Androgen Receptor(AR) and Estrogen Receptor α (ERα) Affect Cell Growth and Metastasis of Non-Small Cell Lung Cancer Through EGFR/PI3K/AKT Axis.

Background/Aims: Androgen receptor (AR), estrogen receptor α (ERα) signaling and their interaction with epidermal growth factor receptor (EGFR) signaling pathway is a potential therapeutic target in non-small cell lung cancer (NSCLC). To explore the cross communication between AR, ERα and EGFR signaling pathway, we used RNA silencing technology and sex hormones intervention in NSCLC cell line. Methods: Model system was the well-characterized A549 adenocarcinoma NSCLC cells which can express AR and ERs well. We used different concentrations of testosterone (T), estradiol (E 2 ) intervention cells and small interfering RNA (siRNA) specically targeting AR, ERα in A549 cells, then examined the expression of AR, ERα and EGFR/PI3K/AKT axis, followed by detection of cells proliferation, apoptosis, migration and invasion. Results: After knocked down the expression of AR, ERα, the EGFR/PI3K/AKT axis was inhibited, and the proliferation decreased, apoptosis increased, migration and invasion decreased in A549 cells. 50µM T and 50µM E 2 intervention inhibited AR, ERα and EGFR/PI3K/AKT axis expression, and the proliferation decreased, apoptosis increased, migration and invasion decreased in A549 cells. 10µM T, 10µM E 2 and 100µM T, 100µM E 2 intervention promoted the expression of AR, ERα and EGFR/PI3K/AKT axis, which enhanced the activity and migration invasiveness of A549 cells. Conclusions: These data provide a rationale for further investigation of the antitumor activity of clinical sex hormones therapy and the development of anticancer drugs targeting sex hormone receptors in the clinic. Future .

Background Non-small cell lung cancer (NSCLC) is the most common cause of cancer mortality throughout the world (Sequist and Lynch, 2008). It remains of the most lethal types of cancer; a diagnosis of NSCLC portends a 5-year survival rate of only 18% (Siegel et al. , 2017). Thus, improvements in treatment strategies for NSCLC are of great interest. The "2017 China Cancer Registry Annual Report" shows that there are about 800,000 new cases of lung cancer in China each year, and there are about 700,000 deaths every year. Cytotoxic chemotherapy is traditionally the basis for the treatment of advanced NSCLC, but even the most radical regimens provides only about 20% -35% of the tumor response opportunities, and the average survival time has increased by 2-4 months (JH et al. , 2002, Sandler et al. ). The high mortality rate of lung cancer patients is mainly due to delays in detection and diagnosis, resulting in the identi cation of disease at an advanced stage and limited treatment options provided to patients (Youlden et al. , 2008). For most patients, chemotherapy for the early stages of the disease is sensitive, but then it develops drug resistance and leads to relapse (Mostertz et al. , 2010). Since the introduction of EGFR tyrosine kinase inhibitors (EGFR-TKIs) 17 years ago in lung cancer clinical trials, they have had a huge impact on the treatment of NSCLC, but most patients will develop resistance within one year of taking the drug(Jackman et al. , 2010). Needless to say, there is no successful anti-cancer drug can effectively prevent and reduce the progression of lung cancer. Therefore, there is an urgent need to develop new molecular mechanisms aimed at better understanding the role of potential anti-cancer chemicals and to further provide more effective basic therapeutic strategies.
Recent medical research shows that sex hormones are involved in the development of lung tissue, the formation of pulmonary in ammation and perhaps the molecular biology of lung cancer (Så Owikowski et al. , 2017, Siegfried, 2014, Zhang et al. , 2009). It has been found that Androgen receptor(AR),estrogen receptor α (ERα) and estrogen receptor β (ERβ) are expressed in normal lung tissue and in lung tumors in human and mouse (Dou et al. , 2017, Raso et al. , 2009. A Chinese study showed that the expression of AR may be related to the disease progression and lymph node metastasis of lung cancer (Yan et al. , 2008). A study from the United States and Norway reported that the prognosis of NSCLC patients was correlated with the level of ERα(Olivo-Marston et al. , 2010). On the contrary, a Canada study showed that elevated ERβ levels were associated with a higher survival rate in NSCLC patients (Navaratnam et al. , 2010). Therefore, reproductive hormones and their receptors may be potential biomarkers for predicting the occurrence, development, treatment and prognosis of NSCLC (Dou, Zhu, 2017).
Mutations in the EGFR gene are widely recognized alterations involved in the pathobiology of lung cancer and have a clinical impact on NSCLC therapy. As an important pathway of EGFR mediated signaling pathway, PI3K/AKT axis has recently been con rmed to be involved in the oncogene gene alteration of NSCLC (Fumarola et al. , 2014). Regarding the function of ERs and EGFR, the stimulation of ERs has been reported to increase the activity of the EGFR signal, and the signal of EGFR increases the activity of ERs.
And there is no correlation between AR and EGFR has been reported so far.
In the present work, our aim was to evaluate whether reproductive hormones and their receptors may inhibit growth and metastasis of NSCLC cells. Model system was the well-characterized A549 adenocarcinoma NSCLC cells which can express AR and ERs well (Garon et al. , 2013, Kaiser et al. , 1996, Weinberg et al. , 2005. We used different concentrations of testosterone (T), estradiol (E 2 ) intervention cells and small interfering RNA (siRNA) speci cally targeting AR, ERα in A549 cells to observe the effect on EGFR/PI3K/AKT axis and cell growth, metastasis of A549 cells. In addition, the mechanism of sex hormones and their receptors on the occurrence and development of lung cancer was also discussed.

