# Oncogenic EFNA4 Amplification Promotes Lung Adenocarcinoma Lymph Node Metastasis
## 5 Conclusions  
This study provides new insights into the link between EFNA4 and lung cancer metastasis. The amplified and up-regulated gene _EFNA4_ is closely associated with lymph node metastasis. Additionally, we experimentally show that EFNA4 promotes the growth and migration of lung cancer cells, and knockout of _EFNA4_ leads to the inhibition of lung tumor cell proliferation and migration. Based on the prognosis and survival analysis, EFNA4 would be a novel biomarker to predict the progression of lung cancer patients. Together, our work suggests that EFNA4 may be a potential therapeutic target and prognostic marker in lung cancer.  
**Supplementary Materials:** The following supporting information can be downloaded at: [[https://www.mdpi.com/article/10.3390/cancers14174226/s1](https://www.mdpi.com/article/10.3390/cancers14174226/s1)]([https://www.mdpi.com/article/10.3390/cancers14174226/s1](https://www.mdpi.com/article/10.3390/cancers14174226/s1)), Figure S1: 1q21.3 amplification in metastatic lung cancer; Figure S2: EFNA4 expression level is associated with cell adhesion; Figure S3: RT-qPCR was utilized to detect expression levels of EFNA4 in tumor xenografts derived from EFNA4 over-expressing or control A549 cells; Figure S4: Knockout EFNA4 in lung cancer cells using CRISPR/Cas9; Figure S5: Downregulation of EFNA4 inhibits the growth and migration of lung tumor cells; Figure S6: EFNA4 is located on the cell membrane in LUAD.  
**Author Contributions:** Conceptualization, X.-S.L. and X.Z.; methodology, Y.C. and X.Z.; software, Y.C. and X.Z.; validation, Y.C. and X.Z.; formal analysis, X.Z. and Y.C.; investigation, X.Z., Y.C., X.S., Z.H., T.W., C.W., J.C., J.W. and K.D.; resources, X.-S.L.; data curation, X.Z. and Y.C.; writing--original draft preparation, X.Z., Y.C. and X.-S.L.; writing--review and editing, X.Z., Y.C. and X.-S.L.; visualization, X.Z. and Y.C.; supervision, X.-S.L.; project administration, X.-S.L.; funding acquisition, X.-S.L. All authors have read and agreed to the published version of the manuscript.  
**Funding:** This work was funded in part by Shanghai Science and Technology Commission (21 ZRI442400), National Natural Science Foundation of China (31771373), and startup funding from ShanghaiTech University.  
**Institutional Review Board Statement:** All mouse studies were carried out in strict accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) at the School of Life Science and Technology, ShanghaiTech University (IACUC No.: 20200918002).  
**Data Availability Statement:** Clinical data, CNA data and mRNA expression data were downloaded from TCGA data portal ([[https://portal.gdc.cancer.gov/](https://portal.gdc.cancer.gov/)]([https://portal.gdc.cancer.gov/](https://portal.gdc.cancer.gov/)) (accessed on 8 October 2021). IHC images were downloaded from HPA database (www:proteinatlas.org (accessed on 10 June 2022). A subset of the clinical data included in the manuscript is publicly available at NCBI GEO: [[https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE41271](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE41271)]([https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE41271](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE41271)) (accessed on 10 June 2022) and [[https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE11969](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE11969)]([https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE11969](https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE11969)) (accessed on 10 June 2022).  
**Acknowledgments:** We thank ShanghaiTech University High Performance Computing Public Service Platform for computing services. We are greatly indebted for the invaluable and timely technical support from the Molecular Imaging Core Facility, the Molecular and Cell Biology Core Facility, and the Multi-Omics Spectrometry Core Facility in the School of Life Science and Technology at ShanghaiTech University.  
**Conflicts of Interest:** The authors declare no conflict of interest.