Estrogen Receptor β1 Expression Patterns Have Different Effects on Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors’ Treatment Response in Epidermal Growth Factor Receptor Mutant Lung Adenocarcinoma

Estrogen receptor β (ERβ) can regulate cellular signaling through non-genomic mechanisms, potentially promoting resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). However, the mechanisms underlying the ERβ-mediated resistance to EGFR TKIs remain poorly understood. In this study, we investigated the role of the interaction between ERβ1 and ERβ5 in non-genomic signaling in lung adenocarcinoma. We established PC9 cell lines stably overexpressing ERβ1 or ERβ1/ERβ5. Immunofluorescence revealed that ERβ5 overexpression partly retained ERβ1 in the cytoplasm. Immunoblotting analyses revealed that EGFR pathway activation levels were higher in PC9/ERβ1/5 cells than those in PC9/ERβ1 or control PC9 cells. In the presence of estradiol, PI3K/AKT/mTOR pathway activation levels were higher in ERβ1/5-expressing cells than those in ERβ1-expressing cells. Additionally, PC9/ERβ1/5 cells were less prone to the cytotoxic and pro-apoptotic effects of gefitinib compared with PC9/ERβ1 or control PC9 cells. Cytoplasmic ERβ1 was associated with poor progression-free survival in lung cancer patients treated with EGFR TKIs. These results suggest that cytoplasmic ERβ1 was responsible for EGFR TKI resistance slightly through non-genomic mechanism in EGFR mutant lung adenocarcinoma.


INTRODUCTION
Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have revolutionized non-small cell lung cancer (NSCLC) personalized treatment and have improved the survival and quality of life of EGFR-mutant NSCLC patients (1)(2)(3). However, the development of primary and acquired resistance to TKIs remains a significant clinical challenge. Several mechanisms underlying acquired resistance to EGFR TKIs have been identified, including the acquisition of EGFR T790M mutation, c-MET amplification, PIK3CA mutations, and phenotypic transformation into small cell lung cancer (4,5). Nevertheless, the mechanisms involved in primary resistance are poorly understood (5)(6)(7)(8).
Estrogen receptor b (ERb) is the primary ER subtype expressed in lung cancer; upon binding to estrogen in the cytoplasm, ERb activates non-genomic signaling pathways, including PI3K/AKT/mTOR and RAS/RAF/MEK/MAPK pathways, promoting cancer cell proliferation and apoptosis evasion (9,10). Importantly, significant overlap exists between ERb-and EGFR-regulated signaling pathways (11). Preclinical studies have shown that EGFR expression was downregulated in response to estradiol (E2); in contrast, ERb antagonists upregulated EGFR expression, highlighting the crosstalk between ERb and EGFR signaling (12). Hence, it is believed that non-genomic signaling events may modulate EGFR TKI resistance. ER belongs to the nuclear receptor superfamily of ligand-activated transcription factors. Since a nuclear localization of ERb in cancer cells has been reported (13), the relevance of cytoplasmic ERb in non-genomic signaling activation in cancer cells has attracted increasing attention in recent years.
Studies on endocrine-related cancers suggested that certain ERb isoforms are associated with ERb protein localization and patient prognosis (14)(15)(16)(17). For example, ERb1 (also known as wild-type ERb) was primarily found in the nucleus of prostate cancer cells, whereas ERb5 localized both in the cytoplasm and nucleus (16,18,19). Although ERb5 lacks the ability to bind estrogen or form homodimers due to the absence of helix 12 in its C-terminal, it can heterodimerize with ERb1 in the presence of estrogen (20).
The aim of this study was to assess the role of the interaction between ERb1 and ERb5 in non-genomic signaling in lung adenocarcinoma. To this end, we overexpressed ERb1 and E R b 5 in EGFR exon 19 deletion-harboring lung adenocarcinoma cells and assessed their ability to form heterodimers, as well as the relevance of ERb1/ERb5 heterodimerization in non-genomic signaling and response to EGFR TKIs.

Cell Culture and Chemicals
The EGFR-mutant lung adenocarcinoma cells PC9, HCC827, H1975, and H1650 were kindly provided by Peking University Cancer Hospital. Cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum and maintained at 37°C in a humidified 5% CO 2 atmosphere.
Gefitinib was purchased from Selleck Chemicals (Selleck, USA) and diluted in dimethyl sulfoxide (DMSO) at a concentration of 10 mmol/L. Estradiol (E2) was purchased from Sigma-Aldrich (Sigma-Aldrich, Germany) and diluted in pure ethanol at a concentration of 10 mmol/L. Both drugs were aliquoted and stored at −80°C.

