br Materials and methods br Results

Materials and methods
Results
Discussion Ajuba was originally identified as an adaptor protein which communicates cell adhesive events with nuclear responses to remodel the epithelium (Langer et al., 2008; Marie et al., 2003). Recently, increasing evidence has shown that Ajuba functions as an oncogene in human cancers including esophageal squamous cell carcinoma, colorectal carcinoma and head and neck squamous cell carcinoma (Jia et al., 2017; Shi et al., 2016; Zhang et al., 2017b). However, the expression pattern and biological roles of Ajuba in gastric cancers remains unclear. Our findings clearly showed that Ajuba protein was upregulated in gastric cancer tissues and cell lines, which was supported by analyses of RNA-seq data from TCGA and Oncomine. We also demonstrated that Ajuba overexpression positively correlated with TNM stage, nodal metastasis and poor prognosis, making Ajuba as a potential cancer bio-marker. The MTT cell viability and colony formation assays indicated that Ajuba overexpression facilitated cell proliferation. Tumor GSK1838705A depend on ATP to support their proliferation and typically shift towards glycolysis to fulfill energy demand (Wang et al., 2018). The shift of glucose metabolism is important during the process of malignant progression, which was investigated in this study. Our results demonstrated that Ajuba robustly increased glucose uptake and consumption, and enhanced lactate and ATP production. We postulated that change of glucose transporter proteins could be the possible cause of metabolic shift after Ajuba overexpression. Our data showed that Ajuba could upregulated GLUT1 mRNA and protein. In addition, TCGA data analysis showed a positive correlation of Ajuba and GLUT1 mRNA in gastric cancer tissues. GLUT1 belongs to the GLUT superfamily of membrane transporters that mediate the transport of small carbon compounds across the cell membranes (Wang et al., 2018). GLUT1 plays a critical role in metabolic reprogramming of tumor cells. Aberrant GLUT family expression, especially GLUT1, has been found in various cancers (Chai et al., 2017; Gu et al., 2006; Hussein et al., 2011; Rastogi et al., 2007; Yang et al., 2016; Yang et al., 2017). GLUT1 has also been reported to support growth, metastasis and glucose metabolism in gastric cancer cells (Lu et al., 2018; Yan et al., 2015). Our results clearly showed that Ajuba upregulated GLUT1 mRNA and protein, which enhanced glucose uptake and, thus, functions as a positive regulator of glycolysis. Our finding is the first to reveal the relationship between Ajuba and the glucose transport/GLUT1. Although the role of Ajuba in reducing chemosensitivity has been previously reported (Bi et al., 2018), its underlying mechanism remains elusive. The pathways of chemotherapeutic drugs, such as cisplatin, to induce apoptosis mainly dependent on changes of the mitochondrial status (Guerra et al., 2017; Kim et al., 2004; Zhao et al., 2013). We first determined changes of apoptosis and mitochondrial membrane potentials after cisplatin treatment. Ajuba prevented caspase 3 and PARP cleavage and inhibited cisplatin induced apoptosis. JC-1 staining showed that Ajuba was able to maintain a normal mitochondrial membrane potential. We also screened related proteins and found that Ajuba upregulated Bcl-xL, an important mitochondrial membrane protein which has been reported to maintain mitochondrial membrane potential, mitochondrial homeostasis and prevent apoptosis (Vander Heiden et al., 1997). We also observed a positive association between Ajuba and Bcl-xL using TCGA data. Overall, our findings linked the anti-apoptotic role of Ajuba to mitochondrial function and Bcl-xL, which has not been previously reported. Ajuba serves as a negative regulator of Hippo pathway by interacting with LATS to inhibit YAP phosphorylation (Das Thakur et al., 2010). Ajuba could limit Hippo regulation of YAP in proliferating cells by sequestering the cytosolic Hippo kinase complex (Jagannathan et al., 2016). Ajuba overexpression upregulated YAP protein in ovarian cancer cells (Bi et al., 2018). In addition, depletion of YAP downregulated Ajuba mRNA in small cell lung cancer (Horie et al., 2016), suggesting a positive feedback loop between Ajuba and YAP in these cells. Importantly, both Bcl-xL and GLUT1 have been shown to be target genes of YAP. YAP depletion suppresses Bcl-xL mRNA, which could rescues YAP-depleted cells from growth suppression by RAF-MEK inhibition(Lin et al., 2015). The YAP-GLUT1 axis has been shown to regulate glycolysis and proliferation in breast cancer cells (Lin and Xu, 2017). In the present study, we showed that Ajuba overexpression upregulated while Ajuba depletion downregulated YAP protein. YAP suppression by siRNA partly abolished the effect of Ajuba on Bcl-xL, GLUT1, cyclin D1 expression and glycolysis, which supported the importance of the Ajuba-YAP-GLUT1/Bcl-xL axis in gastric cancer cells.