STK25 inhibits cancer-associated fibroblast activation to overcome cetuximab resistance in colorectal cancer

Abstract

Background: Cancer-associated fibroblasts (CAFs) within the tumour microenvironment play a pivotal role in colorectal cancer (CRC) progression and therapeutic resistance. Serine/threonine protein kinase 25 (STK25) exerts multiple roles in tumourigenesis; however, its role in mediating tumour–stroma crosstalk remains largely unexplored.

Methods: Primary CAFs were isolated from CRC patient tissues and characterised to confirm their identity. The effects of STK25 expression on CAF activity and CAF-mediated tumour progression were evaluated both in vitro and in vivo. Western blot, qRT-PCR, ChIP and dual-luciferase reporter assays were performed to elucidate the mechanism by which STK25 regulated CAF activation. Moreover, the effect of STK25 expression on CAF-induced cetuximab resistance was assessed in vitro and in vivo. The clinical significance of STK25 expression was determined in CRC patient tissues, tissue microarrays and patient-derived organoids.

Results: Knockdown of STK25 in CRC cells enhanced CAF proliferation, migration and activation, whereas its overexpression exhibited the opposite effect. STK25-knockdown-mediated CAF activation subsequently promoted CRC cell proliferation and metastasis. Moreover, STK25 depletion combined with CAFs significantly enhanced CRC tumour growth in vivo. Mechanistically, STK25 deficiency activated the NF-κB pathway, leading to p50 phosphorylation which directly bound to the AREG promoter, thereby transcriptionally up-regulating AREG expression. In addition, STK25-regulated AREG/EGFR axis mediated the crosstalk between CRC cells and CAFs. More importantly, CAFs conferred resistance to the anti-EGFR antibody cetuximab, which could be reversed either by STK25 overexpression or by AREG-neutralising antibody treatment. Clinically, low STK25 expression correlated with elevated CAFs marker levels and poor cetuximab response in CRC patients.

Conclusions: Our findings identified STK25 as a critical regulator of the NF-κB/AREG/EGFR axis in tumour–CAF communication and highlight its potential as a therapeutic target for overcoming CAF-induced cetuximab resistance in CRC.

Key points:

Keywords

AREG/EGFR axis / cancer-associated fibroblasts / cetuximab resistance / colorectal cancer / NF-κB / STK25

References

[1] Bray F, Laversanne M, Sung H, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024; 74(3): 229-263.

[2] GBD 2023 Cancer Collaborators. The global, regional, and national burden of cancer, 1990–2023, with forecasts to 2050: a systematic analysis for the Global Burden of Disease Study 2023. Lancet. 2025; 406(10512): 1565-1586.

[3] Tong W, Wang T, Bai Y, et al. Spatial transcriptomics reveals tumor-derived SPP1 induces fibroblast chemotaxis and activation in the hepatocellular carcinoma microenvironment. J Transl Med. 2024; 22(1): 840.

[4] Wang C, Dong D, Zhao N, et al. Tumor-derived CCL15 regulates RNA m(6)A methylation in cancer-associated fibroblasts to promote hepatocellular carcinoma growth. Cancer Lett. 2024; 611:217420.

[5] Zhong B, Cheng B, Huang X, et al. Colorectal cancer-associated fibroblasts promote metastasis by up-regulating LRG1 through stromal IL-6/STAT3 signaling. Cell Death Dis. 2021; 13(1): 16.

[6] Koerner L, Schmiel M, Yang TP, Peifer M, Buettner R, Pasparakis M. NEMO- and RelA-dependent NF-κB signaling promotes small cell lung cancer. Cell Death Differ. 2023; 30(4): 938-951.

[7] Wu YH, Huang YF, Chang TH, et al. COL11A1 activates cancer-associated fibroblasts by modulating TGF-β3 through the NF-κB/IGFBP2 axis in ovarian cancer cells. Oncogene. 2021; 40(26): 4503-4519.

[8] Chen B, Liu X, Yu P, et al. H. pylori-induced NF-κB-PIEZO1-YAP1-CTGF axis drives gastric cancer progression and cancer-associated fibroblast-mediated tumour microenvironment remodelling. Clin Transl Med. 2023; 13(11):e1481.

[9] Wang Q, Li D, Ma H, et al. Tumor cell-derived EMP1 is essential for cancer-associated fibroblast infiltration in tumor microenvironment of triple-negative breast cancer. Cell Death Dis. 2025; 16(1): 143.

[10] Wei G, Nie Y, Sun M, et al. Cancer-associated fibroblasts induce almonertinib resistance in non-small cell lung cancer. J Transl Med. 2025; 23(1): 42.

[11] Vaquero J, Lobe C, Tahraoui S, et al. The IGF2/IR/IGF1R Pathway in Tumor Cells and Myofibroblasts Mediates Resistance to EGFR Inhibition in Cholangiocarcinoma. Clin Cancer Res. 2018; 24(17): 4282-4296.

