The Role of Estrogen Receptors in Proliferation of Non-Small Cell Lung Cancer

To explore the expression pattern of ERs in NSCLC tissues and assess their relationship with tumor histopathological variable. In our study, Ers expression was examined using Real-time PCR with specimens of 28 NSCLC patients. It was shown that both ERɑ and Erβ were over expression in NSCLC tissues, and also the Mrna concentration of both ERɑ and Erβ were significantly higher in primary tumor T2 stage than in T1 stage and higher in squamous carcinoma than in adenocarcinoma. However, the activation of ERɑ and Erβ were completely different. To further explore the role of Ers in development and progression of NSCLC, we used Ers selective siRNA or antagonist in vitro experiments. The results showed that Erβ but not ERɑ can mediate E2 induced cell growth, since siRNA targeting Erβ but not ERɑ gene can induce cell cycle arrest at G1 phase by down regulation of cyclinD1 expression, and also cell cycle regulators p21Waf1/Cip1 and p53 were involved in this signaling pathway. tumor histological subtype, pathological stage and histological stage of NSCLC. To elucidate the role of ERɑ and Erβ in NSCLC cell lines were chosen. Materials and Methods

Lung cancer, especially Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality for both men and women worldwide. Estrogen signaling pathways may play an important role in development of lung cancer. The cellular response to estrogen is mainly mediated by ERɑ and Erβ. There was increasing evidence to show that NSCLC express both ERɑ and Erβ in nucleus as well as extra-nuclear sites [19]. It was observed that Erβ but not ERɑ is the dominant form in normal lung cancer cell line and tissues, and loss of Erβ leading to abnormal lung structure and systemic hypoxia [24,25]. Moreover, when expression of either ERɑ or Erβ was suppressed by small interfering RNA (siRNA), the proliferation of NSCLC cells were significantly reduced [20], and also, blockage of Ers by Ers antagonist ICI 182780 lead to the inhibition of NSCLC cell line growth [26]. Therefore, the function of ERɑ and Erβ seems to prompt the cell proliferation since block of both of them lead to arrest of tumor growth. However, the role of ERɑ and Erβ are complicated by found that Era positive and Erβ negative lung cancer patients are associated with poor prognosis [21], and absence of Erβ expression is a marker showing high risk of lung cancer patients even an early clinical stage [21]. While some study found that Era was elevated in tumor but was not predictive of survival [27]. Although these studies reveal the importance of estrogen receptors in promoting the growth of lung cancers, but it is not clear which ER subtype is involved.
In our study, we explored the expression pattern of Ers in NSCLC tissues, and also the relationship between Ers expression with the electrophoresis through pre-cast 10% Tris-HCL polyacrylamide gel and transferred to PVDF membranes. Nonspecific binding site were block by incubation in 1X PBS. Primary antibodies (Santa cruz, USA) were incubated with the membranes and recognized with secondary antibodies (Santa cruz, USA). ECL-Plus Western Blotting Detection Reagents (Amersham Biosciences, Piscataway, NJ, USA) were used to visualize the complexes.

Cell viability analysis
Cells were seeded in phenol red-free RPMI 1640 medium supplemented with 10% 4X dextran-coated, charcoal-treated FBS (SFS) in 24-well plates or 96-well plates. After overnight attachment, the cell culture medium was removed. The adherent cell was washed twice with sterile PBS, and the cells were then treated with the indicated ligands for a total of 48 h. Selective siRNA or antagonist treatment were 1 h before E2. The effect of treatment on cell growth was determined by MTT assay. The MTT assay kits were purchased from ATCC, and assays were according to the manufacturer`s protocol. The percentage of cell viability was calculated using a standard curve and normalized to non-treated control.

Flow cytometric analysis
Cells were cultured overnight in RPMI-1640 medium and then treated with siRNA of ERɑ and ERβ for 48 h. The treated cells were fixed and stained with propidium iodide (Sigma USA). At least 1 × 10 6 stained cells were analyzed using FACSAAria (BD Biosciences USA).

Statistical analysis
All data shown as bar graphs are expressed as the mean ± SE. The statistical significance of differences was calculated by one-way ANOVA and two-tailed t test analysis. The correlation between gene expressions was evaluated by chi-square test. Two-tailed P<0.05 was considered to be statistically significant.

Both ERɑ and ERβ mRNA were over-expression in NSCLC tissues
We examined the expression of ERs in all lung tumor tissues and the corresponding tumor-free lung tissues from same patient. Both the ERɑ and ERβ mRNA transcript expression levels were over-expression in NSCLC tissues Table 3. However, GPR30 mRNA transcript showed RPMI medium 1640 (ATCC, USA) supplemented with 10% fetal calf serum (ATCC, USA) at 37℃ under 5% humidified CO 2 and 100 µg/ml each of streptomycin and penicillin G (GIBCO, USA). The anti-ERɑ, anti-ERβ and anti-GPR30 siRNA (Santa cruz, USA) were transfected by Lipofectamine™ RNAiMAX (Invitrogen, USA) according to the manufacturer`s instructions. Briefly, approximately 2 × 10 4 cells/ well were grown overnight in 24-wells plate. When the cells reached 80% confluence, they were transfected with anti-ERɑ siRNA (30 nM), anti-ERβ siRNA (30 nM) or anti-GPR30 siRNA (30 nM) and negative control siRNA (30 nM) separately in anti-body free medium using lipofectamine™ RNAiMAX. After incubation for 48 h at 37℃ 200 µl RPMI 1640 with 10% FBS was added and then subjected to real-time RT-PCR and western blot analysis.