Materials And Methods
Cell lines and reagents A549 cell lines were obtained from the Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd (Shanghai, China) and cultured in DMEM/HIGH GLUCOSE medium (Hyclone, Logan, Utah, USA) supplemented with 10% fetal bovine serum (FBS, Ausbian, Australia) and 1% Penicillin-Streptomycin (Sigma, St. Louis, USA) at 37℃ in a humidi ed atmosphere containing 5% CO2. Once every four days, the cells were harvested through trypsinization with trypsin-EDTA (Sigma) and under appropriate dilution were cultivated to a future growth in fresh medium.
Cells were prepared as before, after cells have attached overnight and cells adhesion rate were about 50%, add 0, 10, 50 and 100 µM sex hormones in A549 lung cancer cells, after 2 days of incubation, cells were harvested for more research.

Protein extraction and western blotting
Total protein from each experiment A549 cells were washed twice with ice-cold PBS and isolated on ice in lysis buffer (150µl RIPA, 1mM PMSF, Solarbio) with phosphatase inhibitor cocktail (Solarbio)). Isoluble material was cleared by centrifugation at 12,000g for 5minutes. Protein concentration was estimated by using BCA protein assay kit (Solarbio).
Samples containing equal amounts of protein were subjected to SDS-PAGE using 10% polyacrylamide gel. The proteins were then transferred to PVDF membranes (Millipore, Bedford, MA, USA) electrophoretically at 100 V for 90 min at 4°C. The membrane was washed and blocked for 1 h in blocking with TBST for three times and incubation with secondary antibodies for 2 h at room temperature. The membrane was then washed three times with TBST and detected by ECL (Thermo, Meridian Rd., Rockford, USA) and exposed with Protein-simple automated protein analyzer (Protein-simple, USA).

