Upregulation of family with sequence similarity 83 member D expression enhances cell proliferation and motility via activation of Wnt/β-catenin signaling and predicts poor prognosis in gastric cancer

Abstract

Background/aims: Gastric cancer (GC) is the third most common cause of cancer-related death worldwide. The molecular mechanisms underlying the progression of gastric cancer are still not fully elucidated. In this study, we focused on exploring the role of family with sequence similarity 83, member D (FAM83D) in gastric cancer progression.

Methods: The expression of FAM83D in GC tissues was detected by immunohistochemistry (IHC) staining. FAM83D knockdown or overexpression were constructed in AGS and SGC-7901 cells with two distinct siRNA duplexes and lentivirus infection, respectively, to explore the role of FAM83D in gastric cancer progression. Nude mouse xenograft assay was used to further explore the role of FAM83D in tumorigenesis in vivo.

Results: We found that FAM83D mRNA and protein levels were higher in human GC tumor tissues and in GC cell lines, compared with the adjacent normal tissues and non-malignant gastric epithelial cell lines, respectively, and that higher FAM83D expression was correlated with worse overall survival (p<0.0001) and disease-free survival (p<0.0001) in GC patients. Additionally, our results showed that FAM83D overexpression significantly enhanced the proliferation, clonogenicity, and motility of GC cells, whereas FAM83D depletion caused a dramatic increase in the number of cells arrested at the G1 phase of the cell cycle. Consistent with these findings from in vitro experiment, our data also indicated that FAM83D knockdown significantly repressed GC tumor growth in vivo. Furthermore, we demonstrated that FAM83D depletion was associated with reduced Wnt/β-catenin signaling.

Conclusions: This study suggested that FAM83D overexpression enhanced the proliferation, clonogenicity, and motility of GC cells by activating Wnt/β-catenin signaling, and FAM83D may be a promising diagnostic and therapeutic target for human GC.

Keywords: FAM83D; Wnt/β-catenin; gastric cancer; proliferation.

Figure 1

FAM83D is overexpressed in human GC. (A) FAM83D mRNA expression in liver cancer tissues is higher than normal tissues from ONCOMINE database. Dataset 1, Diffuse Gastric Adenocarcinoma vs Normal, Chen Gastric; Dataset 2, Gastric Intestinal Type Adenocarcinoma vs Normal, Chen Gastric; Dataset 3, Diffuse Gastric Adenocarcinoma vs Normal, Cho Gastric; Dataset 4, Gastric Intestinal Type Adenocarcinoma vs Normal, Cho Gastric; Dataset 5, Gastric Mixed Adenocarcinoma vs Normal Cho Gastric; Dataset 6, Gastric Cancer vs Normal, Cui Gastric; Dataset 7, Gastric Intestinal Type Adenocarcinoma vs Normal, DErrico Gastric; Dataset 8, Gastric Cancer vs Normal, Wang Gastric. Cell color and the number above the cell in the lower panel indicate the best gene rank percentile for the analysis. Red, upregulated; blue, downregulated. (B) The prognostic value of FAM83D mRNA expression in gastric cancer (www.oncolnc.org). (C) The prognostic value of FAM83D mRNA expression in gastric cancer (www.kmplot.com). (D) The mRNA levels of FAM83D from 33 patients were tested by quantitative PCR using paired t-test. (E) FAM83D protein expressions in seven GC cell lines (NCL-N87, BGC-823, AGS, SGC-7901, KATO III, MKN-45, SNU-1), one immortalized gastric cell line (GES-1, Hs 738.St/Int) were examined by Western blotting.

Figure 2

Upregulation of FAM83D is associated with poor prognosis in gastric cancer. (A) and (B), low expression of FAM83D in normal gastric specimen. (C) and (D), low expression of FAM83D in gastric carcinoma. (E) and (F), high expression of FAM83D in gastric carcinoma. The scale bar is 100 μm. (G) FAM83D staining was quantified (***p<0.001, compared with normal tissues). (H) FAM83D expression was associated with overall survival (OS) (n=291, p<0.001) (I) FAM83D expression was associated with disease-free survival (DFS) (n=291, p<0.001).

