Dataset Information

Phosphorylation of CAP1 regulates lung cancer proliferation, migration, and invasion.

ABSTRACT:

Purpose

Cyclase-associated protein 1 (CAP1) is a ubiquitous protein which regulates actin dynamics. Previous studies have shown that S308 and S310 are the two major phosphorylated sites in human CAP1. In the present study, we aimed to investigate the role of CAP1 phosphorylation in lung cancer.

Methods

Massive bioinformatics analysis was applied to determine CAP1's role in different cancers and especially in lung cancer. Lung cancer patients' serum and tissue were collected and analyzed in consideration of clinical background. CAP1 shRNA-lentivirus and siRNA were applied to CAP1 gene knockdown, and plasmids were constructed for CAP1 phosphorylation and de-phosphorylation. Microarray analysis was used for CAP1-associated difference analysis. Both in vitro and in vivo experiments were performed to investigate the roles of CAP1 phosphorylation and de-phosphorylation in lung cancer A549 cells.

Results

CAP1 is a kind of cancer-related protein. Its mRNA was overexpressed in most types of cancer tissues when compared with normal tissues. CAP1 high expression correlated with poor prognosis. Our results showed that serum CAP1 protein concentrations were significantly upregulated in non-small cell lung cancer (NSCLC) patients when compared with the healthy control group, higher serum CAP1 protein concentration correlated with shorter overall survival (OS) in NSCLC patients, and higher pCAP1 and CAP1 protein level were observed in lung cancer patients' tumor tissue compared with adjacent normal tissue. Knockdown CAP1 in A549 cells can inhibit proliferation and migration, and the effect is validated in H1975 cells. It can also lead to an increase ratio of F-actin/G-actin. In addition, phosphorylated S308 and S310 in CAP1 promoted lung cancer cell proliferation, migration, and metastasis both in vitro and in vivo. When de-phosphorylated, these two sites in CAP1 showed the opposite effect. Phosphorylation of CAP1 can promote epithelial-mesenchymal transition (EMT).

Conclusion

These findings indicated that CAP1 phosphorylation can promote lung cancer proliferation, migration, and invasion. Phosphorylation sites of CAP1 might be a novel target for lung cancer treatment.

SUBMITTER: Zeng J

PROVIDER: S-EPMC8752530 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:The efficacy of ginsenoside Rh2 (Rh2) in cancer therapy has been reported; however, its function in lung cancer remains unknown. To analyze the role of Rh2 in the inhibition of lung cancer cell proliferation in the present study, protein expression levels of E-cadherin, vimentin, ?-catenin, Smo, Gli1, and ?-catenin were assessed by western blotting, whilst mRNA expression levels of TCF7 FZD8, Smo, Gli1, Gli2, and Gli3 were determined by reverse transcription-quantitative PCR in the A549 cell line. Phosphorylation sites were detected by proteomic methods and cell proliferation was analyzed by MTT assay. The present study revealed that Rh2 treatment significantly inhibited cell proliferation. Western blotting indicated that the expression levels of E-cadherin were increased and vimentin was downregulated in Rh2-treated cells compared with control cells. Treatment of A549 cells with Rh2 suppressed phosphorylation of five distinct proteins and increased phosphorylation of nine proteins. Among them, the phosphorylation of ?-catenin at S641 was significantly induced. Rh2 treatment suppressed the expression levels of key genes involved in Wnt (Wnt3, transcription factor 7 and frizzled class receptor 8) and hedgehog [smoothened, frizzled class receptor (Smo), GLI family zinc finger (Gli)1, Gli2, and Gli3] signaling. Immunoblotting results indicated that ?-catenin, Smo and Gli1 protein expression levels were also suppressed by treatment with Rh2 compared with control treatment. Expression of ?-catenin S641D, a phosphomimetic form of ?-catenin, inhibited the accumulation of ?-catenin and Gli1 and inhibited cell proliferation and invasion. Furthermore, knockdown of ?-catenin (CTNNB1) or Gli1 with specific small interfering RNAs inhibited cell proliferation, whereas overexpression of these genes had an opposite effect. Additionally, overexpression of ?-catenin or Gli1 activated cell proliferation, even in the presence of Rh2, suggesting that Rh2 affects A549 cell proliferation through inhibition of Wnt and hedgehog signaling by phosphorylation of ?-catenin at S641. Together, these data suggested that Rh2 treatment may inhibit the proliferation of A549 lung cancer cells. Further exploration of the underlying mechanism by which Rh2 inhibits cell proliferation is warranted.

Project description:Calponin 3 (CNN3) is known to serve a role in certain types of cancer, such as gastric cancer and colorectal cancer. The present study investigated the clinical significance of CNN3 in non-small cell lung cancer (NSCLC) by evaluating its expression profile and relationship with disease prognosis using the Gene Expression Omnibus repository, Gene Expression Profiling Interactive Analysis 2 (GEPIA2) and Kaplan-Meier plotter analysis. CNN3 mRNA expression was measured using reverse transcription-quantitative PCR, while the protein expression level was measured using western blot analysis. Cell proliferation, cell cycle and apoptosis, and migration and invasion were analyzed using MTS assay, flow cytometry and Transwell assays, respectively. These results revealed that CNN3 mRNA expression was downregulated in NSCLC tissues compared with that in normal tissues. Additionally, CNN3 expression had a high diagnostic value based on the GSE2514 dataset and the data from The Cancer Genome Atlas and the Genotype Tissue Expression database, whereas it had a low diagnostic value based on the GSE10072 dataset. Furthermore, CNN3 expression was associated with survival in patients with lung adenocarcinoma (LUAD), whereas it was not associated with survival in patients with lung squamous cell carcinoma (LUSC) according to the Kaplan-Meier plotter results. According to the data from GEPIA2, and the GSE72094, GSE41271 and GSE31210 datasets, CNN3 expression was not associated with the prognosis of patients with LUAD and LUSC. The mRNA and protein expression levels of CNN3 were lower in two NSCLC cell lines (A549 and SK-MES-1) than in a human bronchial epithelial cell line (BEAS-2B). CNN3 overexpression suppressed cell proliferation, migration and invasion, induced G1-phase arrest, promoted apoptosis and suppressed PI3K/AKT signaling pathway activation in the NSCLC cell lines, whereas CNN3 overexpression had no effect on cell morphology. In conclusion, CNN3 suppressed the proliferation and metastasis of NSCLC cells by downregulating the PI3K/AKT signaling pathway, making it a potential therapeutic target in this disease.