SFRP2 Overexpression Induces an Osteoblast-like Phenotype in Prostate Cancer Cells

Abstract

Prostate cancer bone metastasis is still one of the most fatal cancer diagnoses for men. Survival of the circulating prostate tumor cells and their adaptation strategy to survive in the bone niche is the key point to determining metastasis in early cancer stages. The promoter of SFRP2 gene, encoding a WNT signaling modulator, is hypermethylated in many cancer types including prostate cancer. Moreover, SFRP2 can positively regulate osteogenic differentiation in vitro and in vivo. Here, we showed SFRP2 overexpression in the prostate cancer cell line PC3 induces an epithelial mesenchymal transition (EMT), increases the attachment, and modifies the transcriptome towards an osteoblast-like phenotype (osteomimicry) in a collagen 1-dependent manner. Our data reflect a novel molecular mechanism concerning how metastasizing prostate cancer cells might increase their chance to survive within bone tissue.

Keywords: EMT; PC3; SFRP2; WNT signaling; bone metastasis; osteomimicry; prostate cancer.

Figure 1

Generation and analysis of PC3 cells stably overexpressing SFRP2. The bicistronic expression plasmid contained either the coding sequences of RFP and puromycin for control cells (PC3^RFP) or an additional coding sequence for human SFRP2 (PC3^{SFRP2; RFP}, A). Stably transfected cells were selected with puromycin for 14 days (B) and then enriched for high RFP fluorescence by FACS (B′), resulting in homogenous bright PC3 cell populations (B″). qPCR validated a significantly higher SFRP2 expression in PC3^SFRP2, when compared to PC3 cells, which was not affected by surface conditions (C). In addition, ICC against SFRP2 shows high expression of SFRP2 in the whole cytoplasm of PC3^SFRP2 cells compared to the controls, independent of the surface (D,D′). Significance level **: p ≤ 0.001, ***: p ≤ 0.001, ns: not significant. Scale bars: B/B″: 100 µm, D/D′: 10 µm.

Figure 2

Differentially expressed gene (DEG) analysis of PC3 and PC3^SFRP2 cells. Next-generation RNA sequencing was performed to uncover genes that are affected by SFRP2 overexpression and surface condition. MA plots show gene expression differences between PC3 and PC3^SFRP2 on CCP (A) and COL1 (A′) surfaces. Genes that may influence osteomimicry were labelled in MA-plots. NS: not significant, blue: significantly upregulated in PC3, green: significantly upregulated in PC3^SFRP2. The Venn diagram illustrates the number of unique upregulated genes specific to each group (B). Principal component analysis (PCA) revealed a strong transcriptional response of PC3^SFRP2 cells on COL1, when compared to PC3^SFRP2 on CCP surface (C).

Figure 3

SFRP2 overexpression leads to osteotropic properties in PC3 cells on a COL1 surface. GO biological processes related to adhesion, taxis, and differentiation were significantly increased by SFRP2 overexpression compared to native PC3 cells when the cells were seeded on COL1 (A). Heatmap of osteoblast differentiation-related genes (GO:0001649) shows clear clustering of COL1-PC3^SFRP2 from all other groups (B). Arrows indicate WNT signaling-related gene expression in osteoblast differentiation.

Figure 4

SFRP2 overexpression induces COL1-dependent EMT in PC3 cells. No morphological differences between PC3 and PC3^SFRP2 were observed in FACS analysis when cells were in suspension (A). On the CCP surface, PC3 cells are epithelial-like, while PC3^SFRP2 cells are more roundish (B). The COL1 surface induces an increase in cell size and elongation in both PC3 and PC3^SFRP2 (B′). Quantification of the cell morphology reveals that COL1 significantly increases cell size in PC3^SFRP2 cells (C) and elongation in COL1-PC3^SFRP2 cells (D). RNAseq analysis revealed that COL1-PC3^SFRP2 exhibited significant upregulation of the EMT pathway-related hallmark gene set when compared to CCP-PC3^SFRP2. COL1-PC3^SFRP2 enrichment score < 0, CCP-PC3^SFRP2 enrichment score > 0 (E). * p ≤ 0.05; *** p ≤ 0.001. Scale bar: 200 µm.

Figure 5

SFRP2 overexpression suppresses the reduced proliferation of PC3 cells on the COL1 surface. Cumulative population doubling (cumPD) (A) and population doubling time (PDT) (B) revealed that culturing PC3 cells on a COL1-coated surface reduced their proliferation in comparison to PC3^SFRP2. COL1, and SFRP2 mutually significantly reduced CFU efficiency of PC3 cells (C). Metabolic activity is significantly reduced in PC3^SFRP2 cells on the CCP surface compared to native PC3. However, this significant difference is no longer observed when the cells are cultured on COL1 surface (D). Significance levels: * equals p ≤ 0.05; *** equals p ≤ 0.001.

Figure 6

SFRP2 overexpression increases the osteotropic-like properties of PC3 cells on the COL1 surface. COL1-coating increases the random migration velocity of PC3 cells significantly, whereas overexpression of SFRP2 did not lead to such a significant increase in motility (A). Cellular invasion through a COL1 matrix shows that SFRP2 overexpression significantly increases the invasion of the PC3 cells (B). Cell attachment assay shows an increase in attachment with SFRP2 overexpression in PC3 cells cultured on both surfaces yet increase on COL1 is not significant. (C). Significance level: * equals p ≤ 0.05.

Figure 7

Visual representation of the potential effect of SFRP2 on prostate cancer cells during bone metastasis. After prostate cancer cells enter the circulation, SFRP2 expression leads them to attach and invade bone. PC3^SFRP2 cells start to secrete osteoblast-specific factors for osteomimicry and disturb the balance between bone formation and resorption. Created with BioRender.com (accessed on 14 June 2022, adapted from “Metastasis to Bone Disrupts Bone Homeostasis”).