Figure 3

FAM83D promotes proliferation and clonogenicity of GC cells. (A) The effect of FAM83D knockdown with siRNAs was verified by Western blotting 48 hrs after transfection. (B) The effect of FAM83D overexpression was verified by Western blotting. (C) Viability of FAM83D knockdown cells was assessed with an MTT assay at indicated times. (D) Viability of FAM83D overexpression cells was assessed with an MTT assay at indicated times. Statistically significant difference:*p<0.05. Abbreviation: GC, gastric cancer.

Figure 4

FAM83D promotes the clonogenicity of GC cells. (A) Colony formation assays of AGS and SGC-7901 cells transfected with negative control and FAM83D-targeted siRNAs. Left panel: representative image; right panel: quantification of the colony numbers. (B) Colony formation assays of control and FAM83D overexpressed GC cells. Left panel: representative image; right panel: quantification of the colony numbers. Statistically significant difference: p<0.05. ***p<0.001. Abbreviation: GC, gastric cancer.

Figure 5

Knockout of FAM83D inhibits migration and invasion in vitro. (A) The migration assay using AGS and SGC-7901 transfected with FAM83D siRNA or control. (B) The invasion assay using AGS and SGC-7901 transfected with FAM83D siRNA or control. Left panel: representative image; Right panel: quantification of the colony numbers. Statistical method: independent t-test. Significant difference: p<0.05. ***p<0.001.

Figure 6

Overexpression of FAM83D facilitates migration and invasion in vitro. (A) The migration assay using AGS and SGC-7901 transfected with FAM83D or vector. (B) The invasion assay using AGS and SGC-7901 transfected with FAM83D or vector. Left panel: representative image; Right panel: quantification of the colony numbers. Statistical method: independent t-test. Significant difference: p<0.05. ***p<0.001.

Figure 7

Knockout of FAM83D inhibits progression of cell cycle in GC cells. (A) Representative images of cell cycle analysis by flow cytometry in AGS cells after FAM83D depletion. (B) Representative images of cell cycle analysis by flow cytometry in SGC-7901 cells after FAM83D depletion. Every test was repeated independently three times. Statistically significant difference: p<0.05.***p<0.001. Abbreviation:  GC, gastric cancer.

Figure 8

Overexpression of FAM83D accelerates progression of cell cycle in GC cells. (A) Representative images of cell cycle analysis by flow cytometry in AGS cells after FAM83D overexpression. (B) Representative images of cell cycle analysis by flow cytometry in SGC-7901 cells after FAM83D overexpression. Every test was repeated independently three times. Statistically significant difference: p<0.05. ***p<0.001. Abbreviation: GC, gastric cancer.

Figure 9

FAM83D activates Wnt/β-catenin signaling. Western blotting analysis of expression of p-GSK-3β, β-catenin, cyclin D1 and c-Myc in AGS and SGC-7901 cells after FAM83D depletion. (B) Western blotting analysis after FAM83D overexpression. (C) Western blotting analysis between siControl+LiCL (20mmol/L), siFAM83D#1+LiCL(20mmol/L) and siFAM83D#2+LiCL (20mmol/L) in AGS or SGC-7901 cell line.

Figure 10

Knockdown of FAM83D inhibits tumour growth in nude mice. (A) Images of mice formed by AGS cells transfected with siControl or siFAM83D. (B) Images of tumors formed by AGS cells transfected with siControl or siFAM83D. (C) The growth curves of the tumours formed by AGS cells transfected with siControl or siFAM83D. The data are presented as the mean ± SD (N=5 mice per group). (D) Weight differences in tumours formed by AGS cells transfected with siControl or siFAM83D and injected in nude mice. The data are presented as the mean ± SD (N=5 mice per group). (E) Time course of body weight changes of two different groups. (F) The histological examination of FAM83D, GSK-3β and β-Catenin expressions the tumours formed by AGS cells transfected with siControl or siFAM83D. (G) Expression of FAM83D, GSK-3βandβ-Catenin was assessed by immunohistochemistry in tumors formed by AGS cells transfected with siControl or siFAM83D. Statistically significant difference: *p<0.05.***p<0.001.