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. 2023 Nov 24;149(20):18103–18117. doi: 10.1007/s00432-023-05503-6

EGR3 and estrone are involved in the tamoxifen resistance and progression of breast cancer

Yu Xie 1, Xiao Han 2, Jing Yu 1, Mengci Yuan 1, Yan Yan 3, Junfang Qin 1, Lan Lan 4, Yue Wang 1,3,✉
PMCID: PMC11798252  PMID: 37999751

Abstract

Background

Tamoxifen (Tam) is an effective treatment for estrogen receptor (ER) positive breast cancer. However, a significant proportion of patients develop resistance under treatment, presenting a therapeutic challenge. The study aims to determine the role of early growth response protein (EGR) 3 in tamoxifen resistance (TamR) and elucidate its molecular mechanism.

Methods

TamR cell models were established and NGS was used to screening signaling alternation. Western blot and qRT-PCR were used to analysis the expression of ERα, EGR3, MCL1 and factors associated with apoptosis. CCK8, colony formation and apoptosis assay were used to analysis resistance to Tam. Immunofluorescence, chromatin immunoprecipitation, and dual luciferase assays were used to investigate mechanism of regulation.

Results

We observed that EGR3, a deeply rooted ERα response factor, showed increased upregulation in response to both estrone (E1) and Tam in TamR cells with elevated level of E1 and ERα expression, indicating a potential connection between EGR3 and TamR. Mechanically, manipulating EGR3 expression revealed that it imparted resistance to Tam through increased expression of the downstream molecule MCL1 (apoptosis suppressor gene) that it regulated. Mechanismly, EGR3 directly binds to the promoter of the anti-apoptotic factor MCL1 gene, facilitating its transcription. Furthermore, apoptosis assays revealed that E1 reduces Tam induced apoptosis by upregulating EGR3 expression. Importantly, clinical public database confirmed the high expression of EGR3 in breast cancer tissue and in Tam-treated patients.

Conclusions

These findings shed light on the novel estrogen/EGR3/MCL1 axis and its role in inducing TamR in ER positive breast cancer. EGR3 emerges as a promising target to overcome TamR. The elucidation of this mechanism holds potential for the development of new therapeutic modalities to overcome endocrine therapy resistance in clinical settings.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00432-023-05503-6.

Keywords: Τamoxifen, Εstrone, EGR3, MCL1, Estrogen receptor

Introduction

Breast cancer is a prevalent disease, with approximately 75% of cases being estrogen receptor (ER) positive. Τhe ERα server as a hormone receptor and plays a crucial role in the development of female organs as well as breast cancer (Turner et al. 2017). Estrogens, including estrone (E1) and estradiol (E2), act as ligands for ERα and initiate downstream signaling events, regulating cellular progression. Recent research has highlighted the distinct and even contradictory role of different estrogen subtypes, such as E1 and E2, in inflammatory processes (Qureshi et al. 2020).

Endocrine therapy is the standard treatment for ER positive breast cancer, as it effectively inhibits ERα signaling and impeding tumor progression (Nabieva et al. 2021; Turner et al. 2017). Tamoxifen (Tam), a well-known selective ERα modulator, is the most prescribed endocrine therapy agents for premenopausal ER positive breast cancer patients (Patel et al. 2018). By competing with estrogen for binding to the ERα, Tam suppresses estrogen signaling activity, exhibiting minimal side effects (Jordan 2003; Mandlekar et al. 2001). Although the utilization of endocrine therapy has significantly reduced recurrence rates, resistance to this treatment remains a major contributor to breast cancer mortality (Early Breast Cancer Trialists’ Collaborative 2015). The underlying mechanisms of resistance to Tam has not yet been fully elucidated. Tumor cells often exhibit resistance to cytotoxic chemotherapy and targeted therapies due to various intrinsic and acquired mechanisms, which also contribute to treatment failure in endocrine therapy (Holohan et al. 2013). The aberrant estrogen related signaling plays a pivotal role in both de novo and acquired resistance to endocrine therapy (Nabieva et al. 2021). This study aims to investigate the role of a novel ERα downstream gene, early growth response protein (EGR) 3, in tamoxifen resistance (TamR).

EGR3 is a member of the EGR family, a group of C2H2-type zinc-finger proteins that play crucial roles in various biological processes, including muscle development, lymphocyte development, endothelial cell growth and migration, and neuronal development. Previous studies have primarily focused on the immune regulatory and psychotherapeutic potential of EGR3 (Engeln et al. 2020; Kwon et al. 2021; Morita et al. 2016; Nie et al. 2021; Parkinson et al. 2014). Additionally, EGR3 has been reported to exert inhibitory effects on epithelial–mesenchymal transition (EMT) and cancer cells progression (Li et al. 2021; Pio et al. 2013; Shin et al. 2020). Notably, recent research has highlighted the involvement of EGR3 as an estrogen responsive gene (Vareslija et al. 2016). Furthermore, EGR3, regulated by ERα through estrogen response element (ERE) located on the promoter region, has been associated with the modulation of the FasL pathway (Inoue et al. 2004). Collectively, these findings suggest that EGR3 plays a role in the development of resistance through the ERα pathway.