[12] Yi Y, Zeng S, Wang Z, et al. Cancer-associated fibroblasts promote epithelial-mesenchymal transition and EGFR-TKI resistance of non-small cell lung cancers via HGF/IGF-1/ANXA2 signaling. Biochim Biophys Acta Mol Basis Dis. 2018; 1864(3): 793-803.

[13] Hu Y, Zhu T, Nong S, et al. Dual disruption of EGFR/PI3K signaling: IGF2BP2 targeting reverses anti-EGFR resistance in CAFs-infiltrated oral squamous cell carcinoma. Int J Mol Sci. 2025; 26(9): 3941.

[14] Johansson AC, Ansell A, Jerhammar F, et al. Cancer-associated fibroblasts induce matrix metalloproteinase-mediated cetuximab resistance in head and neck squamous cell carcinoma cells. Mol Cancer Res. 2012; 10(9): 1158-1168.

[15] Son SS, Hwang S, Park JH, et al. In vivo silencing of amphiregulin by a novel effective Self-Assembled-Micelle inhibitory RNA ameliorates renal fibrosis via inhibition of EGFR signals. Sci Rep. 2021; 11(1): 2191.

[16] Amrutkar M, Cansby E, Chursa U, et al. Genetic disruption of protein kinase STK25 ameliorates metabolic defects in a diet-induced type 2 diabetes model. Diabetes. 2015; 64(8): 2791-2804.

[17] Cansby E, Kumari S, Caputo M, et al. Silencing of STE20-type kinase STK25 in human aortic endothelial and smooth muscle cells is atheroprotective. Commun Biol. 2022; 5(1): 379.

[18] Andersson E, Gongye X, Cansby E, et al. Inhibition of GCKIII kinases STK25 and MST3 mitigates organ lipotoxicity and enhances metabolic resilience under nutritional stress. BMC Med. 2025; 23(1): 518.

[19] Rice MR, Matta B, Wang L, et al. TLR-induced STK25 activation promotes IRF5-mediated inflammation. Life Sci Alliance. 2025; 8(9):e202503343.

[20] Wu F, Gao P, Wu W, et al. STK25-induced inhibition of aerobic glycolysis via GOLPH3-mTOR pathway suppresses cell proliferation in colorectal cancer. J Exp Clin Cancer Res. 2018; 37(1): 144.

[21] Chen J, Gao P, Peng L, et al. Downregulation of STK25 promotes autophagy via the Janus kinase 2/signal transducer and activator of transcription 3 pathway in colorectal cancer. Mol Carcinog. 2022; 61(6): 572-586.

[22] Jiang J, Zheng Y, Chen F, Dong L, Guo X. Activation of YAP1 by STK25 contributes to the progression of hepatocellular carcinoma. Tissue Cell. 2022; 76:101797.

[23] Sun X, Li S, Lin H. LIMK1 interacts with STK25 to regulate EMT and promote the proliferation and metastasis of colorectal cancer. J Oncol. 2022; 2022:3963883.

[24] Zhang Y, Xu J, Qiu Z, et al. STK25 enhances hepatocellular carcinoma progression through the STRN/AMPK/ACC1 pathway. Cancer Cell Int. 2022; 22(1): 4.

[25] Chen KQ, Lei GL, Ke BY, Chen L, Wang ZB, Wang SZ. STK25: a viable therapeutic target for cancer treatments? Anticancer Drugs. 2023; 34(9): 995-1001.

[26] Lee YS, Im J, Yang Y, et al. New function annotation of PROSER2 in pancreatic ductal adenocarcinoma. J Proteome Res. 2024; 23(3): 905-915.

[27] Qiao X, Xing P, Hao H, et al. STK25 loss augments anti-PD-1 therapy efficacy by regulating PD-L1 stability in colorectal cancer. Adv Sci (Weinh). 2025; 12(39):e03891.

[28] Chandra Jena B, Sarkar S, Rout L, Mandal M. The transformation of cancer-associated fibroblasts: Current perspectives on the role of TGF-β in CAF mediated tumor progression and therapeutic resistance. Cancer Lett. 2021; 520: 222-232.

[29] Fang Z, Meng Q, Xu J, et al. Signaling pathways in cancer-associated fibroblasts: recent advances and future perspectives. Cancer Commun (Lond). 2023; 43(1): 3-41.

[30] Gu J, Li X, Zhao L, et al. The role of PKM2 nuclear translocation in the constant activation of the NF-κB signaling pathway in cancer-associated fibroblasts. Cell Death Dis. 2021; 12(4): 291.

[31] Wang L, Liu Y, Yin X, et al. Effect of reduced-dose capecitabine plus cetuximab as maintenance therapy for RAS wild-type metastatic colorectal cancer: a phase 2 clinical trial. JAMA Netw Open. 2020; 3(7):e2011036.

[32] Qin S, Jiang J, Lu Y, et al. Emerging role of tumor cell plasticity in modifying therapeutic response. Signal Transduct Target Ther. 2020; 5(1): 228.

[33] Jia H, Chen X, Zhang L, Chen M. Cancer associated fibroblasts in cancer development and therapy. J Hematol Oncol. 2025; 18(1): 36.

