DOI: https://doi.org/10.21203/rs.2.10348/v1
Non-small cell lung cancer (NSCLC) is a major cause of cancer-related death all over the world with poorer five-year survival rate in patients [1, 2]. In fact, cancer metastasis caused more than 90% of deaths from solid tumor, including lung cancer [3]. Hence, it is critical to understand the mechanisms of NSCLC metastasis for improving survival rate of patients.
Epithelial mesenchymal transition (EMT) is essential in embryonic development and transformation of early-stage tumors into invasive malignancies [4, 5]. There is plenty of evidence that TGF-β1 signaling is a potent inducer of EMT in various cancers, including NSCLC [6-8]. It is well known that the transforming growth factor β1 (TGF-β1) superfamily plays crucial roles in cell differentiation, proliferation, apoptosis and angiogenesis [9-11]. In the canonical TGF-β signaling pathway, TGF-β binds tightly to TGF-β receptorⅡ(TGFβR2) on the cell membrane to form a complex and recruits TGF-β receptor I (TGFβR1) causes its phosphorylation. Activated TGFβR1 phosphorylates SMAD2 and SMAD3, and phosphorylated Smad2/3 forms a transcriptional complex with Smad4 into the nucleus to regulate the transcription of specific target genes. [12]. TGF-β plays a dual role in tumor progression, it inhibits tumor growth in the early stage and promotes tumor metastasis and invasion by inducing EMT in the late stage. [13, 14]. Recently, a study demonstrated that Neuropilin-1 (NRP1) acts as a TGF-β1 co-receptor and activates latent TGF-β1 and respond in breast cancer [15]. In line with the report above, Kwiatkowski and his colleagues showed that NRP1 acts as a co-receptor with TGFβR2 to enhance TGF-β1 receptor signaling via Smad3 in Glioblastoma [16]. Thus, the relationship between NRP1 and TGF-β signaling pathways in NSCLC requires to be verified.
The neuropilins (NRPs) are involved in multiple processes of cellular biological function, such as immunity, cell development, and cancer. NRP1 and its homolog neuropilin-2 (NRP2) are co-receptor that bind to and interact with a variety of growth factors. [17, 18]. NRP1 is a transmembrane glycoprotein that binds to various extracellular ligands, including class III/IV semaphorins [19], certain isoforms of vascular endothelial growth factor (VEGF) [20], TGF-β1 [15], and platelet-derived growth factor (PDGF) [21]. Our previous study showed that NRP1 is up-regulated in NSCLC tissues and associated with poorer survival in patients. Meanwhile NRP1 can promote NSCLC cells proliferation and migration via EGFR signaling pathway [22]. Taken together, we hypothesized that dysregualted NRP1 can influence TGF-β1-induced EMT.
In this study, we investigated the function of NRP1 in regulation of TGF-β1-induced EMT and NSCLC cell migration and invasion. We first observed up-regulated expression of NRP1 in metastatic NSCLC tissues. In addition, we established A549 and H226 cell lines with stable knockdown of NRP1. Then transwell assays indicated that knockdown of NRP1 suppressed TGF-β1-induced migration and invasion in NSCLC cells. Our findings demonstrate that repression of NPR1 inhibits TGF-β1-induced EMT in NSCLC.
Fifty-five NSCLC patient tissues and corresponding para-carcinoma lung tissues were collected between 2012 and 2016 at the respiratory department of First people’s Hospital of Soochow University. All participants have been offered with the written informed consent at recruitment. According to the Revised International System for Staging Lung Cancer, all cases have clinically and pathologically confirmed who had not received any other treatment like radiotherapy or chemotherapy before tissue sampling. The tissue samples were frozen at -80°C for storage. This study was approved by the Ethics Committee of the First Affiliated Hospital of Soochow University.
