FAM83H and SCRIB stabilize β-catenin and stimulate progression of gastric carcinoma

Abstract

FAM83H primarily is known for its function in tooth development. Recently, a role for FAM83H in tumorigenesis, conjunction with MYC and β-catenin, has been suggested. Analysis of public data indicates that FAM83H expression is closely associated with SCRIB expression in human gastric cancers. Therefore, this study investigated the roles of FAM83H and SCRIB in 200 human gastric cancers and gastric cancer cells. In human gastric carcinomas, both the individual and combined expression patterns of the nuclear FAM83H and SCRIB were independent indicators of shorter survival of gastric carcinoma patients. In MKN-45 and NCI-N87 gastric cancer cells, the expression of FAM83H and SCRIB were associated with proliferation and invasiveness of cells. FAM83H-mediated in vivo tumor growth was attenuated with knock-down of SCRIB. Moreover, immunoprecipitation indicates that FAM83H, SCRIB, and β-catenin, form a complex, and knock-down of either FAM83H or SCRIB accelerated proteasomal degradation of β-catenin. In conclusion, this study has found that the individual and combined expression patterns of nuclear FAM83H and SCRIB are prognostic indicators of gastric carcinomas and further suggests that FAM83H and SCRIB are involved in the progression of gastric carcinomas by stabilizing β-catenin.

Keywords: FAM83H; SCRIB; cancer; stomach; β-catenin.

Figure 1

Expression and prognostic significance of the expression of FAM83H, SCRIB, and β-catenin in 200 human gastric carcinomas. (A) Immunohistochemical expression of FAM83H, SCRIB, and β-catenin in normal gastric mucosa, gastric dysplasia, and gastric carcinoma tissue. FAM83H and SCRIB are expressed in both the cytoplasm and nuclei of tumor cells. Original magnification: x400. (B) Immunohistochemical staining scores for nuclear FAM83H (FAM83H-N), cytoplasmic FAM83H (FAM83H-C), nuclear SCRIB (SCRIB-N), cytoplasmic SCRIB (SCRIB-C), and nuclear β-catenin (β-catenin-N) in 20 normal gastric mucosa, 40 gastric dysplasia, and 200 gastric carcinomas. (C) The expression of mRNA of FAM83H and SCRIB in normal gastric tissue and gastric cancers from the GEPIA database (http://gepia.cancer-pku.cn. Accessed 10 January 2020). (D) ROC curve analysis to determine cut-off points for the expression of FAM83H-N (red arrow), FAM83H-C (red empty arrow), SCRIB-N (blue arrowhead), SCRIB-C (blue empty arrowhead), and β-catenin-N (green arrowhead). The cut-off points are determined at the point of the highest area under the curve (AUC) to predict the death of gastric carcinoma patients. (E) Kaplan-Meier survival analysis of overall survival and relapse-free survival according to the FAM83H-N, FAM83H-C, SCRIB-N, SCRIB-C, and β-catenin-N expression.

Figure 2

Survival analysis according to the expression of FAM83H, SCRIB, and β-catenin in early gastric cancers and advanced gastric cancers. Kaplan-Meier survival curves for overall survival and relapse-free survival in 50 early gastric cancers (A) and 150 advanced gastric cancers (B).

Figure 3

Kaplan-Meier survival analysis according to co-expression patterns of nuclear FAM83H and nuclear SCRIB in gastric carcinoma patients. (A) Kaplan-Meier survival curves for overall survival and relapse-free survival in four subgroups of gastric carcinomas: FAM83H-N^-/SCRIB-N^-, FAM83H-N^-/SCRIB-N⁺, FAM83H-N⁺/SCRIB-N^-, and FAM83H-N⁺/SCRIB-N⁺ subgroups. (B) Kaplan-Meier survival curves for overall survival and relapse-free survival in three of gastric carcinomas: FAM83H^-/SCRIB-N^- or FAM83H-N^-/SCRIB-N⁺, FAM83H-N⁺/SCRIB-N^-, and FAM83H-N⁺/SCRIB-N⁺ subgroups. 5y-OS; overall survival rate at five years, 10y-OS; overall survival rate at ten years, 5y-RFS; relapse-free survival rate at five years, 10y-RFS; relapse-free survival rate at ten years.

