FLNa negatively regulated proliferation and metastasis in lung adenocarcinoma A549 cells via suppression of EGFR

FLNa negatively regulated proliferation and metastasis in lung adenocarcinoma A549 cells via... Abstract Filamin A (FLNa) is a ubiquitously expressed cytoplasmic protein, which composes of an N-terminal actin binding domain (ABD) followed by 24 Ig-like repeats. FLNa functions as a cytoskeletal protein that links transmembrane receptors, including integrins, to F-actin and serves as a signaling intermediate. Recent studies have identified FLNa as a scaffold protein that interacts with over 90 proteins and plays vital roles in cellular signaling transduction. Mutations or defects in human FLNa gene have been shown to cause numerous developmental defects. Moreover, aberrant expression of FLNa has been observed in many cancers, such as parathyroid tumor, cervical cancer, and breast cancer. However, its role in lung adenocarcinoma has seldom been discussed. In the present study, our in vitro and in vivo studies demonstrated that silencing FLNa expression in lung cancer cell line A549 cells promoted proliferation, migration, and invasiveness of A549 cells by enhancing the activation of epidermal growth factor receptor and ERK signaling pathway. These results shed light on novel functions of FLNa in lung cancer and uncovered novel mechanisms, these results provided possible targets for the prediction and treatment for lung adenocarcinoma. FLNa, lung adenocarcinoma, proliferation, metastasis, EGFR Introduction Lung cancer, one of the most malignant tumors, is the leading cause of cancer-related deaths worldwide [1]. Among lung cancers, 40% patients are diagnosed as lung adenocarcinoma and the 5-years survival rate for lung adenocarcinoma patients is only 5%–20% at later stages. Usually when diagnosed, the cancer has already progressed to late stage with local invasion and/or distant organ metastases [2,3]. Despite recent progress in surgical resection, chemotherapeutic and radiotherapeutic interventions and molecular mechanism studies, the prognosis of lung adenocarcinoma has not been significantly improved [4,5]. Thus, there is an urgent need to identify novel predictive and treatment targets for lung adenocarcinoma. Due to progress in introduction of new drugs and individualized therapy based on different histological subtypes and driver gene mutations that contributed to malignant behaviors and biology of lung cancer, new windows of opportunity have been opened for the comprehensive treatment of lung cancer [6]. The epidermal growth factor receptor (EGFR) gene is one of the best characterized driver gene for lung adenocarcinoma, the most common type of non-small-cell lung cancer (NSCLC) [7,8]. Previous studies have shown that Dys-regulation of EGFR and/or its natural ligand EGF might lead to uncontrolled cell proliferation, differentiation, metastasis, and survival. EGFR is usually overexpressed in patients of lung adenocarcinoma, and can affect the prognosis of lung cancer patients [9,10]. Strategies to target EGFR, especially by using EGFR tyrosine kinase inhibitors (TKIs), have played vital roles in leading lung cancer research, treatment, and outcome prediction [11,12]. Somatic activating mutations of EGFR are found in about 26% of all patients with lung adenocarcinoma and render sensitivity to first-generation of EGFR TKIs including gefitinib and erlotinib [13,14]. Although most patients exhibit good response to these TKIs, acquired resistance to TKIs usually occurs. The acquired secondary mutations in EGFR continue to render dependence on EGFR signaling pathway in lung adenocarcinoma [15,16]. Thus, there is an urgent need to characterize novel molecular mechanisms to intervene EGFR signaling pathway, to identify novel therapeutic targets to overcome both innate and acquired resistance [17,18]. FLNa is a well-characterized actin cross-linking proteins, which functions as a scaffold and interacts with over 90 binding partners [19]. Through interactions with these proteins, FLNa is involved in the regulation of multiple cellular functions, especially migration, invasion, and adhesion [20–22]. In recent years, the functions of FLNa in cancers have received much attention. Numerous studies have demonstrated the contribution of FLNa to cancer proliferation, progression, and metastasis [23]. FLNa was initially identified as a cancer promoting gene, which promotes cancer cell migration, invasion, and metastasis. However, recent studies indicated that FLNa could play negative roles in cancer progression depending on its cellular localization [24]. The contribution of FLNa to cancer migration and invasion depends on its modification of signaling pathways. FLNa has been reported to play both positive and negative roles in the regulation of ERK1/2 and PKB/Akt signaling pathways, and suppression of FLNa could attenuate the K-ras-induced ERK and Akt pathway activation [25]. In melanoma cells, FLNa was reported to interact with and modulate the function of integrin and phosphorylation and localization of PKB/Akt/ERK kinases [26]. However, the FLNa-dependent regulation and activation of EGFR signaling in lung adenocarcinoma, to our knowledge, has seldom been studied. Based on the decisive role of EGFR signaling pathway in lung adenocarcinoma and the important role of FLNa in cancer migration and invasion, we assessed the impact of FLNa on EGFR signaling pathway in lung cancer cell line A549 cells. Our in vitro and in vivo studies demonstrated that silencing FLNa expression promoted cell proliferation, migration, and invasion of A549 cells. Mechanism studies demonstrated that FLNa played negative role in the EGFR signaling pathway, and could attenuate EGF-induced activation of EGFR signaling pathway. Materials and Methods Cell culture The human lung cancer cell line A549 was obtained from ATCC (Manassas, USA) and cultured in Roswell Park Memorial Institute(RPMI) 1640 medium supplemented with 10% fetal bovine serum according to standard ATCC protocol. RNA isolation and quantitative real-time PCR Trizol reagent (Invitrogen, Carlsbad, USA) was used to prepare total RNA. cDNA was obtained by using TaKaRa PrimeScript RT reagent kit (Dalian, China). The expressions of FLNa and β-actin were determined by quantitative real-time PCR using an ABI 7900HT Real-Time PCR system (Applied Biosystems, Foster City, USA). All reactions were run in triplicate. Primers sequences for FLNa were F: 5′-CACCTGGCAGCTACCTCATCTCCA-3′ and R: 5′-CCACCACCTTGCTGGCGTCAGCA-3′. Primer sequences for β-actin were F: 5′-AGAAGAGCTACGAGCTGCCTGACG-3′ and R: 5′-TGATCCACATCTGCTGGAAGGTGG-3′. Protein extraction and western blot analysis Cells were washed with ice-cold PBS and lysed in RIPA buffer (20 mMTris-HCl, pH8.0, 150 mM NaCl, 1%NP-40, 10% glycerol, and 20 μM EDTA) followed by sonication and centrifugation at 13,400 g for 20 min at 4°C to remove debris. BCA assay (Pierce, Rockford, USA) was used to measure protein concentration. Total protein lysate (20 μg) was subject to electrophoresis in denaturing 8% SDS-polyacrylamide gel, and then transferred to a membrane for subsequent blotting with designated antibodies. FLNa antibody was obtained from Millipore (Cat. No. MAB1680; Billerica, USA) and Santa Cruz (Cat. No. sc-17749; Santa Cruz, USA). Antibodies against ERK1/2 and β-actin were purchased from Santa Cruz. pERK1/2 and EGFR antibodies were obtained from Cell Signaling Technology (Danvers, USA). Anti-phosphotyrosine antibody (clone 4G10) was purchased from Millipore. Lentivirus production and stable cell line selection pLKO.1 TRC cloning vector (Addgene plasmid: 10878; Cambridge, USA) was used to generate shRNA expressing construct against FLNa. The 21-bp targets against FLNa were GACCGCCAATAACGACAAGAA. Control vector pLKO.1 scramble shRNA (Addgene plamid: 1864) was obtained from Addgene. Lentivirus was produced by co-transfection of pLKO.1-shFLNa with psPAX2 and pMD2.G into HEK-293T cells. Stable cell lines were obtained after infection of A549 cells with lentivirus, followed by puromycin selection in a concentration of 2 μg/l. MTT cell proliferation assay Cell proliferation was determined by MTT proliferation assay. In brief, 6 × 103 A549 cells per well were seeded into 96-well plates. Cells were starved by serum deprivation for 4 h, and then treated with EGF at a concentration of 0 nM, 4 nM, and 20 nM for 48 h, respectively. A total of 20 μl MTT reagent (Sigma-Aldrich, St Louis, USA; 5 mg/ml) was added to each well and incubated for 4 h. Then the culture medium was discarded and 150 μl DMSO was added to each well. The absorbance was measured with a microplate reader at wavelength of 490 nm. Immunoprecipitation assay To purify endogenous EGFR, immunoprecipitation (IP) assay was performed. A549 cells were lysed with RIPA ice-cold buffer. The protein concentration was measured by BCA assay. Lysates (500 μg) were incubated with EGFR antibody or relevant control IgG overnight at 4°C. Then Protein A/G agarose beads (Thermo Fisher Scientific, Waltham, USA) were added into the lysates and incubated for 2 h. The beads were washed five times with RIPA buffer. Finally, samples on the beads were denatured with SDS-PAGE loading buffer and subject to SDS-PAGE separation and immunoblotting. Scratch wound-healing assay FLNa silenced or the relative control A549 cells were seeded in 6-well plates. The confluent cell monolayers were scratched with a pipette tip and the plates were washed twice with PBS buffer before addition of fresh medium. Then cell migration was observed and measured at the indicated time intervals. Each assay was repeated three times. Migration assay The migration assay was performed by using a Transwell chamber (Corning Co., Corning, USA). FLNa silencing and the relative control cells were collected and transferred to the chamber inserts in serum-free medium. The bottom chamber contained medium with 10% FBS as a chemoattractant. The cells were cultured in a humidified incubator at 37°C for 24 h. The cells that migrated to the underside of the filter were fixed and stained with crystal violet and counted by using bright-field microscopy. Invasion assay A549 cells were seeded onto filters of a 24-well transwell chamber that was coated with Matrigel (BD Biosciences, Franklin Lakes, USA). Invasion of the cells through the Matrigel to the underside of the filter was assessed 24 h later by fixation and staining with crystal violet. The cells were counted by using bright-field microscopy. Tumorigenesis study BALB/c-nu mice (5–6 weeks of age, 18–20 g; Vital River Laboratory Animal Technology, Beijing, China) were housed in sterile filter-capped cages. A total of 1 × 106 cells in 100 μl PBS were injected subcutaneously into the backs of the mice. Tumor volume was calculated by the formula of (length × width2 × 0.52). Tumor size was measured at the indicated time intervals with calipers from the time of the formation of palpable tumors. Four weeks after implantation, the mice were euthanized, and the tumors were surgically dissected. The tumor specimens were fixed with 4% paraformaldehyde. Samples were then processed for histopathological examination. Immunohistochemical staining Immunohistochemical (IHC) staining of paraffin-embedded tissues was performed according to standard procedures that described previously [27]. Mouse monoclonal FLNa antibody was obtained from Santa Cruz and used in a dilution of 1:100. Anti-Ki67 antibody was purchased from Abcam (Cambridge, UK) and used in a dilution of 1:100. Rabbit monoclonal Phospho-EGF receptor (Tyr1068) (D7A5) was obtained from Cell Signaling Technology and used in a dilution factor of 1:100. Statistical analysis Data were expressed as the mean ± SD, and the differences between any two groups were compared by t-tests. Statistical analyses were performed by SPSS software (SPSS Software, Chicago, USA). P < 0.05 was considered significant difference. Results Silencing FLNa enhances proliferation capacity of A549 cells To assess the role of FLNa in the proliferation of lung adenocarcinoma cell line A549 cells, lentivirus-mediated silencing of FLNa expression in A549 cells was carried out. The effect of silencing was determined by immunoblotting and quantitative real-time PCR analysis. As demonstrated, FLNa was effectively silenced in A549 cells as reflected by immunoblotting (Fig. 1A,B). Moreover, real-time PCR results validated the silencing effect (Fig. 1C). Further MTT proliferation assay reflected that silencing of FLNa enhanced growth capacity of A549 cells with or without EGF stimulation (Fig. 1D). Figure 1. View largeDownload slide Silencing FLNa enhances proliferation capacity of A549 cells A549 cells were transfected with either control or FLNa shRNA plasmids and selected by puromycin. Cell lysates and total RNA were prepared to detect the expression of FLNa by western blot analysis (A) and quantitative real-time PCR (C), respectively. (B) Bars represent ratio of FLNa density to β-actin. (D) Knockdown of FLNa enhances proliferation capacity in A549 cells. *P < 0.05 vs. control A549 cells and **P < 0.01 vs. control A549 cells. Figure 1. View largeDownload slide Silencing FLNa enhances proliferation capacity of A549 cells A549 cells were transfected with either control or FLNa shRNA plasmids and selected by puromycin. Cell lysates and total RNA were prepared to detect the expression of FLNa by western blot analysis (A) and quantitative real-time PCR (C), respectively. (B) Bars represent