Yap methylation-induced FGL1 expression suppresses anti-tumor immunity and promotes tumor progression in KRAS-driven lung adenocarcinoma

Abstract

Background: Despite significant strides in lung cancer immunotherapy, the response rates for Kirsten rat sarcoma viral oncogene homolog (KRAS)-driven lung adenocarcinoma (LUAD) patients remain limited. Fibrinogen-like protein 1 (FGL1) is a newly identified immune checkpoint target, and the study of related resistance mechanisms is crucial for improving the treatment outcomes of LUAD patients. This study aimed to elucidate the potential mechanism by which FGL1 regulates the tumor microenvironment in KRAS-mutated cancer.

Methods: The expression levels of FGL1 and SET1 histone methyltransferase (SET1A) in lung cancer were assessed using public databases and clinical samples. Lentiviruses were constructed for transduction to overexpress or silence FGL1 in lung cancer cells and mouse models. The effects of FGL1 and Yes-associated protein (Yap) on the immunoreactivity of cytotoxic T cells in tumor tissues were evaluated using immunofluorescence staining and flow cytometry. Chromatin immunoprecipitation and dual luciferase reporter assays were used to study the SET1A-directed transcriptional program.

Results: Upregulation of FGL1 expression in KRAS-mutated cancer was inversely correlated with the infiltration of CD8⁺ T cells. Mechanistically, KRAS activated extracellular signal-regulated kinase 1/2 (ERK1/2), which subsequently phosphorylated SET1A and increased its stability and nuclear localization. SET1A-mediated methylation of Yap led to Yap sequestration in the nucleus, thereby promoting Yap-induced transcription of FGL1 and immune evasion in KRAS-driven LUAD. Notably, dual blockade of programmed cell death-1 (PD-1) and FGL1 further increased the therapeutic efficacy of anti-PD-1 immunotherapy in LUAD patients.

Conclusion: FGL1 could be used as a diagnostic biomarker of KRAS-mutated lung cancer, and targeting the Yap-FGL1 axis could increase the efficacy of anti-PD-1 immunotherapy.

Keywords

FGL1 / immune evasion / KRAS-driven / lung adenocarcinoma / methylation / SET1A / Yap

References

[1] Prior IA, Lewis PD, Mattos C. A comprehensive survey of Ras mutations in cancer. Cancer Res. 2012; 72(10): 2457–2467.

[2] Simanshu DK, Nissley DV, McCormick F. RAS Proteins and Their Regulators in Human Disease. Cell. 2017; 170(1): 17–33.

[3] Dunnett-Kane V, Nicola P, Blackhall F, Lindsay CJC. Mechanisms of resistance to KRASG12C inhibitors. Cancers (Basel). 2021; 13(1): 151.

[4] Macaya I, Roman M, Welch C, Entrialgo-Cadierno R. Salmon M, Santos A, et al. Signature-driven repurposing of Midostaurin for combination with MEK1/2 and KRASG12C inhibitors in lung cancer. Nat Commun. 2023; 14(1): 6332.

[5] Hui E, Cheung J, Zhu J, Su X, Taylor MJ, Wallweber HA, et al. T cell costimulatory receptor CD28 is a primary target for PD-1-mediated inhibition. Science. 2017; 355(6332): 1428–1433.

[6] Qin S, Xu L, Yi M, Yu S, Wu K, Luo S. Novel immune checkpoint targets: moving beyond PD-1 and CTLA-4. Mol Cancer. 2019; 18(1): 155.

[7] Zou W, Wolchok JD, Chen L. PD-L1 (B7-H1) and PD-1 pathway blockade for cancer therapy: Mechanisms, response biomarkers, and combinations. Sci Transl Med. 2016; 8(328): 328rv4.

[8] Wang X, Wang F, Zhong M, Yarden Y, Fu L. The biomarkers of hyperprogressive disease in PD-1/PD-L1 blockage therapy. Mol Cancer. 2020; 19(1): 81.

[9] Demchev V, Malana G, Vangala D, Stoll J, Desai A, Kang HW, et al. Targeted deletion of fibrinogen like protein 1 reveals a novel role in energy substrate utilization. PLoS One. 2013; 8(3): e58084.

[10] Wu HT, Chen SC, Fan KC, Kuo CH, Lin SY, Wang SH, et al. Targeting fibrinogen-like protein 1 is a novel therapeutic strategy to combat obesity. Faseb j. 2020; 34(2): 2958–2967.