RNA extraction and Real-time PCR
Total RNA was extracted from cells using TRIzol reagent (Ambion, Meridian Rd., Rockford, USA), and RNA concentration and purity were detected by a microplate reader (Eppendorf, Germany). A Prime Script™ RT reagent kit with gDNA Eraser (Takara, Japan) was used for reverse transcription; speci cally, genomic DNA was removed at 42°C for 2 minutes, and the reverse transcription reaction was performed at 37°C for 15 minutes and 85°C for 5 seconds. Using cDNA as the template, Real-time PCR was performed with SYBR® Premix Ex Taq™ (Takara). GAPDH expression levels were used as the internal reference. The relative mRNA expression levels were quantitated by the 2 -ΔΔCt method. The primer sequences were: AR 5'-TGTACACGTGGTCAAGTGGGCCA-3' (forward) and 5'-GGGGCGAAGTAGAGCATCCTGGA-3' (reverse), Cells were seeded into 6-well plates (Corning) at the density of 2×10 5 cells per well. After incubation for 24 h at 37°C, the operation of gene silencing and sex hormones intervention were the same as described previously. After another 48 h, cells were collected and washed twice with ice-cold PBS and then resuspended, and aliquots of 1×10 5 cells were transferred into new 5 mL culture tubes in 500 µL of binding buffer. Then, 5 µL of Annexin V-FITC and 5 µL of propidium iodide were addedto the resuspended cells. After incubation in the dark for 10 min at room temperature. Flow cytometry (Becton Dikinson, USA) was used to assess the apoptotic cells.
Cell migration assay 2×10 5 cells were seeded in 6-well plates (Corning) and the operation of gene silencing and sex hormones intervention were the same as described previously. After cells were completely covered with the plates, in a sterile environment, removed the lid from the 6-well plates, laied a sterilized plastic ruler across the top of the plates. Without removing the media, used a sterile 200 µl pipet tip guided by the sterilized plastic ruler to make a scratche across the plates approximately 50 mm long. Then washed the plates twice with ice-cold PBS, and then photographed at 0,12,24,36 h respectively.

Cell invasion assay
Cell invasion assays were performed with the use of 8 µm pore Transwell inserts (Corning). The upper chambers of the Transwells were pre-coated with diluted Matrigel (BD Biosciences, Sparks, MD). A total of 10,000 cells were seeded onto the upper chamber in serum-free medium, and medium containing 10% serum was added to the lower chamber as a chemoattractant. After 24 h incubation, the upper surface of the insert was wiped with a cotton swab and cells that migrated to the lower surface were xed by anhydrous methanol and stained with crystal violet. And then photographed with a 100x microscope and analyzed.

Statistical analysis
The quantitative date were shown as the mean ± standard deviation. All statistical analyses were performed using SPSS version 24.0, and differences between groups were analyzed by independent sample t-test. And P < 0.05 was considered signi cant.

Results
Differences in AR, ERα and EGFR, PI3k, AKT protein expression levels after gene silencing and sex hormones intervention in A549 cells After Small interfering RNA (siRNA) speci cally targeting AR and ERα in A549 cells for 48 h, according to the western blotting analysis, AR and ERα protein expression levels were signi cantly knocked down ( Fig.1 A, B). We then examined the protein expression of EGFR and its downstream PI3K/AKT axis and found that the EGFR, PI3K, AKT protein expression levels were down-regulated ( Fig.1 A, B). After testosterone (T) and estradiol (E 2 ) at concentrations of 0, 10, 50 and 100μM interfered with A549 cells for 48 hours, according to the western blotting analysis, we found that 10μM T and 10μM E 2 interventions could up-regulate AR and ERα expression and increase protein expression on EGFR/PI3K/AKT axis, while 50μM T and 50μM E 2 interventions inhibited the expression of AR and ERα and inhibited the expression of EGFR/PI3K/AKT axis compared to the Control group. However, compared with the 50μM T and 50μM E 2 groups, 100μM concentration of T and E 2 intervention increased the expression of AR and ERα again, and correspondingly increased the expression of EGFR/PI3K/AKT axis (Fig.1 C, D).

Changes of cell migration and invasion after gene silencing and sex hormone intervention in A549 cells
Knocked down of AR and ERα expression result in the poor migration and invasion compared to control, Lipo, and NC groups (Fig.4 A, B; P<0.001). In the sex hormone intervention experiments, compared with the control group, 10 μM T and 10 μM E 2 intervention might promote cell migration and invasion, while 50 μM T and 50 μM E 2 intervention inhibited cell migration and invasion (Fig.4 A,B; (Fig.4 A, B; P<0.05, P<0.001). When the concentrations of T and E 2 increased to 100 μM, the migration and invasion of the cells were enhanced compared with the concentration of 50 μM (Fig.4 A, B; P<0.001).