A549 and H226 were obtained from the Cell bank of the Chinese Academy of Sciences (Shanghai, China) and grown in RPMI1640 medium (Hyclone, South Logan, UT, USA) containing 1% Penicillin-Streptomycin (Invitrogen, Carlsbad, CA, USA) and 10% heat-inactivated fetal bovine serum (Gibco, Carlsbad, CA, USA). All cells were cultured in a humidified incubator containing 5% CO2 at 37˚C. In some conditions, cells were exposed in 5ng/ml TGF-β1, 3μM SIS3 for 48h for further experiment. NRP1 knockdown of A549 and H226 cells were cultured in same medium supplemented 0.5μg/ml puromycin. In addition, NRP1 overexpressed cells were grown with medium containing G418 for positive selection.
The human NRP1 specific small interfering RNA fragment (NRP1 shRNA-1: 5′-CCAUACCAGAGAAUUAUGATT-3′; NRP1shRNA-2: 5′-GUAUACGGUUGC
AAGAUAATT-3′) were cloned into the lentiviral vector pGMLV-SC5 –Puro (GenePharma, Shagnhai, China) containing the endonucleases EcoR1 and BamH1.Then we co-transfected pGMLV-SC5 –Puro vector along with packaging plasmids into 293T cells by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). A549 and H226 cells were infected with the packaged lentiviruses at a multiplicity of infection (MOI) of 25 along with 8ug/l polybrene as coadjutant and cultured for 2 days , and cells being selected with 0.4 μg/ml of puromycin (Sigma-Aldrich, St Louis, MO, USA). The Transfection efficiency was evaluated by later western blot and qRT-PCR analysis.
For plasmid construction, human NRP1 CDS fragment was cloned into PLVX-IRES-Neo vector between EcoR1 and Xbal by Genewiz. H226 and A549 cells were transfected with UPLVX-IRES-Neo-vector or PLVX-IRES-Neo-NRP1 by Lipofectamine 2000 (Invitrogen) and further cultured for2 days before being selected with G418 reagent (Sigma-Aldrich, St Louis, MO, USA).The NRP1-overexpreeion efficiency was evaluated by later qPCR and western blot analysis.
After NRP-knockdown A549 and H226 cells stimulated with TGF-β1or NRP-overexpression tumor cells treatment with SIS3 for 48h, cells were suspended and reseeded into a 6-well plate. At day 2, when cells grown to 80-90% as a monolayer, we gently scratched the monolayer with 10µl pipette tip. The most important thing is to ensure the tip was perpendicular to the bottom of the plate during the operation. The detached cells were removed by washing with PBS gently two times after scratching. Add refresh medium and continue to culture for 24 h. Observe the gap distance with a microscope and take photos, quantitatively evaluate the gap distance with Photoshop.
2.6 Migration and invasion assay
Cell migration and invasion assays were performed with Transwell chambers (Corning, New York, NY, USA). For migration assay, cells were suspended and plated on chambers that were not coated with Matrigel matrix (BD Science, Sparks, MD, USA). For invasion assay, cells were suspended and plated on chambers pre-coated with Matrigel matrix at 37˚C for 2 h first. After NRP-knockdown tumor cells stimulated with TGF-β1 or NRP-overexpression tumor cells treatment with SIS3 for 48h. 800 µl RPMI1640 medium containing 10% FBS was added into each bottom chamber, meanwhile cells were collected and 5x104 cells diluted in 200ul medium containing 1% FBS were seeded into the upper chamber. After incubation for 24h, cells were fixed with methanol for 30 min and non-invasive cells were removed, air-dried for 15 min, the left cells were stained with 0.1% crystal violet for 1 h and washed with PBS three times. In the end, the cells were photographed and counted. Each experiment was performed in triplicate.
Using cold PBS to wash Cells two times and then lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) containing phosphatase inhibitor and protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). 10% SDS-PAGE was used to separate proteins which then transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA) , the immunoblots were blocked with 5% Skim milk in TBST buffer with 0.1% Tween-20 for 1 h at room temperature and then incubated with corresponding primary antibodies overnight at 4°C and the appropriate secondary antibodies. Two hours later, we performed our detection by chemiluminescence (Pierce, Rockford, IL, USA) after washing three times with TBST. All antibodies used in this research including NRP1(sc-5307, santa cruze biotechbology), anti-p-Smad3(Ser423/425), anti-Smad3(C67H9), anti-Snail (C15D3), anti-MMP2 (D8N9Y), and anti-MMP9 (603H) (Cell Signaling Technology, Danvers, MA, USA), anti-N-cadherin, anti-Vimentin (RV202) (BD Biosciences, USA), anti-β-actin and anti-mouse or anti-rabbit secondary antibodies (Cell Signaling Technology).
