EPHB1 Protein Promoted the Progression of Prostate Adenocarcinoma Through Phosphorylating GSK3B and Activating EPHB1-GSK3B-SMAD3 Pathway

Abstract

Background: The apoptosis affected the prostate adenocarcinoma (PRAD); we aimed to explore the potential pathogenesis of high-risk patients based on the apoptosis features. Method: The RNA-seq data of patients and apoptosis genes were used for apoptosis score calculation via "GSVA" package; then, the weighted gene coexpression network analysis (WGCNA) and Lasso algorithm were performed for a RiskScore model. After that, the "maftools" package was applied for the somatic mutation analysis. By combining the Kaplan-Meier (KM) survival curves in order to compare the prognosis of different subgroups of patients, Cell Counting Kit-8 (CCK-8), EdU staining, and Transwell assays were performed. Protein expression was measured using western blotting. Finally, the activity of PRAD cells in macrophage polarization was detected using coculture and immunofluorescence assays. Results: The PRAD samples had significantly lower apoptosis scores, and the RiskScore supported the risk stratification of patients. In somatic mutation analysis, EPHB1 and KIF13A from the top six mutant genes were overexpressed in 22RV1 and PC-3 tumor cells, and low levels of EPHB1 indicated a better prognosis. Overexpression or knockdown of EPHB1 affected cell viability, proliferation, and invasion. We found that high expression of EPHB1 interacting with GSK3B protein promoted the expression of p-SMAD3 in 22RV1 cells with high levels of antiapoptotic and invasion markers (BCL2, Snail, and N-CAD). Importantly, GSK3B and EPHB1 knockdown inhibited p-SMAD3 activation and promoted proapoptotic features, accompanied by a reduction in macrophage M2 polarization. Conclusion: This study revealed that EPHB1 plays a pivotal role in activating the EPHB1-GSK3B-SMAD3 pathway to facilitate PRAD progression.

Keywords: apoptotic characteristics; cell coculture; co-immunoprecipitation; prostate adenocarcinoma; western blot.

Figure 1

Construction and validation of model. (a) LASSO coefficient path plot. (b) LASSO regularization path plot. (c) Model gene and its coefficient. (d–g) KM survival analysis and ROC analysis in the training set, test set, TCGA cohort, and MSKCC cohort.

Figure 2

qPCR for the gene expression. (a, b) The expression of EPHB1, DCHS2, KIF13A, FBN3, CDH23, and CDK12 in RWPE-2, 22RV1, and PC-3 cells. (c–e) The expression of EPHB1 in RWPE-2, C4-2, DU145, and C4-2B cells. (f) KM survival analysis of EPHB1 expression on PRAD prognosis (ns is p > 0.05, ⁣^∗p < 0.05, ⁣^∗∗p < 0.01, ⁣^∗∗∗p < 0.001).

Figure 3

Cell viability, invasion, and proliferation test. (a, b) The expression of EPHB1 in 22RV1 and PC-3 cells after EPHB1 silencing. (c, d) CCK-8 for cell viability test after EPHB1 silencing. (e, f) Trans-well for cell invasion test after EPHB1 silencing. (g, h) Cell proliferation test after EPHB1 silencing. (i, j) The cell viability test after overexpression of EPHB1. (k, l) Cell invasion test after overexpression of EPHB1. (m) Cell proliferation test after overexpression of EPHB1 (⁣^∗p < 0.05, ⁣^∗∗p < 0.01, ⁣^∗∗∗p < 0.001, ⁣^∗∗∗∗p < 0.0001).

Figure 4

Phosphorylation function verification of EPHB1. (a, b) Co-IP for the interaction between EPHB1 and GSK3B protein. (c) Phosphorylation model of EPHB1 and GSK3B protein. (d, e) The phosphorylation levels of GSK3B and SMAD3 protein in the 22RV1 cells. (f, g) The phosphorylation levels of GSK3B and SMAD3 protein in the PC-3 cells (⁣^∗p < 0.05, ⁣^∗∗p < 0.01).

Figure 5

Detection of apoptosis-related protein expression. (a, b) Western blot of BCL2, BAX, Snail, E-CAD, N-CAD, and GAPDH protein expression in 22RV1 cells. (c, d) Western blot of BCL2, BAX, Snail, E-CAD, N-CAD, and GAPDH protein expression in PC-3 cells (⁣^∗p < 0.05, ⁣^∗∗p < 0.01, ⁣^∗∗∗p < 0.001).

Figure 6

Viability, invasion, and proliferation assays on PRAD cells after GSK3B inhibition. (a, b) CCK-8 for assessing the effect on the proliferative capacity of PRAD cell lines after inhibition of GSK3B. (c, d) Trans-well for assessing the effect on the invasive capacity of PRAD cell lines after inhibition of GSK3B. (e, f) EdU assay to be used to assess the effect on the proliferation level of 22RV1 and PC-3 cells after inhibition of GSK3B (⁣^∗p < 0.05, ⁣^∗∗p < 0.01, ⁣^∗∗∗p < 0.001, ⁣^∗∗∗∗p < 0.0001).

Figure 7

Phosphorylation function verification of GSK3B inhibition. (a, b) Western blot of p-GSK3B, GSK3B, p-SMAD3, SMAD3, and GAPDH protein expression after GSK3B inhibition in 22RV1 cells. (c, d) Western blot of p-GSK3B, GSK3B, p-SMAD3, SMAD3, and GAPDH protein expression after GSK3B inhibition in PC-3 cells (⁣^∗p < 0.05, ⁣^∗∗p < 0.01, ⁣^∗∗∗∗p < 0.0001).

Figure 8

Detection of apoptosis and invasion-related protein expression after GSK3B inhibition. (a, b) Western blot of BCL2, BAX, Snail, E-CAD, N-CAD, and GAPDH protein expression in 22RV1 cells after GSK3B inhibition in 22RV1 cells. (c, d) Western blot of BCL2, BAX, Snail, E-CAD, N-CAD, and GAPDH protein expression in 22RV1 cells after GSK3B inhibition in PC-3 cells (⁣^∗p < 0.05, ⁣^∗∗p < 0.01).

Figure 9

Coculture assay for testing the effect of EPHB1 silencing on macrophage polarization. (a–d) The coculture of 22RV1/PC-3 and macrophage after EPHB1 silencing. (e, f) The CD86 and CD206 fluorescence intensity detection (⁣^∗∗p < 0.01).

Figure 10

Coculture assay for testing the effect of GSK3B inhibition on macrophage polarization. (a–d) The coculture of 22RV1/PC-3 and macrophage after GSK3B inhibition. (e, f) The CD86 and CD206 fluorescence intensity detection after GSK3B inhibition (⁣^∗∗p < 0.01).