Verification of novel Wnt/B-catenin pathway targets

One of the most fundamental signal transduction cascades, the Wnt/β-catenin signaling pathway, is an evolutionary conserved pathway in vertebrates which controls embryonic development and adult homeostasis. Signaling through secreted glycoproteins of Wnt family results in translocation of the transcriptional activator β-catenin into the nucleus where it controls key developmental gene expression programs. Having an important role in tissue homeostasis, aberrant activation of Wnt/β-catenin signaling pathway is often linked to a wide variety of cancers. Mutations in target molecules of this pathway are identified to have tumorigenic effect. Previous studies confirmed BRI3 (Brain Protein 3) and MGAT1 (Mannosyl (Alpha-1,3-)-Glycoprotein Beta-1,2-N-Acetylglucosaminyltransferase) genes to be novel transcriptional targets of Wnt/β-catenin pathway. Huh7 cells (hepatocellular cancer cell line) stably expressing either BRI3 or MGAT1 have greater proliferative and invasive capabilities compared to wildtype Huh7 cells, and when subcutaneously injected into NUDE/SCID mice resulted in larger tumor formation. In the light of these findings, tumors were subjected to RNA-Sequencing for further investigation. We observed differential expression of certain genes involved in several cellular pathways. Among these genes, we continued with YAP1, CSNK2B, and AFP in BRI3 expressing cells; and EDNRB, ADAM17, and HSPA8 in MGAT1 expressing cells. We validated differential expression levels by using Western Blot and qRT-PCR techniques. We further aim to shed light into the crosstalk between different signaling pathways involved in cancer progression.