Figure 4

The effect of knock-down or overexpression of FAM83H on the proliferation and invasiveness of gastric cancer cells. (A) The effect of knock-down or overexpression of FAM83H on proliferation were evaluated with an MTT assay, cell counting, and a colony-forming assay in MKN-45 and NCI-N87 cells. The MTT assay was performed after seeding 3,000 cells per well of a 96-well plate. Cell counting was performed after seeding 3,000 cells per well of a 6-well plate. The colony-forming assay was performed by plating 1,000 cells per well in a 6-well plate for ten days. The knock-down or overexpression of FAM83H was assessed via western blot for FAM83H. (B) The migration assay was performed by seeding 1x10⁵ MKN-45 or 1x10⁵ NCI-N87 cells in the upper chamber for 48 hours. (C) The invasion assay was performed by seeding 2x10⁵ MKN-45 or 2x10⁵ NCI-N87 cells in the upper chamber with Matrigel for 48 hours. The chambers were stained with DIFF-Quik staining solution, and the number of migrated or invaded cells were counted in five x200 microscopic fields in each well. *; P < 0.05, **; P < 0.001.

Figure 5

Western blot, quantitative reverse-transcription polymerase chain reaction, and luciferase reporter assay after knock-down or overexpression of FAM83H in gastric cancer cells. (A) Western blot was performed for FAM83H, SCRIB, β-catenin, active β-catenin, cyclin D1, E-cadherin, N-cadherin, TGF-β1, snail, vimentin, MMP2, MMP9, GSK3β, phosphorylated GSK3β, and actin after knock-down or overexpression of FAM83H in MKN-45 and NCI-N87 gastric cancer cells. (B) Quantitative reverse-transcription polymerase chain reaction was performed for FAM83H, SCRIB, β-catenin, cyclin D1, E-cadherin, N-cadherin, TGF-β1, snail, vimentin, MMP2, MMP9, and GSK3β after knock-down or overexpression of FAM83H in MKN-45 and NCI-N87 gastric cancer cells. (C) TOPflash luciferase reporter assay was performed by transfecting TOPflash or FOPflash plasmid DNA with pRL-TK Renilla Luciferase plasmid DNA in MKN-45 and NCI-N87 cells with induction of knock-down or overexpression of FAM83H. *; P < 0.05, **; P < 0.001.

Figure 6

The effect of knock-down or overexpression of SCRIB on the proliferation and invasiveness of gastric cancer cells. (A, B) The effect of knock-down or overexpression of SCRIB on proliferation was evaluated with an MTT assay and colony-forming assay in MKN-45 (A) and NCI-N87 cells (B). The MTT assay was performed after seeding 3,000 cells per well of a 96-well plate. The colony-forming assay was performed by plating 1,000 cells per well in a 6-well plate for ten days. The knock-down or overexpression of SCRIB was assessed via western blot for SCRIB. (C) The migration assay was performed by seeding 1x10⁵ MKN-45 or 1x10⁵ NCI-N87 cells in the upper chamber for 48 hours. (D) The invasion assay was performed by seeding 2x10⁵ MKN-45 or 2x10⁵ NCI-N87 cells in the upper chamber with Matrigel for 48 hours. The chambers were stained with DIFF-Quik staining solution, and the number of migrated or invaded cells were counted in five x200 microscopic fields in each well. **; P < 0.001.