Apoptosis is a crucial pathway of cell death induced by anti-cancer drug treatments, including Tam, for inhibition of cancer cells progression (Obrero et al. 2002). However, some cancer cells develop alternations in apoptosis related pathways, leading to resistance to drugs such as Tam (Holohan et al. 2013; Viedma-Rodriguez et al. 2013). Such as BCL2 conferring resistance to endocrine treatment (Merino et al. 2016). Therefore, targeting genes involved in apoptosis has shown significant potential in restoring sensitivity to therapy in cancer cells (Shahbandi et al. 2020).

Therefore, this study aims to investigate the role of the estrogen responsive gene EGR3 in TamR. Our findings indicate that both Tam and estrogen, particularly E1, upregulate the expression of EGR3. The increased expression of EGR3, in turn, acts as a transcriptional factor for the survival related gene MCL1, leading to the development of TamR. The novel mechanism we have discovered provides a crucial insight into addressing TamR, which could go some way towards overcoming TamR and improve its efficacy in the clinical setting.

Methods

Cell models and reagents

The human breast cancer cell lines MCF7 and T47D cells were obtained from lab stocks and used as the parental cell models (MCF7P and T47DP) in this study. To establish TamR cell models, TamR cells MCF7 (MCF7R) and T47D (T47DR) cells were generated using the following protocol: the cells were continuously cultured with 0.1 µM Tam for 2 months, followed by 0.5 µM Tam for an additional 2 months, and finally maintained in 1 µM Tam (Gu et al. 2017). For all experiments, the cells were cultured in RPMI-1640 medium containing L-glutamine, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin cocktail. The MCF7R and T47DR cells were further supplemented with 1 µM Tam. The culture medium was refreshed every 2 days, and the cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2.

The RPMI-1640 medium (cat#01-100-1A) and fetal bovine serum (cat#04-001-1A) were purchased from Biological Industries, Israel. All reagents used in the experiments were dissolved in DMSO and stored at − 80 °C. The relevant reagents were added to the cell medium at least 12 h after cell seeding. Tamoxifen (cat#S1238) was purchased from Selleck Chemicals, USA. Estrone (cat#IE0240) was purchased from Solarbio, Beijing, China. Estradiol (cat#ST1101) was purchased from Beyotime Biotechnology, Shanghai, China.

Next generation sequencing (NGS)

Total RNA extraction was performed using Trizol reagent from MCF7P and MCF7R cells. The obtained RNA samples were then processed and subjected to RNA sequencing analysis services provides by Gene Denovo Biotechnology Co (Guangzhou, China). The sequencing was performed on an Illumina Novaseq 6000 platform.

Plasmid construction

Plasmids for forcing expression were constructed using the following procedure. The full-long transcripts of EGR3 and MCL1 were amplified via PCR, basing the complementary DNA extracted from MCF7R cells. The PCR primers were designed based on the corresponding sequences from the National Center for Biotechnology Information GenBank (accession numbers: EGR3, NM_004430.3; MCL1, NM_021960.5). Then the products were purified using the EasyPure PCR Purification Kit and then ligated into the pcDNA3.1 vector, which had been double digested with the appropriate restriction enzymes. We would like to express our gratitude to the Lian lab at the Third Military Medical University, Chongqing, China, for kindly providing us with the pcDNA3.1 vector plasmid.

The MCL1 sh plasmids were generated as follows: the sh primer pair was annealed to form duplexes, then digested with EcoR I restriction enzymes and ligated into the EcoR I-digested pLKO.1 vector. We would like to acknowledge the Qi Lab at the School of Medicine, Nankai University, and Tianjin, China, for kindly providing us with the pLKO-puro vector plasmid. The relevant sequences of the plasmids are provided in Table S1.

Additionally, the EGR3 sh plasmids were acquired from Tsingke Biotechnology, Beijing, China, and the relevant sequences are presented in Table S1. The EasyPure PCR Purification Kit (cat#EP101-1) was purchased from TransGen Biotech, Beijing, China. The specific details of the constructed plasmids are provided in Table S1. The insertion of the sequence was verified through Sanger sequencing performed by Sangon, Shanghai, China.

Plasmid transfection

The cells were plated in the appropriate culture plates prior to transfection. For plasmids transfection, DNA-Lipofectamine complexes were prepared in Opti-MEM following the manufacturer's instructions. After 8 h of transfection, the medium was replaced with fresh medium and subsequent experiments were conducted. Lipofectamine™ 3000 Transfection Reagent (cat#L3000015) and Opti-MEM (cat#31985070) were purchased from ThermoFisher Scientific, USA, for use in the transfection process.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from the cells using TRIzol reagent according to the manufacturer's instructions. Complementary DNA was synthesized through reverse transcription using relative synthesis kits, following the provided protocols. QRT-PCR was conducted using Hieff UNICON® qPCR SYBR Green Master Mix and performed on the LightCycler 96 Instrument, Roche. The mRNA expression level of the target gene was normalized to ACTB. Raw data were calculated and analyzed with the LightCycler 96 v1.1.0.1320 software provided by Roche. The TRIzol reagent (cat#AC0101-B) was purchased from Sparkjade®, Shandong, China. The cDNA synthesis kit (cat#11141ES10) and the Hieff UNICON® qPCR SYBR Green Master Mix (cat#11198ES03) were purchased from Yeasen Biotechnology, Shanghai, China. The synthesis of relative primers was conducted by Sangon, Shanghai, China. The sequences of the primers utilized in this study are provided in Table S2.