ratio of FLNa density to β-actin. (D) Knockdown of FLNa enhances proliferation capacity in A549 cells. *P < 0.05 vs. control A549 cells and **P < 0.01 vs. control A549 cells. FLNa silencing promotes migration capacity of A549 cells FLNa is a well-characterized gene that regulates cell behaviors including migration, invasion, and adhesion that are associated with metastasis. To assess the contribution of FLNa to lung adenocarcinoma cell metastasis, migration, and invasion assays were performed through silencing FLNa expression. As demonstrated by scratch wound-healing assay, FLNa silencing promotes cell migration. In addition, FLNa silencing also enhances EGF-induced cell migration (Fig. 2A,B). Moreover, cell migration assay indicated that FLNa silencing could promote A549 cell migration capacity in the presence or absence of EGF (Fig. 2C,D). These results suggest that FLNa functions as a negative regulator of metastasis in A549 cells. Figure 2. View largeDownload slide FLNa silencing promotes migration capacity of A549 cells Wound-healing assay were performed in control A549 cells and FLNa-knockdown A549 cells in the absence or presence of EGF (20 nM) for the indicated time. Images were taken under an inverted microscope (A) (Magnification, ×10), and migration rate was calculated (B). Transwell chambers were used to determine migration capacity of control A549 cells and FLNa-knockdown A549 cells. Images were taken (C) and migrated cells were counted (D). **P < 0.01 vs. control A549 cells. Figure 2. View largeDownload slide FLNa silencing promotes migration capacity of A549 cells Wound-healing assay were performed in control A549 cells and FLNa-knockdown A549 cells in the absence or presence of EGF (20 nM) for the indicated time. Images were taken under an inverted microscope (A) (Magnification, ×10), and migration rate was calculated (B). Transwell chambers were used to determine migration capacity of control A549 cells and FLNa-knockdown A549 cells. Images were taken (C) and migrated cells were counted (D). **P < 0.01 vs. control A549 cells. FLNa silencing increases invasiveness of A549 cells To analyze the impact of FLNa on the invasion capacity of lung adenocarcinoma, cell invasion assay was performed. As shown by Matrigel invasion assay, FLNa silencing enhanced the invasiveness of A549 cells (Fig. 3A,B). These results further validate the suppressive function of FLNa on A549 invasiveness. Figure 3. View largeDownload slide FLNa silencing increases invasiveness of A549 cells Transwell chambers were used to determine the invasive capacity of control A549 cells and FLNa-knockdown A549 cells. Images were taken (A) and cells were counted (B). **P < 0.01 vs. control A549 cells. Figure 3. View largeDownload slide FLNa silencing increases invasiveness of A549 cells Transwell chambers were used to determine the invasive capacity of control A549 cells and FLNa-knockdown A549 cells. Images were taken (A) and cells were counted (B). **P < 0.01 vs. control A549 cells. Role of FLNa in ligand-induced EGFR phosphorylation in human A549 cells To explore the underlying molecular mechanism through which FLNa silencing induces proliferation, migration, and invasion, the effect of FLNa on EGF-induced EGFR signaling pathway activation was analyzed. As demonstrated, FLNa silencing could enhance EGF-induced activation of EGFR. EGF-induced activation of EGFR could activate the ERK signaling pathway, which promotes cancer cell proliferation, migration, and invasion. An increase in activated ERK1/2 in FLNa-silenced cells treated with EGF was also observed (Fig. 4A,C). FLNa is a scaffold protein that interacts with many transmembrane proteins, such as integrin, which results in variation in transmembrane protein signal transduction. Thus, we analyzed the impact of FLNa on phosphorylation of tyrosine on EGFR. As observed, silencing FLNa changed the phosphorylation status of tyrosine on EGFR when cells were treated with EGF (Fig. 4D). Figure 4. View largeDownload slide Role of FLNa in ligand-induced EGFR phosphorylation in human A549 cells (A) Western blot analysis was performed using antibodies against FLNa, phosphotyrosine, EGFR, phosphorylated and total ERK1/2. β-actin is the loading control. (B) Ratio of pY-EGFR to total EGFR. (C) Ratio of p-ERK to total ERK. (D) Lysates from control A549 cells and FLNa-knockdown A549 cells were immunoprecipitated (IP) with anti-EGFR antibody and analyzed by western blot analysis with antibodies against phosphotyrosine and EGFR. NI, nonimmune IgG. **P < 0.01 vs. control A549 cells. Figure 4. View largeDownload slide Role of FLNa in ligand-induced EGFR phosphorylation in human A549 cells (A) Western blot analysis was performed using antibodies against FLNa, phosphotyrosine, EGFR, phosphorylated and total ERK1/2. β-actin is the loading control. (B) Ratio of pY-EGFR to total EGFR. (C) Ratio of p-ERK to total ERK. (D) Lysates from control A549 cells and FLNa-knockdown A549 cells were immunoprecipitated (IP) with anti-EGFR antibody and analyzed by western blot analysis with antibodies against phosphotyrosine and EGFR. NI, nonimmune IgG. **P < 0.01 vs. control A549 cells. Silencing FLNa increases tumorigenic capacity of A549 cells To examine the effect of FLNa on the tumorigenic capacity of A549 cells, tumorigenesis assay was performed by injecting tumor cells subcutaneously into nude mice. It was found that silencing FLNa expression could increase tumor volume and tumor growth (Fig. 5A,B). IHC staining of the tumor samples from sacrificed mice by using anti-FLNa, anti-EGFR, and anti-Ki67 antibodies further supported our in vitro observations of the negative role of FLNa in tumor cell growth (Fig. 5C). Furthermore, immunoblotting results also demonstrated that silencing FLNa could increase the activation of EGFR and ERK1/2 (Fig. 5D). Taken together, these results are in consistent with in vitro observation, which supports the negative role of FLNa in tumor growth. Figure 5. View largeDownload slide Silencing FLNa increases tumorigenic capacity of A549 cells Tumor-bearing mice were generated by subcutaneous injection of control A549 cells and FLNa-knockdown A549 cells. Mice were sacrificed 28 days after inoculation. (A) Representative photographs of tumors after surgical resection. (B) Tumor growth in mice injected with control A549 cells and FLNa-knockdown A549 cells. Tumor volume in each group was expressed as the mean ± SD (n = 6). (C) IHC staining with antibodies against FLNa, pY-EGFR, and Ki-67 of tumor tissue sections. (D) Western blot analysis was performed with tumor lysates using antibodies against FLNa, phosphotyrosine, EGFR, phosphorylated and total ERK1/2. β-actin is the loading control. **P < 0.01 vs. control A549 cells. Figure 5. View