Suppression of FAM83D Inhibits Glioma Proliferation, Invasion and Migration by Regulating the AKT/mTOR Signaling Pathway

Abstract

Objective: To explore the mechanism by which the family with sequence similarity 83, member D (FAM83D)-mediated AKT/mTOR signaling pathway activation affects the proliferation and metastasis of glioma cells.

Methods: FAM83D protein expression in glioma cells and tissues was detected by western blotting. Glioma U87 and U251 cells were selected and divided into the Mock, siNC, siFAM83D, FAM83D, MK2206 and FAM83D + MK2206 groups. Cell proliferation was assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) and clone formation assays, while invasion and migration were evaluated by Transwell assays and wound healing tests. The protein expression of members of the AKT/mTOR pathway was determined via western blotting. Xenograft models were also established in nude mice to observe the in vivo effect of FAM83D on the growth of glioma.

Results: FAM83D was upregulated in glioma patients, especially in those with Stage III-IV. In addition, cells treated with siFAM83D had significant downregulation of p-AKT/AKT and p-mTOR/mTOR, with decreased proliferation and colony numbers, as well as decreased invasion and migration compared to the Mock group. However, FAM83D overexpression could activate the Akt/mTOR pathway and promote the proliferation, invasion and migration of glioma cells. Moreover, treatment with MK2206, an inhibitor of AKT, reversed the promoting effect of FAM83D on the growth of glioma cells. The in vivo experiments demonstrated that silencing FAM83D could inhibit the in vivo growth of glioma cells CONCLUSION: FAM83D was upregulated in glioma and silencing FAM83D suppressed the proliferation, invasion and migration of glioma cells via inhibition of the AKT/mTOR pathway.

Keywords: AKT/mTOR; FAM83D; Glioma; Invasion; Migration; Proliferation.

Fig. 1

Expression of FAM83D in glioma cells and tissues. A, TCGA database demonstrated that FAM83 was highly expressed in GBM patients; * P < 0.05 vs. normal tissues; B, Western blotting was conducted to determine the protein expression of FAM83D in glioma and adjacent tissues; * P < 0.05 vs. tumor-adjacent normal tissues; # P < 0.05 vs. glioma patients in grade I or II; C, Western blotting was conducted to determine the protein expression of FAM83D in glioma cell lines (U87, U251, T98G, SHG44 and U118) and normal human astrocytes (NHAs); * P < 0.05 vs. NHA cells.

Fig. 2

Expression of FAM83D and the AKT/mTOR pathway in U251 and U87 cells. A-B, Western blotting was conducted to determine the protein expression of FAM83D, p-AKT/AKT and p-mTOR/mTOR in glioma U251 (A) and U87 (B) cell lines; * P < 0.05 vs. the Mock group; # P < 0.05 vs. the siFAM83D group; & P < 0.05 vs. the FAM83D group; % P < 0.05 vs. the MK2206 group.

Fig. 3

Effect of the FAM83D-mediated AKT/mTOR pathway on the proliferation of glioma cells. A, MTT assays were conducted to evaluate the proliferation ability of U87 and U251 cells; B, Colony formation assays were conducted to determine the colony formation of U87 and U251 cells; C, Comparison of the colony numbers between U87 and U251 cells; * P < 0.05 vs. the Mock group; # P < 0.05 vs. the siFAM83D group; & P < 0.05 vs. the FAM83D group; % P < 0.05 vs. the MK2206 group.

Fig. 4

Effect of the FAM83D-mediated AKT/mTOR pathway on the invasion and migration abilities of glioma cells. A, Transwell assays were conducted to determine the invasive abilities of U87 and U251 cells; B, Comparison of the invaded cell numbers between U87 cells and U251 cells; C, Wound healing tests were conducted to determine the migration abilities of U87 and U251 cells; B, Comparison of the wound closure between U87 cells and U251 cells; * P < 0.05 vs. the Mock group; # P < 0.05 vs. the siFAM83D group; & P < 0.05 vs. the FAM83D group; % P < 0.05 vs. the MK2206 group.

Fig. 5

Effect of silencing FAM83D on the growth of xenograft tumors in subcutaneous (A-D) and intracranial (E-G) mouse models. A, Gross specimens of xenograft tumors of nude mice; B, Growth curve of xenograft tumors in nude mice; C, Comparison of the weight of xenograft tumors among groups; D, Immunohistochemistry analysis of Ki67 protein levels in xenograft tumor tissues; E, Mouse survival shown in Kaplan–Meier curves; F, Representative images of H&E staining of mouse brain sections; G, The tumor volume in the siNC and siFAM83D groups were analyzed.