Carbonic Anhydrase IV Inhibits Colon Cancer Development by Inhibiting WNT Signaling Pathway Through Targeting WTAP-WT1-TBL1 Axis

Abstract

Using Methylated DNA Immunoprecipitation (MeDIP) combined with microarray assay, Carbonic Anhydrase IV (CA4) was identified to be preferentially methylated in colorectal cancer (CRC). CA4 is a member of carbonic anhydrases family which catalyzes the reversible hydration of carbondioxide. However, the functional role of CA4 in CRC is still unclear. CA4 promoter methylation status was evaluated by bisulfite genomic sequencing. The biological functions of CA4 were determined in vitro cell viability, colony formation, cell apoptosis, and cell cycle assays and in vivo tumorigenicity assay. The cooperators of CA4 were identified by co-immunoprecipitation assay, mass spectrometry, promoter luciferase reporter assay, chromatin immunoprecipitation assay and gel shift assay. Clinical application of CA4 methylation was assessed in 115 primary CRC tissues. We first screened the expression status of CA4 in 9 CRC cell lines and primary CRC tissues. CA4 was silenced in all 9 cell lines and the mean protein expression level of CA4 was significantly lower in CRC tissues as compared to their adjacent normal tissues (P<0.01). Silence of CA4 was mediated by promoter methylation. Methylation status of CA4 was detected in 75.7% (87/115) of CRC patients. Multivariate analysis and recurrence curve revealed that patients with CA4 methylation had significantly higher opportunities to suffer from cancer recurrence (P < .05). Re-expression of CA4 in CRC cell lines suppressed cell viability (P < .001) and colony formation (P < .01), and caused cell cycle arrest in G1 phase (P < .05). CA4 also induced cell apoptosis (P < .01), concomitant with enhanced protein expression of pro-apoptotic factors cleaved caspase3, caspase7, caspase8 and PARP. The in vivo growth of CRC cells in nude mice was also markedly inhibited by stable transfection with vector harboring CA4 (P < .05). We further tested the molecular mechanisms of CA4 in CRC as a tumor suppressor. Co-immunoprecipitation assay revealed that Wilms tumor 1-associating protein (WTAP) is directly interacted with CA4. Transfection of CA4 in CRC cell lines induced WTAP protein degradation through poly-ubiquitination. Moreover, CA4 promoted transcriptional activity of Wilms tumor 1 (WT1), a negative regulator of Wnt pathway, leading to induction of transducing β-like protein 1(TBL1). TBL1 induced β-catenin degradation through a non-phosphorylated way which finally inhibit Wnt signaling pathway activity. Epigenetic inactivation of CA4 is a common event in CRC which may play a pivotal role as a novel tumor suppressor in CRC carcinogenesis. CA4 inhibit Wnt signaling pathway by targeting WTAP-WT1-TBL1 axis. Detection of CA4 methylation may serve as an independent biomarker for the recurrence of CRC.