ZEB2 upregulation modulates the polarization of TAMs toward the immunosuppressive state in EGFR-TKI-resistant NSCLC

Abstract

Aim: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment approach for NSCLC. However, the effectiveness of ICI therapy in patients with EGFR-driven NSCLC, particularly those resistant to EGFR-TKI, has been disappointing. The immunosuppressive tumor microenvironment (TME) following EGFR-TKI therapy has been proved to significantly affected the effectiveness of ICIs. Therefore, studying the mechanism behind the development of a suppressive TME and exploring potential interventions is crucial for research on EGFR-TKI-resistant NSCLC.

Methods: ZEB2 levels were quantified in human NSCLC cell lines and in tumor specimens from NSCLC patients by quantitative RT-PCR (qRT-PCR), WB, and immunohistochemical staining. To examine how ZEB2 affected macrophage polarization, M1/M2 marker profiles were measured with qRT-PCR and flow cytometry. Changes in cytokine production triggered by altered ZEB2 expression were determined with qRT-PCR, ELISA, and Meso Scale Discovery electrochemiluminescence assays. The direct binding of ZEB2 to cytokine-gene promoters was tested using a dual-luciferase reporter system. Upstream regulatory pathways were investigated by correlating LUAD transcriptomic data from TCGA with ZEB2 expression and validating key findings via western blotting. Finally, cell-derived xenograft (CDX) models were generated by subcutaneously implanting pre-treated PC9 or HCC827 cells into BALB/c nude mice to verify the impact of EGFR-TKI resistance and ZEB2 on tumor-associated macrophage (TAM) polarization in vivo.

Results: It was elucidated that EGFR-TKI resistance upregulated the M2 polarization biomarkers, Arg-1 (PC9-GR: P < 0.01; HCC827-GR: P < 0.05) and IL4 (PC9-GR: P < 0.01; HCC827-GR: P < 0.01), while downregulated the M1 polarization biomarkers, TNF-α (PC9-GR: P < 0.01; HCC827-GR: P < 0.01), IL1β (PC9-GR: P < 0.01; HCC827-GR: P < 0.01), and IL6(PC9-GR: P < 0.001; HCC827-GR: P < 0.001) in NSCLC cell lines. Meanwhile, CD206⁺ TAMs (PC9-GR: P < 0.05; HCC827-GR: P < 0.01) were increased and CD86⁺ TAMs (PC9-GR: P < 0.05; HCC827-GR: P < 0.05) were decreased in both EGFR-TKI-resistant mice models. Apart from the formation of suppressive TME, ZEB2 was found to be upregulated in PC9-GR (qRT-PCR: P < 0.0001; WB: P < 0.05) and HCC827-GR (qRT-PCR: P < 0.0001; WB: P < 0.05) cells. The same trend was also noticed in clinical samples, with ZEB2 upregulated after gefitinib resistance in NSCLC patients (P < 0.0001). Based on these findings, ZEB2 knockdown was proved to downregulate Arg-1 (PC9-GR: P < 0.01; HCC827-GR: P < 0.05) and IL4 (PC9-GR: P < 0.01; HCC827-GR: P < 0.001), while upregulate the TNF-α (PC9-GR: P < 0.0001; HCC827-GR: P < 0.0001), IL1β (HCC827-GR: P < 0.001), and IL6 (PC9-GR: P < 0.01; HCC827-GR: P < 0.001), indicating its role in M1/M2 polarization in both EGFR-TKI-resistant NSCLC cell lines. The downregulation of CD206⁺ TAMs (PC9-GR: P < 0.05; HCC827-GR: P < 0.01) and the upregulation of CD86⁺ TAMs (PC9-GR: P < 0.001; HCC827-GR: P < 0.05) also demonstrated the reversion of suppressive TME after ZEB2 knockout in EGFR-TKI-resistant mice models. Additionally, after the intervention of MK2206, which was an Akt inhibitor, ZEB2 expression was suppressed at both low (PC9-GR: P < 0.001; HCC827-GR: P < 0.001) and high concentrations (PC9-GR: P < 0.001; HCC827-GR: P < 0.0001). Finally, the mechanism underlying ZEB2’s regulation on TAM polarization was proved to be associated with cytokine secretion. According to the results of ELISA, apart from its inducement on TGF-β1 secretion (PC9-GR: P < 0.0001; HCC827-GR: P < 0.0001), ZEB2 could directly bind to the promoter region of CSF-1 to elevate its secretion (PC9-GR: P < 0.0001; HCC827-GR: P < 0.0001).