Discussion
There is no doubt that lung cancer is still the number one cause of cancer deaths of the world today, and its morbidity and mortality are increasing every year (Siegel, Miller, 2017). NSCLC accounts for nearly 85% to 90% of lung cancer cases (Wakelee et al. , 2014), and EGFR is expressed in up to 80%-90% of NSCLC (Laskin and Sandler, 2004). Sex hormones may signal through their receptors, and their receptors are involved in a variety of malignancies, including hormone-dependent tumors such as breast cancer (Harbeck and Gnant, 2017) and prostate cancer (Latil et al. , 2001). Endocrine therapy is the main treatment for adjuvant treatment of patients with hormone-sensitive breast cancer and prostate cancer, such as ovarian castration in breast cancer and androgen deprivation therapy in prostate cancer (Rachner et al. , 2018, Roche et al. , 2011. Lung cancer has long been considered a non-hormone-dependent tumor. However, experimental and population-based evidence has been steadily accumulating that steroid hormones are fundamentally involved in the biology of the lung (Schwartz et al. , 2015, Siegfried, 2014, which provided the possibility of hormones-treated lung cancer and their receptors-targeted intervention for lung cancer therapy. Thus, we designed this experiment to observe the effects of sex hormones and their receptors on non-small cell lung cancer, and selected the EGFR/PI3K/AKT signaling pathway closely related to the development of lung cancer as the research object. In the gene silencing experiment, the Lipo group and the NC group were set to exclude the in uence of Lipo-2000 and NC-siRNA intervention on the experimental results. Western blotting and qPCR con rmed that AR and ERα were signi cantly knocked down in A549 cells AR: (0.96 ± 0.03),(0.99 ± 0.02),(1.02 ± 0.03) vs (0.08 ± 0.01) and ERα: (1.07 ± 0.06),(1.01 ± 0.04),(1.05 ± 0.07) vs (0.14 ± 0.02); P<0.001; Fig.1  A,B; Fig.2 A,B , we then detected the protein and mRNA expression levels of EGFR/PI3K/AKT axis closely related to lung cancer, and found that EGFR/PI3K/AKT axis was inhibited (Fig.1 A,B; Fig.2 A,B; P<0.001 ). Cell proliferation and apoptosis assays showed that A549 cells decreased activity and increased apoptosis after AR and ERα were knocked down (Fig.3 A,C; P<0.001). Cell migration and invasion assays also showed reduced migration and invasion of A549 cells after knockdown of AR and ERα (Fig.4 A, B).
Garon EB 17 reported anti-estrogen fulvestrant adds to effects of EGFR inhibitors, and sensitivity to fulvestrant correlates with greater baseline ERα gene expression. Some reports indicated that ERβ expression in lung cancer as a negative prognosticator in women, and patients expressing high levels of AR have an unfavorable prognostic outcome compared with low AR expressing patients , Skjefstad et al. , 2016, Stabile et al. , 2011. Thus, we speculate that AR and ERα promote the development of NSCLC through EGFR/PI3K/AKT axis, and they could be treatment targets in NCSLC, as it has been successfully used in breast cancer and prostate cancer(Lancet, 1998, Rachner, Coleman, 2018, Roche, Niu, 2011).
In the sex hormone intervention experiment, sex hormone intervention with different concentration gradients was set to observe the effects of different concentrations of sex hormones on lung cancer. By Western blotting and qPCR assays, we found that 10μM T and 10μM E 2 promoted the expression of AR Cell proliferation and apoptosis assays showed that 10μM T and10μM E 2 promoted cell growth and reduced apoptosis (Fig.3 B, C; P<0.05, P<0.01, P<0.001), 50μM T and 50μM E 2 inhibited cell growth and promoted apoptosis (Fig.3 B, C; P<0.001), when the concentrations of T and E 2 were increased to 100μM, the activity of the cells was enhanced and apoptosis was decreased (Fig.3 B, C; P<0.001). Cell migration and invasion assays showed that 10μM T and 10μM E 2 promoted cells migration and invasion, and 50μM T and 50μM E 2 inhibited cells migration