Tumor Suppressive Role of the PRELP Gene in Ovarian Clear Cell Carcinoma

Abstract

Ovarian clear cell carcinoma (OCCC) has a poor prognosis, and its therapeutic strategy has not been established. PRELP is a leucine-rich repeat protein in the extracellular matrix of connective tissues. Although PRELP anchors the basement membrane to the connective tissue and is absent in most epithelial cancers, much remains unknown regarding its function as a regulator of ligand-mediated signaling pathways. Here, we obtained sets of differentially expressed genes by PRELP expression using OCCC cell lines. We found that more than 1000 genes were significantly altered by PRELP expression, particularly affecting the expression of a group of genes involved in the PI3K-AKT signaling pathway. Furthermore, we revealed the loss of active histone marks on the loci of the PRELP gene in patients with OCCC and how its forced expression inhibited cell proliferation. These findings suggest that PRELP is not only a molecule anchored in connective tissues but is also a signaling molecule acting in a tumor-suppressive manner. It can serve as the basis for early detection and novel therapeutic approaches for OCCC toward precision medicine.

Keywords: PRELP; epigenetics; gene expression; ovarian clear cell carcinoma.

Figure 1

Correlation between PRELP gene expression and genetic mutation and copy number alteration (CNA) in ovarian cancer. (a) PRELP mRNA expression (y-axis) is plotted against normal tissue (n = 88), primary tumor (n = 419), recurrent tumor (n = 8). Statistical analysis was performed using Student’s t-test. **** p < 0.0001, n.s.: not significant. (b) PRELP mRNA expression (z-scores relative to diploid samples (y-axis) is plotted against CNAs in the PRELP gene (n = 316). Shallow deletion (CNA = −1), diploid (CNA = 0), gain (CNA = +1), and amplification (CNA = +2) are shown. (c) PRELP mRNA expression (z-scores relative to diploid samples; y-axis) is plotted against a wild-type (n = 315) or missense mutation (n = 1; x-axis).

Figure 2

PRELP gene expression in ovarian cancer. (a) RT-PCR analysis using ovarian cancer tissue and matched normal ovarian tissue as a comparison. (b) RT-PCR analysis using HMOsisEC10, an immortalized human ovarian endometriotic epithelial cell line, and ovarian clear cell carcinoma (OCCC) cell lines. Relative mRNA expression is shown. RT-PCR was performed in triplicate for each sample. Statistical analysis was performed using Student’s t-test. * p < 0.05; ** p < 0.01.

Figure 3

Reduced cell viability associated with induced PRELP gene expression in ovarian clear cell carcinoma (OCCC) cell lines. PRELP gene expression was induced by adding doxycycline (DOX) (1 μg/mL) to the lenti-viral expression system. The expression of myc-tagged PRELP protein was analyzed using whole-cell extracts from (a) RMG-I, (b) OVTOKO, and (c) SKOV3 cells with or without DOX. The left side indicates the protein size marker. Alpha-tubulin; loading control. The cell viability of (a) RMG-I, (b) OVTOKO, and (c) SKOV3 cells was evaluated using Cell-Counting Kit-8 assays. Black lines; without DOX, red lines; with DOX. Error bars indicate biological replicates (n = 3 or 4). Statistical analysis was performed using Student’s t-test. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 4

Loss of epigenetic marks at the PRELP locus in clinical tissues of ovarian clear cell carcinomas (OCCC). The integrative genomics viewer (IGV) tracks of H3K4me3 (blue), H3K27ac (red), and CTCF (green) peak at the PRELP locus, illustrating the loss of epigenetic marks around the PRELP gene promoter upstream region in OCCC (n = 5). Normal ovarian tissues (n = 2) on the opposite healthy ovary were used as a comparison. All data ranges are standardized as 0–2. The upstream region of the PRELP gene promoter is indicated by a black underline at the bottom of the figure.

Figure 5

PRELP regulates the PI3K-AKT signaling pathway. (a) MA plots illustrating differentially expressed genes (FDR < 0.05, |logFC| > 1, red dots) between conditionally expressed OVTOKO cells with (n = 3) and without DOX (n = 3). The overexpression of PRELP is shown as green dots. The average logCPM is plotted on the x-axis. The logFC is plotted on the y-axis. (b) KEGG pathway enrichment analysis of differentially expressed genes in OVTOKO cells that overexpressed PRELP. Dot plot showing GeneRatio on the x-axis and terms sorted by GeneRatio on the y-axis. The p-value is displayed as a gradient from red to blue. The number of counts is indicated by the size of the black circle. (c) Volcano plots illustrating differentially expressed genes (FDR < 0.05, |logFC| > 1, red dots) between the same set of OVTOKO cells as (a). Differences in Log₂ fold change in gene expression values are plotted on the x-axis. Adjusted p-values calculated using the Benjamin–Hochberg method are plotted on the y-axis. Genes corresponding to the PI3K-AKT signaling pathway are shown as large red circles.