[11] Wang J, Sanmamed MF, Datar I, Su TT, Ji L, Sun J, et al. Fibrinogen-like protein 1 is a major immune inhibitory ligand of LAG-3. Cell. 2019; 176(1): 334–347. e12.

[12] Maruhashi T, Sugiura D, Okazaki IM, Okazaki T. LAG-3: from molecular functions to clinical applications. J Immunother Cancer. 2020; 8(2): e001014.

[13] Woo SR, Turnis ME, Goldberg MV, Bankoti J, Selby M, Nirschl CJ, et al. Immune inhibitory molecules LAG-3 and PD-1 synergistically regulate T-cell function to promote tumoral immune escape. Cancer Res. 2012; 72(4): 917–927.

[14] Workman CJ, Cauley LS, Kim IJ, Blackman MA, Woodland DL, Vignali DA. Lymphocyte activation gene-3 (CD223) regulates the size of the expanding T cell population following antigen activation in vivo. J Immunol. 2004; 172(9): 5450–5455.

[15] Maruhashi T, Sugiura D, Okazaki IM, Shimizu K, Maeda TK, Ikubo J, et al. Binding of LAG-3 to stable peptide-MHC class II limits T cell function and suppresses autoimmunity and anti-cancer immunity. Immunity. 2022; 55(5): 912–924.e8.

[16] Guo M, Yuan F, Qi F, Sun J, Rao Q, Zhao Z, et al. Expression and clinical significance of LAG-3, FGL1, PD-L1 and CD8(+)T cells in hepatocellular carcinoma using multiplex quantitative analysis. J Transl Med. 2020; 18(1): 306.

[17] Tsai HI, Wu Y, Liu X, Xu Z, Liu L, Wang C, et al. Engineered Small Extracellular Vesicles as a FGL1/PD-L1 Dual-Targeting Delivery System for Alleviating Immune Rejection. Adv Sci (Weinh). 2022; 9(3): e2102634.

[18] Hong AW, Meng Z, Yuan HX, Plouffe SW, Moon S, Kim W, et al. Osmotic stress-induced phosphorylation by NLK at Ser128 activates YAP. EMBO Rep. 2017; 18(1): 72–86.

[19] Zanconato F, Cordenonsi M, Piccolo S. YAP/TAZ at the Roots of Cancer. Cancer Cell. 2016; 29(6): 783–803.

[20] Lo Sardo F, Pulito C, Sacconi A, Korita E, Sudol M, Strano S, et al. YAP/TAZ and EZH2 synergize to impair tumor suppressor activity of TGFBR2 in non-small cell lung cancer. Cancer Lett. 2021; 500: 51–63.

[21] Guo J, Wu Y, Yang L, Du J, Gong K, Chen W, et al. Repression of YAP by NCTD disrupts NSCLC progression. Oncotarget. 2017; 8(2): 2307.

[22] Ye XY, Luo QQ, Xu YH, Tang NW, Niu XM, Li ZM, et al. 17-AAG suppresses growth and invasion of lung adenocarcinoma cells via regulation of the LATS1/YAP pathway. J Cell Mol Med. 2015; 19(3): 651–663.

[23] Hsu PC, Tian B, Yang YL, Wang YC, Liu S, Urisman A, et al. Cucurbitacin E inhibits the Yes associated protein signaling pathway and suppresses brain metastasis of human non small cell lung cancer in a murine model. Oncol Rep. 2019; 42(2): 697–707.

[24] Wang Y, Zhu Y, Gu Y, Ma M, Wang Y, Qi S, et al. Stabilization of Motin family proteins in NF2-deficient cells prevents full activation of YAP/TAZ and rapid tumorigenesis. Cell Rep. 2021; 36(8): 109596.

[25] Pan S, Chen R. Pathological implication of protein post-translational modifications in cancer. Mol Aspects Med. 2022; 86: 101097.

[26] Fang L, Teng H, Wang Y, Liao G, Weng L, Li Y, et al. SET1A-Mediated Mono-Methylation at K342 Regulates YAP Activation by Blocking Its Nuclear Export and Promotes Tumorigenesis. Cancer Cell. 2018; 34(1): 103–118.e9.

[27] Kapoor A, Yao W, Ying H, Hua S, Liewen A, Wang Q, et al. Yap1 Activation Enables Bypass of Oncogenic Kras Addiction in Pancreatic Cancer. Cell. 2019; 179(5): 1239.