RNA Extraction and Quantitative Real-Time PCR
Total RNA was extracted using the RNAsimple Total RNA kit (Tiangen, China), and first-strand cDNA synthesis was performed using the PrimeScript ™ RT Master Mix (Takara, Japan). The relative mRNA levels of ERb1 and ERb5 were measured using SYBR green PCR assays (Thermo Fisher Scientific, USA). The sequences of the primers used in qRT-P C R w e r e a s f o l l o w s : E R b 1 forward primer: 5′ -GTCAGGCATGCGAGTAACAA-3′ , reverse primer: GGGAGCCCTCTTTGCTTTTA; ERb5 forward primer: 5′-TGGTCACAGCGACCCAGGATG-3′, reverse primer: 5′-TTAGGGCGCGTACCTCGCATG-3′; GAPDH forward primer: 5′-GACCCCTTCATTGACCTCAAC-3′, reverse primer: 5′-CTTCTCCATGGTGGTGAAGA-3′. Cycle threshold (Ct) values were determined using the system and analysis software. The relative mRNA levels were determined by normalizing to the GAPDH mRNA levels.

Establishment of Cell Lines Stably Expressing Estrogen Receptor b1
and Estrogen Receptor b1/5 Lentiviral vectors expressing ERb1 and ERb5 were purchased from GenePharma (Shanghai, China). PC9 cells were infected with lentiviruses (MOI = 50) for three days. Subsequently, transduced cells were selected with 2 mg/ml of puromycin for one week. ERb1-overexpressing single-cell clones were established (hereafter referred to as PC9/ERb1 cells), and stable ERb1 overexpression was confirmed by Western blot and qRT-PCR. PC9/ERb1 cells were then infected with viruses carrying ERb5 open reading frame (ORF), followed by selection with neomycin (600 mg/ml) for one week.

Cell Viability and Colony Formation Assays
Cells were treated with estradiol (20 nM) during the experiment. Cell viability was assessed using a cell counting kit-8 (CCK8; Dojindo, Japan). Briefly, cells were seeded (3 × 10 3 cells/well) in sextuplicate in 96-well plates containing 100 ml medium and incubated for 24 h. Subsequently, cells were treated with increasing concentrations of the indicated drugs for an additional 72 h. After treatment, 10 ml of water-soluble tetrazolium salt (WST-8) was added to each well and incubated for 2 h. Optical absorbance at 450 nm was measured using a microplate reader. Relative viability was calculated using the following formula: Relative viability (%/control) = [A450 (treated) − A450 (blank)]/[A450 (control) − A450 (blank)].
For colony formation assays, cells were seeded into 6 cm cell culture dishes (500 cells/dish) and treated for two weeks with 40 nM gefitinib or DMSO (1/1,000 dilution). After washing twice with phosphate-buffered saline (PBS), cells were stained with crystal violet (Beyotime, China) for 20 min and washed with PBS.

Patients
The data from 103 Chinese patients with advanced lung adenocarcinoma were retrospectively reviewed. The inclusion criteria used for patient enrollment were as follows: (1) Pathological diagnosis of adenocarcinoma; (2) sufficient tissue for both EGFR and KRAS mutation detection and ERb1 immunohistochemistry; (3) presence of EGFR mutations associated with sensitivity to EGFR TKIs, including 19 exon deletion and 21 exon point mutation, and absence of EGFR T790M or KRAS mutations; (4) patients treated with EGFR TKIs, including erlotinib, gefitinib, and icotinib; (5) available clinicopathological characteristics, including sex, age, disease stage, and smoking history. Treatment responses were classified according to the response evaluation criteria in solid tumors (RECIST), version 1.1. Progression-free survival (PFS) time was defined as the time between the first day of EGFR TKI treatment until radiologic progression or death. The study was approved by the Ethics Review Committee of the Shandong Cancer Hospital.

Epidermal Growth Factor Receptor and KRAS Mutation Detection and Immunohistochemistry for Estrogen Receptor b1
Amplification refractory mutation system (ARMS) was employed to detect different genetic variants, including EGFR (exon 19 deletions, L858R, and T790M) and KRAS mutations.
ERb1 expression in lung adenocarcinoma tissue samples was assessed by immunohistochemistry (IHC). Informed consent to use biopsy tissues was obtained from all patients. Briefly, formalin-fixed, paraffin-embedded tissue sections (3 mm) were deparaffinized and stained according to standard procedures. Sections were probed with anti-ERb1 mouse antibody (1:200; Abcam, USA); a biotinylated anti-mouse IgG secondary antibody was used. Brown staining in over 10% cancer cells with cytoplasm or/and nucleus was considered positive. No staining was observed in negative controls, including lung tissues probed with a non-immune primary antibody. Based on the localization of "positive" immunoreactivity in the cytoplasm, nucleus, or both, patients were grouped as cERb1-, n/cERb1-, or nERb1positive. IHC staining was evaluated independently by two investigators (LZ and MT) and a pathologist (JZ).