largeDownload slide Silencing FLNa increases tumorigenic capacity of A549 cells Tumor-bearing mice were generated by subcutaneous injection of control A549 cells and FLNa-knockdown A549 cells. Mice were sacrificed 28 days after inoculation. (A) Representative photographs of tumors after surgical resection. (B) Tumor growth in mice injected with control A549 cells and FLNa-knockdown A549 cells. Tumor volume in each group was expressed as the mean ± SD (n = 6). (C) IHC staining with antibodies against FLNa, pY-EGFR, and Ki-67 of tumor tissue sections. (D) Western blot analysis was performed with tumor lysates using antibodies against FLNa, phosphotyrosine, EGFR, phosphorylated and total ERK1/2. β-actin is the loading control. **P < 0.01 vs. control A549 cells. Discussion In the present study, we have uncovered a novel function of FLNa in lung adenocarcinoma cell line A549 cells. Our in vitro and in vivo results suggest that FLNa functions as a negative regulator of proliferation and metastasis in A549 cells. Molecular mechanism studies indicate that silencing FLNa expression in A549 cells promotes the activation of EGFR signaling pathway. FLNa was initially identified as a structure protein that participates in actin cross-linking. FLNa was reported to interact with many proteins, including membrane channels, receptors, intracellular signaling molecules, and even transcription factors. Due to the diversity of FLNa-interacting partners, FLNa is involved in many intracellular physiological functions. Recently, FLNa has received much attention because of its involvement in cancers. FLNa plays dual roles in cancers, depending on its subcellular localization and interacting partners. For example, in prostate cancer and breast cancer, FLNa displays bifunctional roles, by promoting or inhibiting the growth and metastasis of these cancer cells [28–31]. In breast cancer, when interacting with caveolin1, FLNa regulates focal adhesion disassembly and suppresses breast cancer cell migration and invasion [32]. On the other hand, through interaction with cyclinD1, FLNa functions as a cancer promoting gene by promoting migrations and invasion [33]. In human lung cancer, proteomic studies indicated that FLNa expression is up-regulated during the EMT process, indicating its positive role in cell migration and invasiveness [34]. Moreover, IHC staining analysis also showed that FLNa is overexpressed in lung cancer, but no significance was observed between its expression and metastasis or prognosis indications [35]. However, few experiments were carried out in cell lines to verify the role of FLNa in lung cancer. To explore the underlying molecular mechanism, we turn to examine the impact of FLNa on the EGFR signaling pathway, a well-established pathway that contributes to cancer cell proliferation, migration, and invasion, especially in lung adenocarcinoma [36,37]. Since the discovery of EGFR, new functions of EGFR have gradually been revealed. The traffic of EGFR from cell membrane to intracellular compartments could amplify or quench EGFR signaling [38–40]. For example, EGFR can undergo endocytosis and subsequent nuclear transport through importin β1 [41]. Cellular traffic and endocytosis of EGFR depend on its interactions with structure proteins like actin and caveolin, which are well-established FLNa-interacting proteins [42,43]. These processes could result in the phosphorylation of EGFR. EGFR was reported to bind to F-actin cytoskeleton in intact cell, leading to sequestration of EGFR transduction. Based on the role of FLNa in actin cross-linking, it is conceivable that FLNa might also participate in EGFR signal transduction [44]. In support of this hypothesis, we observed the impact of FLNa on the activation status of EGFR and its downstream ERK1/2 pathway activation. The negative role of FLNa on EGFR signaling pathway accounts for its suppressive impact on A549 cell growth and metastasis. Moreover, these observations laid the foundation for further studies to uncover the role of FLNa in the trafficking of endogenous EGFR to organelles, as nuclear-localized FLNa has been proposed to possess tumor suppressive functions. The negative role of FLNa in EGFR pathway might due to its influence on EGFR trafficking or its suppression of EGFR activation in the nucleus. In summary, we have revealed a novel tumor suppressive role of FLNa in lung adenocarcinoma cell line A549 cells. Mechanism studies indicated that FLNa is involved in the activation of EGFR pathway, the key signaling event in lung adenocarcinoma proliferation, progression, metastasis, and resistance to chemotherapies (Fig. 6). This study may provide novel predictive and treatment targets for lung adenocarcinoma, and facilitate further researches to elucidate novel molecular mechanisms in lung cancer. Figure 6. View largeDownload slide Schematic representation of the working model The activation of the EGF/EGRF/ERK1/2 axis plays vital roles in the oncogenesis and progression of cancer cells. In the present study, we have identified FLNa as a negative regulator of the axis, leading to decreased metastasis, proliferation, and tumorigenesis capacity of lung cancer cell line A549 cells. Figure 6. View largeDownload slide Schematic representation of the working model The activation of the EGF/EGRF/ERK1/2 axis plays vital roles in the oncogenesis and progression of cancer cells. In the present study, we have identified FLNa as a negative regulator of the axis, leading to decreased metastasis, proliferation, and tumorigenesis capacity of lung cancer cell line A549 cells. Funding This work was supported by the grants from the National Natural Science Foundation of China (No. 81071846), Natural Science Foundation of Hebei Province of China (No. H2013505059), Department of Science and Technology of Hebei Province of China (Nos. 12396107D, 14397707D, 09966114D, and 092461102D), and Wu Jieping Foundation (Nos. 320.6750.12604, 320.6750.14063, and 320.6799.15005). References 1 Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin  2015, 65: 5– 29. Google Scholar CrossRef Search ADS PubMed  2 Travis WD. Pathology of lung cancer. Clin Chest Med  2002, 23: 65– 81. viii. Google Scholar CrossRef Search ADS PubMed  3 Spira A, Ettinger DS. Multidisciplinary management of lung cancer. N Engl J Med  2004, 350: 379– 392. Google Scholar CrossRef Search ADS PubMed  4 Sangodkar J, Katz S, Melville H, Narla G. Lung adenocarcinoma: lessons in translation from bench to bedside. Mt Sinai J Med  2010, 77: 597– 605. 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For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Biochimica et Biophysica Sinica Oxford University Press

FLNa negatively regulated proliferation and metastasis in lung adenocarcinoma A549 cells via suppression of EGFR

