FAM83A is amplified and promotes tumorigenicity in non-small cell lung cancer via ERK and PI3K/Akt/mTOR pathways

Abstract

Family with sequence similarity 83A (FAM83A) is a newly-found over-expressed oncogene in several types of cancers and associates with poor prognosis. However, the role that FAM83A may play in the carcinogenesis of non-small cell lung cancer (NSCLC) still needs to be defined. The present study aimed to investigate the function of FAM83A in NSCLC progression and to investigate the possible mechanism. Analysis of Gene Expression Omnibus (GEO) database and rt-PCR showed up-regulated expression of FAM83A in NSCLC. GEO and the Cancer Genome Atlas (TCGA) data analysis revealed that high expression level of FAM83A in NSCLC was associated with poor prognosis. In vitro experiments showed that depleting FAM83A by siRNA/shRNA significantly inhibited cell proliferation and induced cell apoptosis. Cell motility was also retarded after silencing FAM83A, as demonstrated by Transwell assay. FAM83A depletion in A549 cells also inhibited subcutaneous tumor growth and lung metastasis in vivo. Western blotting showed that silencing FAM83A decreased the phosphorylation of ERK and PI3K/Akt/mTOR. On the other hand, overexpressing FAM83A in vitro enhanced cell proliferation and invasiveness, which was repressed by PI3K inhibitor and ERK inhibitor separately. Taken together, our study suggests that FAM83A promotes tumorigenesis of NSCLC at least partly via ERK and PI3K/Akt/mTOR pathways, making it a promising therapeutic target.

Keywords: FAM83A; metastasis; non-small cell lung cancer; tumorigenesis.

Figure 1

FAM83A is overexpressed in NSCLC and correlates with poorer prognosis. (A) Analysis of GEO database (GSE18842, GSE19188, GSE43458, GSE75037) shows higher expression level of FAM83A in NSCLC compared with normal tissues. (B) FAM83A mRNA expression in 4 NSCLC cell lines is higher than in normal bronchial epithelial cell line BEAS2B as tested by rt-PCR. (C) Prognosis analysis of TCGA and GEO databases shows that higher expression of FAM83A in NSCLC correlates with poorer overall survival and progression-free survival. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

FAM83A promotes NSCLC cell growth in vitro and in vivo. (A) FAM83A is successfully depleted by siRNA in A549 and H1299 cells. (B) Knocking down FAM83A in vitro inhibits growth of A549 and H1299 cells as demonstrated by CCK8 assay. (C) Depleting FAM83A decreased number of clones formed in both A549 and H1299 cell lines. (D) FAM83A is successfully overexpressed in H460 cells. (E) Overexpressing FAM83A in H460 cells promotes clone formation, which is reversed by wortmanin and SCH772984. (F) Silencing of FAM83A decreases protein expression of PCNA, Bcl-xL, MMP-9 and increases expression of Bad and Bax. In vivo experiments shows FAM83A depletion in A549 inhibited subcutaneous tumor volume (G) and weight (H), and representative figures are shown (I). *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Depleting FAM83A promotes NSCLC cell apoptosis. (A, C) Rt-PCR and WB show that FAM83A is successfully knocked down by 2 shRNAs in A549 and H1299 cells. (B, D) Cell apoptosis is induced by specified concentration of H₂O₂ and stained by PI for detection of apoptosis. RNAi groups show significant cell apoptosis compared with Con and NC groups (Magnification: 200×).

Figure 4

FAM83A promotes NSCLC metastasis and functions through ERK and PI3K/Akt/mTOR pathways. (A, B) Depleting FAM83A in A549 and H1299 cells restrains wound healing percentage in Scratch assay (Magnification: 200×). (C, D) Depleting FAM83A in A549 and H1299 cells decreases number of migrated and invaded cells in Transwell assays (Magnification: 200×). (E) FAM83A is overexpressed in H460 cells. (F) Overexpressing FAM83A increases number of migrated H460 cells in Transwell assay, which is reversed by wortmanin and SCH772984. (G) Representative images of H460 Transwell assay are shown (Magnification: 200×). Lung metastasis model of mice is established by tail vein injection of A549 cells. RNAi group shows less metastatic nodules on the lung surface (H), which is also illustrated in lung sections (I, magnification: 200×). (J) WB shows that depleting FAM83A in A549 and H1299 cells mitigated protein expression of p-ERK, p-PI3K p85, p-Akt and p-mTOR. **P < 0.01, ***P < 0.001.