and invasion, when T and E2 concentrations were increased to 100μM, cells migration and invasion were promoted again (Fig.4 A,B). A recent study reported higher T predict increased incidence of lung cancer over the next decade in older men (Chan et al. , 2017). Marsaud V reported that the effect of E 2 on ERα expression in lung cancer was also related to intervention time (Marsaud et al. , 2003). Our animal experiments have shown that the internal environment of sex hormone imbalance promotes the development of mouse lung cancer xenografts 11 , and Clinical studies also support the conclusion that there is an imbalance in sex hormones and their receptors in patients with lung cancer (Xu et al. , 2016, Zhou et al. , 2002. Combined with our experimental data, sex hormones may have a positive effect in the treatment of lung cancer, but the dose and time of administration of sex hormones still need to be explored, which is also the requirement to maintain environmental balance and stability in the body.
Although platinum-based chemotherapy has been clearly established in the treatment of lung cancer, there is still debate about the choice of chemotherapy regimen (Wakelee, Kelly, 2014). And in recent years, EGFR-TKIs have played an active role in the treatment of advanced lung cancer, but most patients will develop resistance within one year of taking the drug (Jackman, Pao, 2010). All of these indicate that it is necessary to explore treatments for lung cancer other than chemotherapy and to nd new therapeutic targets for lung cancer. New data from randomized, prospective clinical trials show a signi cant impact of hormone replacement therapy (HRT) in lung cancer (Chlebowski et al. , 2009). Furthermore, a prospective cohort study con rmed an increased, dose-dependent lung cancer risk among women who received HRT (Slatore et al. , 2010). Both Mah V (Mah, Marquez, 2011) and Marquez-Garban DC (Marquezgarban et al. , 2011) reported that estrogen and ERs can activate the Src family of nonreceptor tyrosine kinases, MAPKs (mitogen-activated protein kinases) and PI3K, which affected cancer cell growth, proliferation and apoptosis. A study from Lee SH (Lee et al. , 2015) showed an interaction between androgen and the PI3K/AKT pathway in prostate cancer. And the PI3K/AKT signalling pathway mediates the process of EMT and has attracted widespread attention as a potential target for the prevention and treatment of metastatic tumours (Bakin AV, 2000, Xu Q, 2014. And the project leader has achieved good clinical e cacy by using traditional Chinese medicine Bushenshugan formula to treat lung cancer by enhancing the patient's gonadal function (Fan Z, 2018   Cells proliferation and apoptosis after gene silencing and sex hormones intervention. A: After knocked down of AR and ERα expression, the proliferation of cells was inhibited compared to Control, Lipo and NC groups respectively. B: After sex hormones intervention for 48 h, compared with the control group, 10μM T and 10μM E2 intervention promoted cells proliferation, and 50μM T and 50μM E2 intervention inhibited cells proliferation, when the concentrations of T and E2 were increased to 100μM, the proliferation of cells were enhanced compared to 50μM T and 50μM E2 groups. C: Knocked down of AR and ERα expression promoted cells apoptosis; 10μM T and 10μM E2 intervention inhibited cells apoptosis and 50μM T and 50μM E2 intervention promoted cells apoptosis, when the concentrations of T and E2 were increased to 100μM, the apoptosis of cells were inhibited again. Control: cells without any intervention; Lipo: cells were transfected only with Lipo-2000; NC: cells were transfected with NC-siRNA; AR: cells were transfected with AR-siRNA; ERα: cells were transfected with ERα-siRNA; 10, 50 and 100μΜ T and E2: cells were intervened with 10, 50 and 100μΜ T and E2 respectively. *P<0.05, **P<0.01, ***P<0.001: compared with Control group; △△△P<0.001: compared with 50μM T and 50μM E2 groups.