RGL1 overexpression promotes metastasis of colorectal cancer by upregulating motile focal adhesion assembly via activating the CDC42/RAC1 complex

doi:10.12122/j.issn.1673-4254.2025.05.16

Abstract

Abstract:

Objective To investigate the regulatory role of Ral guanine nucleotide dissociation stimulator-like 1 (RGL1) in metastasis of colorectal cancer (CRC). Methods We analyzed the differential expression of RGL1 between metastatic and non-metastatic CRC in GEO database, and examined its expression in 25 patients with metastatic CRC and 25 patients with non-metastatic CRC treated in Zhujiang Hospital between January, 2020 and December, 2022 using quantitative PCR (qPCR) and immunohistochemistry. HCT116 cell lines with stable RGL1 overexpression and SW480 cells with RGL1 knockdown were established using lentiviral vecors, and the changes in invasion and migration abilities of the cells were assessed using Transwell invasion and migration assays. The transduced cells were injected into the serosa of the cecum of nude mice, and tumor growth and liver metastasis were observed 8 weeks later. Fibronectin adhesion assays and immunofluorescence experiments were employed to assess the relationship between RGL1 and focal adhesion formation, and co-immuno-precipitation assays were performed to explore the interaction between RGL1 and GTPase activation. Results Compared with non-metastatic CRC, metastatic CRC showed significantly upregulated expression of RGL1. HCT116 cells overexpressing RGL1 exhibited obviously enhanced migration and invasion in vitro with increased capacity for liver metastasis in nude mice. RGL1 overexpression strongly accelerated focal adhesion assembly, facilitated the formation of motile focal adhesions, and enhanced the binding of activated CDC42/RAC1 complex to RGL1. Conclusion RGL1 is highly expressed in metastatic CRC and promotes distant metastasis of CRC by activating the CDC42/RAC1 complex to facilitate the formation of motile focal adhesions. These findings suggest that RGL1 can potentially serve as a therapeutic target for CRC metastasis.

Key words: Ral guanine nucleotide dissociation stimulator-like 1, metastatic colorectal cancer, motile focal adhesion, CDC42, RAC1

Nuozhou WENG, Bin TAN, Wentao ZENG, Jiayu GU, Lianji WENG, Kehong ZHENG. RGL1 overexpression promotes metastasis of colorectal cancer by upregulating motile focal adhesion assembly via activating the CDC42/RAC1 complex[J]. Journal of Southern Medical University, 2025, 45(5): 1031-1038.

Figures/Tables 5

Fig.1 RGL1 is highly expressed in metastatic colorectal cancer (CRC). A: Expression of RGL1 in non-metastatic CRC (nmCRC) and metastatic CRC (mCRC) based on data from GSE39582 and GSE87211 datasets. B: RGL1 mRNA levels in clinical specimens of nmCRC and mCRC. C: Immunohistochemical staining of RGL1 in nmCRC and mCRC (Original magnification: ×400). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig.2 Construction of RGL1-overexpressing and knockdown cell lines. A: Expression of RGL1 in NCM460 and CRC cell lines detected by Western blotting. B, C: Verification of RGL1 overexpression and knockdown efficiency in different CRC cell lines using Western blotting.

Fig.3 Overexpression of RGL1 promotes metastasis and invasion of CRC. A: Transwell migration assay of CRC cells with RGL1 overexpression or knockdown. B: Transwell invasion assay of CRC cells with RGL1 overexpression or knockdown. C: Orthotopical tumor and liver metastasis of SW480 cell xenografts with and without RGL1 knockdown implanted in the cecum of nude mice. D: Survival curve analysis of mice in shNC and shRGL1 groups. E: HE staining of liver tissues from mice in shNC and shRGL1 groups. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig.4 RGL1 overexpression promotes motile focal adhesion assembly. A: Relationship between RGL1 and focal adhesion in CRC based on data from Gene Set Enrichment Analysis (GSEA). B: Fibronectin adhesion assays for detecting adherent cells between veh and RGL1+HCT116 cells. C: FN adhesion assays for detecting adherent cells between shNC and shRGL1 SW480 cells (***P<0.001, ****P<0.0001). D: Immunofluorescence assay showing cellular expression of FAK (scale bar=10 µm). E: Immunofluorescence assay showing cellular expression of paxillin (scale bar=10 µm).

Fig.5 RGL1 facilitates focal adhesion assembly by activating the CDC42/RAC1 complex. A: Western blotting for Rho GTPase family (CDC42, RAC1 and RhoA) in RGL1+ or shRGL1 cells. B-D: Co-immunoprecipitation assay of RhoA, RhoAGTP, CDC42, CDC42GTP, RAC1 and RAC1GTP in RGL+ or shRGL1 cells.

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