Figure 7

Western blot and quantitative reverse-transcription polymerase chain reaction after knock-down or overexpression of SCRIB in gastric cancer cells. (A) Western blot was performed for SCRIB, FAM83H, β-catenin, active β-catenin, cyclin D1, E-cadherin, N-cadherin, TGF-β1, snail, vimentin, MMP2, MMP9, GSK3β, phosphorylated GSK3β, and actin after knock-down or overexpression of SCRIB in MKN-45 and NCI-N87 gastric cancer cells. (B) Quantitative reverse-transcription polymerase chain reaction was performed for SCRIB, FAM83H, β-catenin, cyclin D1, E-cadherin, N-cadherin, TGF-β1, snail, vimentin, MMP2, MMP9, and GSK3β after knock-down or overexpression of SCRIB in MKN-45 and NCI-N87 gastric cancer cells. (C) TOPflash luciferase reporter assay was performed by transfecting TOPflash or FOPflash plasmid DNA with pRL-TK Renilla Luciferase plasmid DNA in MKN-45 and NCI-N87 cells with induction of knock-down or overexpression of SCRIB. *; P < 0.05, **; P < 0.001.

Figure 8

The combined effect of overexpression of FAM83H and knock-down of SCRIB on the proliferation and tumor formation of gastric cancer cells. (A, B) The effect of overexpression of FAM83H and/or knock-down of SCRIB on the proliferation of MKN45 and NCI-N87 gastric cancer cells were evaluated with an MTT assay (A) and a colony-forming assay (B). The MTT assay was performed after seeding 3,000 cells per well of a 96-well plate. The colony-forming assay was performed by plating 1,000 cells per well in a 6-well plate for ten days. The number of colonies was determined with GeneTools analysis software. (C, D) In vivo tumor growth was evaluated by subcutaneously implanting 2x10⁶ NCI-N87 cells with overexpression of FAM83H and/or knock-down of SCRIB. The tumor volume was measured every week after tumor implantation by the equation V = LxWxHx0.52 mm³ (C). At five weeks after tumor inoculation, the mice were euthanized and tumor weight was measured (D). (E) The mice were evaluated for metastasis and histologic findings of the lung. The arrows indicate metastatic NCI-N87 cells in lung. *; P < 0.05, **; P < 0.001.

Figure 9

FAM83H and SCRIB are involved in the stabilization of β-catenin from proteasomal ubiquitin degradation. (A) Western blot for β-catenin and active β-catenin after overexpression of FAM83H and/or knock-down of SCRIB. (B) The protein lysate obtained from MKN-45 cells after immunoprecipitation with FAM83, SCRIB, or β-catenin. Thereafter, the immunoprecipitated protein was immunoblotted for FAM83H, SCRIB, and β-catenin. (C) Western blot for FAM83H, SCRIB, β-catenin, actin, and lamin B1 with the cytoplasmic membrane, cytoplasm, and nuclear protein lysates fractionated according to subcellular localization after inducing knock-down or overexpression of FAM83H or SCRIB in MKN-45 cells. (D) The MKN-45 cells were transfected with empty vector, shRNA for FAM83H, or hSCRIB CRISPR/Cas9 KO plasmid and treated with 30 μM cycloheximide or 30 μM MG132 for 0.5 to 4.0 hours. Thereafter, the total protein was immunoblotted for β-catenin and actin. (E) The MKN-45 cells were transfected with empty vector, shRNA for FAM83H, or hSCRIB CRISPR/Cas9 KO plasmid, and were treated with 30 μM MG132 for two hours. The protein lysate was immunoprecipitated with anti-β-catenin antibodies and immunoblotted with anti-ubiquitin antibodies. The immunoblot was performed on total protein lysate. (F) The MKN-45 cells were transfected with empty vectors, shRNA for FAM83H, or an overexpression vector for FAM83H. The protein lysate was immunoprecipitated with anti-β-catenin antibodies and immunoblotted with anti-β-TrCP, anti-USP47, or anti-GAPDH antibodies. The immunoblots were performed on total protein lysate.