Conclusion: In EGFR-TKI-resistant NSCLC, activation of the PI3K-Akt cascade drove a marked rise in ZEB2 expression. The elevated ZEB2 increased CSF-1 and TGF-β1 release, steering macrophages toward an M2 phenotype while impeding M1 polarization. Accordingly, suppressing ZEB2 had the potential to reshape the TME and enhance the effectiveness of ICIs once EGFR-TKI resistance had emerged.

Keywords

EGFR-TKI resistance / tumor-associated macrophages (TAMs) / ZEB2 / tumor microenvironment (TME) / immunotherapy resistance / PI3K-Akt pathway

References

[1] Siegel RL,Jemal A.Cancer statistics, 2024.CA Cancer J Clin2024;74:12-49

[2] Chen P,Wen Y.Non-small cell lung cancer in China.Cancer Commun2022;42:937-70 PMCID:PMC9558689

[3] Thai AA,Sequist LV,Heist RS.Lung cancer.Lancet2021;398:535-54

[4] Maemondo M,Kobayashi K.North-East Japan Study GroupGefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR.N Engl J Med2010;362:2380-8

[5] Akinleye A.Immune checkpoint inhibitors of PD-L1 as cancer therapeutics.J Hematol Oncol2019;12:92 PMCID:PMC6729004

[6] Reck M,Robinson AG.KEYNOTE-024 InvestigatorsPembrolizumab versus chemotherapy for PD-L1-positive non-small-cell lung cancer.N Engl J Med2016;375:1823-33

[7] Mok TSK,Kudaba I.KEYNOTE-042 InvestigatorsPembrolizumab versus chemotherapy for previously untreated, PD-L1-expressing, locally advanced or metastatic non-small-cell lung cancer (KEYNOTE-042): a randomised, open-label, controlled, phase 3 trial.Lancet2019;393:1819-30

[8] Qin BD,Yuan LY,Ling Y.Immunotherapy-based regimens for patients with EGFR-mutated non-small cell lung cancer who progressed on EGFR-TKI therapy.J Immunother Cancer2024;12:e008818 PMCID:PMC11029279

[9] Mok T,Park K.Nivolumab plus chemotherapy in epidermal growth factor receptor-mutated metastatic non-small-cell lung cancer after disease progression on epidermal growth factor receptor tyrosine kinase inhibitors: final results of CheckMate 722.J Clin Oncol2024;42:1252-64 PMCID:PMC11095864

[10] Lai GGY,Jain A.A randomized phase 2 trial of nivolumab versus nivolumab-ipilimumab combination in EGFR-mutant NSCLC.JTO Clin Res Rep2022;3:100416 PMCID:PMC9679031

[11] Kamphorst AO,Nasti T.Rescue of exhausted CD8 T cells by PD-1-targeted therapies is CD28-dependent.Science2017;355:1423-7 PMCID:PMC5595217

[12] Overacre-Delgoffe AE,Dadey RE.Interferon-γ drives T_reg fragility to promote anti-tumor immunity.Cell2017;169:1130-1141.e11 PMCID:PMC5509332

[13] Tavazoie MF,Tanqueco R.LXR/ApoE activation restricts innate immune suppression in cancer.Cell2018;172:825-840.e18 PMCID:PMC5846344

[14] Gebhardt C,Jiang H.Myeloid cells and related chronic inflammatory factors as novel predictive markers in melanoma treatment with ipilimumab.Clin Cancer Res2015;21:5453-9

[15] De Henau O,Winkler D.Overcoming resistance to checkpoint blockade therapy by targeting PI3Kγ in myeloid cells.Nature2016;539:443-7 PMCID:PMC5634331

[16] Kaneda MM,Ralainirina N.PI3Kγ is a molecular switch that controls immune suppression.Nature2016;539:437-42 PMCID:PMC5479689

[17] Peng W,Liu C.Loss of PTEN promotes resistance to T cell-mediated immunotherapy.Cancer Discov2016;6:202-16 PMCID:PMC4744499

[18] Gainor JF,Sequist LV.EGFR mutations and ALK rearrangements are associated with low response rates to PD-1 pathway blockade in non-small cell lung cancer: a retrospective analysis.Clin Cancer Res2016;22:4585-93 PMCID:PMC5026567