To establish experimental lung metastasis model, we resuspended cells in PBS (1x106 cells/100μL /mouse) and injected cells into each mouse (6 weeks old, female, BALB/c,) via the tail vein on day 0. The mice were then injected with TGF-β1 drugs（400ng/μL）into their abdominal cavity every five days, and the total injection was 5 times. All mice were sacrificed 50 days after tail vein injection, and the numbers of pulmonary metastasis nodules were counted under microscope after the appropriate tissues were stained with hematoxylin and eosin (H&E).
All results we obtained are presented as mean ± standard deviation (SD). Statistical significance was analyzed with Student's ttest and P < 0.05 was regarded as significant. All statistical analyses were performed using SPSS 7.0 software (SPSS, Chicago, IL, USA) and GraphPad Prism 7.0 (GraphPad, San Diego, CA, USA).
We first elucidate the role of NRP1 in NSCLC metastasis. A549 and H226 cell lines were stably knocked down NRP1. The mRNA and protein expression of NRP1 was significantly reduced in A549 and H226 cells transfected with two NRP1 short hairpin RNAs (shRNAs) compared with control group. (Fig. 1A), and knockdown of NRP1 (sh-NRP1) significantly inhibited the expression of Snail, N-cadherin, Vimentin, MMP2 and MMP9. Next, the effect of NRP1 on migration in NSCLC cells was evaluated by wound healing assay. The A549 and H226 cells transfected with sh-NRP1 migrated towards the scratch more slowly than the control cells (Fig. 1B). Further, transwell assays indicated that knockdown of NRP1 considerably inhibited the migration and invasion in NSCLC cells (Fig. 2C).
To further investigate the role of NRP1 in NSCLC cells, we also established NRP1 stably over-expressed A549 and H226 cell lines (Fig. 2A). The mRNA and protein expression levels of NRP1 were increased in stable NRP1-overexpressing A549 and H226 cells compared to control groups. Moreover, the results of transwell and wound healing assays indicated that overexpression of NRP1 promoted the migration and invasion in A549 and H226 cells (Fig. 2B-C).
To explore the mechanism of NRP1 promoting the metastasis of NSCLC. We injected stable NRP1-knockdown A549 cells and negative control cells into BALB/c athymic nude mice via the tail vein. As shown in Figure 3A-B, the pulmonary metastasis nodules in mice injected with NRP1-knockdown A549 cells were fewer than control group. Further, we classified 55 lung tissues according to presence or absence of lymph node metastasis and compared the expression of NRP1 mRNA (additional file1, Table S1). We found that mRNA level of NRP1 was up-regulated in NSCLC tissues associated with lymph node metastasis compared to NSCLC tissues without metastasis (Fig. 3C). These results indicate that the expression of NRP1 may contribute to tumor metastasis in NSCLC tissues. Consistent with the hypothesis that NRP1 was correlated with TGF-β1 signaling or several EMT transcription factors in advanced lung cancer. From public data deposited in LinkedOmics (http://www.linkedomics.org/login.php), NRP1 was correlated with SNAI1, SNAI2, MMP2 and TGFBR2 (Fig. 3D-G). Moreover, as a co-receptor of TGF-β1, NRP1 can modulate TGF-β1 receptor signaling in various cell types [21, 16]. In present study, we observed that the interaction between NRP1 and TGFβRⅡ by co-immunoprecipitation experiments (Fig. 3H), this finding was also reported by Kwiatkowski et al [15].