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© The Author(s) 2017. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
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Abstract

Abstract Filamin A (FLNa) is a ubiquitously expressed cytoplasmic protein, which composes of an N-terminal actin binding domain (ABD) followed by 24 Ig-like repeats. FLNa functions as a cytoskeletal protein that links transmembrane receptors, including integrins, to F-actin and serves as a signaling intermediate. Recent studies have identified FLNa as a scaffold protein that interacts with over 90 proteins and plays vital roles in cellular signaling transduction. Mutations or defects in human FLNa gene have been shown to cause numerous developmental defects. Moreover, aberrant expression of FLNa has been observed in many cancers, such as parathyroid tumor, cervical cancer, and breast cancer. However, its role in lung adenocarcinoma has seldom been discussed. In the present study, our in vitro and in vivo studies demonstrated that silencing FLNa expression in lung cancer cell line A549 cells promoted proliferation, migration, and invasiveness of A549 cells by enhancing the activation of epidermal growth factor receptor and ERK signaling pathway. These results shed light on novel functions of FLNa in lung cancer and uncovered novel mechanisms, these results provided possible targets for the prediction and treatment for lung adenocarcinoma. FLNa, lung adenocarcinoma, proliferation, metastasis, EGFR Introduction Lung cancer, one of the most malignant tumors, is the leading cause of cancer-related deaths worldwide [1]. Among lung cancers, 40% patients are diagnosed as lung adenocarcinoma and the 5-years survival rate for lung adenocarcinoma patients is only 5%–20% at later stages. Usually when diagnosed, the cancer has already progressed to late stage with local invasion and/or distant organ metastases [2,3]. Despite recent progress in surgical resection, chemotherapeutic and radiotherapeutic interventions and molecular mechanism studies, the prognosis of lung adenocarcinoma has not been significantly improved [4,5]. Thus, there is an urgent need to identify novel predictive and treatment targets for lung adenocarcinoma. Due to progress in introduction of new drugs and individualized therapy based on different histological subtypes and driver gene mutations that contributed to malignant behaviors and biology of lung cancer, new windows of opportunity have been opened for the comprehensive treatment of lung cancer [6]. The epidermal growth factor receptor (EGFR) gene is one of the best characterized driver gene for lung adenocarcinoma, the most common type of non-small-cell lung cancer (NSCLC) [7,8]. Previous studies have shown that Dys-regulation of EGFR and/or its natural ligand EGF might lead to uncontrolled cell proliferation, differentiation, metastasis, and survival. EGFR is usually overexpressed in patients of lung adenocarcinoma, and can affect the prognosis of lung cancer patients [9,10]. Strategies to target EGFR, especially by using EGFR tyrosine kinase inhibitors (TKIs), have played vital roles in leading lung cancer research, treatment, and outcome prediction [11,12]. Somatic activating mutations of EGFR are found in about 26% of all patients with lung adenocarcinoma and render sensitivity to first-generation of EGFR TKIs including gefitinib and erlotinib [13,14]. Although most patients exhibit good response to these TKIs, acquired resistance to TKIs usually occurs. The acquired secondary mutations in EGFR continue to render dependence on EGFR signaling pathway in lung adenocarcinoma [15,16]. Thus, there is an urgent need to characterize novel molecular mechanisms to intervene EGFR signaling pathway, to identify novel therapeutic targets to overcome both innate and acquired resistance [17,18]. FLNa is a well-characterized actin cross-linking proteins, which functions as a scaffold and interacts with over 90 binding partners [19]. Through interactions with these proteins, FLNa is involved in the regulation of multiple cellular functions, especially migration, invasion, and adhesion [20–22]. In recent years, the functions of FLNa in cancers have received much attention. Numerous studies have demonstrated the contribution of FLNa to cancer proliferation, progression, and metastasis [23]. FLNa was initially identified as a cancer promoting gene, which promotes cancer cell migration, invasion, and metastasis. However, recent studies indicated that FLNa could play negative roles in cancer progression depending on its cellular localization [24]. The contribution of FLNa to cancer migration and invasion depends on its modification of signaling pathways. FLNa has been reported to play both positive and negative roles in the regulation of ERK1/2 and PKB/Akt signaling pathways, and suppression of FLNa could attenuate the K-ras-induced ERK and Akt pathway activation [25]. In melanoma cells, FLNa was reported to interact with and modulate the function of integrin and phosphorylation and localization of PKB/Akt/ERK kinases [26]. However, the FLNa-dependent regulation and activation of EGFR signaling in lung adenocarcinoma, to our knowledge, has seldom been studied. Based on the decisive role of EGFR signaling pathway in lung adenocarcinoma and the important role of FLNa in cancer migration and invasion, we assessed the impact of FLNa on EGFR signaling pathway in lung cancer cell line A549 cells. Our in vitro and in vivo studies demonstrated that silencing FLNa expression promoted cell proliferation, migration, and invasion of A549 cells. Mechanism studies demonstrated that FLNa played negative role in the EGFR signaling pathway, and could attenuate EGF-induced activation of EGFR signaling pathway. Materials and Methods Cell culture The human lung cancer cell line A549 was obtained from ATCC (Manassas, USA) and cultured in Roswell Park Memorial Institute(RPMI) 1640 medium supplemented with 10% fetal bovine serum according to standard ATCC protocol. RNA isolation and quantitative real-time PCR Trizol reagent (Invitrogen, Carlsbad, USA) was used to prepare total RNA. cDNA was obtained by using TaKaRa PrimeScript RT reagent kit (Dalian, China). The expressions of FLNa and β-actin were determined by quantitative real-time PCR using an ABI 7900HT Real-Time PCR system (Applied Biosystems, Foster City, USA). All reactions were run in triplicate. Primers sequences for FLNa were F: 5′-CACCTGGCAGCTACCTCATCTCCA-3′ and R: 5′-CCACCACCTTGCTGGCGTCAGCA-3′. Primer sequences for β-actin were F: 5′-AGAAGAGCTACGAGCTGCCTGACG-3′ and R: 5′-TGATCCACATCTGCTGGAAGGTGG-3′. Protein extraction and western blot analysis Cells were washed with