The Roles Of LisH2 In Canonical Wnt Signaling

Signaling by the Wnt family of secreted glycolipoproteins is one of the fundamental mechanisms that direct cell proliferation, cell differentiation, and dorsoventral patterning during embryonic development and maintain adult tissue homeostasis. Dysregulation of Wnt signaling is often linked to a variety of human diseases including developmental defects, tumorigenesis, metabolic disorders, and neuronal degeneration. The research into the molecular regulatory mechanism of Wnt signaling has an important theoretical significance and a potential clinical application value on diagnosis and therapy of related diseases.A critical and heavily studied Wnt pathway is the canonical Wnt pathway. The hallmark of canonical Wnt signaling is the translocation of β-catenin into the nucleus, In the absence of Wnt, cytoplasmic β-catenin is efficiently maintained at a low level by Axin complex, which comprises two scaffolding proteins Axin and adenomatous polyposis coli (APC), and the kinases casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). Phosphorylation by GSK3β and CK1 targets β-catenin for ubiquitination and subsequent proteasomal degradation. Upon Wnt stimulation, Wnt binding to Frizzle (Fz) and LDL receptor-related protein 6 (LRP6) receptors initiates a signaling cascade that results in β-catenin stabilization via inactivating Axin complex, thereby the stabilized β-catenin enters the nucleus to form a transcriptional complex with T cell factor (TCF) or lymphoid enhancer factor (LEY) to activate Wnt target genes expression. However, the mechanisms that control β-catenin nuclear localization are not fully understood.LisH2 (Lisl Homology 2) is an evolutionarily conserved protein, which was found to interact with RanBPM. RanBPM serves as a scaffolding protein and plays important roles in cell cycle progression and signaling transduction; however, the biological functions of LisH2 are still unknown.In this study, we sought to elucidate the functions of LisH2 in human diseases, particularly in colorectal cancer (CRC). We initially analyzed multiple sets of human colorectal cancer tissue samples using the publicly available database TCGA (The Cancer Genome Atlas). RNA-seq data showed that the mRNA expression level of LisH2 was significantly higher in tumor tissues than in corresponding adjacent normal tissues. We then detected LisH2 mRNA level in our collected CRC samples, and found that LisH2 was indeed highly expressed in colorectal tumor, implying a role for LisH2 in colorectal tumorigenesis. Since signaling pathways controlling normal cell growth, differentiation, and embryonic development are almost invariably altered in cancer, we tested a panel of well-established signaling reporters monitoring the activities of several major signaling pathways. Unexpectedly, we found that knockdown of LisH2 selectively reduced Wnt3a-stimulated Lef-1 reporter activity. Furthermore, loss of LisH2 decreased the mRNA level of Axin2 and CyclinD1, two of the most established Wnt readouts. These suggest that LisH2 is a positive regulator in canonical Wnt signaling.To investigate how LisH2 modulates canonical Wnt signaling, we first examined β-catenin level in LisH2-depleted HEK-293 cells. Western analyses showed that LisH2 knockdown did not change the total level of P-catenin upon Wnt stimulation, but strikingly abolished β-catenin nuclear accumulation, and the amount of β-catenin that bound to TCF4. Meanwhile, we found that Wnt probably promotes LisH2 nuclear accumulation via inactivation of Axin complex.To clarify the mechanisms how LisH2 modulates β-catenin nuclear accumulation, we determined the interaction between LisH2 and β-catenin. GST-pull down and CO-IP analyses revealed that LisH2 interacted with P-catenin through CRA domain. Ectopic expression of wild-type LisH2, LisH2 mutant either deleted LisH domain or CTLH domain successfully reversed the defects induced by LisH2 knockdown, but LisH2 mutant deprived of CRA domain failed to rescue those defects. Moreover, NLS-LisH2 or NLS-LisH2 mutants harboring CRA domain sufficiently induced P-catenin accumulation in the nucleus even without Wnt stimulation. But NLS-LisH2-ΔCRA, in spite of predominately accumulating in the nucleus, is not capable of promoting P-catenin nuclear localization. These suggest that LisH2 facilitates P-catenin nuclear localization through CRA domain.To determine the physiological relevance of LisH2 in canonical Wnt signaling, we extended our analyses to zebrafish embryos where nuclear P-catenin is essential for dorsal development during early embryogenesis. Depletion of LisH2 suppressed Wnt target gene boz expression, and led to ventralization of the zebrafish embryos, resembling β-catenin2 knockdown defects. Co-injection of wild-type LisH2 or NLS-β-catenin mRNA were able to rescue the defects caused by LisH2 deficiency, but LisH2-ΔCRA failed to reversed the LisH2 knockdown phenotypes. These data suggest that LisH2 facilitates β-catenin nuclear accumulation in vivo.Elevated nuclear P-catenin accumulation is a hallmark downstream of oncogenic Wnt signaling for colorectal cancer development, we sought to determine the role of LisH2 in colorectal tumorigenesis.We testified LisH2 function in DLD1 and SW480, two colorectal cancer cell lines harboring a constitutively high level of nuclear P-catenin due to APC mutation. Depletion of LisH2 not only reduced β-catenin nuclear localization and Wnt activity, but also attenuated the growth and tumorigenecity of CRC cells. These data suggest that LisH2-mediated β-catenin nuclear localization probably contributes to human colorectal tumorigenesis.In conclusion, LisH2 regulates canonical Wnt signaling by facilitating β-catenin nuclear accumulation, which is essential for dorsoventral patterning and colorectal tumorigenesis. Our findings contributes to understanding the roles of LisH2 in canonical Wnt signaling, and LisH2 may represent a potential therapeutic target for human diseases related to the dysfunction of canonical Wnt pathway.