RNA extraction and reverse transcription
Lung tumor tissue (N=28) and the corresponding tumor-free tissue (N=28) were homogenized and total RNA extraction from tissues was performed using RNeasy Mini Kit (Qiagen, Valencia, CA). RNA quality was checked with Spectrophotometer (NanoDrop 1000). Almost all samples had high-quality RNA. 1 µg RNA was reverse transcribed into cDNA with Omniscript RT kit (QIAGEN, Germany) according to the manufacturer`s protocol.

Quantitative real-time PCR
Primers (IDT, USA) for estrogen receptors and other genes were designed to locate into two exons, Sequences of the primers used in the Real-Time PCR analysis were shown in Table 2. The 18s gene was chosen as an endogenous house-keeping control gene. Quantitative real-time PCR was performed using Cyler iQ Real-time PCR Detection System (BIO RAD iQ5 Optical Module. Reaction mixtures contained 1 × SYBR Green PCR supermix (Bio-rad, USA), 0.1 µl Primer, 1 µl cDNA template in total volume of 25 µl. Thermal cycling conditions included activation at 95℃ (10 min) followed by 40 cycles each of denaturation at 95℃ (15 sec) and annealing at 60℃ (1 min).
Each reaction was performed in triplicate and negative controls were included in each experiment. To compare the expression levels among different samples, the relative mRNA levels were calculated using the comparative delta CT (∆Ct) method.

Western blot analysis
After transfection, cells were lysed in lysis buffer. Equivalent amounts of protein lysates from each sample were separated by

RNAs Primer sequences
Human estrogen receptor a mRNA Human estrogen receptor β mRNA

ERβ but not ERɑ mediates estrogen-dependent growth of NSCLC
To evaluate the role of ERɑ and ERβ in non-small cell lung cancer, we used receptor selective siRNA to down regulate the ERɑ, ERβ and GPR30 expression separately in H1650 cell lines, as well as A549 cell lines. and then Real-time RT-PCR and western blotting were performed to determine ERɑ, ERβ and GPR30 expression levels in transfected NSCLC cell lines (Figure 1). When H1650 cell line were transfected with anti-ERɑ siRNA, anti-ERβ siRNA or anti-GPR30 siRNA, the mRNA concentration ( Figure 1) and proteins level ( Figure 2) were down-regulated. The results obtained from A549 cells were same.
To determine whether the expression of ERɑ or ERβ affect the NSCLC cells proliferation, Cell viability was detected using MTT method. The cell growth in 10 nM E2 treatment group had no significant difference from that in control group. However, blocking of estrogen receptors before E2 treatment significantly decreased the proliferation of cells compared with that in E2-treatment group. Furthermore, the role of ERβ was detected by blocking both ERɑ and GPR30 expression by selective siRNA but left ERβ expression, and the result shown that the cell growth was increased compared with all ERs blockage group. However, no difference was found in ERβ and GPR30 blockage but ERα expression group compared with all ERs blockage group ( Figure  3A). It indicated that up-expression and down-expression of ERβ but not ERɑ can accelerate and inhibit the cellular proliferation.
To further confirm that ERβ but not ERɑ is involved in NSCLC cells proliferation, we performed cell proliferation assays with ERɑ-, ERβand GPR30 selective antagonist. Combined with G15 and MPP, but not PHTPP can stimulate the growth of H1650 cells, while combined with PHTPP and G15 shows no difference ( Figure 3B)

Inhibition of ERβ induced a G1-phase cell-cycle arrest
Flow cytometric analysis (Figures 4A-4D) showed a significant increase in the number of cells in G0/G1 phase after treatment of H1650 with anti-ERβ siRNA for 48 hr, as compared with that in control cells. It indicated that the induction of a cell cycle arrest at G1 phase was induced by ERβ siRNA in H1650 cells. Further, the role of ERɑ was further determined by transfection with ERɑ siRNA, and the result showed no significantly difference in G0/G1 cell population compared with control group.

Variables
ERɑ mRNA ERβ mRNA      To determine whether the anti-ERβ siRNA treated H1650 were arrested at the G0/G1 phase; we examined changes in the expression of panel cell-cycle-specific markers after 48 h. The mRNA concentration as well as the protein level of cyclin-D1 which functions in the transition from G1 to S phase of the cell cycle decreased (Figures 5A and 5B). The mRNA concentration and the protein level of cyclin-A2, which is involved in transition from S to G2, did not change significantly ( Figure  5C and 5D). Collectively, these data support the notion that inhibition of ERβ induces cell-cycle arrest at the G0/G1 phase in H1650 cells, which is consistent with flow cytometric analysis. The results obtained from A549 also shown the changes of cyclinD1 and cyclinA2 when the expression of ERβ was blocked ( Figure 5B).