[19] Rizvi NA,Snyder A.Cancer immunology. Mutational landscape determines sensitivity to PD-1 blockade in non-small cell lung cancer.Science2015;348:124-8 PMCID:PMC4993154

[20] Yarchoan M,Jaffee EM.Tumor mutational burden and response rate to PD-1 inhibition.N Engl J Med2017;377:2500-1 PMCID:PMC6549688

[21] McGranahan N,Rosenthal R.Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade.Science2016;351:1463-9 PMCID:PMC4984254

[22] Mladkova N,Brown PD,Palmer JD.Hippocampal avoidance prophylactic cranial irradiation: interpreting the evidence.J Thorac Oncol2021;16:e60-3

[23] Lin A,Meng H,Zhang J.Role of the dynamic tumor microenvironment in controversies regarding immune checkpoint inhibitors for the treatment of non-small cell lung cancer (NSCLC) with EGFR mutations.Mol Cancer2019;18:139 PMCID:PMC6745797

[24] Jia Y,Jiang T.EGFR-targeted therapy alters the tumor microenvironment in EGFR-driven lung tumors: implications for combination therapies.Int J Cancer2019;145:1432-44

[25] Poh AR.Targeting macrophages in cancer: from bench to bedside.Front Oncol2018;8:49 PMCID:PMC5858529

[26] Cavnar MJ,Kim TS.KIT oncogene inhibition drives intratumoral macrophage M2 polarization.J Exp Med2013;210:2873-86 PMCID:PMC3865475

[27] Yan K,Lu Y.Coexpressed genes that promote the infiltration of M2 macrophages in melanoma can evaluate the prognosis and immunotherapy outcome.J Immunol Res2021;2021:6664791 PMCID:PMC7959968

[28] Venugopalan A,Niu G.EGFR-targeted therapy results in dramatic early lung tumor regression accompanied by imaging response and immune infiltration in EGFR mutant transgenic mouse models.Oncotarget2016;7:54137-56 PMCID:PMC5338915

[29] Ayeni D,Kuhlmann A.Tumor regression mediated by oncogene withdrawal or erlotinib stimulates infiltration of inflammatory immune cells in EGFR mutant lung tumors.J Immunother Cancer2019;7:172 PMCID:PMC6617639

[30] Wang DH,Yoon D.Progression of EGFR-mutant lung adenocarcinoma is driven by alveolar macrophages.Clin Cancer Res2017;23:778-88

[31] Maynard A,Rotow JK.Therapy-induced evolution of human lung cancer revealed by single-cell RNA sequencing.Cell2020;182:1232-1251.e22 PMCID:PMC7484178

[32] Comijn J,Vermassen P.The two-handed E box binding zinc finger protein SIP1 downregulates E-cadherin and induces invasion.Mol Cell2001;7:1267-78

[33] Mowat DR,Cass DT.Hirschsprung disease, microcephaly, mental retardation, and characteristic facial features: delineation of a new syndrome and identification of a locus at chromosome 2q22-q23.J Med Genet1998;35:617-23 PMCID:PMC1051383

[34] Wakamatsu N,Yamada K.Mutations in SIP1, encoding Smad interacting protein-1, cause a form of Hirschsprung disease.Nat Genet2001;27:369-70

[35] Dai D,Han X.The transcription factor ZEB2 drives the formation of age-associated B cells.Science2024;383:413-21 PMCID:PMC7616037

[36] Goto M,Yoshida R.Age-associated CD4⁺ T cells with B cell-promoting functions are regulated by ZEB2 in autoimmunity.Sci Immunol2024;9:eadk1643

[37] Gao X,Roco JA.Zeb2 drives the formation of CD11c⁺ atypical B cells to sustain germinal centers that control persistent infection.Sci Immunol2024;9:eadj4748

[38] Sreekumar R,Moutasim K.Assessment of nuclear ZEB2 as a biomarker for colorectal cancer outcome and TNM risk stratification.JAMA Netw Open2018;1:e183115 PMCID:PMC6324431

[39] Yi H,Li S.Oncogenic circular RNA circ_0007534 contributes to paclitaxel resistance in endometrial cancer by sponging miR-625 and promoting ZEB2 expression.Front Oncol2022;12:985470 PMCID:PMC9386306

