Wnt/β-Catenin Inhibition by CWP232291 as a Novel Therapeutic Strategy in Ovarian Cancer

Abstract

The poor prognosis of ovarian cancer patients mainly results from a lack of early diagnosis approaches and a high rate of relapse. Only a very modest improvement has been made in ovarian cancer patient survival with traditional treatments. More targeted therapies precisely matching each patient are strongly needed. The aberrant activation of Wnt/β-catenin signaling pathway plays a fundamental role in cancer development and progression in various types of cancer including ovarian cancer. Recent insight into this pathway has revealed the potential of targeting Wnt/β-catenin in ovarian cancer treatment. This study aims to investigate the effect of CWP232291, a small molecular Wnt/β-catenin inhibitor on ovarian cancer progression. Various in vitro, in vivo and ex vivo models are established for CWP232291 testing. Results show that CWP232291 could significantly attenuate ovarian cancer growth through inhibition of β-catenin. Noticeably, CWP232291 could also s suppress the growth of cisplatin-resistant cell lines and ovarian cancer patient-derived organoids. Overall, this study has firstly demonstrated the anti-tumor effect of CWP232291 in ovarian cancer and proposed Wnt/β-catenin pathway inhibition as a novel therapeutic strategy against ovarian cancer.

Keywords: CWP232291; Wnt/β-catenin; organoids; ovarian cancer; targeted therapy.

Figure 1

Wnt/β-catenin pathway in ovarian cancer progression. GSEA was performed using the GSEA tool provided by the Broad Institute (http://software.broadinstitute.org/gsea). Enriched plots were generated to compare the expression of a set of genes in GO canonical Wnt signaling and Hallmark Wnt/β-catenin signaling between different groups of datasets (X axis means the gene hits and Y axis means the enrichment score. Red part means genes that positively correlated with the pathway and blue part means genes that negatively correlated with the pathway). (A) The gene expression profiling of 185 primary ovarian cancer tissues was included in GSE26712 dataset. Samples were divided according to the overall survival (OS) of patients into the long (OS >= 36 months) and short survival group (OS < 36 months). Wnt/β-catenin pathway was shown to be enriched in the short OS group than the long OS group by GO analysis (left) and Hallmark analysis (right). (B) The gene expression profiling of 88 high-grade serous ovarian cancer tissues was included in GSE156699 dataset. Samples were divided into the chemotherapy responder group and non-responder group according to patients’ response to chemotherapy. Wnt/β-catenin pathway was enriched in the chemotherapy non-responder group by GO analysis (left) and Hallmark analysis (right). (C) The gene expression profiling of 18 paired ovarian cancer primary tissues and omental metastases was included in GSE30587 dataset. Wnt/β-catenin pathway was enriched in omental metastases than primary sites by GO analysis (left) and Hallmark analysis (right). (D) Top 20 frequently mutated genes were listed in ovarian cancer in COSMIC (https://cancer.sanger.ac.uk/cosmic/). Among these, CTNNB1 mutation frequency accounted for 6% among all ovarian cancer patients. (E) Most of the CTNNB1 mutation types were copy number variation (CNV) gain or gene overexpression. CTNNB1 expression was negatively related to the overall survival of ovarian cancer patients by Kaplan-Meier Plotter (https://kmplot.com/analysis/). Nuclear β-catenin expression was significantly associated with the survival of ovarian cancer patients (F). Nuclear β-catenin expression was assessed using the high-grade serous ovarian cancer tissue microarray (N=56). Samples were scored as negative (above) or positive (below) according to the β-catenin staining intensity in the nucleus (G). (H) The positive nuclear β-catenin expression was correlated with a worse overall survival (Log-rank test, p value=0.0004).

Figure 2

CWP232291 suppressed the growth of various ovarian cancer cell lines through β-catenin inhibition. Five ATCC cell lines, A2780/S, A2780/CP, CAOV3, PA1 and OVCAR3 (A) and three SNU cell lines, SNU119, SNU251, SNU840 (B) were used in this study. Cells were treated with different doses (0, 0.001, 0.01, 0.1, 0.2, 0.5, 1.0 µM) of CWP232291 for 24h and 48h. Cell viability was measured through MTT assay. (C) The representative ATCC cell line OVCAR3 and SNU cell line SNU251 were treated with different doses of CWP232291 (0, 0.1, 0.5, 1µM) for 24h and the expression of active β-catenin form, total β-catenin form and apoptotic markers was determined by Western blotting. CWP232291 inhibited tumor growth in the in vivo mouse model. (D) The tumor size was measured and calculated using the formula: 0.5 × length × (width)². (E) The body weight of mice was also examined together with the tumor size. Results were shown as mean ± SEM (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Figure 3

CWP232291 inhibited the growth of ovarian cancer patient-derived organoids. (A) Morphologic comparison of cell lines and organoids (up: low-power field; down: high-power field). (B) The representative morphology of organoid in bright field. (C) Organoid F-actin and Ki-67 staining. Twenty patient-derived organoids were established and treated with CWP232291 (1 µM) and cisplatin (20 µM). Organoids with over 50% growth inhibition were defined as sensitive. Nine of twenty (P1, P2, P3, P5, P9, P12, P15, P16, P19) was sensitive to CWP232291 (D) while four (P3, P5, P15, P19) of twenty was sensitive to cisplatin (E). (F) Six organoids were treated with CWP232291 (1 µM), cisplatin (20 µM) or the combination of these two compounds. The cell viability was determined by MTT assay.CDDP: cisplatin. Results were shown as mean ± SEM (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).