Statistical Analysis
Differences in the relative mRNA levels, cell viability, and apoptosis between different cell lines were analyzed using twotailed Student's t-tests. Difference of ERb1 expression between male and female was analyzed using chi-square test. Patients' survival was estimated using the Kaplan-Meier method, and comparisons between groups were conducted using log-rank tests. All statistical tests were two-tailed, and P-values <0.05 were considered statistically significant. All statistical analyses were performed using GraphPad Prism 8.0 (Prism Software Inc., San Diego, USA).

Estrogen Receptor b5 Affects Estrogen Receptor b1 Localization in Epidermal Growth Factor Receptor-Mutant Lung Adenocarcinoma Cancer Cells
ERb5 has been identified as the predominant ERb splice variant in non-malignant lung tissues. In this study, we found that ERb5 mRNA levels were elevated in four lung adenocarcinoma cell lines harboring EGFR mutations ( Figure 1A). We further assessed the role of ERb5 in lung adenocarcinoma using PC9 cells, which harbor EGFR exon 19 deletions. Immunofluorescence analyses revealed that endogenous ERb predominantly localized in the cell cytoplasm, and only low ERb levels were detected in the nucleus ( Figure 1B).
Next, we overexpressed ERb1 in PC9 cells (PC9/ERb1); we also overexpressed ERb5 in PC9/ERb1 cells (hereafter referred to as PC9/ERb1/5). ERb1 and ERb5 overexpression was confirmed at the mRNA and protein levels by qRT-PCR and immunoblotting, respectively ( Figure 1C; Table 1). Immunofluorescence using a non-variant specific antibody revealed that ERb levels were elevated both in PC9/ERb1 and PC9/ERb1/5 cells; however, ERb localization differed between the two cell lines. Although ERb primarily localized in the cell nucleus in PC9/ERb1 cells, in PC9/ERb1/5 cells, it was found both in the cytoplasm and nucleus (Figures 2A, B).
ERb1 has the highest affinity for estradiol among all ERb splice variants. Hence, we used an ERb1-specific antibody to determine ERb1 localization. ERb1 predominantly localized in the cell nucleus in PC9/ERb1 cells. However, in PC9/ERb1/5 cells, we observed that ERb1 was partly detained in the cytoplasm, suggesting that the expression of ERb5 suppressed ERb1 translocation from the cytoplasm to the nucleus ( Figures 2C, D).

The Interaction Between Estrogen Receptor b1 and Estrogen Receptor b5 Regulates Downstream Signaling Events in the Presence of Estradiol
Next, we assessed the role of nuclear and cytoplasmic ERb in transcriptional regulation and non-genomic signaling, respectively. The expression of the cell cycle regulator P21 is induced by the nuclear ERb (18). In this study, we found that P21 expression levels were profoundly higher in PC9/ERb1 cells compared to those in PC9/NC or PC9/ERb1/5 cells, especially after stimulation with estradiol ( Figure 3A).
PI3K/AKT/mTOR signaling pathway is regulated by both EGFR and ERb (11). To determine the PI3K/AKT/mTOR pathway activation status, we assessed both total and phosphorylated levels of EGFR, AKT, and RPS6. We found that phospho-EGFR levels were lower in PC9/ERb1/5 cells than those in PC9/NC or PC9/ERb1 cells. Although total and phospho-EGFR levels decreased in all groups after estradiol treatment, the decrease in phospho-EGFR levels was stronger in PC9/ERb1 and PC9/ERb1/5 cells than that in PC9/NC cells. The phospho-AKT levels were higher in PC9/ERb1/5 cells than those in PC9/NC or PC9/ERb1 cells, both at baseline and after estradiol treatment. The levels of phospho-RPS6, which functions downstream of mTOR, were similar among the groups (Figures 3A, B).
We also found that ERb1 but not ERb5 was upregulated in PC9/NC cells after estradiol treatment. Interestingly, qRT-PCR showed no changes in the ERb1 mRNA levels after estradiol treatment, suggesting that the estradiol-mediated ERb1 upregulation occurs at the post-transcriptional level ( Figure 3C).
When cells were treated with gefitinib in addition to estradiol, phospho-EGFR levels were decreased in all groups, whereas phosphor-AKT levels were increased, especially in PC9/ERb1 and PC9/ERb1/5 cells. Similar to phospho-EGFR, phospho-RPS6 levels were decreased in all groups after gefitinib treatment. P21 was also downregulated in gefitinib-treated cells ( Figures 3D, E).