Colony formation assay

Cells were plated in triplicate into 6-well culture plates and transfected with the appropriate plasmids. After 1 week of incubation, the cells were washed twice with PBS, fixed with methanol for 30 min, and stained with 1% crystal violet for an additional 30 min. The colonies were then counted using ImageJ analysis software.

Cell viability assay

Cells were plated in triplicate in 96-well plates and transfected with the relevant plasmids. After the completion of treatments, cell viability assay was performed using CCK8-kits. The cells were incubated with 10 µL CCK8 per well at 37 °C for 0.5 to 1 h, following the instructions provided by the manufacturer. The absorbance of the reaction was then measured using a DNM-9606 plate reader (Perlong Medical, Beijing, China). The CCK8-kit (cat#CK04) was purchased from Dojindo, Rockville, MD.

Western blot (WB)

After the relevant treatment, cells were collected and washed with PBS. Subsequently, the cells were lysed with RIPA buffer. The proteins were separated by SDS gel electrophoresis and transferred to PVDF membranes. The membranes were subsequently incubated with specific primary and secondary antibodies. The protein bands were visualized using the Immobilon Western Chemiluminescent HRP Substrate and assessed using the Tanon 5200 Image System and Image Analysis software from Biotanon, Shanghai, China.

The following primary antibodies were used: Anti-ERα antibody (cat#8644) was purchased from Cell Signaling Technology, USA. Anti-EGR3 antibody (cat#sc-390967) was purchased from Santa Cruz Biotechnology, CA, USA; anti-MCL1 antibody (cat#16225-1-AP) was purchased from Proteintech, Wuhan, China; Anti-BCL2 antibody (cat#AF6139), anti-cleaved caspase 3 antibody (cat#AF7022), and anti-BAX antibody (cat#AF0120) were purchased from Affinity Biosciences, Jiangsu, China. The Immobilon Western Chemiluminescent HRP Substrate (cat#WBKLS0500) and PVDF membrane (cat#IPVH00010) were purchased from Merck Millipore, Darmstadt, Germany.

Immunofluorescence

Cells were plated onto glass coverslips and allowed to adhere. Subsequently, the cells were fixed in 4% paraformaldehyde and permeabilizated using permeabilization reagent. Following a blocking step with 3% BSA, the cells were incubated overnight at 4 °C with a specific primary antibody. After thorough washing, the cells were incubated with a suitable secondary antibody conjugated to a fluorophore. Nuclei were visualized by staining with DAPI. Immunofluorescence images were acquired using the Pannoramic MIDI system (3DHISTECH). The permeabilization reagent (cat#G1204) was purchased from Servicebio, Wuhan, China. The relative antibody was listed before.

Chromatin immunoprecipitation (ChIP) assay

At the conclusion of the treatment, cells were harvested and washed with PBS. Fixation of the cells was performed using 1% formaldehyde. Subsequently, cell lysis and nuclear extraction were carried out using the appropriate kits. The cell lysate was centrifuged to remove the sediment, and the resulting suspension was sonicated in an ice bath using a probe sonicator (JY92-IIN, Xinzhi Biotechnology, Ningbo, China) to shear the cross-linked DNA into fragments of approximately 200–1000 base pairs in length. Sonication was performed for 10 min at 60% power. Immunoprecipitation was conducted using an anti-EGR3 antibody or mouse IgG, following the instructions provided by the manufacturer. The chromosomal DNA was then purified and subjected to qRT-PCR analysis, as described earlier. The MCL1 promoter region, located within the upstream 2000 base pairs from the genomic MCL1 transcription start site, was selected based on the UCSC database. A pair of qRT-PCR primers was designed for this region, and an upstream region beyond 5000 base pair were selected as a negative control for the ChIP-qRT-PCR analysis (Fig. 4C). The Chromatin Immunoprecipitation Kits (cat#17-10086) were purchased from Merck Millipore, Darmstadt, Germany. The sequences of the qRT-PCR primers are provided in Table S3.

Fig. 4.

Fig. 4

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EGR3 promoted MCL1 transcription. A WB analysis of MCL1 manipulating plasmids efficiency in MCF7R. B WB analysis of regulatory relationship between EGR3 and MCL1 expression. Manipulation of EGR3 expression regulated MCL1 expression, but no reciprocal regulation of MCL1 on EGR3 expression. C ChIP-qRT-PCR analysis of EGR3 binding to MCL1 promoter. The EGR3 binding sites on the human MCL1 genomic locus was showed by transcriptional motif prediction, and the binding of EGR3 to the MCL1 promoter was validated in MCF7R and T47DR cells. Mouse IgG served as a negative control. D Dual luciferase reporter analysis of the transcriptional promotion role of EGR3 to the MCL1 promoter. EGR3 promoted transcriptional activity of the promoter of MCL1 both in MCF7R and T47DR cells. MCF7R tamoxifen resistant MCF7, T47DR tamoxifen resistant T47D, MCL1-p pGL4.1-MCL1 promoter. Data are shown as the means ± SDs. P values were calculated with two-tailed unpaired Student’s t test; *P < 0.05, **P < 0.01