Next, the function of NRP1 in TGF-β1-induced metastasis in NSCLC was confirmed. The wound healing assay showed that the speed was inhibited in cells stimulated with exogenous TGF-β1 moved towards the scratch in sh-NRP1 group than control group in both A549 and H226 cell lines (Fig. 4C). The transwell assay further indicated that the increased migratory and invasive abilities of cells with exogenous TGF-β1 stimulation for 24h were considerably suppressed in cells with stable knockdown of NRP1 when compared to control group (Fig. 4D-E). In consistence with the phenotype, western blot analysis was conducted to clarify the underlying mechanism. We can obviously find that the TGF-β1-induced increase in the p-Smad3 level was inhibited in the stable cell line with NRP1 knockdown. In addition, the downstream signaling molecules associated with MMPs family like MMP2, MMP9 and EMT markers like Snail, N-cadherin, Vimentin showed similar tendency (Fig. 4A-B).
Based on previous data, we know that knockdown of NRP1 inhibits TGF-β1-induced cell migration and invasion in NSCLC cells. Next, we further validated the association between NRP1 and TGF-β/Smad3 pathway in NRP1 overexpressed cell lines. SIS3 is a permeable, selective Smad3 inhibitor which can inhibit the activation of TGF-β1/Smad3 signaling pathway and thus inhibits cell metastasis. Data from the wound healing assay showed that treatment with SIS3 can slow down the speed in both control and NRP1 overexpressed cells which moved towards the scratch. However, the inhibitory trend was weakened in NRP1 overexpressed cells compared to control cells (Fig. 5C). Transwell assays showed that overexpression of NRP1 attenuated the inhibitory effect of SIS3 on cell migration and invasion compared with the control group (Fig. 5D-E). And western blot analysis confirmed that overexpression of NRP1 noticeably inhibited the SIS3-induced downregulation of p-Smad3, Snail, MMP2, MMP9, N-cadherin and Vimentin in A549 and H226 cells (Fig. 5A-B).
Neuropilins are a class of cell surface glycoprotein, which consist of two family members NRP1 and NRP2 .The extracellular structure of NRP1 can be divided into three individual components. The a1/a2 domains can function as cubilin homology domain, b1/b2 domain contained TGF-β1 binding site and thus can function as a co-receptor for TGF-β1. In addition to TGF-β1, the b1 domain contained a negatively charged cleft, which may account for other ligands or receptors to bind to NRP1 like VEGF and its receptor, hepatocyte growth factor (HGF) and its receptor and so on. And c domain is also named as A5-protein [23-25]. Neuropilins are originally implicated in axon guidance and vascular development on the basis of interaction with semaphorins and VEGF family [23]. Later in development, NRP1 is reported to be frequently up-regulated in human cancer tissues and functions to contribute to tumor progression via interaction with various extracellular growth factor and its receptors [26-28]. On the one hand, NRP1 has been reported to modulate tumor microenvironment, particularly in regulating the function of dendritic cells (DCs) and regulatory T cells (Tregs) [29, 30].
EMT plays a vital role in tumor metastasis, accompanied by the up-regulated expression of N-cadherin and Vimentin, meanwhile the expression level of E-cadherin is down-regulated [31]. TGFβ/Smad signaling pathway is an important driver in promoting EMT process via activating canonical pathway like Smad family members or non-canonical signaling molecules like Rho kinase [32, 33]. TGF-β1-induced EMT has been implicated in diabetic kidney diseases, fibrosis phenotype and tumor cell metastasis [34-36]. And the functional associated research between NRP1 and TGFβ1-induced EMT has been carried out recently. In the immune system, NRP1 can activate TGFβ latent form to promote regulatory T cell activity [37]. In addition, small molecule NRP1 antagonists can block TGFβ1 production in regulatory T cells [38]. In tumor cells, NRP1 expression is upregulated along with GBM tumor progression. Meanwhile NRP1 can bind with TGFβR2 to activate Smad3 signaling to drive GBM development and the TGFβ/Smad3 signaling is NRP1 dependent during the process [16]. In the central nervous system, NRP1 can balance integrin β8-activated TGFβ signaling to control sprouting angiogenesis [39]. In breast cancer, NRP1 can collaborate with TGFβR1 to capture and activate (LAP)-TGF-β1 [15]. However functions for NRP1 in modulating TGFβ signaling in NSCLC cells have not been extensively investigated.