PC9/Estrogen Receptor b1/5 Cells Are Less Prone to the Cytotoxic Effects of Gefitinib
To determine the effects of different ERb splice variants in response to gefitinib, we performed cell viability and colony formation assays. We found that PC9/ERb1/5 cells were less prone to the cytotoxic effects of gefitinib (40 nM) compared with PC9/NC or PC9/ERb1 cells, although we found no significant differences in cell viability at low concentrations of gefitinib  ( Figure 4A). Additionally, the ability of gefitinib (40 nM) to inhibit colony formation was stronger in PC9/ERb1 and PC9/ NC than in PC9/ERb1/5 cells ( Figure 4B). We found that cPARP levels were increased in all three groups after gefitinib treatment. However, the increase in cPARP levels was more substantial in PC9/NC and PC9/ERb1 cells than in PC9/ERb1/5 cells. Consistently, the decrease in cyclin D3 levels was more profound in PC9/NC and PC9/ERb1 cells, while almost no change in cyclin D3 levels was observed in PC9/ERb1/5 cells after gefitinib treatment (Figures 4C, D). These results suggest that PC9/ERb1/5 cells are less sensitive to EGFR TKIs than PC9/NC and PC9/ERb1 cells.

Estrogen Receptor b1 Expression and Intracellular Distribution Affect Progression-Free Survival in Patients With Advanced Epidermal Growth Factor Receptor-Mutant Lung Adenocarcinoma
In this study, we retrospectively analyzed the data from 103 stage IIIb-IV lung adenocarcinoma patients treated with EGFR TKIs at the Shandong Cancer Hospital between January 2014 and November 2017. All patients harbored EGFR mutations affecting response to EGFR TKIs, including exon 19 deletions (47; 45.6%) and exon 21 point mutations (55; 53.4%); one    Table 2.
At the time of data collection (Nov 20, 2017), four patients were lost to follow-up, and 79 patients (76.7%) presented with progressive disease. The median survival of the 99 patients was 13.3 months ( Figure 5B). There was no significant difference in PFS between patients with ERb1-positive and ERb1-negative tumors (13.3 months vs. 14 months, P = 0.944) ( Figure 5C). Interestingly, we also found significant differences in the median PFS of patients with different intracellular ERb1distribution pattern (nERb1, 17.5 months; n/cERb1, 11.4 months; cERb1, 6.5 months; negative, 14 months, P = 0.008) ( Figure 5D). Because cytoplasmic ERb1 is key for non-genomic signaling activation, we combined patients with n/cERb1 expression and those with cERb1 (cytoERb1). Survival analysis showed that patients with cytoERb1 expression (n = 43) had a shorter median PFS after EGFR TKI treatment (9.5 months) compared to those with nERb1 expression (n =37; PFS, 17.5months; P = 0.0006) ( Figure 5E).

