Long noncoding RNA DLGAP1-AS2 facilitates Wnt1 transcription through physically interacting with Six3 and drives the malignancy of gastric cancer

The long noncoding RNA (lncRNA) DLGAP1-AS2 has recently been characterized as an oncogenic lncRNA in several cancers. However, its biological roles and clinical significance in gastric cancer (GC) remains barely understood. In this study, we performed a systematic analysis of DLGAP1-AS2 expression with data from the TCGA and GEO database as well as our clinic GC samples. In agreement with previous studies, our findings demonstrated that DLGAP1-AS2 was significantly up-regulated in GC and its high expression was associated with poor prognosis, suggesting that DLGAP1-AS2 might be a putative oncogenic lncRNA of GC. Loss of DLGAP1-AS2 restricted cell proliferation, migration, and invasion in GC cell lines. Mechanically, Wnt1 was identified as the downstream target of DLGAP1-AS2 by using bioinformatics analysis coupled with qPCR and Western blot assays. Furthermore, DLGAP1-AS2 was found to directly interact with the transcriptional repressor Six3, and this interaction hampered Six3 binding to the promoter regions of the Wnt1 gene, thereby leading to transcriptional activation of Wnt1. Consequently, GC cells lacking DLGAP1-AS2 showed a decreased Wnt1 expression and weakened Wnt/β-catenin signaling. Further, Six3 silencing could reverse the above effects, highlighting a pivotal role of Six3 in the DLGAP1-AS2-mediated activation of Wnt/β-catenin signaling. Either genetic (Wnt1 knockdown) or pharmacological (LF3) inhibition of Wnt/β-catenin signaling could effectively abolish the activation of Wnt/β-catenin signaling by Six3 depletion, thereby preventing GC cell malignant transformation. Taken together, our results suggest that DLGAP1-AS2 functions as an oncogenic factor by directly interacting with Six3 to relieve its suppression on Wnt1 expression, thereby driving the malignancy of GC. DLGAP1-AS2/Six3/Wnt1/β-catenin signaling axis might serve as a promising diagnostic and therapeutic target for GC.

Wnt1 Promotes Cementum and Alveolar Bone Growth in a Time-Dependent Manner

The WNT/β-catenin signaling pathway plays a central role in the biology of the periodontium, yet the function of specific extracellular WNT ligands remains poorly understood. By using a Wnt1-inducible transgenic mouse model targeting Col1a1-expressing alveolar osteoblasts, odontoblasts, and cementoblasts, we demonstrate that the WNT ligand WNT1 is a strong promoter of cementum and alveolar bone formation in vivo. We induced Wnt1 expression for 1, 3, or 9 wk in Wnt1Tg mice and analyzed them at the age of 6 wk and 12 wk. Micro–computed tomography (CT) analyses of the mandibles revealed a 1.8-fold increased bone volume after 1 and 3 wk of Wnt1 expression and a 3-fold increased bone volume after 9 wk of Wnt1 expression compared to controls. In addition, the alveolar ridges were higher in Wnt1Tg mice as compared to controls. Nondecalcified histology demonstrated increased acellular cementum thickness and cellular cementum volume after 3 and 9 wk of Wnt1 expression. However, 9 wk of Wnt1 expression was also associated with periodontal breakdown and ectopic mineralization of the pulp. The composition of this ectopic matrix was comparable to those of cellular cementum as demonstrated by quantitative backscattered electron imaging and immunohistochemistry for noncollagenous proteins. Our analyses of 52-wk-old mice after 9 wk of Wnt1 expression revealed that Wnt1 expression affects mandibular bone and growing incisors but not molar teeth, indicating that Wnt1 influences only growing tissues. To further investigate the effect of Wnt1 on cementoblasts, we stably transfected the cementoblast cell line (OCCM-30) with a vector expressing Wnt1-HA and performed proliferation as well as differentiation experiments. These experiments demonstrated that Wnt1 promotes proliferation but not differentiation of cementoblasts. Taken together, our findings identify, for the first time, Wnt1 as a critical regulator of alveolar bone and cementum formation, as well as provide important insights for harnessing the WNT signal pathway in regenerative dentistry.

Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

Endothelial Wnt/β-catenin signaling is necessary for angiogenesis of the central nervous system and blood–brain barrier (BBB) differentiation, but its relevance for glioma vascularization is unknown. In this study, we show that doxycycline-dependent Wnt1 expression in subcutaneous and intracranial mouse glioma models induced endothelial Wnt/β-catenin signaling and led to diminished tumor growth, reduced vascular density, and normalized vessels with increased mural cell attachment. These findings were corroborated in GL261 glioma cells intracranially transplanted in mice expressing dominant-active β-catenin specifically in the endothelium. Enforced endothelial β-catenin signaling restored BBB characteristics, whereas inhibition by Dkk1 (Dickkopf-1) had opposing effects. By overactivating the Wnt pathway, we induced the Wnt/β-catenin–Dll4/Notch signaling cascade in tumor endothelia, blocking an angiogenic and favoring a quiescent vascular phenotype, indicated by induction of stalk cell genes. We show that β-catenin transcriptional activity directly regulated endothelial expression of platelet-derived growth factor B (PDGF-B), leading to mural cell recruitment thereby contributing to vascular quiescence and barrier function. We propose that reinforced Wnt/β-catenin signaling leads to inhibition of angiogenesis with normalized and less permeable vessels, which might prove to be a valuable therapeutic target for antiangiogenic and edema glioma therapy.