TSZAF monomer combination downregulates the Wnt/β-catenin signaling pathway and inhibits neutrophil recruitment to prevent lung cancer metastasis

Abstract

Metastasis remains the primary cause of cancer-related mortality worldwide. Circulating tumor cells (CTCs) represent critical targets for metastasis prevention and treatment. Traditional Chinese medicine may prevent lung cancer metastasis through long-term intervention in CTC activity. Tiao-Shen-Zhi-Ai Formular (TSZAF) represents a Chinese medicine compound prescription utilized clinically for lung cancer treatment. This study combined three principal active ingredients from TSZAF into a novel TSZAF monomer combination (TSZAF mc) to investigate its anti-metastatic effects and mechanisms. TSZAF mc demonstrated significant inhibition of proliferation, migration, and invasion in CTC-TJH-01 and LLC cells, while inducing cellular apoptosis in vitro. Moreover, TSZAF mc substantially inhibited LLC cell growth and metastasis in vivo. Mechanistically, TAZSF mc significantly suppressed the Wnt/β-catenin signaling pathway and CXCL5 expression in lung cancer cells and tissues. Additionally, TAZSF mc notably reduced neutrophil infiltration in metastatic lesions. These findings indicate that TSZAF mc inhibits lung cancer growth and metastasis by suppressing the Wnt/β-catenin signaling pathway and reducing CXCL5 secretion, thereby decreasing neutrophil recruitment and infiltration. TSZAF mc demonstrates potential as an effective therapeutic agent for lung cancer metastasis.

Keywords

Circulating tumor cells (CTCs) / Lung cancer / Metastasis / Wnt/β-catenin / Neutrophil / CXCL5

References

[1] Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. CA Cancer J Clin.2024; 74(1):12-49. https://doi.org/10.3322/caac.21820.

[2] Xia C, Dong X, Li H, et al. Cancer statistics in China and United States, 2022: profiles, trends, and determinants. Chin Med J. 2022; 135(5):584-590. https://doi.org/10.1097/CM9.0000000000002108.

[3] Feng R, Su Q, Huang X, et al. Cancer situation in China: what does the China cancer map indicate from the first national death survey to the latest cancer registration? Cancer Commun (Lond). 2023; 43(1):75-86. https://doi.org/10.1002/cac2.12393.

[4] Salas S, Cottet V, Dossus L, et al. Nutritional factors during and after cancer: impacts on survival and quality of life. Nutrients. 2022; 14(14):2958. https://doi.org/10.3390/nu14142958.

[5] Deng Z, Wu S, Wang Y, et al. Circulating tumor cell isolation for cancer diagnosis and prognosis. EBioMedicine. 2022;83:104237. https://doi.org/10.1016/j.ebiom.2022.104237.

[6] Pereira-Veiga T, Schneegans S, Pantel K, et al. Circulating tumor cell-blood cell crosstalk: biology and clinical relevance. Cell Rep. 2022; 40(9):111298. https://doi.org/10.1016/j.celrep.2022.111298.

[7] Sun Y, Li T, Ding L, et al. Platelet-mediated circulating tumor cell evasion from natural killer cell killing through immune checkpoint CD155-TIGIT. Hepatology. 2025; 81(3):791-807. https://doi.org/10.1097/HEP.0000000000000934.

[8] Chemi F, Rothwell DG, McGranahan N, et al. Pulmonary venous circulating tumor cell dissemination before tumor resection and disease relapse. Nat Med. 2019; 25(10):1534-1539. https://doi.org/10.1038/s41591-019-0593-1.

[9] Crist SB, Ghajar CM. When a house is not a home: a survey of antimetastatic niches and potential mechanisms of disseminated tumor cell suppression. Annu Rev Pathol. 2021; 16:409-432. https://doi.org/10.1146/annurev-pathmechdis-012419-032647.

[10] Adachi H, Ito H, Sawabata N. Circulating tumor cells and the non-touch isolation technique in surgery for non-small-cell lung cancer. Cancers (Basel). 2022; 14(6):1448. https://doi.org/10.3390/cancers14061448.

[11] Li Z, Xu K, Tartarone A, et al. Circulating tumor cells can predict the prognosis of patients with non-small cell lung cancer after resection: a retrospective study. Transl Lung Cancer Res. 2021; 10(2):995-1006. https://doi.org/10.21037/tlcr-21-149.

[12] Bidard FC, Kiavue N, Jacot W, et al. Overall survival with circulating tumor cell count-driven choice of therapy in advanced breast cancer: a randomized trial. J Clin Oncol. 2024; 42(4):383-389. https://doi.org/10.1200/JCO.23.00456.

[13] Li Y, Sheng H, Ma F, et al. RNA m(6)A reader YTHDF2 facilitates lung adenocarcinoma cell proliferation and metastasis by targeting the AXIN1/Wnt/β-catenin signaling. Cell Death Dis. 2021; 12(5):479. https://doi.org/10.1038/s41419-021-03763-z.

