ANKRD49 promotes the epithelial-mesenchymal transition of non-small cell lung cancer via the PKNOX1/TGF-β1/SMAD axis.

BACKGROUND: Ankyrin repeat domain 49 (ANKRD49) is highly expressed in non-small cell lung cancer (NSCLC) and promotes tumor cell invasion and metastasis by activating the p38/ATF-2/MMP or JNK/ATF-2/c-Jun/MMP pathways. Although epithelial-mesenchymal transition (EMT) is critical for tumor dissemination, the role of ANKRD49 in EMT remains unclear. This study aimed to investigate the effects of ANKRD49 on EMT in NSCLC and to elucidate its underlying molecular mechanisms. METHODS: The expression levels of ANKRD49 and EMT markers were analyzed in lung adenocarcinoma (LUAD) tissues using tissue microarrays (TMAs) combined with immunohistochemistry (IHC). The role of ANKRD49 in EMT was assessed in vitro and in vivo through real-time qPCR, Western blot, and IHC. Key signaling molecules were quantified by real-time qPCR, Western blot, enzyme-linked immunosorbent assay (ELISA) and IHC. Transcriptional regulation of TGF-β1 by PKNOX1 was verified using luciferase reporter assays and chromatin immunoprecipitation (ChIP). Protein-protein interactions between ANKRD49 and PKNOX1 were examined by co-immunoprecipitation (Co-IP) and immunofluorescence microscopy. RESULTS: TMA analysis revealed a significant negative correlation between ANKRD49 and E-cadherin expression, whereas positive correlations were observed with α-smooth muscle actin (α-SMA) and TGF-β1. ANKRD49 overexpression promoted EMT, whereas its knockdown suppressed EMT in both cellular and animal models. Furthermore, ANKRD49 upregulation increased TGF-β1 levels, which are transcriptionally regulated by PKNOX1, while ANKRD49 silencing had the opposite effect. Mechanistically, ANKRD49 physically interacts with PKNOX1, and PKNOX1 binds to the TGF-β1 promoter, elevating TGF-β1 expression and subsequently activating SMAD signaling. Consistently, ANKRD49 and PKNOX1 levels were positively correlated in LUAD tissues. CONCLUSION: ANKRD49 facilitates EMT in NSCLC by interacting with PKNOX1 to activate the TGF-β1/SMAD pathway. PKNOX1 mediates this process through transcriptional regulation of TGF-β1. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12935-026-04240-3.