KLHL6 drives colorectal cancer liver metastasis through ubiquitin-mediated degradation of MBD2 that triggers CLDN4 activation

Background Liver metastasis is the leading cause of death among patients with colorectal cancer (CRC) and remains a major clinical challenge because durable therapeutic options are limited. Metastatic progression depends on extensive transcriptional plasticity, yet how epigenetically silenced prometastatic genes are reactivated remains unclear. Methods We performed integrative analyses of bulk RNA sequencing from a CRC liver metastasis (CRLM) mouse model and single-cell RNA sequencing datasets from human CRC samples to identify metastasis-associated candidates. The expression pattern and clinical significance of Kelch-like protein 6 (KLHL6) were assessed in public datasets and independent clinical cohorts. Functional roles of KLHL6 were investigated using gain- and loss-of-function assays in vitro and liver metastasis models in vivo. The underlying mechanism was explored through RNA sequencing, chromatin immunoprecipitation, luciferase reporter assays, pyrosequencing, whole-genome bisulfite sequencing, co-immunoprecipitation, GST pull-down, surface plasmon resonance, cycloheximide chase, and ubiquitination analyses. Therapeutic efficacy was evaluated using lipid nanoparticle-mediated siRNA delivery targeting KLHL6, alone or in combination with anti-PD-1 therapy. Results KLHL6 was identified as a metastasis-associated gene that was upregulated in CRLM and related with poor prognosis. Functionally, KLHL6 promotes CRC cell migration, invasion, and liver metastasis in an E3 ligase-dependent manner. Mechanistically, KLHL6 directly interacts with the DNA methylation reader MBD2 and induces its proteasomal degradation by mediating K27-linked polyubiquitination at MBD2 lysine 185, thereby relieving methylation-dependent transcriptional repression. This leads to activation of the tight junction protein CLDN4, which drives metastatic progression. Genetic and functional rescue experiments establish the KLHL6/MBD2/CLDN4 axis as a critical regulator of metastasis both in vitro and in vivo. Systemic delivery of KLHL6 siRNA suppresses liver metastasis, prolongs survival and shows enhanced antitumor activity when combined with PD-1 blockade in mice. Conclusions We identify a previously unrecognized KLHL6/MBD2/CLDN4 regulatory axis that drives CRLM. Our findings reveal that proteostasis-driven destabilization of epigenetic repressors enables metastatic transcriptional plasticity, uncovering a mechanistic link between the ubiquitin system and DNA methylation-dependent gene regulation in CRC.