ice-cold PBS and lysed in RIPA buffer (20 mMTris-HCl, pH8.0, 150 mM NaCl, 1%NP-40, 10% glycerol, and 20 μM EDTA) followed by sonication and centrifugation at 13,400 g for 20 min at 4°C to remove debris. BCA assay (Pierce, Rockford, USA) was used to measure protein concentration. Total protein lysate (20 μg) was subject to electrophoresis in denaturing 8% SDS-polyacrylamide gel, and then transferred to a membrane for subsequent blotting with designated antibodies. FLNa antibody was obtained from Millipore (Cat. No. MAB1680; Billerica, USA) and Santa Cruz (Cat. No. sc-17749; Santa Cruz, USA). Antibodies against ERK1/2 and β-actin were purchased from Santa Cruz. pERK1/2 and EGFR antibodies were obtained from Cell Signaling Technology (Danvers, USA). Anti-phosphotyrosine antibody (clone 4G10) was purchased from Millipore. Lentivirus production and stable cell line selection pLKO.1 TRC cloning vector (Addgene plasmid: 10878; Cambridge, USA) was used to generate shRNA expressing construct against FLNa. The 21-bp targets against FLNa were GACCGCCAATAACGACAAGAA. Control vector pLKO.1 scramble shRNA (Addgene plamid: 1864) was obtained from Addgene. Lentivirus was produced by co-transfection of pLKO.1-shFLNa with psPAX2 and pMD2.G into HEK-293T cells. Stable cell lines were obtained after infection of A549 cells with lentivirus, followed by puromycin selection in a concentration of 2 μg/l. MTT cell proliferation assay Cell proliferation was determined by MTT proliferation assay. In brief, 6 × 103 A549 cells per well were seeded into 96-well plates. Cells were starved by serum deprivation for 4 h, and then treated with EGF at a concentration of 0 nM, 4 nM, and 20 nM for 48 h, respectively. A total of 20 μl MTT reagent (Sigma-Aldrich, St Louis, USA; 5 mg/ml) was added to each well and incubated for 4 h. Then the culture medium was discarded and 150 μl DMSO was added to each well. The absorbance was measured with a microplate reader at wavelength of 490 nm. Immunoprecipitation assay To purify endogenous EGFR, immunoprecipitation (IP) assay was performed. A549 cells were lysed with RIPA ice-cold buffer. The protein concentration was measured by BCA assay. Lysates (500 μg) were incubated with EGFR antibody or relevant control IgG overnight at 4°C. Then Protein A/G agarose beads (Thermo Fisher Scientific, Waltham, USA) were added into the lysates and incubated for 2 h. The beads were washed five times with RIPA buffer. Finally, samples on the beads were denatured with SDS-PAGE loading buffer and subject to SDS-PAGE separation and immunoblotting. Scratch wound-healing assay FLNa silenced or the relative control A549 cells were seeded in 6-well plates. The confluent cell monolayers were scratched with a pipette tip and the plates were washed twice with PBS buffer before addition of fresh medium. Then cell migration was observed and measured at the indicated time intervals. Each assay was repeated three times. Migration assay The migration assay was performed by using a Transwell chamber (Corning Co., Corning, USA). FLNa silencing and the relative control cells were collected and transferred to the chamber inserts in serum-free medium. The bottom chamber contained medium with 10% FBS as a chemoattractant. The cells were cultured in a humidified incubator at 37°C for 24 h. The cells that migrated to the underside of the filter were fixed and stained with crystal violet and counted by using bright-field microscopy. Invasion assay A549 cells were seeded onto filters of a 24-well transwell chamber that was coated with Matrigel (BD Biosciences, Franklin Lakes, USA). Invasion of the cells through the Matrigel to the underside of the filter was assessed 24 h later by fixation and staining with crystal violet. The cells were counted by using bright-field microscopy. Tumorigenesis study BALB/c-nu mice (5–6 weeks of age, 18–20 g; Vital River Laboratory Animal Technology, Beijing, China) were housed in sterile filter-capped cages. A total of 1 × 106 cells in 100 μl PBS were injected subcutaneously into the backs of the mice. Tumor volume was calculated by the formula of (length × width2 × 0.52). Tumor size was measured at the indicated time intervals with calipers from the time of the formation of palpable tumors. Four weeks after implantation, the mice were euthanized, and the tumors were surgically dissected. The tumor specimens were fixed with 4% paraformaldehyde. Samples were then processed for histopathological examination. Immunohistochemical staining Immunohistochemical (IHC) staining of paraffin-embedded tissues was performed according to standard procedures that described previously [27]. Mouse monoclonal FLNa antibody was obtained from Santa Cruz and used in a dilution of 1:100. Anti-Ki67 antibody was purchased from Abcam (Cambridge, UK) and used in a dilution of 1:100. Rabbit monoclonal Phospho-EGF receptor (Tyr1068) (D7A5) was obtained from Cell Signaling Technology and used in a dilution factor of 1:100. Statistical analysis Data were expressed as the mean ± SD, and the differences between any two groups were compared by t-tests. Statistical analyses were performed by SPSS software (SPSS Software, Chicago, USA). P < 0.05 was considered significant difference. Results Silencing FLNa enhances proliferation capacity of A549 cells To assess the role of FLNa in the proliferation of lung adenocarcinoma cell line A549 cells, lentivirus-mediated silencing of FLNa expression in A549 cells was carried out. The effect of silencing was determined by immunoblotting and quantitative real-time PCR analysis. As demonstrated, FLNa was effectively silenced in A549 cells as reflected by immunoblotting (Fig. 1A,B). Moreover, real-time PCR results validated the silencing effect (Fig. 1C). Further MTT proliferation assay reflected that silencing of FLNa enhanced growth capacity of A549 cells with or without EGF stimulation (Fig. 1D). Figure 1. View largeDownload slide Silencing FLNa enhances proliferation capacity of A549 cells A549 cells were transfected with either control or FLNa shRNA plasmids and selected by puromycin. Cell lysates and total RNA were prepared to detect the expression of FLNa by western blot analysis (A) and quantitative real-time PCR (C), respectively. (B) Bars represent ratio of FLNa density to β-actin. (D) Knockdown of FLNa enhances proliferation capacity in A549 cells. *P < 0.05 vs. control A549 cells and **P < 0.01 vs. control A549 cells. Figure 1. View largeDownload slide Silencing FLNa enhances proliferation capacity of A549 cells A549 cells were transfected with either control or FLNa shRNA plasmids and selected by puromycin. Cell lysates and total RNA were prepared to detect the expression of FLNa by western blot analysis (A) and quantitative real-time PCR (C), respectively. (B) Bars represent ratio of FLNa density to β-actin. (D) Knockdown of FLNa enhances proliferation capacity in A549 cells. *P < 0.05 vs. control A549 cells and **P < 0.01 vs. control A549 cells. FLNa silencing promotes migration capacity of A549 cells FLNa is a well-characterized gene that regulates cell behaviors including migration, invasion, and adhesion that are associated with metastasis. To assess the contribution of FLNa to lung adenocarcinoma cell metastasis, migration, and invasion assays were performed through silencing FLNa expression. As demonstrated by scratch wound-healing assay, FLNa silencing promotes cell migration. In addition, FLNa silencing also enhances EGF-induced cell migration (Fig. 2A,B). Moreover, cell migration assay indicated that FLNa silencing could promote A549 cell migration capacity in the presence or absence of EGF (Fig. 2C,D). These results suggest that FLNa functions as a negative regulator of metastasis in A549 cells. Figure 2. View largeDownload slide FLNa silencing promotes migration capacity of A549 cells Wound-healing assay were performed in control A549 cells and FLNa-knockdown A549 cells in the absence or presence of EGF (20 nM) for the indicated time. Images were taken under an inverted microscope (A) (Magnification, ×10), and migration rate was calculated (B). Transwell chambers were used to determine migration capacity of control A549 cells and FLNa-knockdown A549 cells. Images were taken (C) and migrated cells were counted (D). **P < 0.01 vs. control A549 cells. Figure 2. View largeDownload slide FLNa silencing promotes migration capacity of A549 cells Wound-healing assay were performed in control A549 cells and FLNa-knockdown A549 cells in the absence or presence of EGF (20 nM) for the indicated time. Images were taken under an inverted microscope (A) (Magnification, ×10), and migration rate was calculated (B). Transwell chambers were used to determine migration capacity of control A549 cells and FLNa-knockdown A549 cells. Images were taken (C) and migrated cells were counted (D). **P < 0.01 vs. control A549 cells. FLNa silencing increases invasiveness of A549 cells To analyze the impact of FLNa on the invasion capacity of lung adenocarcinoma, cell invasion assay was performed. As shown by Matrigel invasion assay, FLNa silencing enhanced the invasiveness of A549 cells (Fig. 3A,B). These results further validate the suppressive function of FLNa on A549 invasiveness. Figure 3. View largeDownload slide FLNa silencing increases invasiveness of A549 cells Transwell chambers were used to determine the invasive capacity of control A549 cells and FLNa-knockdown A549 cells. Images were taken (A) and cells were counted (B). **P < 0.01 vs. control A549 cells. Figure 3. View largeDownload slide FLNa silencing increases invasiveness of A549 cells Transwell chambers were used to determine the invasive capacity of control A549 cells and FLNa-knockdown A549 cells. Images were taken (A) and cells were counted (B). **P < 0.01 vs. control A549 cells. Role of FLNa in ligand-induced EGFR phosphorylation in human A549 cells To explore the underlying molecular mechanism through which FLNa silencing induces proliferation, migration, and invasion, the effect of FLNa on EGF-induced EGFR signaling pathway activation was analyzed. As demonstrated, FLNa silencing could enhance EGF-induced activation of EGFR. EGF-induced activation of EGFR could activate the ERK signaling pathway, which promotes cancer cell proliferation, migration, and invasion. An increase in activated ERK1/2 in FLNa-silenced cells treated with EGF was also observed (Fig. 4A,C). FLNa is a scaffold protein that interacts with many transmembrane proteins, such as integrin, which results in variation in transmembrane protein signal transduction. Thus, we analyzed the impact of FLNa on phosphorylation of tyrosine on EGFR. As observed, silencing FLNa changed the phosphorylation status of tyrosine on EGFR when cells were treated with EGF (Fig. 4D). Figure 4. View largeDownload slide Role of FLNa in ligand-induced EGFR phosphorylation in human A549 cells (A) Western blot analysis was performed using antibodies against FLNa, phosphotyrosine, EGFR, phosphorylated and total ERK1/2. β-actin is the loading control. (B) Ratio of pY-EGFR to total EGFR. (C) Ratio of p-ERK to total ERK. (D) Lysates from control A549 cells and FLNa-knockdown A549 cells were immunoprecipitated (IP) with anti-EGFR antibody and analyzed by western blot analysis with antibodies against phosphotyrosine and EGFR. NI, nonimmune IgG. **P < 0.01 vs. control A549 cells. Figure 4. View largeDownload slide Role of FLNa in ligand-induced EGFR phosphorylation in human A549 cells (A) Western blot analysis was performed using antibodies against FLNa, phosphotyrosine, EGFR, phosphorylated and total ERK1/2. β-actin is the loading control. (B) Ratio of pY-EGFR to total EGFR. (C) Ratio of p-ERK to total ERK. (D) Lysates from control A549 cells and FLNa-knockdown A549 cells were immunoprecipitated (IP) with anti-EGFR antibody and analyzed by western blot analysis with antibodies against phosphotyrosine and EGFR. NI, nonimmune IgG. **P < 0.01 vs. control A549 cells. Silencing FLNa increases tumorigenic capacity of A549 cells To examine the effect of FLNa on the tumorigenic capacity of A549 cells, tumorigenesis assay was performed by injecting tumor cells subcutaneously into nude mice. It was found that silencing FLNa expression could increase tumor volume and tumor growth (Fig. 5A,B). IHC staining of the tumor samples from sacrificed mice by using anti-FLNa, anti-EGFR, and anti-Ki67 antibodies further supported our in vitro observations of the negative role of FLNa in tumor cell growth (Fig. 5C). Furthermore, immunoblotting results also demonstrated that silencing FLNa could increase the activation of EGFR and ERK1/2 (Fig. 5D). Taken together, these results are in consistent with in vitro observation, which supports the negative role of FLNa in tumor growth. Figure 5. View largeDownload slide Silencing FLNa increases tumorigenic capacity of A549 cells Tumor-bearing mice were generated by subcutaneous injection of control A549 cells and FLNa-knockdown A549 cells. Mice were sacrificed 28 days after inoculation. (A) Representative photographs of tumors after surgical resection. (B) Tumor growth in mice injected with control A549 cells and FLNa-knockdown A549 cells. Tumor volume in each group was expressed as the mean ± SD (n = 6). (C) IHC staining with antibodies against FLNa, pY-EGFR, and Ki-67 of tumor tissue sections. (D) Western blot analysis was performed with tumor lysates using antibodies against FLNa, phosphotyrosine, EGFR, phosphorylated and total ERK1/2. β-actin is the loading control. **P < 0.01 