Dual luciferase reporter assay

The MCL1 promoter region was inserted into the pGL4.1 dual luciferase system plasmids following the construction method, and pGL4.7 served as the reference control. Following co-transfection of relative plasmids, the cells were harvested, and luciferase activity was detected using the Dual Luciferase Reporter Assay System. The plate reader DNM-9606 (Perlong Medical, Beijing, China) was employed for collection luciferase activity. The Dual Luciferase Reporter Assay System (cat#E1910) was purchased from Promega, Madison, WI. The primer sequences used for amplifying the MCL1 promoter are provided in Table S1.

Apoptosis assay

After the designated treatment period, the cells were detached and converted into single-cell suspensions. Subsequently, the cell suspensions were adjusted to a density of 5 × 105 cells per tube and stained with Annexin V-FITC/PI, following the instructions provided by the manufacturer. The fluorescence was collected using the BD FACScalibur. The flow cytometry data were exported and analyzed using FlowJo v10.07 software from LLC, OR. The Annexin V-FITC/PI Apoptosis Detection Kit (cat#40302ES20) was purchased from Yeasen Biotechnology, Shanghai, China.

Online database

The breast cancer cell lines mRNA expression matrix of tumors was obtained from the CCLE dataset (https://portals.broadinstitute.org/ccle) (Ghandi et al. 2019). Gene co-expression relationships were calculated using the GEPIA database (http://gepia.cancer-pku.cn/index.html) (Tang et al. 2017). The EGR3 transcriptional binding motif was predicted using JASPAR2022 database (https://jaspar.genereg.net) (Castro-Mondragon et al. 2022).

Statistical analysis

The data were analyzed from at least three separate experiments, and the results are presented as means ± SDs (standard deviations). Statistical significance was determined using unpaired Student’s t test or one-way ANOVA properly in all relevant experiments. Statistical analyses were performed using Prism 8.0 software (GraphPad Software, USA).

Results

Estrone and tamoxifen increased expression of EGR3

Using concentration-dependent induction of Tam, breast cancer cell lines MCF7 and T47D with acquired TamR (MCF7R and T47DR) were established (Fig. S1). The underlying mechanisms of TamR were investigated by performing NGS on MCF7P and MCF7R cells. An enrichment of the estrogen metabolic process was observed in MCF7R cells through GSEA of differentially expressed genes (finished NGS data are shown in supplementary files) (Fig. 1A). Furthermore, the differential expression of enzymes involved in estrogen synthesis or decomposition was detected, especially HSD17B2, HSD17B14 and CYP3A5, which inhibited E1 degradation, were increased in MCF7R cells. The expression of the enzymes AKR1C1 and CYP1A1, which promoted the degradation of E1, were decreased in MCF7R cells (Fig. 1B). This suggests a potential role of elevated E1 level in maintaining TamR. Additionally, increased protein level of ERα in MCF7R cells was confirmed (Fig. 1C).It is known that changes in ER protein levels can lead to the upregulation of multiple responsive genes in the context of TamR (Jeffreys et al. 2020), prompting further investigation into the underlying mechanism.

Fig. 1.

Fig. 1

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Estrone and tamoxifen increased expression of EGR3. A, B NGS analysis of MCF7P and MCF7R cells. A Estrogen metabolic process signaling was enriched in MCF7R cells. B Estrogen metabolizing enzymes, especially HSD17B2, HSD17B14 and CYP3A5, which inhibited E1 degradation, were increased in MCF7R cells. The expression of the enzymes AKR1C1 and CYP1A1, which promoted the degradation of E1, were decreased in MCF7R cells. C WB analysis of ERα expression. ERα protein level was increased in MCF7R cells. D Analysis of EGR3 expression in 57 breast cancer cell lines according to CCLE database. EGR3 expression was increased in ER positive cell lines. E Re-analysis of EGR3 expression in TamR cells according to GSE96670 and GSE14986 datasets. EGR3 expression was increased in TamR cells. F, G QRT-PCR and WB analysis of EGR3 expression with E1, E2 and Tam treatment in MCF7P and MCF7R cells. F EGR3 mRNA level was increased in MCF7R comparing to MCF7P cells. EGR3 mRNA level was increased with 10 nM E1 and 2 µM Tam treatment for 6 h in MCF7P cells, and increased with 10 nM E1 with or without 2 µM Tam treatment for 6 h in MCF7R cells. G EGR3 protein level was increased in MCF7R comparing to MCF7P cells, EGR3 protein level was increased with 10 nM E1/E2 with or without 2 µM Tam treatment for 48 h in MCF7P cells, and EGR3 exhibited high expression protein level across all treatment of MCF7R cells. MCF7P, parental MCF7. MCF7R, tamoxifen resistant MCF7. Tam tamoxifen, E1 estrone, E2 estradiol. Data are shown as the means ± SDs. P values were calculated with one-way ANOVA or two-tailed unpaired Student’s t test; ns non-significance, *P < 0.05, **P < 0.01, ***P < 0.001

The focus shifted to a novel estrogen responsive gene, EGR3, a bona fide target of ERα, as a potential contributor to TamR. Analysis revealed that EGR3 contains an ERE, indicating its significance in ERα response (Fig. S2) (Inoue et al. 2004; Vareslija et al. 2016). Evaluation of EGR3 expression in various breast cancer cell lines with different ERα expression level, according CCLE database, showed significantly higher expression in ER positive cell lines (Fig. 1D) (Ghandi et al. 2019). Additionally, publicly available datasets (GSE96670 and GSE14986) were re-analyzed, revealing an increase in EGR3 expression in TamR cells (Fig. 1E).

