Knockdown of the EEF1A2 gene reduces lung cancer brain metastasis by downregulating the BCL10/NFκB pathway

Abstract

Aim: Brain metastases (BM) in patients with lung cancer (LC) are linked to unfavorable outcomes. The eukaryotic translation elongation factor 1 alpha 2 (EEF1A2) is notably overexpressed across various cancer types and plays a role in promoting tumor initiation and progression. This research aimed to clarify the function of EEF1A2 in the context of lung cancer brain metastasis (LCBM) and to explore the mechanisms underlying its effects.

Methods: To identify genes with differential expression between LC and LCBM samples, transcriptomic microarray analyses were conducted, confirming that EEF1A2 expression is elevated in LCBM. EEF1A2 expression levels were validated in multiple LC cell lines. PC9 and SPCA1 cells were transfected with lentiviral vectors carrying siRNAs targeting EEF1A2 to assess its role both in vitro and in vivo. Tandem mass tag proteomics was employed to identify proteins regulated by EEF1A2. The expression of EEF1A2, BCL10, and phosphorylated NF-κB in tumor tissues from LC and LCBM patients was analyzed.

Results: Compared to the LC samples, the LCBM samples exhibited significantly higher levels of EEF1A2 expression. EEF1A2 knockdown in PC9 and SPCA1 cells resulted in substantial reductions in cell proliferation, migration, and invasion. Proteomic profiling revealed that BCL10 protein levels were markedly reduced in EEF1A2-knockdown cells. Additionally, there was a decrease in phosphorylated NF-κB, EGFR, and mesenchymal markers (N-cadherin, Twist, Snail, Slug, and Cdc42), along with an increase in E-cadherin expression. In a mouse model, EEF1A2 knockdown in PC9 cells significantly inhibited brain metastasis. Furthermore, patient samples presented elevated levels of EEF1A2, BCL10, and phosphorylated NF-κB in LCBM tissues than in LC tissues.

Conclusion: Our research revealed that EEF1A2 is upregulated in LCBM, and that its knockdown suppresses brain metastasis by decreasing BCL10 expression, inhibiting NF-κB signaling, and reducing epithelial-mesenchymal transition markers. These results suggest that targeting EEF1A2 may be a promising therapeutic approach for preventing and treating brain metastasis in lung cancer patients.

Keywords

Eukaryotic translation elongation factor 1 alpha 2 (EEF1A2) / lung cancer brain metastasis / BCL10 / NFκB / epithelial-mesenchymal transition (EMT)

References

[1] Sung H,Siegel RL.Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.CA Cancer J Clin2021;71:209-49

[2] Lamba N,Aizer AA.Epidemiology of brain metastases and leptomeningeal disease.Neuro Oncol2021;23:1447-56 PMCID:PMC8408881

[3] Vogelbaum MA,Messersmith H.Treatment for brain metastases: ASCO-SNO-ASTRO guideline.J Clin Oncol2022;40:492-516

[4] Negrutskii BS.Eukaryotic translation elongation factor 1α: structure, expression, functions, and possible role in aminoacyl-tRNA channeling. In: Progress in Nucleic Acid Research and Molecular Biology. Elsevier; 1998. pp. 47-78.

[5] Dever TE,Green R.Translation elongation and recoding in eukaryotes.Cold Spring Harb Perspect Biol2018;10:a032649 PMCID:PMC6071482

[6] Anand N,Amann G.Protein elongation factor EEF1A2 is a putative oncogene in ovarian cancer.Nat Genet2002;31:301-5

[7] Pinke DE,Francetic T,Lee JM.The prognostic significance of elongation factor eEF1A2 in ovarian cancer.Gynecol Oncol2008;108:561-8

[8] Tomlinson VA,Wray NR.Translation elongation factor eEF1A2 is a potential oncoprotein that is overexpressed in two-thirds of breast tumours.BMC Cancer2005;5:113 PMCID:PMC1236916

[9] Lam DC,Suen W.Establishment and expression profiling of new lung cancer cell lines from chinese smokers and lifetime never-smokers.J Thorac Oncol2006;1:932-42

[10] Schlaeger C,Schiller C.Etiology-dependent molecular mechanisms in human hepatocarcinogenesis.Hepatology2008;47:511-20

