 shRNA) and apoptosis ELISA (C and D). Different superscript letters denote groups that are statistically different (P < 0.05). (E) Effects of NR2F6 depletion on in vivo DTX sensitivity were evaluated using a BCa cell-derived xenograft model, as described in Materials and methods. Tumor volumes were recorded accordingly on a weekly basis (*P < 0.05 and **P < 0.01 when comparing DTX + Scramble to DTX + NR2F6 shRNA). (F) Ectopic of exogenous NR2F6 in BCa cells was validated using immunoblotting. BCa cells with different transfections were seeded onto a 96-well plate at the density of 1 × 104 cells/well. Cells were then treated with different doses of DTX for 48 h, followed by MTT assay (G) (*P < 0.05 and **P < 0.01 when comparing Scramble to NR2F6 shRNA) and apoptosis ELISA (H-I). Different superscript letters denote groups that are statistically different (P < 0.05). (J) Effects of NR2F6 overexpression on in vivo DTX sensitivity were evaluated using a BCa cell-derived xenograft model, as described in Materials and methods. Tumor volumes were recorded accordingly on a weekly basis (*P < 0.05 and **P < 0.01 when comparing DTX + Vector to DTX + DDK-NR2F6). A full color version of this figure is available at https://doi.org/10.1530/ERC-19-0229.

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    Ablation of endogenous NR2F6 confers resistance to paclitaxel (PTX) treatment in BCa cells. BCa cells were seeded onto a 96-well plate at the density of 1 × 104 cells/well. Cells were then treated with different doses of PTX for 48 h, followed by MTT assay (A) (*P < 0.05 and **P < 0.01 when comparing Scramble to NR2F6 shRNA) and apoptosis ELISA (B). Different superscript letters denote groups that are statistically different (P < 0.05). (C) Effects of NR2F6 depletion on in vivo PTX sensitivity (20 mg/kg, twice per week) were evaluated using a BCa cell-derived xenograft model, as described in Materials and methods. Tumor volumes were recorded accordingly on a weekly basis (*P < 0.05 and **P < 0.01 when comparing PTX + Scramble to PTX + NR2F6 shRNA). (D and E) Inhibition of tubulin polymerization by NR2F6 overexpression using an in vitro tubulin polymerization assay kit (Sigma-Aldrich). A full color version of this figure is available at https://doi.org/10.1530/ERC-19-0229.

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    NR2F6 regulates PDGFRB transcription in BCa cells. (A) Association between PDGFRB and NR2F6 mRNA levels was assessed in 79 cases of BCa using RT-qPCR, followed by Pearson Chi-Square test. (B) Co-localization of PDGFRB and NR2F6 in DTX-sensitive and DTX-resistant BCa biopsies were evaluated using double immunofluorescence staining plus confocal microscopy analysis. Bar = 10 μm (C) Prediction of a putative NR2F6-binding site in the promoter of PDGFRB gene. (D) 48 h after shRNA transfection, expression levels of PDGFRB mRNA were assayed using RT-qPCR in different BCa cells. (E) 48 h after pCMV6-DDK-NR2F6 transfection, expression levels of PDGFRB mRNA were assayed using RT-qPCR in different BCa cells. (F) Schematic of WT or mutated pGL3-PDGFRB-Luc reporter plasmids. (G) 0.5 μg reporter plasmids (WT or Mu pGL3-Luc reporter plasmids), pCMV6-DDK-NR2F6 and pRL-TK Renilla reporter plasmid were co-transfected into HeLa cells using Lipofectamine 3000. Forty-eight hours later, cells were stimulated for 6 h with 1.2 nM of DTX, in the presence or absence of co-treatment with 100 ng/mL of trichostatin A (TSA), followed by measurement of the relative luciferase activity using the dual luciferase reporter assay system. Different superscript letters denote groups that are statistically different (P < 0.05). (H) ChIP-qPCR analysis showing recruitment of NR2F6 onto a specific region of the PDGFRB promoter. This recruitment was further enhanced by treatment with DTX. The direct binding of NR2F6 onto the PDGFRB chromatin was totally abolished in the presence of TSA co-treatment or NR2F6 shRNA. Different superscript letters denote groups that are statistically different (P < 0.05). (I) Double ChIP-qPCR analysis of NR2F6-HDAC2 complex onto the PDGFRB promoter in MCF-7 cells upon challenge for 12 h with 1.2 nM of DTX. The direct binding of NR2F6-HDAC2 complex onto the PDGFRB chromatin was totally abolished in the presence of TSA co-treatment or NR2F6 shRNA. Different superscript letters denote groups that are statistically different (P < 0.05). A full color version of this figure is available at https://doi.org/10.1530/ERC-19-0229.

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    Effects of stable PDGFRB depletion on cell viability and DTX sensitivity in NR2F6-depleted cells. (A) MCF-7 cells stably deprived of endogenous PDGFRB expression were established as described in Materials and methods. These cells were then transiently transfected with NR2F6 shRNA or Scramble shRNA for 48 h, followed by immunoblotting analysis. MCF-7 cells with different transfections were seeded onto a 96-well plate at the density of 1 × 104 cells/well. Cells were then treated with different doses of DTX for 48 h, followed by MTT assay (B) (*P < 0.05 and **P < 0.01 when comparing NR2F6 shRNA + Ctrl shRNA to NR2F6 shRNA + PDGFRB shRNA) and apoptosis ELISA (C). Different superscript letters denote groups that are statistically different (P < 0.05). (D) Effects of PDGFRβ manipulation on in vivo DTX sensitivity were evaluated using a NR2F6-depleted MCF-7 cell-derived xenograft model, as described in Materials and methods. Tumor volumes were recorded accordingly on a weekly basis (*P < 0.05 and **P < 0.01 when comparing DTX + NR2F6 shRNA + Ctrl shRNA to DTX + NR2F6 shRNA + PDGFRB shRNA). (E) Proposed working model for the current study. A full color version of this figure is available at https://doi.org/10.1530/ERC-19-0229.

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