[14] Xiang D, Gu M, Liu J, et al. m6A RNA methylation-mediated upregulation of HLF promotes intrahepatic cholangiocarcinoma progression by regulating the FZD4/β-catenin signaling pathway. Cancer Lett. 2023;560:216144. https://doi.org/10.1016/j.canlet.2023.216144.

[15] Tang Y, Tian W, Zheng S, et al. Dissection of FOXO1-induced LYPLAL1-DT Impeding triple-negative breast cancer progression via mediating hnRNPK/β-catenin complex. Research (Wash D C). 2023;6:0289. https://doi.org/10.34133/research.0289.

[16] Liu J, Xiao Q, Xiao J, et al. Wnt/β-catenin signalling: function, biological mechanisms, and therapeutic opportunities. Signal Transduct Target Ther. 2022; 7(1):3. https://doi.org/10.1038/s41392-021-00762-6.

[17] Zhang Y, Wang X. Targeting the Wnt/β-catenin signaling pathway in cancer. J Hematol Oncol. 2020; 13(1):165. https://doi.org/10.1186/s13045-020-00990-3.

[18] Stewart DJ. Wnt signaling pathway in non-small cell lung cancer. J Natl Cancer Inst. 2014; 106(1):djt356. https://doi.org/10.1093/jnci/djt356.

[19] Yang F, Xu J, Li H, et al. FBXW2 suppresses migration and invasion of lung cancer cells via promoting β-catenin ubiquitylation and degradation. Nat Commun. 2019; 10(1):1382. https://doi.org/10.1038/s41467-019-09289-5.

[20] Yu F, Yu C, Li F, et al. Wnt/β-catenin signaling in cancers and targeted therapies. Signal Transduct Target Ther. 2021; 6(1):307. https://doi.org/10.1038/s41392-021-00701-5.

[21] Xiong S, Dong L, Cheng L. Neutrophils in cancer carcinogenesis and metastasis. J Hematol Oncol. 2021; 14(1):173. https://doi.org/10.1186/s13045-021-01187-y.

[22] Huang X, Nepovimova E, Adam V, et al. Neutrophils in Cancer immunotherapy: friends or foes? Mol Cancer. 2024; 23(1):107. https://doi.org/10.1186/s12943-024-02004-z.

[23] Sun D, Tan L, Chen Y, et al.CXCL5 impedes CD8(+) T cell immunity by upregulating PD-L1 expression in lung cancer via PXN/AKT signaling phosphorylation and neutrophil chemotaxis. J Exp Clin Cancer Res. 2024; 43(1):202. https://doi.org/10.1186/s13046-024-03122-8.

[24] Ren W, Liang P, Ma Y, et al.Research progress of traditional Chinese medicine against COVID-19. Biomed Pharmacother. 2021;137:111310. https://doi.org/10.1016/j.biopha.2021.111310.

[25] Que Z, Luo B, Yu P, et al. Polyphyllin VII induces CTC anoikis to inhibit lung cancer metastasis through EGFR pathway regulation. Int J Biol Sci. 2023; 19(16):5204-5217. https://doi.org/10.7150/ijbs.83682.

[26] Wang Z, Wu W, Wang Z, et al. Ex vivo expansion of circulating lung tumor cells based on one-step microfluidics-based immunomagnetic isolation. Analyst. 2016; 141(12):3621-3625. https://doi.org/10.1039/C5AN02554K.

[27] Lyden D, Ghajar CM, Correia AL, et al. Metastasis. Cancer Cell. 2022; 40(8):787-791. https://doi.org/10.1016/j.ccell.2022.07.010.

[28] Gerstberger S, Jiang Q, Ganesh K. Metastasis. Cell. 2023; 186(8):1564-1579. https://doi.org/10.1016/j.cell.2023.03.003.

[29] Chen X, Zhou F, Li X, et al. Folate receptor-positive circulating tumor cell detected by LT-PCR-based method as a diagnostic biomarker for non-small-cell lung cancer. J Thorac Oncol. 2015; 10(8):1163-1171. https://doi.org/10.1097/JTO.0000000000000606.

[30] Jin F, Zhu L, Shao J, et al. Circulating tumour cells in patients with lung cancer universally indicate poor prognosis. Eur Respir Rev. 2022; 31(166):220151. https://doi.org/10.1183/16000617.0151-2022.

[31] Que Z, Luo B, Zhou Z, et al. Establishment and characterization of a patient-derived circulating lung tumor cell line in vitro and in vivo. Cancer Cell Int. 2019;19:21. https://doi.org/10.1186/s12935-019-0735-z.

[32] Fan L, Li Y, Sun Y, et al. Paris saponin VII inhibits the migration and invasion in human A549 lung cancer cells. Phytother Res. 2015; 29(9):1366-1372. https://doi.org/10.1002/ptr.5389.

[33] Xiang YC, Shen J, Si Y, et al. Paris saponin VII, a direct activator of AMPK, induces autophagy and exhibits therapeutic potential in non-small-cell lung cancer. Chin J Nat Med. 2021; 19(3):195-204. https://doi.org/10.1016/S1875-5364(21)60021-3.

