Dishevelled (DVL) proteins serve as crucial regulators that transduce canonical Wnt signals to the GSK3β-destruction complex, resulting in the stabilization of β-catenin. Emerging evidence underscores the nuclear functions of DVLs, which are critical for Wnt/β-catenin signaling. However, the mechanism underlying DVL nuclear localization remains poorly understood. Here we discovered two Forkhead box (FOX) transcription factors, FOXK1 and FOXK2, as bona fide DVL-interacting proteins. FOXK1 and FOXK2 positively regulate Wnt/β-catenin signaling by translocating DVL into the nucleus. Moreover, FOXK1 and FOXK2 protein levels are elevated in human colorectal cancers and correlate with DVL nuclear localization. Conditional expression of Foxk2 in mice induced intestinal hyper-proliferation that featured enhanced DVL nuclear localization and upregulated Wnt/β-catenin signaling. Together, our results not only reveal a mechanism by which DVL is translocated into the nucleus but also suggest unexpected roles of FOXK1 and FOXK2 in regulating Wnt/β-catenin signaling.

Despite the implication of Wnt signalling in radioresistance, the underlying mechanisms are unknown. Here we find that high Wnt signalling is associated with radioresistance in colorectal cancer (CRC) cells and intestinal stem cells (ISCs). We find that LIG4, a DNA ligase in DNA double-strand break repair, is a direct target of β-catenin. Wnt signalling enhances non-homologous end-joining repair in CRC, which is mediated by LIG4 transactivated by β-catenin. During radiation-induced intestinal regeneration, LIG4 mainly expressed in the crypts is conditionally upregulated in ISCs, accompanied by Wnt/β-catenin signalling activation. Importantly, among the DNA repair genes, LIG4 is highly upregulated in human CRC cells, in correlation with β-catenin hyperactivation. Furthermore, blocking LIG4 sensitizes CRC cells to radiation. Our results reveal the molecular mechanism of Wnt signalling-induced radioresistance in CRC and ISCs, and further unveils the unexpected convergence between Wnt signalling and DNA repair pathways in tumorigenesis and tissue regeneration.

Epithelial integrity is maintained by the cytoskeleton and through cell adhesion. However, it is not yet known how a deregulated cytoskeleton is associated with cancer. We identified cancer-related regulator of actin dynamics (CRAD) as frequently mutated or transcriptionally downregulated in colorectal cancer. We found that CRAD stabilizes the cadherin-catenin-actin complex via capping protein inhibition. The loss of CRAD inhibits F-actin polymerization and subsequently disrupts the cadherin-catenin-actin complex, which leads to β-catenin release and Wnt signalling hyperactivation. In mice, CRAD knockout induces epithelial cell integrity loss and Wnt signalling activation, resulting in the development of intestinal mucinous adenoma. With APC mutation, CRAD knockout initiates and accelerates mucinous and invasive adenoma development in the colorectum. These results define CRAD as a tumour suppressor, the inactivation of which deregulates the cytoskeleton and hyperactivates Wnt signalling thus initiating mucinous colorectal cancer. Our study reveals the unexpected roles of an actin cytoskeletal regulator in maintaining epithelial cell integrity and suppressing tumorigenesis.

Vesicular acidification and trafficking are associated with various cellular processes. However, their pathologic relevance to cancer remains elusive. We identified transmembrane protein 9 (TMEM9) as a vesicular acidification regulator. TMEM9 is highly upregulated in colorectal cancer. Proteomic and biochemical analyses show that TMEM9 binds to and facilitates assembly of vacuolar-ATPase (v-ATPase), a vacuolar proton pump, resulting in enhanced vesicular acidification and trafficking. TMEM9-v-ATPase hyperactivates Wnt/β-catenin signalling via lysosomal degradation of adenomatous polyposis coli (APC). Moreover, TMEM9 transactivated by β-catenin functions as a positive feedback regulator of Wnt signalling in colorectal cancer. Genetic ablation of TMEM9 inhibits colorectal cancer cell proliferation in vitro, ex vivo and in vivo mouse models. Moreover, administration of v-ATPase inhibitors suppresses intestinal tumorigenesis of APC mouse models and human patient-derived xenografts. Our results reveal the unexpected roles of TMEM9-controlled vesicular acidification in hyperactivating Wnt/β-catenin signalling through APC degradation, and propose the blockade of TMEM9-v-ATPase as a viable option for colorectal cancer treatment.