[28] Shao DD, Xue W, Krall EB, Bhutkar A, Piccioni F, Wang X, et al. KRAS and YAP1 converge to regulate EMT and tumor survival. Cell. 2014; 158(1): 171–184.

[29] Lin L, Sabnis AJ, Chan E, Olivas V, Cade L, Pazarentzos E, et al. The Hippo effector YAP promotes resistance to RAF-and MEK-targeted cancer therapies. Nat Genet. 2015; 47(3): 250–256.

[30] Xia L, Liu Y, Wang Y. PD-1/PD-L1 Blockade Therapy in Advanced Non-Small-Cell Lung Cancer: Current Status and Future Directions. Oncologist. 2019; 24(Suppl 1): S31–s41.

[31] Zhang X, Wang Y, Fan J, Chen W, Luan J, Mei X, et al. Blocking CD47 efficiently potentiated therapeutic effects of anti-angiogenic therapy in non-small cell lung cancer. J Immunother Cancer. 2019; 7(1): 346.

[32] Kloten V, Lampignano R, Krahn T, Schlange T. Circulating Tumor Cell PD-L1 Expression as Biomarker for Therapeutic Efficacy of Immune Checkpoint Inhibition in NSCLC. Cells. 2019; 8(8): 809.

[33] Xu C, Jin G, Wu H, Cui W, Wang YH, Manne RK, et al. SIRPγ-expressing cancer stem-like cells promote immune escape of lung cancer via Hippo signaling. J Clin Invest. 2022; 132(5): e141797.

[34] Riley SE, Feng Y, Hansen CG. Hippo-Yap/Taz signalling in zebrafish regeneration. NPJ Regen Med. 2022; 7(1): 9.

[35] Park HW, Kim YC, Yu B, Moroishi T, Mo JS, Plouffe SW, et al. Alternative Wnt Signaling Activates YAP/TAZ. Cell. 2015; 162(4): 780–794.

[36] Wu C, Liu Y, Liu W, Zou T, Lu S, Zhu C, et al. NNMT-DNMT1 Axis is Essential for Maintaining Cancer Cell Sensitivity to Oxidative Phosphorylation Inhibition. Adv Sci (Weinh). 2022; 10(1): e2202642.

[37] Brahmer J, Reckamp KL, Baas P, Crinò L, Eberhardt WE, Poddubskaya E, et al. Nivolumab versus Docetaxel in Advanced Squamous-Cell Non-Small-Cell Lung Cancer. N Engl J Med. 2015; 373(2): 123–135.

[38] Chen D, Menon H, Verma V, Guo C, Ramapriyan R, Barsoumian H, et al. Response and outcomes after anti-CTLA4 versus anti-PD1 combined with stereotactic body radiation therapy for metastatic non-small cell lung cancer: retrospective analysis of two single-institution prospective trials. J Immunother Cancer. 2020; 8(1): e000492.

[39] Yan J, Yu Y, Wang N, Chang Y, Ying H, Liu W, et al. LFIRE-1/HFREP-1, a liver-specifi. gene, is frequently downregulated and has growth suppressor activity in hepatocellular carcinoma. Oncogene. 2004; 23(10): 1939–1949.

[40] Gong C, Yu X, Zhang W, Han L, Wang R, Wang Y, et al. Regulating the immunosuppressive tumor microenvironment to enhance breast cancer immunotherapy using pH-responsive hybrid membrane-coated nanoparticles. J Nanobiotechnology. 2021; 19(1): 58.

[41] Zhang Y, Qiao HX, Zhou YT, Hong L, Chen JH. Fibrinogen like protein 1 promotes the invasion and metastasis of gastric cancer and is associated with poor prognosis. Mol Med Rep. 2018; 18(2): 1465–1472.

[42] Wu M, Yuan Y, Pan YY, Zhang Y. Combined gefitinib and pemetrexed overcome the acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer. Mol Med Rep. 2014; 10(2): 931–938.

[43] Lehtiö J, Arslan T, Siavelis I, Pan Y, Socciarelli F, Berkovska O, et al. Proteogenomics of non-small cell lung cancer reveals molecular subtypes associated with specific therapeutic targets and immune evasion mechanisms. Nat Cancer. 2021; 2(11): 1224–1242.

[44] Yan Q, Lin HM, Zhu K, Cao Y, Xu XL, Zhou ZY, et al. Immune Checkpoint FGL1 Expression of Circulating Tumor Cells Is Associated With Poor Survival in Curatively Resected Hepatocellular Carcinoma. Front Oncol. 2022; 12: 810269.

