SHOX2 promotes prostate cancer proliferation and metastasis through disruption of the Hippo-YAP pathway

Abstract

The transcription factor SHOX2 gene is critical in regulating gene expression and the development of tumors, but its biological role in prostate cancer (PCa) remains unclear. In this study, we found that SHOX2 expression was significantly raised in PCa tissues and was associated with clinicopathological features as well as disease-free survival (DFS) of PCa patients. Phenotypic tests showed that the absence of SHOX2 inhibited PCa growth and invasion, while SHOX2 overexpression promoted these effects. Mechanistically, SHOX2 was found to activate the transcription of nephronophthisis type 4 (NPHP4), a gene located downstream of SHOX2. Further analysis revealed that SHOX2 could potentially interfere with the Hippo-YAP signaling pathway through NPHP4 activation, facilitating the oncogenic behavior of PCa cells. These findings highlight SHOX2 as an oncogene in PCa and provide a basis for developing potential therapeutic approaches against this disease.

Keywords: Biological sciences; Cancer; Molecular biology.

Graphical abstract

Figure 1

SHOX2 showed upregulated expression in PCa tissues and cell lines (A) The TCGA database was utilized to produce graphs that depict the expression of SHOX2 in normal and prostate cancer tissues. (B) The TCGA database was utilized to produce graphs that depict the correlation between SHOX2 and prostate cancer Gleason grade. (C) The GEO database GSE35988 was utilized to produce graphs that depict the correlation between SHOX2 expression and prostate cancer metastasis. (D) SHOX2 protein levels in PCa tumor tissues and corresponding adjacent nontumor tissues. (E) Representative IHC staining of SHOX2 in PCa tumor tissues as compared with peritumor tissues. Scale bar = 50 μm. (F and G) SHOX2 mRNA and protein expression levels in PCa cell lines and normal RWPE-1 cells by qRT-PCR and WB analyses. The data are presented as the mean ± standard deviation (SD) of experiments performed in triplicate. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 2

SHOX2 expression was correlated with advanced progression and poor prognosis (A) Kaplan-Meier analysis was used to compare disease-free survival (DFS) curves between PCa patients with low SHOX2 expression and high SHOX2 expression (n = 106) through a log rank test. (B) Cox-regression analysis was utilized for multivariate analysis of DFS in a group of 106 PCa patients.

Figure 3

Knockdown of SHOX2 suppressed the proliferation of PCa cells (A and B) QRT-PCR and WB analysis of SHOX2 expression in the indicated cell lines. β-actin was used as a loading control. (C) The CCK-8 assay results of LNCaP, MDA PCa 2b, C4-2B and PC-3 cells following silencing or overexpression of SHOX2. (D) Colony formation assay results of LNCaP, MDA PCa 2b, C4-2B and PC-3 cells after knockdown or overexpression of SHOX2. (E) Flow cytometry was performed to assess the apoptosis rate of C4-2B and PC-3 cells following SHOX2 knockdown. The data are presented as the mean ± standard deviation (SD) of experiments performed in triplicate. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 4

SHOX2 promotes the migration and invasion of PCa cells (A–C) The impact of SHOX2 knockdown and overexpression on the migration of PCa cells was respectively shown using Transwell migration assays, with a scale bar of 200 μm. (D–F) The effects of SHOX2 knockdown and overexpression on PCa cell migration were evaluated using wound healing assays, with a scale bar of 200 μm. The data are presented as the mean ± standard deviation (SD) of experiments performed in triplicate. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 5

SHOX2 inhibits Hippo signaling leading to YAP activation (A) Through analysis of the TCGA PCa protein dataset, it was observed that p-YAP-S127 expression was considerably reduced in PCa samples belonging to the highest quartiles of SHOX2 in comparison to those in the lowest quartiles, as determined by a two-tailed Student’s t-test. (B) The expression of the specified proteins was assessed via WB assay in the mentioned cells, with β-actin utilized as a loading control for total extracts and P84 used as a loading control for nuclear extracts. (C) The expression and subcellular localization of YAP were examined in the mentioned cells with modified SHOX2 expression and visualized through fluorescence and laser confocal microscopy. The scale bars of 20 μm were used. (Left panel); The proportion of YAP subcellular localization was determined as a percentage and categorized as either nucleus (N) or cytoplasm (C). (Right panel). (D) The activity of HOP/HIP luciferase was examined in the specified cells. HOP-Flash luciferase assay was employed to determine the transcriptional activity of YAP/TAZ-TEAD. (E) The qRT-PCR analysis was performed to compare the mRNA expression fold change of the specified genes between cells overexpressing SHOX2 and those with vector, as well as shSHOX2 versus shNC. The fold change values were generated by log2 transformation. The data are presented as the mean ± standard deviation (SD) of experiments performed in triplicate. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 6

NPHP4, an essential suppressor of Hippo signaling, is upregulated transcriptionally by SHOX2 (A and B) QRT-PCR and WB analysis NPHP4 expression in SHOX2 silenced-, SHOX2 overexpressing-, and control cells. (C) A schematic diagram of the NPHP4 promoter was presented, including the sequences of the wild-type and mutant SHOX2 binding sites. (Left panel); The luciferase activity of the SHOX2 promoter reporter was quantified in the mentioned cells. (Right panel). (D) The enrichment of SHOX2 on the NPHP4 gene promoter was evaluated through ChIP analysis. A negative control was established using IgG. (E) The mentioned cells were subjected to ChIP assays using anti-p300 acetyltransferase, anti-RNA POL II (RNAP II), and anti-H3K4me3 antibodies. The data are presented as the mean ± standard deviation (SD) of experiments performed in triplicate. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 7

SHOX2 promotes PCa proliferation and metastasis by transactivating NPHP4 (A) Rescuing CCK-8 assays in C4-2B and PC-3 cells. (B) Rescuing colony-forming assays in C4-2B and PC-3 cells. (C) Rescuing wound healing assay in C4-2B and PC-3 cells. (D) Rescuing Transwell migration assays in C4-2B and PC-3 cells. The scale bar for migration images is 200 μm. The data are presented as the mean ± standard deviation (SD) of experiments performed in triplicate. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 8

Clinical relevance of the SHOX2 /NPHP4/ YAP axis in human PCa (A) The IHC staining of SHOX2, NPHP4, YAP, and p-YAP was performed in 106 prostate cancer patient specimens, and representative images were presented. The scale bars in the images were 50 μm. (B) Proportion of PCa samples displaying SHOX2 expression in relation to the levels of NPHP4 and YAP. (C) Survival analysis of patients with PCa (n = 106) using Kaplan-Meier method. The p-values obtained from log rank test are presented.