P53 and P21 expression was involved in cell growth inhibition through ERβ-mediated signaling
Inhibition of ERβ expression by anti-ERβ siRNA markedly increased P53 expression at both mRNA level and protein level in H1650 cells as well as A549 cells. The overexpression was completely induced by anti-ERβ siRNA but not by a scramble siRNA control (Figures 6A-6D). Furthermore, the cyclin-dependent kinase cyclin inhibitors gene, P21 mRNA concentration were also upregulated by inhibition of ERβ siRNA. These finding establish that inhibition of ERβ induced upregulation of P53 and P21 as a direct mediator of the growth-inhibitory response.

Discussion
It is well established that ERs play a major role in pathological processes of lung cancer, while the results remain inconsistent. Early studies detected for the presence of the classical ERɑ only, and then with the development of ERβ antibody, the expression of ERβ was also found in lung cancer [28][29][30]. Many studied have shown the expression of ERβ in majority of human NSCLC cell lines as well as primary  tissues, however, the role of ERɑ in lung cancer is less clear, and also the relationship between ERs expression and tumor pathological stage, histology subtype and histologic grade are not clear. Our findings used NSCLC tissues and its corresponding tumor-free lung tissues to explore ERs expression in NSCLC, and the result is consistent with those in cell findings, which shown that both ERɑ and ERβ mRNA concentration of NSCLC tissues are higher than the corresponding normal lung tissues.
The other main question addressed in this study was whether expression of ERɑ or ERβ have relationships with tumor pathological stage, histology subtype or histologic grade. There is evidence suggests that in breast cancer, ERs is an important prognostic factor, which is the same case in lung cancer [31,32]. Our results showed that both ERɑ and ERβ were overexpressed in NSCLC tissues, and their mRNA concentration was related to the tumor pathological stage and histology subtype. Higher concentration of ERs mRNA has been found in T2 stage, and also that ERɑ and ERβ mRNA expression were higher in squamous carcinoma than in adenocarcinoma. In addition, evidence suggests that mRNA concentration of ERɑ was correlate with ERβ, since we found that the number of patients who have either low level of ERɑ but high level of ER β or high level of ERɑ but low level of ERβ were relatively small in present study.
In order to explain the activation of ERɑ and ERβ in the development of NSCLC separately, we selected a NSCLC cell line] which deprived from lung adenocarcinoma patients. Selected ERs specific siRNAs were successfully transfected into H1650 cell lines separately. ERβ but not ERa affects NSCLC cells proliferation. MTT method was used in this study for cell viability. The results showed that inhibitory ERs by selective siRNA or antagonist can block the cell proliferation induced by 10 nM E2, however only the presence of ERβ but not ERɑ can accelerate cell growth. This is indicated that ERβ but not ERɑ was involved in cell proliferation. These results were consistent with other reports. A549 cells transfected with ERβ have increased proliferation in response to estrogen [33].
The progression of cells proliferation is associated with cell cycle, which is known to be positively regulated by a series of cyclin-dependent kinases (CDKs), as well as cyclins, and is negatively controlled by specific CDK inhibitors (CDKIs) [34][35][36]. CDKIs are separate into two classes, including this, the CIP/KIP family ((p21Waf1/Cip1, p27Kip1, and p57Kip2) which inhibits all CDKs by directly binding CDK complexes [37]. Wild-type P53 regulates cell transition from G1 to S phase in cell cycle and induce G1 arrest in response to DNA damage [38]. The p21Waf1/Cip1 gene, which is transcriptional target of wildtype P53 [39,40], is activated by wild-type P53 and is downstream effectors for P53 function by inducing G1 arrest if cells are exposed to DNA damaging agents [41]. We found that inhibitory of ERβ but not ERɑ by selective siRNA can leading to upregulation of P53 and P21, which in turn downregulated cyclinD1, which is G1-checkpoint regulator [42], and led to cell-cycle arrest at the G1 phase in H1650 cells. These findings provide credence for P21-mediation of ERβ -induced-cell cycle arrest at the G1 phase in H1650 cells.
One major limitation of the present study was small tissue samples. Therefore, it cannot be test the level of ERs protein in both lung cancer tissues as well as tumor free tissues. A further limitation was that a more detailed demonstration of the relationship between the level of GPR30 and lung cancer proliferation was not carried out. This was not completed because of the relatively low expression of GPR30 in lung cancer tissue. A further study with more detail on the correlation between GPR30 and lung cancer should be performed.

Conclusion
In conclusion, we showed that both ERɑ and ERβ play important role in development of NSCLC. ERβ were involved in proliferation of NSCLC induced by estrogen separately. The discovery of the expression pattern of ERs may open up an opportunity for development of estrogen-based therapy.