Fbxw7 regulates tumor apoptosis, growth arrest and the epithelial-to-mesenchymal transition in part through the RhoA signaling pathway in gastric cancer

Highlights

• Fbxw7 regulates apoptosis, growth arrest and EMT in gastric cancer.

• Fbxw7 regulates RhoA abundance via ubiquitination and proteasomal degradation.

• Fbxw7 may exert its tumor suppressive function by regulating the stability of RhoA.

Abstract

F-box and WD repeat domain-containing7 (Fbxw7), a member of the F-box family of proteins, which are components of an E3 ubiquitin ligase complex, plays an important role as a general tumor suppressor in regulating the effects of various oncoproteins. Recently, accumulating studies have shown that Fbxw7 plays an important role in tumor cell motility, invasion and cancer metastasis. However, little is known about the signaling mechanisms that regulate tumor apoptosis, growth arrest and the epithelial-to-mesenchymal transition (EMT) in gastric cancer. In our study, we confirmed that Fbxw7 expression was decreased in gastric cancer tissues, and that Fbxw7 inhibited gastric cancer progression by inducing apoptosis and growth arrest. Furthermore, gastric cancer migration and invasion were decreased or increased following Fbxw7 overexpression or knockdown, respectively, and the expressions of various EMT markers, such as E-cadherin, N-cadherin and vimentin, were altered after Fbxw7 inhibition or overexpression. Furthermore, we demonstrated that Fbxw7 inhibits the EMT via the down-regulation of Snail 1 and ZEB 1, which are upstream transcription factors that promote this process. Additionally, RhoA showed higher expression in the same gastric cancer tissues than in normal tumor-adjacent samples. We found that Fbxw7 expression was negatively correlated with RhoA protein expression in gastric cancer tissues based on Pearson's correlation coefficient analysis. Moreover, we found that RhoA protein abundance was regulated by Fbxw7 via ubiquitination and proteasomal degradation in gastric cancer. We further demonstrated the effects of RhoA re-expression or inhibition on stable Fbxw7-overexpressing or Fbxw7-silenced cell lines in vitro and in vivo. These results suggest that Fbxw7 induces apoptosis and growth arrest and inhibits the EMT in part by down-regulating the RhoA signaling pathway.

Introduction

Gastric cancer is the fourth most common malignancy and the second most common cause of cancer-related deaths worldwide [1]. It is thought to result from a combination of environmental factors and the accumulation of generalized and specific genetic alterations; these factors mainly affect older patients after a long period of atrophic gastritis [2]. However, there are no significant therapeutics that can successfully treat progressive or metastatic gastric cancer. Therefore, the molecular mechanisms that regulate gastric cancer cell apoptosis, growth arrest and migratory behavior should be further explored because this information is necessary for the development of new therapies.

The epithelial-to-mesenchymal transition (EMT), a highly conserved cellular program that is characterized by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype, plays a crucial role in tumor progression and metastasis [3], [4], [5], [6]. Many lines of evidence derived from the analysis of prostate cancer have demonstrated that an increasing Gleason score is associated with loss of the basement membrane and cell polarity and increasingly invasive cancer behavior [7]. The EMT has been observed in various gastric cell lines, including MGC-803, BGC-823, SGC-7901 and MKN-45 cells [8], [9]. However, the signaling mechanisms involved in the EMT in gastric cancer cells are unclear.

F-box and WD repeat domain-containing7 (Fbxw7), a member of the F-box family of proteins, which are components of the SKIP-CUL1-F-box protein (SCF) E3 ubiquitin ligase complex, determines target specificity by recognizing and binding to proteins, leading to their ubiquitination and proteasomal degradation [10]. Fbxw7 targets several oncoproteins for degradation, including Cyclin E, c-Myc, c-Jun, mTOR, SREBP-1, MCL-1, KLF5, Notch 1 and Notch 4, and its tumor suppressor function is thought to be mediated by these substrates [11], [12], [13]. Fbxw7 displays reduced expression in multiple human cancers, including breast cancer, colorectal cancer, gastric cancer, prostate cancer, pancreatic cancer and hepatocellular carcinoma [11]. Many reports have shown that mutation of the Fbxw7 gene, which displays an overall 6% point mutation frequency, leads to inactivation of Fbxw7 in multiple human cancers [11], [12], [14]. Recent studies have reported that Fbxw7 inhibits cholangiocarcinoma and colorectal cancer cell migration and serves as a prognostic marker [15], [16]. However, the mechanisms that regulate the effects of Fbxw7 on apoptosis, growth arrest and the EMT suppression in gastric cancer are unknown.

RhoA is a member of the Rho family of small GTPases, which are Ras-like proteins that act as intermediaries between cell surface receptors and different intracellular signaling proteins [17]. Similar to other GTPases, RhoA cycles between an inactive GDP-bound configuration and an active GTP-bound configuration that interacts with downstream effectors, such as ROCK, to impact actin cytoskeletal structure and dynamics, cell migration, cytokinesis, and the cell cycle [17], [18]. RhoA overexpression has been observed in various cancers, and the activity of RhoA has been implicated in tumorigenesis and tumor cell invasion [17], [19]. A recent study reported that FBXL19, an E3 ubiquitin ligase, mediates RhoA ubiquitination and proteasomal degradation in lung epithelial cells [20].

In this study, we demonstrated that Fbxw7 acted as a tumor suppressor by promoting apoptosis, growth arrest and anti-EMT activities and that its expression was inversely correlated with RhoA protein expression in gastric cancer. Fbxw7 regulated RhoA protein abundance by inducing RhoA ubiquitination and proteasomal degradation. Importantly, we further showed that Fbxw7 regulated apoptosis, growth arrest and the EMT in gastric cancer through the RhoA signaling pathway.