vs. control A549 cells. Figure 5. View largeDownload slide Silencing FLNa increases tumorigenic capacity of A549 cells Tumor-bearing mice were generated by subcutaneous injection of control A549 cells and FLNa-knockdown A549 cells. Mice were sacrificed 28 days after inoculation. (A) Representative photographs of tumors after surgical resection. (B) Tumor growth in mice injected with control A549 cells and FLNa-knockdown A549 cells. Tumor volume in each group was expressed as the mean ± SD (n = 6). (C) IHC staining with antibodies against FLNa, pY-EGFR, and Ki-67 of tumor tissue sections. (D) Western blot analysis was performed with tumor lysates using antibodies against FLNa, phosphotyrosine, EGFR, phosphorylated and total ERK1/2. β-actin is the loading control. **P < 0.01 vs. control A549 cells. Discussion In the present study, we have uncovered a novel function of FLNa in lung adenocarcinoma cell line A549 cells. Our in vitro and in vivo results suggest that FLNa functions as a negative regulator of proliferation and metastasis in A549 cells. Molecular mechanism studies indicate that silencing FLNa expression in A549 cells promotes the activation of EGFR signaling pathway. FLNa was initially identified as a structure protein that participates in actin cross-linking. FLNa was reported to interact with many proteins, including membrane channels, receptors, intracellular signaling molecules, and even transcription factors. Due to the diversity of FLNa-interacting partners, FLNa is involved in many intracellular physiological functions. Recently, FLNa has received much attention because of its involvement in cancers. FLNa plays dual roles in cancers, depending on its subcellular localization and interacting partners. For example, in prostate cancer and breast cancer, FLNa displays bifunctional roles, by promoting or inhibiting the growth and metastasis of these cancer cells [28–31]. In breast cancer, when interacting with caveolin1, FLNa regulates focal adhesion disassembly and suppresses breast cancer cell migration and invasion [32]. On the other hand, through interaction with cyclinD1, FLNa functions as a cancer promoting gene by promoting migrations and invasion [33]. In human lung cancer, proteomic studies indicated that FLNa expression is up-regulated during the EMT process, indicating its positive role in cell migration and invasiveness [34]. Moreover, IHC staining analysis also showed that FLNa is overexpressed in lung cancer, but no significance was observed between its expression and metastasis or prognosis indications [35]. However, few experiments were carried out in cell lines to verify the role of FLNa in lung cancer. To explore the underlying molecular mechanism, we turn to examine the impact of FLNa on the EGFR signaling pathway, a well-established pathway that contributes to cancer cell proliferation, migration, and invasion, especially in lung adenocarcinoma [36,37]. Since the discovery of EGFR, new functions of EGFR have gradually been revealed. The traffic of EGFR from cell membrane to intracellular compartments could amplify or quench EGFR signaling [38–40]. For example, EGFR can undergo endocytosis and subsequent nuclear transport through importin β1 [41]. Cellular traffic and endocytosis of EGFR depend on its interactions with structure proteins like actin and caveolin, which are well-established FLNa-interacting proteins [42,43]. These processes could result in the phosphorylation of EGFR. EGFR was reported to bind to F-actin cytoskeleton in intact cell, leading to sequestration of EGFR transduction. Based on the role of FLNa in actin cross-linking, it is conceivable that FLNa might also participate in EGFR signal transduction [44]. In support of this hypothesis, we observed the impact of FLNa on the activation status of EGFR and its downstream ERK1/2 pathway activation. The negative role of FLNa on EGFR signaling pathway accounts for its suppressive impact on A549 cell growth and metastasis. Moreover, these observations laid the foundation for further studies to uncover the role of FLNa in the trafficking of endogenous EGFR to organelles, as nuclear-localized FLNa has been proposed to possess tumor suppressive functions. The negative role of FLNa in EGFR pathway might due to its influence on EGFR trafficking or its suppression of EGFR activation in the nucleus. In summary, we have revealed a novel tumor suppressive role of FLNa in lung adenocarcinoma cell line A549 cells. Mechanism studies indicated that FLNa is involved in the activation of EGFR pathway, the key signaling event in lung adenocarcinoma proliferation, progression, metastasis, and resistance to chemotherapies (Fig. 6). This study may provide novel predictive and treatment targets for lung adenocarcinoma, and facilitate further researches to elucidate novel molecular mechanisms in lung cancer. Figure 6. View largeDownload slide Schematic representation of the working model The activation of the EGF/EGRF/ERK1/2 axis plays vital roles in the oncogenesis and progression of cancer cells. In the present study, we have identified FLNa as a negative regulator of the axis, leading to decreased metastasis, proliferation, and tumorigenesis capacity of lung cancer cell line A549 cells. Figure 6. View largeDownload slide Schematic representation of the working model The activation of the EGF/EGRF/ERK1/2 axis plays vital roles in the oncogenesis and progression of cancer cells. In the present study, we have identified FLNa as a negative regulator of the axis, leading to decreased metastasis, proliferation, and tumorigenesis capacity of lung cancer cell line A549 cells. Funding This work was supported by the grants from the National Natural Science Foundation of China (No. 81071846), Natural Science Foundation of Hebei Province of China (No. H2013505059), Department of Science and Technology of Hebei Province of China (Nos. 12396107D, 14397707D, 09966114D, and 092461102D), and Wu Jieping Foundation (Nos. 320.6750.12604, 320.6750.14063, and 320.6799.15005). References 1 Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin  2015, 65: 5– 29. Google Scholar CrossRef Search ADS PubMed  2 Travis WD. Pathology of lung cancer. Clin Chest Med  2002, 23: 65– 81. viii. Google Scholar CrossRef Search ADS PubMed  3 Spira A, Ettinger DS. Multidisciplinary management of lung cancer. N Engl J Med  2004, 350: 379– 392. Google Scholar CrossRef Search ADS PubMed  4 Sangodkar J, Katz S, Melville H, Narla G. Lung adenocarcinoma: lessons in translation from bench to bedside. Mt Sinai J Med  2010, 77: 597– 605. 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Acta Biochimica et Biophysica SinicaOxford University Press

Published: Feb 1, 2018

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