To investigate the potential induction of EGR3 upregulation by estrogen signaling, EGR3 expression was assessed at mRNA and protein level after treating MCF7P and MCF7R cells with E1, E2, or Tam for 6 h and 48 h. A significant increase in EGR3 mRNA level, particularly in response to E1, after 6 h of treatment was observed in MCF7R cells (Fig. 1F). Additionally, Tam treatment resulted in elevated EGR3 mRNA level in both MCF7P and MCF7R cells (Fig. 1F). At the 6-h time point, no significant effect on EGR3 protein level was observed with the treatment (data not shown). However, after 48 h of treatment, a noteworthy increase in EGR3 expression was observed in MCF7P cells treated with estrogen (significantly in E1) and Tam, and in all groups of MCF7R cells (Fig. 1G). These findings collectively suggest that both E1 and Tam treatment contribute to the upregulation of EGR3 in TamR cells, implicating EGR3 elevation as a potential novel mechanism underlying TamR.

EGR3 expression related to tamoxifen resistance

The relationship between EGR3 expression and TamR was further validated in T47DR cells. Consistently, increased protein level of ERα was observed in T47DR (Fig. 2A). Additionally, a significant upregulation of EGR3 at mRNA and protein levels was detected in TamR cells (Fig. 2B), confirming the elevation of EGR3 in the context of TamR.

Fig. 2.

Fig. 2

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EGR3 expression related to tamoxifen resistance. A WB analysis of ERα expression. ERα protein level was increased in T47DR cells. B QRT-PCR and WB analysis of EGR3 expression. EGR3 mRNA and protein levels were increased in TamR cells. C, D QRT-PCR and WB analysis of EGR3 manipulating plasmids efficiency in MCF7R cells. E CCK8 analysis of cell viability with EGR3 expression manipulating in MCF7R cells. Cell viability was not affected by EGR3 expression manipulating in the absence of Tam treatment in MCF7R cells. F CCK8 analysis of cell viability with EGR3 expression manipulating under Tam treatment in MCF7R cells. Resistance to Tam was increased by EGR3 overexpression under 2 µM and 5 µM Tam treatment for 48 h, and was impaired by EGR3 knockdown under 1 µM, 2 µM and 5 µM Tam treatment for 48 h in MCF7R cells. G Colony formation assay of cells with EGR3 expression manipulating under Tam treatment. Colony formation ability was increased by EGR3 overexpression under 1 µM Tam treatment for 1 week in MCF7P cells, and was impaired by EGR3 knockdown under 1 µM Tam treatment for 1 week in MCF7R and T47DR cells. MCF7P parental MCF7, MCF7R tamoxifen resistant MCF7, T47DP parental T47D, T47DR tamoxifen resistant T47D, Tam tamoxifen. Data are shown as the means ± SDs. P values were calculated with one-way ANOVA or two-tailed unpaired Student’s t test; ns non-significance, *#P < 0.05, **P < 0.01, ***###P < 0.001

To elucidate the functional significance of EGR3, relative plasmids were developed to manipulate EGR3 expression, and its efficiency was confirmed at both the mRNA and protein levels in MCF7R cells (Fig. 2C, D). Cell viability was assessed over time to ensure that any observed effects were specific to Tam treatment and not due to alterations in cell growth (Fig. 2E). Notably, EGR3 had minimal impact on cell viability, both with overexpression and knockdown, in the absence of Tam.

Subsequently, the influence of EGR3 expression on the tolerance of TamR cells to different concentrations of Tam treatment was examined (Fig. 2F). Results showed that EGR3 overexpression increased the viability of MCF7R cells at Tam concentrations of 2 µM and 5 µM, and EGR3 knockdown impaired cell viability specifically at of 1 µM, 2 µM and 5 µM. These findings indicate that EGR3 expression correlates with cell viability under Tam treatment, suggesting its involvement in conferring resistance to Tam. To further validate these observations, a colony formation assay was performed under 1 µM Tam treatment (Fig. 2G). The results demonstrated that increased EGR3 expression enhanced the colony formation ability of MCF7P cells in the presence of Tam, while EGR3 knockdown decreased the colony formation ability in MCF7R and T47DR cells.

Taken together, these results provide compelling evidence for the involvement of EGR3 in TamR. The increased expression of EGR3 is associated with enhanced cell viability and colony formation ability under Tam treatment, suggesting its role in promoting resistance to Tam.