[34] Xu Q, Yang C, Ning J, et al. SMOC2 (high) myofibroblastic cancer-associated fibroblast drives primary cilia-associated tumor microenvironment remodeling and poor prognosis in gastric cancer. Chin J Cancer Res. 2025; 37(4): 603-623.

[35] Shi X, Zhu W, Zhang J, Fan C, Zhang J. Cancer-associated fibroblasts in hepatocellular carcinoma: origins, heterogeneity, and therapeutic implications. Front Immunol. 2025; 16:1620075.

[36] Kobayashi H, Gieniec KA, Lannagan TRM, et al. The origin and contribution of cancer-associated fibroblasts in colorectal carcinogenesis. Gastroenterology. 2022; 162(3): 890-906.

[37] Huang J, Tsang WY, Li ZH, Guan XY. The origin, differentiation, and functions of cancer-associated fibroblasts in gastrointestinal cancer. Cell Mol Gastroenterol Hepatol. 2023; 16(4): 503-511.

[38] Liu J, Huang Z, Chen HN, et al. ZNF37A promotes tumor metastasis through transcriptional control of THSD4/TGF-β axis in colorectal cancer. Oncogene. 2021; 40(19): 3394-3407.

[39] Li Q, Yuan H, Zhao G, et al. ZNF32 prevents the activation of cancer-associated fibroblasts through negative regulation of TGFB1 transcription in breast cancer. Faseb j. 2023; 37(4):e22837.

[40] Mucciolo G, Araos Henríquez J, Jihad M, et al. EGFR-activated myofibroblasts promote metastasis of pancreatic cancer. Cancer Cell. 2024;42(1):101-118. e111.

[41] Sun R, Zhao H, Gao DS, et al. Amphiregulin couples IL1RL1(+) regulatory T cells and cancer-associated fibroblasts to impede antitumor immunity. Sci Adv. 2023; 9(34):eadd7399.

[42] Jung CH, Seo M, Otto NM, Kim DH. ULK1 inhibits the kinase activity of mTORC1 and cell proliferation. Autophagy. 2011; 7(10): 1212-1221.

[43] Kameshita I, Ishida A, Fujisawa H. Phosphorylation and activation of Ca2+/calmodulin-dependent protein kinase phosphatase by Ca2+/calmodulin-dependent protein kinase II. FEBS Lett. 1999; 456(2): 249-252.

[44] Ferris RL, Lenz HJ, Trotta AM, et al. Rationale for combination of therapeutic antibodies targeting tumor cells and immune checkpoint receptors: Harnessing innate and adaptive immunity through IgG1 isotype immune effector stimulation. Cancer Treat Rev. 2018; 63: 48-60.

[45] Correale P, Botta C, Cusi MG, et al. Cetuximab ± chemotherapy enhances dendritic cell-mediated phagocytosis of colon cancer cells and ignites a highly efficient colon cancer antigen-specific cytotoxic T-cell response in vitro. Int J Cancer. 2012; 130(7): 1577-1589.

[46] Cremolini C, Rossini D, Dell'Aquila E, et al. Rechallenge for patients with RAS and BRAF wild-type metastatic colorectal cancer with acquired resistance to first-line cetuximab and irinotecan: a phase 2 single-arm clinical trial. JAMA Oncol. 2019; 5(3): 343-350.

[47] Yao Y, Wang Y, Chen L, et al. Clinical utility of PDX cohorts to reveal biomarkers of intrinsic resistance and clonal architecture changes underlying acquired resistance to cetuximab in HNSCC. Signal Transduct Target Ther. 2022; 7(1): 73.

[48] Yegodayev KM, Novoplansky O, Golden A, et al. TGF-beta-activated cancer-associated fibroblasts limit cetuximab efficacy in preclinical models of head and neck cancer. Cancers (Basel). 2020; 12(2): 339.

[49] Busser B, Sancey L, Josserand V, et al. Amphiregulin promotes BAX inhibition and resistance to gefitinib in non-small-cell lung cancers. Mol Ther. 2010; 18(3): 528-535.

[50] Kim SA, Park H, Kim KJ, et al. Amphiregulin can predict treatment resistance to palliative first-line cetuximab plus FOLFIRI chemotherapy in patients with RAS wild-type metastatic colorectal cancer. Sci Rep. 2021; 11(1):23803.

[51] Mason JD, Marks E, Fan SJ, et al. Stress-induced Rab11a-exosomes induce amphiregulin-mediated cetuximab resistance in colorectal cancer. J Extracell Vesicles. 2024; 13(6):e12465.

[52] Höpfner M, Sutter AP, Huether A, Schuppan D, Zeitz M, Scherübl H. Targeting the epidermal growth factor receptor by gefitinib for treatment of hepatocellular carcinoma. J Hepatol. 2004; 41(6): 1008-1016.

[53] Piffkó A, Yang K, Panda A, et al. Radiation-induced amphiregulin drives tumour metastasis. Nature. 2025; 643(8072): 810-819.

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2026 The Author(s). Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.