FAM83A Promotes Lung Cancer Progression by Regulating the Wnt and Hippo Signaling Pathways and Indicates Poor Prognosis

Abstract

FAM83A (family with sequence similarity 83, member A) has been found to be highly expressed in cancers. The purpose of this study was to clarify the role and mechanism of FAM83A in lung cancers. The expression of FAM83A in lung cancer cells was enhanced by gene transfection or knocked down by small interfering RNA interference. The key proteins of the Wnt signaling pathway, the Hippo signaling pathway, and epithelial-mesenchymal transition (EMT) were examined using Western blot. The proliferation and invasion of lung cancer cells were examined using cell proliferation, colony formation, and invasion assays. The expression of FAM83A in lung cancer tissues was significantly increased and was correlated with advanced tumor-node-metastasis (TNM) stage and poor prognosis. Overexpression of FAM83A enhanced the proliferation, colony formation, and invasion of lung cancer cells. Meanwhile, FAM83A overexpression increased the expression of active β-catenin and Wnt target genes and the activity of EMT. Furthermore, in FAM83A-overexpressed cells, the activity of Hippo pathway was downregulated, whereas the expression of yes-associated protein (YAP) and its downstream targets cyclin E and CTGF were upregulated. The inhibitor of the Wnt signaling pathway, XAV-939, reversed the promoting effect of FAM83A on YAP, cyclin E, and CTGF. On knocking down the expression of FAM83A, we obtained the opposite results. However, the inhibitor of GSK3β, CHIR-99021, restored the expression of YAP, cyclin E, and CTGF after FAM83A was knocked down. FAM83A is highly expressed in lung cancers and correlated with advanced TNM stage and poor prognosis. FAM83A promotes the proliferation and invasion of lung cancer cells by regulating the Wnt and Hippo signaling pathways and EMT process.

Keywords: FAM83A; Hippo signaling pathway; Wnt signaling pathway; epithelial–mesenchymal transition; invasion; lung cancer; proliferation.

Figure 1

Expression of FAM83A in lung cancers and its correlation with prognosis. Expression levels of FAM83A in lung cancers and normal lung tissues, and their significant relation to the prognosis of patients with lung cancer. Box plots of FAM83A expression levels in lung adenocarcinomas (LUADs) (A) and lung squamous cell carcinomas (LUSC) (B) compared to normal lung tissues, which were retrieved from the UALCAN database. Kaplan–Meier curves of FAM83A expression in LUAD (C) and LUSC (D), as retrieved from the UALCAN database.

Figure 2

Effects of FAM83A on the proliferation, colony formation, and invasion of lung cancer cells. (A,B) The expression level of FAM83A of H1299 and A549 cells transfected with FAM83A or SiFAM83A. (C) The cell growth curve of H1299 and A549 cells transfected with FAM83A or SiFAM83A, and their control cells. (D–G) Representative images of the colony formation assay for H1299 and A549 cells transfected with FAM83A (D) or SiFAM83A (F), and their control cells. The number of colonies formed by each group is shown in the histogram (E,G). Representative images of the Matrigel invasion assay for H1299 and A549 cells transfected with FAM83A (H) or SiFAM83A (J) and their control cells. The invasive cell number for each group is shown in the histogram (I,K). *P < 0.05, **P < 0.01, ***P < 0.001, NC, negative control cells; FAM83A, cells transfected with FAM83A; SiNC, cells interfered with control SiRNA; SiFAM83A, cells interfered with SiFAM83A.

Figure 3

Expression of Wnt- and EMT-related genes under the regulation of FAM83A in lung cancer cells. Expression of Wnt signaling pathway- and EMT-related proteins after FAM83A overexpression (A) or knockdown (D) and relative protein levels in H1299 cells (B,E) and A549 cells (C,F). GAPDH served as an internal control (*P < 0.05, **P < 0.01, NC, negative control cells; FAM83A, cells transfected with FAM83A; SiNC, cells interfered with control SiRNA; SiFAM83A, cells interfered with SiFAM83A).

Figure 4

FAM83A inhibits the activation of the Hippo signaling pathway. Representative results of Western blot analysis (A,D) and relative protein levels in H1299 cells (B,E) and A549 cells (C,F). GAPDH served as an internal control (*P < 0.05, **P < 0.01, NC, negative control cells; FAM83A, cells transfected with FAM83A; SiNC, cells interfered with control SiRNA; SiFAM83A, cells interfered with SiFAM83A).

Figure 5

FAM83A enhances the proliferative and invasive abilities of lung cancer cells by the Wnt signaling pathway. (A) The cell growth curve of H1299 cells transfected with FAM83A, and their control cells were added with DMSO or XAV-939. (B,C) Representative images of the colony formation assay for H1299 cells transfected with FAM83A, and their control cells were added with DMSO or XAV-939 (B). The number of colonies formed by each group is shown in the histogram (C). (D,E) Representative images of the Matrigel invasion assay for H1299 cells transfected with FAM83A, and their control cells were added with DMSO or XAV-939 (D). The invasive cell number for each group is shown in the histogram (E). *P < 0.05, **P < 0.01, ***P < 0.001, NC, negative control cells; FAM83A, cells transfected with FAM83A; DMSO, dimethyl sulfoxide; XAV-939, an inhibitor of Wnt/β-catenin signaling; *NC+DMSO vs. FAM83A+DMSO; *FAM83A+DMSO vs. FAM83A+XAV-939.

Figure 6

GSK3β and the Wnt signaling pathway contribute to FAM83A induced YAP activation. Expression of YAP and downstream proteins after FAM83A overexpression and adding Wnt inhibitor (A) or knocking down and adding GSK-3α/β inhibitor (B) in H1299 cells. Relative expression levels of proteins for each group are shown in the histogram (C,D). *P < 0.05, **P < 0.01, NC, negative control cells; FAM83A, cells transfected with FAM83A; SiNC, cells interfered with control SiRNA; SiFAM83A, cells interfered with SiFAM83A; XAV-939, an inhibitor of Wnt/β-catenin signaling; CHIR-99021, a GSK-3α/β inhibitor.

Figure 7

A proposed model to illustrate the role of FAM83A in the Wnt and Hippo signaling pathways. GSK3β phosphorylates β-catenin and results in the degradation of β-catenin, which inhibits the activity of the Wnt signaling pathway. FAM83A can inhibit GSK3β activity and increase the level of active unphosphorylated β-catenin; active β-catenin then transports into the nucleus and activates the Wnt signaling pathway. Meanwhile, similar to β-catenin, FAM83A may also inhibit the phosphorylation and degradation of YAP, which is induced by the Hippo signaling pathway and enhance the activity of YAP through repressing GSK3β. In addition, FAM83A could downregulate the upstream MST to inhibit the activation of the Hippo signaling pathway. Figures were produced using Servier Medical Art (https://smart.servier.com).