EGR3 regulated the expression of MCL1

To elucidate the underlying mechanism of EGR3 in TamR promotion, a comprehensive analysis was conducted using transcriptome sequencing data from 1211 breast cancer patients in the TCGA database. Through GSEA grouped by EGR3 expression level, a significant enrichment in the apoptosis pathway was identified (Fig. 3A). For further targeting investigation, qRT-PCR analysis was performed with EGR3 overexpression, both in MCF7R and MCF7P cells. The screening of survival related genes under EGR3 overexpression led to the focus on MCL1 (Fig. 3B). Subsequent data analysis from the GEO dataset (GSE150997) confirmed elevated MCL1 expression in the TamR cells (Fig. 3C). Furthermore, a positive correlation (R = 0.41) between EGR3 and MCL1 was established using the GEPIA database (Tang et al. 2017) (Fig. 3D). Notably, both high level of MCL1 and EGR3 expression were associated with poor survival outcomes in ER positive breast cancer patients (Fig. 3E).

Fig. 3.

Fig. 3

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EGR3 regulated the expression of MCL1. A GSEA of different express genes from 1211 breast cancer patients’ transcriptome grouped by EGR3 expression in the TCGA database. Apoptosis signaling pathway was enriched. B Heatmap of survival-related gene screen with EGR3 overexpression in MCF7P and MCF7R cells. MCL1 was increased with EGR3 overexpression and focused. C Re-analysis of MCL1 expression in TamR cells according to GSE150997 datasets. MCL1 was increased in TamR cells. D GEPIA analysis of co-expression trend between EGR3 and MCL1 in breast cancer. The co-expression trend was validated (R = 0.41). E Kaplan–Meier plotter analysis of EGR3 and MCL1 in ER positive breast cancer patients. Both MCL1 and EGR3 related to poor survival. F QRT-PCR analysis of MCL1 expression with EGR3 overexpression in MCF7P and MCF7R cells. MCL1 mRNA level was increased in MCF7R compared to MCF7P cells, and increased with EGR3 overexpression in MCF7R cells. G WB analysis of EGR3 and MCL1 expression in MCF7P, T47DP, MCF7R and T47DR cells. EGR3 and MCL1 protein level was increased in TamR cells. H Immunofluorescence analysis of EGR3 and MCL1 expression in MCF7P and MCF7R cells. EGR3 and MCL1 expression increased in MCF7R cells. EGR3 partially localized in the cell nucleus in MCF7R cells. MCF7P parental MCF7, MCF7R tamoxifen resistant MCF7, T47DP parental T47D, T47DR tamoxifen resistant T47D, Tam Tamoxifen. Data are shown as the means ± SDs. P values were calculated with one-way ANOVA or two-tailed unpaired Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001

To further validate the regulatory relationship, overexpression of EGR3 were conducted, resulting in increased MCL1 mRNA expression in MCF7R cells (Fig. 3F). Notably, this increase was consistent with higher MCL1 mRNA level in MCF7R compared to MCF7P cells (Fig. 3F). Subsequently, an investigation of EGR3 and MCL1 expression revealed elevated protein level of both in MCF7R and T47DR cells compared to MCF7P and T47DP cells (Fig. 3G). Immunofluorescence analysis confirmed a substantial upregulation of both EGR3 and MCL1 expression in MCF7R compared to MCF7P cells (Fig. 3H). Moreover, the observation of EGR3 partially localized in the nucleus supports a potential functional model of transcriptional promotion (Fig. 3H). Taken together, these findings strongly support the notion that EGR3 positively regulates the expression of MCL1, shedding light on its role in mediating TamR in breast cancer.

EGR3 promoted MCL1 transcription

Having observed increased MCL1 expression in TamR cells with high EGR3 expression, our focus was to comprehend the mechanism of the regulatory relationship between EGR3 and MCL1. To investigate this relationship, a system for manipulating MCL1 expression was established and its efficiency was validated at protein level in MCF7R cells (Fig. 4A). Through simultaneous manipulation of MCL1 and EGR3 expression in MCF7R cells, it was determined that EGR3 regulates MCL1 expression, whereas MCL1 does not exert any effect on EGR3 expression (Fig. 4B). These findings suggest a direct regulatory role of EGR3 in MCL1 expression.

As a member of the early growth response family and a functioning transcription factor, with the visualized nuclear location (Fig. 3H), EGR3's potential role in promoting MCL1 transcription was explored. Employing a transcription factor binding motif from the JASPAR2022 database, two EGR3 binding sites were identified on the MCL1 promoter region, situated upstream of the transcription start site at positions -171 to -156 and -165 to -150, respectively (Fig. 4C). ChIP-qRT-PCR analysis demonstrated EGR3 binding to the predicted sites on the MCL1 promoter in both MCF7R and T47DR cells (Fig. 4C). To further confirm the transcriptional regulatory function of EGR3 on MCL1 promoter, dual luciferase reporter system containing the MCL1 promoter were established. It was observed that EGR3 exhibited transcription factor activity at the MCL1 promoter (Fig. 4D). Collectively, these results provide compelling evidence supporting the direct transcriptional promoting function of EGR3 on MCL1 in TamR cells.

