SUMMARY 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 (CRC). Proteomic and biochemical analyses show that TMEM9 binds to and facilitates assembly of v-ATPase, a vacuolar proton pump, resulting in enhanced vesicular acidification and trafficking. TMEM9-v-ATPase hyperactivates Wnt/β-catenin signaling via lysosomal degradation of APC. Moreover, TMEM9 transactivated by β-catenin functions as a positive feedback regulator of Wnt signaling in CRC. Genetic ablation of TMEM9 inhibits CRC 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 signaling through APC degradation, and propose the blockade of TMEM9-v-ATPase as a viable option for CRC treatment.
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.
The epithelial integrity is maintained by the cytoskeleton and cell adhesion. However, it remains unknown how deregulated cytoskeleton is associated with cancer. We identified Cancer-related Regulator of Actin Dynamics (CRAD) as frequently mutated or transcriptionally downregulated in colorectal cancer (CRC). We found that CRAD stabilizes the cadherin-catenin-actin (CCA) complex via capping protein inhibition. CRAD loss inhibits F-actin polymerization and subsequently disrupts the CCA complex, which leads to β-catenin release and Wnt signaling hyperactivation. In mice, CRAD knockout induces the epithelial cell integrity loss and Wnt signaling activation, resulting in intestinal mucinous adenoma development. With APC mutation, CRAD knockout initiates and accelerates mucinous and invasive adenoma development in the colorectum. These results define CRAD as a tumor suppressor, of which inactivation deregulates the cytoskeleton and hyperactivates Wnt signaling, initiating mucinous CRC. Our study reveals the unexpected roles of an actin cytoskeletal regulator in maintaining epithelial cell integrity and suppressing tumorigenesis.
SUMMARY Fine control of stem cell maintenance and activation is crucial for tissue homeostasis and regeneration. However, the mechanism of quiescence exit of Tert+ intestinal stem cells (ISCs) remains unknown. Employing a Tert knock-in (TertTCE/+) mouse model, we found that Tert+ cells are long-term label-retaining self-renewing cells, which are partially distinguished from the previously identified +4 intestinal stem cells (ISCs). Tert+ cells become mitotic upon irradiation (IR) injury. Conditional ablation of Tert+ cells impairs IR-induced intestinal regeneration but not intestinal homeostasis. Upon IR injury, Wnt signaling is specifically activated in Tert+ cells via the ROS-HIFs-transactivated Wnt2b signaling axis. Importantly, conditional knock-out of β-catenin/Ctnnb1 in Tert+ cells undermines IR-induced quiescence exit of Tert+ cells, which subsequently impedes intestinal regeneration. Our results strongly suggest that Wnt signaling-induced activation of Tert+ ISCs is indispensable for intestinal regeneration, which unveils the underlying mechanism of how Tert+ stem cells undergo quiescence exit upon tissue injury.
The underlying mechanisms of how self-renewing cells are controlled in regenerating tissues and cancer remain ambiguous. PCNA-associated factor (PAF) modulates DNA repair via PCNA. Also, PAF hyperactivates Wnt/β-catenin signaling independently of PCNA interaction. We found that PAF is expressed in intestinal stem and progenitor cells (ISCs and IPCs) and markedly upregulated during intestinal regeneration and tumorigenesis. Whereas PAF is dispensable for intestinal homeostasis, upon radiation injury, genetic ablation of PAF impairs intestinal regeneration along with the severe loss of ISCs and Myc expression. Mechanistically, PAF conditionally occupies and transactivates the c-Myc promoter, which induces the expansion of ISCs/IPCs during intestinal regeneration. In mouse models, PAF knockout inhibits Apc inactivation-driven intestinal tumorigenesis with reduced tumor cell stemness and suppressed Wnt/β-catenin signaling activity, supported by transcriptome profiling. Collectively, our results unveil that the PAF-Myc signaling axis is indispensable for intestinal regeneration and tumorigenesis by positively regulating self-renewing cells.
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