[34] Cheng X, Gu J, Zhang M, et al. Astragaloside IV inhibits migration and invasion in human lung cancer A549 cells via regulating PKC-α-ERK1/2-NF-κB pathway. Int Immunopharmacol. 2014; 23(1):304-313. https://doi.org/10.1016/j.intimp.2014.08.027.

[35] Xu F, Cui WQ, Wei Y, et al. Astragaloside IV inhibits lung cancer progression and metastasis by modulating macrophage polarization through AMPK signaling. J Exp Clin Cancer Res. 2018; 37(1):207. https://doi.org/10.1186/s13046-018-0878-0.

[36] Wang W, Huang Q, Chen Y, et al. The novel FAT4 activator jujuboside A suppresses NSCLC tumorigenesis by activating HIPPO signaling and inhibiting YAP nuclear translocation. Pharmacol Res. 2021;170:105723. https://doi.org/10.1016/j.phrs.2021.105723.

[37] Que Z, Zhou Z, Luo B, et al. Jingfukang induces anti-cancer activity through oxidative stress-mediated DNA damage in circulating human lung cancer cells. BMC Complement Altern Med. 2019; 19(1):204. https://doi.org/10.1186/s12906-019-2601-x.

[38] Que ZJ, Yang Y, Liu HT, et al. Jinfukang regulates integrin/Src pathway and anoikis mediating circulating lung cancer cells migration. J Ethnopharmacol. 2021;267:113473. https://doi.org/10.1016/j.jep.2020.113473.

[39] Que ZJ, Yao JL, Zhou ZY, et al. Jinfukang inhibits lung cancer metastasis by upregulating CX3CL1 to recruit NK cells to kill CTCs. J Ethnopharmacol. 2021;275:114175. https://doi.org/10.1016/j.jep.2021.114175.

[40] Yang S, Liu Y, Li MY, et al. FOXP3 promotes tumor growth and metastasis by activating Wnt/β-catenin signaling pathway and EMT in non-small cell lung cancer. Mol Cancer. 2017; 16(1):124. https://doi.org/10.1186/s12943-017-0700-1.

[41] Sarode P, Zheng X, Giotopoulou GA, et al. Reprogramming of tumor-associated macrophages by targeting β-catenin/FOSL2/ARID5A signaling: A potential treatment of lung cancer. Sci Adv. 2020; 6(23):eaaz6105. https://doi.org/10.1126/sciadv.aaz6105.

[42] Liu Y, Lv H, Liu X, et al. The RP11-417E7.1/THBS 2 signaling pathway promotes colorectal cancer metastasis by activating the Wnt/β-catenin pathway and facilitating exosome-mediated M2 macrophage polarization. J Exp Clin Cancer Res. 2024; 43(1):195. https://doi.org/10.1186/s13046-024-03107-7.

[43] Leppänen N, Kaljunen H, Takala E, et al. SIX2 promotes cell plasticity via Wnt/β-catenin signalling in androgen receptor independent prostate cancer. Nucleic Acids Res. 2024; 52(10):5610-5623. https://doi.org/10.1093/nar/gkae206.

[44] Muto S, Enta A, Maruya Y, et al. Wnt/β-catenin signaling and resistance to immune checkpoint inhibitors: from non-small-cell lung cancer to other cancers. Biomedicines. 2023; 11(1):190. https://doi.org/10.3390/biomedicines11010190.

[45] Guan S, Chen X, Chen Y, et al. FOXM1 variant contributes to gefitinib resistance via activating Wnt/β-catenin signal pathway in patients with non-small cell lung cancer. Clin Cancer Res. 2022; 28(17):3770-3784. https://doi.org/10.1158/1078-0432.CCR-22-0791.

[46] Szczerba BM, Castro-Giner F, Vetter M, et al. Neutrophils escort circulating tumour cells to enable cell cycle progression. Nature. 2019; 566(7745):553-557. https://doi.org/10.1038/s41586-019-0915-y.

[47] Gungabeesoon J, Gort-Freitas NA, Kiss M, et al. A neutrophil response linked to tumor control in immunotherapy. Cell. 2023; 186(7):1448-1464. e1420. https://doi.org/10.1016/j.cell.2023.02.032.

[48] Gentles AJ, Newman AM, Liu CL, et al. The prognostic landscape of genes and infiltrating immune cells across human cancers. Nat Med. 2015; 21(8):938-945. https://doi.org/10.1038/nm.3909.

[49] Jaillon S, Ponzetta A, Di Mitri D, et al. Neutrophil diversity and plasticity in tumour progression and therapy. Nat Rev Cancer. 2020; 20(9):485-503. https://doi.org/10.1038/s41568-020-0281-y.

[50] Milosevic V, Kopecka J, Salaroglio IC, et al.Wnt/IL-1β/IL-8 autocrine circuitries control chemoresistance in mesothelioma initiating cells by inducing ABCB5. Int J Cancer. 2020; 146(1):192-207. https://doi.org/10.1002/ijc.32419.