In current study, NRP1 is identified to be highly expressed 55 paired tissues when compared with corresponding noncancerous lung tissues from patients. After we pay our attention to lymph node metastasis subgroup, it is interesting to demonstrate that NRP1 expression is higher in metastatic NSCLC tissues. Public database also showed that NRP1 in positively correlated with snail, slug, MMP2 and TGFBRⅡin 515 patients samples. In vitro, we constructed NRP1-stable knockdown cell lines and made transwell and would healing assays. Data indicated that NRP1 overexpression can promote the migratory and invasive abilities of NSCLC cells. We can also observe less pulmonary metastasis nodules in the metastasis model injected with NRP1-silenced cells than control cells. Research into mechanism suggested that snail, N-cadherin, Vimentin, MMP2 and MMP9 was changed, displaying that NRP1 can induce EMT to promote NSCLC metastasis.
Given that NRP1 can induce EMT in NSCLC cell metastasis, we next analyzed links between NRP1 and TGF-β1-induced EMT following activating or blocking TGF-β1 /Smad3 pathway. First, we made verification in stable NRP1-knockdown cell lines with exogenous TGF-β1 stimulation. In agreement with previous findings, the transwell assay and wound healing assay indicated that the increased migratory and invasive abilities of cells with exogenous TGF-β1 stimulation were considerably suppressed in cells with stable knockdown of NRP1 when compared to control group. And the inside mechanism also showed that TGF-β1-induced increase in the p-Smad3 level was inhibited in the stable cell line with NRP1 knockdown. In contrast, cells with NRP1 overexpression were treated with SIS3 inhibitor to block Smad3 phosphorylation. SIS3 treatment blocked TGF-β1 signaling in NSCLC cells and also partially diminished TGF-β1-induced EMT and metastasis in NSCLC cells with NRP1 overexpression compared to NC group. In addition, the downstream signaling molecules associated with MMPs family like MMP2, MMP9 and EMT markers like Snail, N-cadherin, Vimentin showed similar tendency. Interestingly, co-immunoprecipitation data showed that NRP1 can bind with TGF-βRⅡ in tumor cells, which may affects TGF-β1 activation and signaling.
In summary, we showed that NRP1 is overexpressed in metastatic NSCLC tissues. And that NRP1 can contribute to TGF-β1-induced EMT and metastasis in NSCLC cells. In addition, as shown in figure 6, our findings illuminate the inner binding interaction between NRP1 and TGF-βRⅡ in NSCLC metastasis, and the affinity for NRP1 to bind with TGFβRⅡ may explain, at least in part, how they contribute to cancer metastasis.
NSCLC: non-small cell lung cancer; EMT: epithelial mesenchymal transition; TGF-β1: transforming growth factor β1; NRPs: neuropilins; NRP1: Neuropilin-1; NRP2: neuropilin-2; VEGF: vascular endothelial growth factor; PDGF: platelet-derived growth factor; HGF: hepatocyte growth factor; DCs: dendritic cells; Tregs: regulatory T cells.
All participants were provided with written informed consent at the time of recruitment. And this study was approved by the Ethics Committee of the First Affiliated Hospital of Soochow University.
Not applicable.
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
The authors declare that they have no competing interests.
This work was supported by grants from the Jiangsu Provincial Medical Youth Talent (No. QNRC2016746), the Science and Technology Development Foundation of Nanjing Medical University (No. 2016NJMU017), the Postgraduate Research & Practice Innovation Program of Jiangsu Province (No. KYCX18_2525), the Suzhou Key Laboratory for Respiratory Medicine (No. SZS201617), the Clinical Medical Center of Suzhou (No. Szzx201502), the Jiangsu Provincial Key Medical Discipline (No. ZDXKB2016007). The funding sources played no role in the design of the study and collection, analysis, and interpretation of data, nor in the writing of the manuscript.
ZLD, WWD, and ZL performed the experiments. JJZ and YYZ collected the patient’s data, and provided pathologic evaluation. YZ and SJW analyzed all data. ZYL and JAH contributed to the design of this work and ZYL participate in drafting this article. All authors read and approved the final manuscript.
We thank all patients who participated in this study for their cooperation.
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Additional file 1: Table S1. Clinicopathological features of NSCLC tissues from 27 patients with non-lymph node and metastasis and 28 patients with lymph node and metastasis.