[40] Nilsson MB,Robichaux J.A YAP/FOXM1 axis mediates EMT-associated EGFR inhibitor resistance and increased expression of spindle assembly checkpoint components.Sci Transl Med2020;12 PMCID:PMC8269000

[41] Li X,Zhao Q,Wang L.Physiological, biochemical, and genomic elucidation of the ensifer adhaerens M8 strain with simultaneous arsenic oxidation and chromium reduction.J Hazard Mater2023;441:129862

[42] Li T,Zeng Z.TIMER2.0 for analysis of tumor-infiltrating immune cells.Nucleic Acids Res2020;48:W509-14 PMCID:PMC7319575

[43] Tan CS,Huang YQ.Third generation EGFR TKIs: current data and future directions.Mol Cancer2018;17:29 PMCID:PMC5817792

[44] Patsialou A,Wang Y,Stanley ER.Invasion of human breast cancer cells in vivo requires both paracrine and autocrine loops involving the colony-stimulating factor-1 receptor.Cancer Res2009;69:9498-506 PMCID:PMC2794986

[45] Yoshimura T,Wang Y.The chemokine monocyte chemoattractant protein-1/CCL2 is a promoter of breast cancer metastasis.Cell Mol Immunol2023;20:714-38 PMCID:PMC10310763

[46] Xie T,Li ZM.CircSMARCC1 facilitates tumor progression by disrupting the crosstalk between prostate cancer cells and tumor-associated macrophages via miR-1322/CCL20/CCR6 signaling.Mol Cancer2022;21:173 PMCID:PMC9434883

[47] Augimeri G,Morelli C.The omega-3 docosahexaenoyl ethanolamide reduces CCL5 secretion in triple negative breast cancer cells affecting tumor progression and macrophage recruitment.Cancers2023;15:819 PMCID:PMC9913844

[48] Sattiraju A,Giotti B.Hypoxic niches attract and sequester tumor-associated macrophages and cytotoxic T cells and reprogram them for immunosuppression.Immunity2023;56:1825-1843.e6 PMCID:PMC10527169

[49] Wang Y,Qin Y.FOXO1 promotes tumor progression by increased M2 macrophage infiltration in esophageal squamous cell carcinoma.Theranostics2020;10:11535-48. DOI: 10.7150/thno.45735 PMCID:PMC7546008

[50] Chen L,Wang S.SCCA1/SERPINB3 suppresses antitumor immunity and blunts therapy-induced T cell responses via STAT-dependent chemokine production.J Clin Invest2023;133 PMCID:PMC10378164

[51] Chen X,Zhang F.ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation.Theranostics2021;11:3392-416 PMCID:PMC7847666

[52] Liu K,Yuan S.Structural basis of CXC chemokine receptor 2 activation and signalling.Nature2020;585:135-40

[53] van Elsas MJ,Labrie C.Immunotherapy-activated T cells recruit and skew late-stage activated M1-like macrophages that are critical for therapeutic efficacy.Cancer Cell2024;42:1032-1050.e10

[54] Zhang L,Zhou S.Endothelial DGKG promotes tumor angiogenesis and immune evasion in hepatocellular carcinoma.J Hepatol2024;80:82-98

[55] Gregory PA,Smith E.An autocrine TGF-beta/ZEB/miR-200 signaling network regulates establishment and maintenance of epithelial-mesenchymal transition.Mol Biol Cell2011;22:1686-98 PMCID:PMC3093321

[56] Doroshow DB,Hastings K.Immunotherapy in non-small cell lung cancer: facts and hopes.Clin Cancer Res2019;25:4592-602 PMCID:PMC6679805

[57] Gajewski TF.The next hurdle in cancer immunotherapy: overcoming the non-T-cell-inflamed tumor microenvironment.Semin Oncol2015;42:663-71 PMCID:PMC4555998

[58] Mellman I,Dranoff G.Cancer immunotherapy comes of age.Nature2011;480:480-9 PMCID:PMC3967235

[59] Smyth MJ,Ribas A.Combination cancer immunotherapies tailored to the tumour microenvironment.Nat Rev Clin Oncol2016;13:143-58

[60] Janco JM, Lamichhane P, Karyampudi L, Knutson KL. Tumor-infiltrating dendritic cells in cancer pathogenesis.J Immunol2015;194:2985-91 PMCID:PMC4369768