[11] Amiri A,Jeganathan S,Pinke DE.eEF1A2 activates Akt and stimulates Akt-dependent actin remodeling, invasion and migration.Oncogene2007;26:3027-40

[12] Xu C,Zhu Q.eEF1A2 promotes cell migration, invasion and metastasis in pancreatic cancer by upregulating MMP-9 expression through Akt activation.Clin Exp Metastasis2013;30:933-44

[13] Giudici F,Nanni O.Elevated levels of eEF1A2 protein expression in triple negative breast cancer relate with poor prognosis.PLoS One2019;14:e0218030 PMCID:PMC6586289

[14] Qiu FN,Chen DY.Eukaryotic elongation factor-1α 2 knockdown inhibits hepatocarcinogenesis by suppressing PI3K/Akt/NF-κB signaling.World J Gastroenterol2016;22:4226-37 PMCID:PMC4837440

[15] Biswas DK.Linkage between EGFR family receptors and nuclear factor kappaB (NF-kappaB) signaling in breast cancer.J Cell Physiol2006;209:645-52

[16] Achrol AS,Anders C.Brain metastases.Nat Rev Dis Primers2019;5:5

[17] Bhutia YD.Glutamine transporters in mammalian cells and their functions in physiology and cancer.Biochim Biophys Acta2016;1863:2531-9 PMCID:PMC4919214

[18] Zhang B,Shi X.Regulation of branched-chain amino acid metabolism by hypoxia-inducible factor in glioblastoma.Cell Mol Life Sci2021;78:195-206 PMCID:PMC8112551

[19] Ngo B,Osorio-Vasquez V.Limited environmental serine and glycine confer brain metastasis sensitivity to PHGDH inhibition.Cancer Discov2020;10:1352-73 PMCID:PMC7483776

[20] Liu J,Li Q.Phosphoglycerate dehydrogenase induces glioma cells proliferation and invasion by stabilizing forkhead box M1.J Neurooncol2013;111:245-55 PMCID:PMC3565087

[21] Horzum U,Pesen-Okvur D.Differentiation of normal and cancer cell adhesion on custom designed protein nanopatterns.Nano Lett2015;15:5393-403

[22] Tilghman RW.Focal adhesion kinase as a regulator of cell tension in the progression of cancer.Semin Cancer Biol2008;18:45-52 PMCID:PMC2267763

[23] Lauko A,Gutmann DH,Lathia JD.Junctional adhesion molecules in cancer: a paradigm for the diverse functions of cell-cell interactions in tumor progression.Cancer Res2020;80:4878-85 PMCID:PMC7669553

[24] Hirohashi S.Cell adhesion system and human cancer morphogenesis.Cancer Sci2003;94:575-81 PMCID:PMC11160151

[25] Lafrenie RM,Bewick MA.Cell adhesion and cancer: is there a potential for therapeutic intervention?.Expert Opin Ther Targets2007;11:727-31

[26] Eke I.Focal adhesion signaling and therapy resistance in cancer.Semin Cancer Biol2015;31:65-75

[27] Patel SA,Naik M.EEF1A2 promotes HIF1A mediated breast cancer angiogenesis in normoxia and participates in a positive feedback loop with HIF1A in hypoxia.Br J Cancer2024;130:184-200 PMCID:PMC10803557

[28] Yang S,Chen Y.Overexpression of eukaryotic elongation factor 1 alpha-2 is associated with poorer prognosis in patients with gastric cancer.J Cancer Res Clin Oncol2015;141:1265-75

[29] Patel SA,Dixit M.Oncogenic activation of EEF1A2 expression: a journey from a putative to an established oncogene.Cell Mol Biol Lett2024;29:6 PMCID:PMC10765684

[30] Duanmin H,Qi Z.eEF1A2 protein expression correlates with lymph node metastasis and decreased survival in pancreatic ductal adenocarcinoma.Hepatogastroenterology2013;60:870-5

[31] Sun Y,Guan Y.The eukaryotic translation elongation factor eEF1A2 induces neoplastic properties and mediates tumorigenic effects of ZNF217 in precursor cells of human ovarian carcinomas.Int J Cancer2008;123:1761-9 PMCID:PMC2606039