[45] Ma LG, Bian SB, Cui JX, Xi HQ, Zhang KC, Qin HZ, et al. LKB1 inhibits the proliferation of gastric cancer cells by suppressing the nuclear translocation of Yap and β-catenin. Int J Mol Med. 2016; 37(4): 1039–1048.

[46] Nguyen HB, Babcock JT, Wells CD, Quilliam LA. LKB1 tumor suppressor regulates AMP kinase/mTOR-independent cell growth and proliferation via the phosphorylation of Yap. Oncogene. 2013; 32(35): 4100–4109.

[47] Mohseni M, Sun J, Lau A, Curtis S, Goldsmith J, Fox VL, et al. A genetic screen identifies an LKB1-MARK signalling axis controlling the Hippo-YAP pathway. Nat Cell Biol. 2014; 16(1): 108–117.

[48] Boeschen M, Kuhn CK, Wirtz H, Seyfarth HJ, Frille A, Lordick F, et al. Comparative bioinformatic analysis of KRAS, STK11 and KEAP1 (co-)mutations in non-small cell lung cancer with a special focus on KRAS G12C. Lung Cancer. 2023; 184: 107361.

[49] Zanconato F, Cordenonsi M, Piccolo SJNRC. YAP and TAZ: a signalling hub of the tumour microenvironment. Nat Rev Cancer. 2019; 19(8): 454–464.

[50] Sanchez-Vega F, Mina M, Armenia J, Chatila WK, Luna A, La KC, et al. Oncogenic Signaling Pathways in The Cancer Genome Atlas. Cell. 2018; 173(2): 321–337.e10.

[51] Dubois F, Keller M, Calvayrac O, Soncin F, Hoa L, Hergovich A, et al. RASSF1A Suppresses the Invasion and Metastatic Potential of Human Non-Small Cell Lung Cancer Cells by Inhibiting YAP Activation through the GEF-H1/RhoB Pathway. Cancer Res. 2016; 76(6): 1627–1640.

[52] Matallanas D, Romano D, Al-Mulla F. O’Neill E, Al-Ali W. Crespo P, et al. Mutant K-Ras activation of the proapoptotic MST2 pathway is antagonized by wild-type K-Ras. Mol Cell. 2011; 44(6): 893–906.

[53] Jin D, Guo J, Wu Y, Yang L, Wang X, Du J, et al. m(6)A demethylase ALKBH5 inhibits tumor growth and metastasis by reducing YTHDFs-mediated YAP expression and inhibiting miR-107/LATS2-mediated YAP activity in NSCLC. Mol Cancer. 2020; 19(1): 40.

[54] Chao YC, Lee KY, Wu SM, Kuo DY, Shueng PW, Lin CW. Melatonin Downregulates PD-L1 Expression and Modulates Tumor Immunity in KRAS-Mutant Non-Small Cell Lung Cancer. Int J Mol Sci. 2021; 22(11): 5649.

[55] Stampouloglou E, Cheng N, Federico A, Slaby E, Monti S, Szeto GL, et al. Yap suppresses T-cell function and infiltration in the tumor microenvironment. PLoS Biol. 2020; 18(1): e3000591.

[56] Wang G, Lu X, Dey P, Deng P, Wu CC, Jiang S, et al. Targeting YAP-Dependent MDSC Infiltration Impairs Tumor Progression. Cancer Discov. 2016; 6(1): 80–95.

[57] Miao J, Hsu PC, Yang YL, Xu Z, Dai Y, Wang Y, et al. YAP regulates PD-L1 expression in human NSCLC cells. Oncotarget. 2017; 8(70): 114576–114587.

[58] Keir ME, Butte MJ, Freeman GJ, Sharpe AH. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol. 2008; 26: 677–704.

[59] Yang H, Chen H, Luo S, Li L, Zhou S, Shen R, et al. The correlation between programmed death-ligand 1 expression and driver gene mutations in NSCLC. Oncotarget. 2017; 8(14): 23517–23528.

[60] Liu C, Zheng S, Wang Z, Wang S, Wang X, Yang L, et al. KRAS-G12D mutation drives immune suppression and the primary resistance of anti-PD-1/PD-L1 immunotherapy in non-small cell lung cancer. Cancer Commun (Lond). 2022; 42(9): 828–847.

RIGHTS & PERMISSIONS

2024 2024 The Author(s). Cancer Communications published by John Wiley & Sons Australia, Ltd on behalf of Sun Yat-sen University Cancer Center.