Estrone enhanced tamoxifen resistance through EGR3 by decreasing tamoxifen induced apoptosis

Tam is recognized for its inhibitory effect on the progression of ER positive breast cancer through apoptosis induction (Obrero et al. 2002). Hence, we sought to investigate the impact of Tam induced apoptosis in the context of elevated expression of E1 related enzymes in TamR cells and the transcriptional promotion of MCL1 by estrogen induced EGR3, previously established findings (Fig. S3)). Apoptosis analysis revealed that E1 treatment significantly reduced Tam induced apoptosis in MCF7R cells, with a similar trend observed in T47DR cells (Fig. 5A). As expected, the resistance to Tam induced apoptosis induced by E1 was rescued by the knockdown of EGR3 or MCL1, both in MCF7R and T47DR cells (Fig. 5A). To validate these findings further, detection protein expression of apoptosis related genes was conducted under parallel conditions in MCF7R cells. Consistently, EGR3 knockdown, under E1 and Tam treatment, increased the expression of apoptotic proteins, including cleaved caspase 3 and BAX, while decreasing the anti-apoptotic proteins BCL2 and MCL1 (Fig. 5B). These results further support the roles of E1, EGR3, and MCL1 in Tam induced apoptosis in TamR cells. The signaling pathway of TamR was elucidated in our study, whereby E1 and Tam treatment upregulated the ERα response gene EGR3, which, in turn, increased MCL1 expression by functioning as a transcriptional factor, ultimately leading to a decrease in apoptosis induced by Tam treatment (Figs. 5C, S3).

Fig. 5.

Fig. 5

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Estrone enhanced tamoxifen resistance through EGR3 by decreasing tamoxifen induced apoptosis. A Apoptosis analysis by flow cytometry with Tam and E1 treatment, and with EGR3 and MCL1 knockdown in TamR cells. 2 µM Tam treatment induced apoptosis was significantly decreased with 10 nM E1 treatment, which was rescued by knockdown of EGR3 or MCL1 in MCF7R and T47DR cells. B WB analysis of apoptosis related gene expression Tam and E1 treatment, and EGR3 knockdown in MCF7R cells. Apoptotic proteins level was increased, and anti-apoptotic proteins were decreased by EGR3 knockdown, with 2 µM Tam and 10 nM E1 treatment for 48 h in MCF7R cells. C The schematic of this study showed in TamR cells, the upregulation of EGR3 induced by E1 and Tam treatment promotes MCL1 expression by functioning as a transcriptional factor. This inhibition of Tam induced apoptosis facilitates TamR. D Microarray re-analysis of breast cancer patients with Tam treatment according to GSE147271 datasets. EGR3 expression is increased in tissue of breast cancer patient with Tam treatment. E Expression analysis of EGR3 in the Human Protein Atlas database. EGR3 expression is increased in breast cancer tissue. MCF7R tamoxifen resistant MCF7, T47DR tamoxifen resistant T47D, Tam tamoxifen, E1 estrone. Data are shown as the means ± SDs. P values were calculated with one-way ANOVA or two-tailed unpaired Student’s t test; *P < 0.05, **P < 0.01, ***P < 0.001

The relevance of EGR3 as a potential therapeutic target for combating TamR in breast cancer patients is reinforced by the clinical data. EGR3 gene expression was re-analyzed from tumor gene expression data (microarray from FFPE) obtained from breast cancer patients enrolled in a pre-operative window trial (Clinical Trials. gov Identifier: NCTO0738777). A significant increase in EGR3 expression with Tam treatment was observed, validating the potential role of EGR3 in acquiring resistance to Tam (Fig. 5D). Furthermore, a high expression of EGR3 was observed in breast cancer tissue compared to the normal breast tissue and the non-hormone related cancer, in the Human Protein Atlas database (Figs. 5E, S4). These findings further support the rationale for targeting EGR3 in endocrine treatment to overcome drug resistance.

Discussion

The activity of ERα plays a crucial role in driving the oncogenic progression of ER positive breast cancer (Turner et al. 2017). Altered estrogen signaling is a well-known contributing factor to TamR in endocrine therapy (Nabieva et al. 2021; Turner et al. 2017). Furthermore, the functional regulation of ERα is influenced by its relatively short half-life (Jeffreys et al. 2020). Previous studies have indicated that stabilized ERα protein leads to breast cancer progression and reduces the effectiveness of ERα blockers (Tang et al. 2021a, b; Tang et al. 2021a, b). EGR3, significantly upregulated in TamR cells as a response gene of ERα, played a crucial role in the survival of TamR cells. Notably, EGR3 exhibited no impact in non-Tam treatment, suggesting its specific role in cell survival closely associated with Tam treatment. Therefore, targeting EGR3 could be a precise strategy to restore relative sensitivity to ERα inhibitors in clinical settings.

As a nuclear receptor, ERα promotes downstream gene expression by directly binding to the ERE present in the target gene's genome, such as EGR3 in our study (Inoue et al. 2004; Vareslija et al. 2016). In addition to the previously reported role of ERα in improving EGR3 expression, we observed that different ERα ligands had varying effects on increasing EGR3 expression. ERα transcriptional coregulators have been acknowledged for their pivotal roles in modulating ERα transcriptional activity, contributing to the induction of TamR (Hanker et al. 2020). Nonetheless, our study reminded ERα coregulators NCOA3 and HOXB7 had no significant expression difference (Fig. S5), but FOXA1, NCOA2 and NCOA1-exhibited statistically significant expression difference in MCF7R cells when compared to MCF7P cells. Therefore, further investigation on ER coregulators, specifically focusing on recruitment mechanisms that exert regulatory control over EGR3 will be next consideration, to better understand the mechanism of ER coregulators interacting with ER and regulating ER activity.

