Epigenetic silencing of PDLIM4 promotes colorectal cancer by impairing BTF3 autophagic degradation and activating PI3K/Akt signalling

BACKGROUND: Epigenetic silencing of tumour suppressor genes is a pivotal early event in colorectal cancer (CRC) pathogenesis. PDZ and LIM Domain 4 (PDLIM4) is a member of the PDZ-LIM family with context-dependent roles in tumour progression across different malignancies. However, its specific role and epigenetic regulation in colorectal cancer (CRC) remain elusive. METHODS: We integrated multi-omics datasets and clinical cohorts to evaluate PDLIM4 expression and promoter methylation. Stool DNA was analyzed to assess the feasibility of non-invasive detection of PDLIM4 promoter methylation. PDLIM4 knockout and overexpression cell lines were constructed to perform functional assays including proliferation, apoptosis, and stemness. In vivo experiments were validated utilizing xenograft mice or an azoxymethane (AOM)/ dextran sulphate sodium salt (DSS) CRC mouse model with intestinal epithelial cell-specific overexpression of PDLIM4. Mechanistic investigations were conducted using coimmunoprecipitation coupled with mass spectrometry, RNA sequencing, ChIP-qPCR and pharmacological rescue experiments. RESULTS: We identified a progressive loss of PDLIM4 protein along the normal-adenoma-carcinoma sequence, driven by DNMT3A-mediated promoter hypermethylation, and lower PDLIM4 expression was associated with adverse patient prognosis. PDLIM4 methylation was also significantly increased in stool DNA from CRC patients compared with healthy controls. Mechanistically, genetic ablation of PDLIM4 significantly blunted the therapeutic efficacy of the DNA demethylating agent 5-Azacytidine against CRC. Functionally, PDLIM4 restoration suppressed CRC cell proliferation and stemness while enhancing apoptosis in vitro, and reduced tumour growth in both AOM/DSS and xenograft models in vivo. At the molecular level, PDLIM4 interacted with BTF3 and promoted its autophagic-lysosomal degradation, thereby limiting PI3K/Akt signalling, with NTRK2 identified as a potential transcriptional target linking BTF3 to PI3K/Akt activation. CONCLUSION: DNMT3A-mediated silencing of PDLIM4 is an early epigenetic event that facilitates CRC progression. PDLIM4 suppresses tumour growth by promoting BTF3 degradation, thereby restricting PI3K/Akt signalling. Thus, PDLIM4 methylation may have potential as a biomarker for early CRC detection. KEY POINTS: DNMT3A-mediated PDLIM4 promoter hypermethylation is an early event in colorectal tumorigenesis. Restoring PDLIM4 suppresses CRC proliferation and stemness while promoting apoptosis in vitro and in vivo. PDLIM4 promotes the autophagic-lysosomal degradation of BTF3 and restrains PI3K/Akt signalling. Stool DNA analysis supports the potential of PDLIM4 methylation as a non-invasive CRC biomarker.