[61] Bertrand F,Marcheteau E.TNFα blockade overcomes resistance to anti-PD-1 in experimental melanoma.Nat Commun2017;8:2256 PMCID:PMC5741628

[62] Robert C,Long GV.KEYNOTE-006 investigatorsPembrolizumab versus ipilimumab in advanced melanoma.N Engl J Med2015;372:2521-32

[63] Han R,Shi J.Tumour microenvironment changes after osimertinib treatment resistance in non-small cell lung cancer.Eur J Cancer2023;189:112919

[64] Fardi M,Baradaran B,Solali S.The crucial role of ZEB2: from development to epithelial-to-mesenchymal transition and cancer complexity.J Cell Physiol2019;234:14783-99

[65] Cacheux V,Kääriäinen H.Loss-of-function mutations in SIP1 Smad interacting protein 1 result in a syndromic Hirschsprung disease.Hum Mol Genet2001;10:1503-10

[66] Adam MP,Gallagher R.Clinical features and management issues in Mowat-Wilson syndrome.Am J Med Genet A2006;140:2730-41

[67] Ivanovski I,Caraffi SG.Phenotype and genotype of 87 patients with Mowat-Wilson syndrome and recommendations for care.Genet Med2018;20:965-75

[68] di Gennaro A,Brisotto G.Correction to: a p53/miR-30a/ZEB2 axis controls triple negative breast cancer aggressiveness.Cell Death Differ2019;26:2493 PMCID:PMC6889402

[69] Francescangeli F,De Angelis ML.A pre-existing population of ZEB2⁺ quiescent cells with stemness and mesenchymal features dictate chemoresistance in colorectal cancer.J Exp Clin Cancer Res2020;39:2 PMCID:PMC6947904

[70] Li Y,Lin Q.ZEB2 facilitates peritoneal metastasis by regulating the invasiveness and tumorigenesis of cancer stem-like cells in high-grade serous ovarian cancers.Oncogene2021;40:5131-41 PMCID:PMC8363099

[71] Li QS.ESRRb inhibits the TGFβ signaling pathway to drive cell proliferation in cervical cancer.Cancer Res2023;83:3095-114 PMCID:PMC10502452

[72] Erkeland SJ,Schilperoord-Vermeulen J.The miR-200c/141-ZEB2-TGFβ axis is aberrant in human T-cell prolymphocytic leukemia.Haematologica2022;107:143-53 PMCID:PMC8719092

[73] Qu Y,Cordeddu L.Cancer-specific changes in DNA methylation reveal aberrant silencing and activation of enhancers in leukemia.Blood2017;129:e13-25

[74] Tian Y,Du X.The interplay between noncoding RNAs and insulin in diabetes.Cancer Lett2018;419:53-63

[75] Wu DM,Liu YB.The PAX6-ZEB2 axis promotes metastasis and cisplatin resistance in non-small cell lung cancer through PI3K/AKT signaling.Cell Death Dis2019;10:349 PMCID:PMC6483988

[76] Li J,Goossens S.The EMT transcription factor Zeb2 controls adult murine hematopoietic differentiation by regulating cytokine signaling.Blood2017;129:460-72

[77] Ren J,Li L.OAMDP, a novel podophyllotoxin derivative, induces apoptosis, cell cycle arrest and autophagy in hepatoma HepG2 cells.Cell Biol Int2018;42:194-204

[78] Chen X,Feng J.Combination of lysine-specific demethylase 6A (KDM6A) and mismatch repair (MMR) status is a potential prognostic factor in colorectal cancer.Cancer Med2021;10:317-24 PMCID:PMC7826484

[79] Pyo KH,Park CW.Comprehensive analyses of immunodynamics and immunoreactivity in response to treatment in ALK-positive non-small-cell lung cancer.J Immunother Cancer2020;8:e000970 PMCID:PMC7394300

[80] Han JJ,Koh J.Change in PD-L1 expression after acquiring resistance to gefitinib in EGFR-mutant non-small-cell lung cancer.Clin Lung Cancer2016;17:263-70.e2

[81] Kim H,Kim MJ.EGFR inhibitors enhanced the susceptibility to NK cell-mediated lysis of lung cancer cells.J Immunother2011;34:372-81

[82] He S,Li D.Enhanced interaction between natural killer cells and lung cancer cells: involvement in gefitinib-mediated immunoregulation.J Transl Med2013;11:186 PMCID:PMC3766712