ARL4C Regulates the Progression of Clear Cell Renal Cell Carcinoma by Affecting the Wnt/β-Catenin Signaling Pathway

Abstract

Purpose. To investigate the expression of the ADP-ribosylation factor (ARF)-like proteins (ARLs) and ARL4C in clear cell renal cell carcinoma (ccRCC) based on bioinformatics analysis and experimentally determine the effect and mechanism of ARL4C on cellular properties involved in ccRCC progression. Methods. After downloading the data of cancer patients from the TCGA database, we used various bioinformatics analysis websites and methods to analyze the expression and function of ARLs and ARL4C. The differential expression of ARL4C in clinical renal cancer tissues versus adjacent normal tissues was further verified using immunohistochemistry and real-time quantitative reverse-transcription (qRT-PCR). qRT-PCR was used to explore the expression of ARL4C mRNA in normal renal cells versus different ccRCC cell lines, and the protein expression of ARL4C was further verified using western blotting. CCK-8, colony formation, and EdU assays were used to determine the effect of ARL4C knockdown on ccRCC cell proliferation. We also used wound healing and Transwell assays to analyze the changes in ccRCC cell migration and invasion following ARL4C knockdown. Finally, we used western blotting to probe the molecular mode of action of ARL4C in ccRCC cells after exposure to Wnt signaling pathway agonists. Results. Biological function analysis showed that methylation of ARL4C and changes in immune cell infiltration and targeted drug sensitivity caused by altered ARL4C expression affected the prognosis of ccRCC. Further bioinformatics analysis suggested that the expression of ARL4C mRNA was increased in ccRCC, and this was associated with a poor prognosis in ccRCC patients. Increased expression of ARL4C was further verified using qRT-PCR and western blotting of human ccRCC tissue samples. Downregulation of ARL4C significantly inhibited the proliferation, migration, and invasion of ccRCC cells, and activation of the Wnt/β-catenin pathway promoted the expression of ARL4C. As an essential downstream effector of the Wnt signaling pathway, ARL4C increased the expression of cyclin D1 and c-myc, thereby increasing the ability of the cells to undergo epithelial-mesenchymal transition (EMT) and ccRCC progression. Conclusions. As a critical factor in the Wnt/β-catenin pathway, ARL4C regulates EMT and progression in ccRCC.

The role of Wnt, ARL4C, and Sema3A in developmental process and disease pathogenesis

“…Regarding the function of ARL4C, the expression of ARL4C controls the activation of Rac, the inhibition of Rho and the nuclear localization of YAP and TAZ in HCT116 colon cancer cells and A549 lung adenocarcinoma cells 108 . Consistently, ARL4C‐depleted tumor cells, including HCT116, A549, NCI‐H520 lung SCC cells, HLE HCC cells, H1975 lung adenocarcinoma cells, AGS gastric cancer cells, S2‐CP8 pancreatic cancer cells and ACHN clear cell renal cell carcinoma cells, exhibited reduced migration, invasion and proliferation capabilities both in vitro and in vivo 108,109,114–116,120,121 . In addition, ARL4C reportedly enhances the rate of cholesterol efflux in HeLa cells, 122 a human cervical carcinoma cell line, and is involved in the transport of transferrin from early endosomes to recycling endosomes by interacting with α‐tubulin in human renal carcinoma cells 123 …”

“…108 Consistently, ARL4C-depleted tumor cells, including HCT116, A549, NCI-H520 lung SCC cells, HLE HCC cells, H1975 lung adenocarcinoma cells, AGS gastric cancer cells, S2-CP8 pancreatic cancer cells and ACHN clear cell renal cell carcinoma cells, exhibited reduced migration, invasion and proliferation capabilities both in vitro and in vivo. 108,109,[114][115][116]120,121 In addition, ARL4C reportedly enhances the rate of cholesterol efflux in HeLa cells, 122 a human cervical carcinoma cell line, and is involved in the transport of transferrin from early endosomes to recycling endosomes by interacting with α-tubulin in human renal carcinoma cells. 123 The expression of ARL4C as a molecular target for anti-cancer therapy…”

Ursolic acid inhibits the metastasis of colon cancer by downregulating ARL4C expression

“…Another study demonstrated that the downregulation of ARL4C significantly inhibits the proliferation, migration and invasion of clear cell renal cell carcinoma cells. Moreover, ARL4C promoted the progression of clear cell renal cell carcinoma and the occurrence of epithelial-mesenchymal transformation (EMT) ( 39 ). By contrast, other studies have showed that ARL4C is a tumour suppressor in ovarian and breast cancer and low expression of ARL4C was associated with poor prognosis in these patients ( 40 , 41 ).…”

“…Zhang et al ( 50 ) reported that the PI3K/AKT/mTOR signalling pathway is an important regulator of the proliferation, migration and invasion of LoVo colon cancer cells. Previous studies demonstrated that the regulation of ARL4C expression is involved in multiple carcinogenic signalling pathways, such as Wnt/β-catenin ( 39 ), p53 ( 13 ), RAF1-MEK/ERK ( 51 ) and AKT/mTOR ( 34 ). In the present study, AKT or mTOR inhibitors regulated ARL4C protein expression in HCT-116 and SW480 colon cancer cells, which further identifies the critical role of AKT in regulating the ARL4C protein.…”

Wnt/β-catenin-driven EMT regulation in human cancers

“…Another malignant urological tumor is renal cancer, although only one study has evaluated function of Wnt in the modulation of metastasis. In this case, Wnt increases levels of ARL4C to enhance cyclin D1 and c-Myc levels, leading to EMT induction and enactment in progression of tumor cells [ 120 ].…”