Estrogen signaling is regulated by ligands that bind to the receptor, such as E1 and E2, including artificial ligands (Vincze et al. 2015). Elevated estrogen level not only promote breast cancer progression but also induce endocrine resistance (Liang et al. 2013). Previous research has reported contradictory role of two subtypes of estrogen, E1, and E2, in NF-κB signaling (Qureshi et al. 2020). In our study, we observed that E1 induced a higher expression of EGR3 compared to E2, suggesting that EGR3 may respond differently to different estrogen subtypes. Additionally, the expression level of EGR3 may contribute to various changes observed between pre- and post-menopausal states, such as inflammatory responses. Further investigation is needed to elucidate the precise mechanisms and roles of different estrogen subtypes and their effects on EGR3 expression.

Inflammation regulation plays a crucial role not only in immune activity but also in cancer cell progression. Previous studies have reported the involvement of inflammatory activity, including NF-κB signaling and progressive allergic inflammation, in the regulation of EGR3 (Kwon et al. 2021). Furthermore, EGR3 has been implicated in other types of cancer, such as prostate cancer, gastric cancer, and nasopharyngeal carcinoma, where it inhibits cancer cell EMT progression and migration (Li et al. 2021; Pio et al. 2013; Shin et al. 2020). Considering the promotion of inflammation by E1, these findings highlight the crucial role of EGR3 in inflammation related endocrine resistance. However, further confirmation through in vivo experiments is necessary to elucidate the precise relationship between inflammation regulation, endocrine resistance, and the role of EGR3 as a potentially stronger regulator in this process.

Tam is important apoptosis inducer for breast cancer. Previous research has demonstrated that EGR3 inhibits caspase 3 induced apoptosis and promotes the viability and angiogenesis of prostate cancer cells (Zhang et al. 2019). In our study, we provide evidence that EGR3 regulates caspase 3 induced apoptosis through its role as a transcription factor for MCL1. MCL1, a member of the BCL2 family, is known to be highly expressed in ER positive breast cancer (Curtis et al. 2012). Studies by Clara Alcon have shown that MCL1 promotes resistance to apoptosis in ER positive breast cancer cells (Alcon et al. 2021). Additionally, other genes that regulate MCL1, such as S6K1, have been demonstrated to promote TamR (Hong et al. 2013). MCL1 not only inhibits apoptosis but also plays a role in maintaining stemness (Campbell et al. 2021). Our discovery of a direct binding between EGR3 and MCL1 extends the understanding of the molecular mechanisms underlying EGR3 mediated TamR. The EGR3-MCL1 axis identified in our study may also be applicable to other cancer types, considering the functions of EGR3 in prostate cancer and gastric cancer, as reported by other researchers, including EMT, migration, invasion, and differentiation (Liao et al. 2013; Pio et al. 2013; Shin et al. 2020; Zhang et al. 2019). Therefore, our study highlights the significant role of EGR3 in the apoptosis process, particularly in cancer cells, and opens avenues for further research into targeted therapies aimed the EGR3.

Conclusion

In conclusion, our study provides compelling evidence supporting the involvement of EGR3 and E1 in promoting MCL1 transcription and inhibiting Tam induced apoptosis in TamR breast cancer cells. Through comprehensive elucidation of the molecular mechanisms underlying the interaction between EGR3 and MCL1 in TamR, we have gained valuable insights that can potentially contribute to the development of novel and effective therapeutic strategies for endocrine treatment on breast cancer.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 963 KB) (919KB, docx)
Supplementary file2 (TIF 8506 KB) (8.3MB, tif)
Supplementary file3 (XLS 498 KB) (498KB, xls)
Supplementary file4 (PDF 93 KB) (92.6KB, pdf)
Supplementary file5 (DOC 44 KB) (44KB, doc)

Author contributions

YX: investigation, data curation, conceptualization, original draft. XH: cell model establishment. JY: experiment assistance. MY & YY & JQ & LL: review and editing. YW: project administration, funding acquisition, review and editing.

Funding

This study was supported by the Natural Science Foundation of China (nos. 31770968 and 31800661), the Science Fund Project of Tianjin (nos. 21JCYBJC00240 and 20JCYBJC01130).

Data availability

Data will be made available on request.

Declarations

Conflict of interest

The authors declare that they have no conflicts of interest.

Ethical approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Footnotes

Publisher's Note

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (DOCX 963 KB) (919KB, docx)
Supplementary file2 (TIF 8506 KB) (8.3MB, tif)
Supplementary file3 (XLS 498 KB) (498KB, xls)
Supplementary file4 (PDF 93 KB) (92.6KB, pdf)
Supplementary file5 (DOC 44 KB) (44KB, doc)

Data Availability Statement

Data will be made available on request.

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