[32] Jia L,Du C.EEF1A2 interacts with HSP90AB1 to promote lung adenocarcinoma metastasis via enhancing TGF-β/SMAD signalling.Br J Cancer2021;124:1301-11 PMCID:PMC8007567

[33] Thornton S,Purcell D.Not just for housekeeping: protein initiation and elongation factors in cell growth and tumorigenesis.J Mol Med2003;81:536-48

[34] Mendoza MB,Ortiz R.The elongation factor eEF1A2 controls translation and actin dynamics in dendritic spines.Sci Signal2021;14:eabf5594

[35] Bodman JAR,Logan MR.Yeast translation elongation factor-1A binds vacuole-localized Rho1p to facilitate membrane integrity through F-actin remodeling.J Biol Chem2015;290:4705-16 PMCID:PMC4335209

[36] Li Z,Shin DM.Eef1a2 promotes cell growth, inhibits apoptosis and activates JAK/STAT and AKT signaling in mouse plasmacytomas.PLoS One2010;5:e10755 PMCID:PMC2873962

[37] Pellegrino R,Neumann O.EEF1A2 inactivates p53 by way of PI3K/AKT/mTOR-dependent stabilization of MDM4 in hepatocellular carcinoma.Hepatology2014;59:1886-99 PMCID:PMC4115286

[38] Willis TG,Du MQ.Bcl10 is involved in t(1;14)(p22;q32) of MALT B cell lymphoma and mutated in multiple tumor types.Cell1999;96:35-45

[39] Ekambaram P,Hubel NE.The CARMA3-Bcl10-MALT1 signalosome drives NFκB activation and promotes aggressiveness in angiotensin II receptor-positive breast cancer.Cancer Res2018;78:1225-40 PMCID:PMC6436094

[40] Holzmann K,Schwaenen C.Genomic DNA-chip hybridization reveals a higher incidence of genomic amplifications in pancreatic cancer than conventional comparative genomic hybridization and leads to the identification of novel candidate genes.Cancer Res2004;64:4428-33

[41] Yeh PY,Yeh KH.A pathway for tumor necrosis factor-alpha-induced Bcl10 nuclear translocation. Bcl10 is up-regulated by NF-kappaB and phosphorylated by Akt1 and then complexes with Bcl3 to enter the nucleus.J Biol Chem2006;281:167-75

[42] Wang D,Lin PC.Bcl10 plays a critical role in NF-kappaB activation induced by G protein-coupled receptors.Proc Natl Acad Sci USA2007;104:145-50

[43] Jiang T,Zhu Y.CARMA3 is crucial for EGFR-Induced activation of NF-κB and tumor progression.Cancer Res2011;71:2183-92 PMCID:PMC3059846

[44] Martin D,Gutkind JS.CXCL8/IL8 stimulates vascular endothelial growth factor (VEGF) expression and the autocrine activation of VEGFR2 in endothelial cells by activating NFkappaB through the CBM (Carma3/Bcl10/Malt1) complex.J Biol Chem2009;284:6038-42 PMCID:PMC2649103

[45] Staudt LM.Oncogenic activation of NF-κB.Cold Spring Harb Perspect Biol2010;2:a000109 PMCID:PMC2869521

[46] Mirzaei S,Bassiri F.NF-κB as a regulator of cancer metastasis and therapy response: a focus on epithelial-mesenchymal transition.J Cell Physiol2022;237:2770-95

[47] Thiery JP.Complex networks orchestrate epithelial-mesenchymal transitions.Nat Rev Mol Cell Biol2006;7:131-42

[48] Grünert S,Beug H.Diverse cellular and molecular mechanisms contribute to epithelial plasticity and metastasis.Nat Rev Mol Cell Biol2003;4:657-65

[49] Yilmaz M.EMT, the cytoskeleton, and cancer cell invasion.Cancer Metastasis Rev2009;28:15-33

[50] Yang S,Li MY.FOXP3 promotes tumor growth and metastasis by activating Wnt/β-catenin signaling pathway and EMT in non-small cell lung cancer.Mol Cancer2017;16:124 PMCID:PMC5514503