DISCUSSION
Approximately 20-30% of patients with EGFR activating mutations exhibit primary resistance to EGFR TKIs. The mechanism underlying resistance to EGFR TKIs, and primary resistance, in particular, are extremely complex and remain poorly understood. ERb expression has been associated with response to EGFR TKIs. Notably, in a Japanese cohort study, strong ERb expression predicted favorable clinical outcomes in patients with lung adenocarcinoma after treatment with EGFR TKIs. In contrast, we previously identified high cytoplasmic ERb expression as a predictor of poor PFS (21,22). Therefore, further elucidation of the expression pattern and intracellular distribution of ERb is required to determine the effects of non-genomic signaling on EGFR signal transduction and clinical outcomes.
Several ERb splicing variants have been identified, the most important of which are ERb1 (wild-type ERb), and ERb2-5 (20,23). ERb1 is the only fully functional receptor in the ERb family, and has the highest affinity for estradiol; other ERb family members have weak to no ligand binding capacity, despite maintaining their ability to heterodimerize with  ERb1 (20). Therefore, assessing the function of ERb splice variants other than ERb1 is equally important. Notably, the crucial role of ERb5 in lung cancer is becoming increasingly evident (17,24). In this study, we focused on the role of ERb1 and ERb5 in lung adenocarcinoma. Previous studies demonstrated that ERb1 was predominantly localized in the cell nucleus and exerted antiproliferative effects. In contrast, ERb5 was found both in the cytoplasm and nucleus, and it has been implicated in cancer cell migration and invasion (17,22,25). Our results confirmed the elevated ERb5 levels in EGFR-mutant lung cancer cells; in contrast, ERb1 was lowly expressed. These results were consistent with those of a previous study showing that ERb5 was the primary ERb isoform expressed in non-malignant lung cells, and heterodimerized with ERb1 (20). Similarly, we previously showed that ERb5 formed complexes with ERb1, confirming their ability to interact (22).
In this study, we also found that ERb1 was predominantly localized in the cell nucleus. However, the forced overexpression of ERb5 partly retained ERb1 in the cytoplasm. Hence, the presence of ERb5 can explain previous findings of ERb1 localization in the nucleus and cytoplasm in cancer cells.
Total and phospho-EGFR levels were decreased after estradiol treatment, highlighting the crosstalk between EGFR and ERb signaling pathways (12). P21 is an essential cell cycle regulator, playing important tumor-suppressing roles (26). Importantly, P21 expression was induced by ERb (18,25). In this study, we confirmed that ERb1 increased P21 levels, suggesting a role of ERb1 in transcriptional regulation in lung cancer cells. Consistently, ERb1 exerted anti-proliferative effects in other cancer cells (15,18). However, when ERb1 and ERb5 were coexpressed, P21 levels were lower compared with those in PC9/ ERb1 cells, suggesting that ERb5 impairs the transcriptional abilities of ERb1. However, in the presence of estradiol, PI3K/ AKT/mTOR signaling pathway activation levels were higher in ERb1/5-expressing lung cancer cells than those in ERb1expressing cells, suggesting that the interaction between ERb1 and ERb5 potentiated the effects of ERb1 in non-genomic signaling. Hence, we believe that ERb1 translocation from the nucleus to cytoplasm in the presence of ERb5 was essential in determining its biological function, reflecting the bi-faceted role of ERb in cancer (27). mTOR signaling was inhibited by gefitinib treatment in all groups, although phospho-AKT levels were increased. Consistently, gefitinib treatment exerted cytotoxic effects in all cell groups. However, ERb1/ERb5 co-expression rendered cells less prone to the cytotoxic and pro-apoptotic effects of gefitinib. These results confirmed the critical role of ERb1/ERb5 complexes in estrogen receptor-mediated non-genomic signaling. We also found that estradiol upregulated ERb1 but not ERb5 at the posttranscriptional level, confirming the high affinity of ERb1 for estradiol.
In this study, we also investigated the effect of the ERb1 expression pattern on PFS in EGFR-mutant lung adenocarcinoma patients. We found that patients with nuclear ERb1 expression exhibited a relatively longer PFS after EGFR TKI treatment, whereas cytoplasmic ERb1 was associated with shorter PFS after EGFR TKI treatment. These results highlight the clinical relevance of our findings from in vitro experiments in EGFR-mutant lung cancer cell lines. Importantly, nuclear ERb1 expression in lung cancer tissues was associated with tumor-suppressing effects, whereas cytoplasmic ERb1 promoted EGFR TKI resistance to some extent. Although cytoERb1 was associated with a lower response to EGFR TKIs, the median PFS of patients with cytoERb1 was 9.5 months, suggesting that EGFR mutations remain the most powerful predictor for EGFR TKI treatment response. The findings reported here need to be confirmed in large cohort prospective studies. Additionally, the relationship between ERb1/ERb5 ratio and response to EGFR TKIs merits further investigation. Our study has potential clinical relevance for guiding anti-estrogen agents' treatment in future. Recently two clinical trials evaluating efficacy and safety of EGFR TKI plus anti-estrogen treatment showed no survival improvement in combination group compared to that of EGFR TKI alone (28,29). However, the status of ERb expression and its predictive value for anti-estrogen therapy were not evaluated in both trials.
In conclusion, we showed that ERb1 localized in the cell cytoplasm by interacting with ERb5, inducing non-genomic signaling activation, and promoting EGFR TKI treatment resistance in EGFR-mutant lung adenocarcinoma. Hence, these results suggest that cytoplasmic ERb1 was responsible for EGFR TKI resistance slightly through non-genomic mechanism in EGFR mutant lung adenocarcinoma.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Ethics Committee of the Shandong Cancer Hospital and Institute. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
LZ and MT did the experimental work and participated in the analysis of the results. ZL supervised the research and secured