Rho GTPase-activating protein 35 suppresses gastric cancer metastasis by regulating cytoskeleton reorganization and epithelial-to-mesenchymal transition

ABSTRACT

Cytoskeletal reorganization and epithelial-to-mesenchymal transition (EMT) are key processes and typical characteristics of metastatic cancer cells. Rho GTPase‑activating protein 35 (ARHGAP35) is a GTPase-activating protein, which has a significant effect on cell motility. However, the particular function of ARHGAP35 in gastric cancer (GC) remains unknown. In the present study, the role of ARHGAP35 in GC was investigated by in vitro loss-of-function and gain-of-function experiments. Cytoskeletal reorganization in GC cells was evaluated using immunofluorescence staining and the protein expression levels of key molecules and active RhoA were detected by western blot analysis. Additionally, the clinical evaluation of proteins in human GC tissues was assessed by immunohistochemistry. The results showed that ARHGAP35, a tumor suppressor, was downregulated in GC tissues and its decreased expression was associated with the metastatic status of GC. Additionally, Transwell and wound healing assays demonstrated that ARHGAP35 knockdown promoted cell motility in vitro. However, the above effects were abrogated following ectopic ARHGAP35 expression. Furthermore, ARHGAP35 could affect cytoskeletal reorganization via directly regulating RhoA activation. In addition, ARHGAP35 upregulated E-cadherin and attenuated EMT in GC cells. Both ARHGAP35 and E-cadherin were associated with overall survival in patients with GC, while their combination allowed for an even greater capacity for distinguishing GC patients with different prognosis. Overall, the results of the current study suggested that ARHGAP35 could directly regulate cell morphology and motility via affecting cytoskeletal reorganization and EMT via targeting RhoA and E-cadherin, respectively. Targeting the ARHGAP35/RhoA/E-cadherin pathway could be a potential approach for treating GC.

KEYWORDS:

• ARHGAP35
• metastasis
• cytoskeleton
• gastric cancer
• Epithelial-mesenchymal transition

Abbreviations

GC: Gastric cancer; GEFs: Guanine nucleotide exchange factors; EMT: Epithelial-to-mesenchymal transition; ROCK: Rho‑associated kinase; LIMK: LIM kinase; NIMA: Never in mitosis gene; RIPA: Radioimmunoprecipitation; IHC: Immunohistochemical; IF: Immunofluorescence analysis

Author’s Contributions

Yi Sun and Rui Du performed the functional experiments and analysed data. Yulong Shang performed critical bioinformatics analyses. Changhao Liu provided technical support and analysed data. Yuanyong Wang provided academic advice and revised the manuscript. Guofang Lu designed the study, wrote and revised the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethics approval

Inclusion of the human samples in the tissue array was approved by Xijing Hospital review board (KY20173286-1, approved on 03/07/2017).

Additional information

Funding

The study was supported by grants from National Natural Science Foundation of China (nos. 81773072 and 82173241), Shaanxi Foundation for innovation capacity supporting program (no. 2020-KJXX-058), Youth Project, Lingyun Program of Fourth Military Medical University (2019cyjhsyl) and Supported by Fourth Military Medical University Doctoral Science Foundation (2021D04lgf).