A Pharmacological Investigation of Eph-Ephrin Antagonism in Prostate Cancer: UniPR1331 Efficacy Evidence

Abstract

The Eph kinases are the largest receptor tyrosine kinases (RTKs) family in humans. PC3 human prostate adenocarcinoma cells are a well-established model for studying Eph-ephrin pharmacology as they naturally express a high level of EphA2, a promising target for new cancer therapies. A pharmacological approach with agonists did not show significant efficacy on tumor growth in prostate orthotopic murine models, but reduced distal metastasis formation. In order to improve the comprehension of the pharmacological targeting of Eph receptors in prostate cancer, in the present work, we investigated the efficacy of Eph antagonism both in vitro and in vivo, using UniPR1331, a small orally bioavailable Eph-ephrin interaction inhibitor. UniPR1331 was able to inhibit PC3 cells' growth in vitro in a dose-dependent manner, affecting the cell cycle and inducing apoptosis. Moreover, UniPR1331 promoted the PC3 epithelial phenotype, downregulating epithelial mesenchymal transition (EMT) markers. As a consequence, UniPR1331 reduced in vitro PC3 migration, invasion, and vasculomimicry capabilities. The antitumor activity of UniPR1331 was confirmed in vivo when administered alone or in combination with cytotoxic drugs in PC3-xenograft mice. Our results demonstrated that Eph antagonism is a promising strategy for inhibiting prostate cancer growth, especially in combination with cytotoxic drugs.

Keywords: Eph receptors; EphA2 antagonism; PC3 xenograft mice; ephrin ligands; prostate cancer.

Figure 1

(A) Cell growth of PC3 cells treated with UniPR1331 at 30, 10, and 3 µM for 24, 48, and 72 h. Two-way ANOVA followed by Tukey’s post-test was used to compare all the groups to control group (DMSO 0.3%) at each time point, * p < 0.05 ** p < 0.01. (B) Western blot analysis of UniPR1331-treated PC3 cells for cyclin D1, CDK4, p21, p27, and chK1, as well as for ERK1/2/p-ERK and p38/p-p38 (C). (D) Enzymatic detection of caspases activity 3 and 9 in UniPR1331-treated PC3 cells. For both the caspases, one-way ANOVA followed by Dunnett’s post-test was used to compare all the groups to control group (DMSO 0.3%), ** p < 0.01. (E) JC1 (apoptosis index) and acridine orange (autophagy index) staining of UniPR1331-treated PC3 cells.

Figure 2

The EphA2 levels of PC3 cells stimulated with ephrin-A1-Fc 0.25 µg/mL or treated with UniPR1331 30 µM are relative to untreated PC3 cells. For both the treatments, one-way ANOVA followed by Dunnett’s post-test were used to compare all the groups to untreated group, ** p < 0.01.

Figure 3

(A) Western blotting analysis of UniPR1331-treated PC3 cells for CD44, SNAIL and FASN. (B) Western blotting analysis of UniPR1331-treated PC3 cells for E-Cadherin, N-Cadherin and ZO-1.

Figure 4

(A) Migration, (B) invasion, and (C) vasculomimicry capabilities of PC3 cells treated with UniPR1331 at 30, 10, and 3 µM. One-way ANOVA followed by Dunnett’s post-test was used to compare all the groups to control group (DMSO 0.3%), * p < 0.05 ** p < 0.01.

Figure 5

Comparisons amongst treatments in terms of final tumor weight in PC3-xenograft nude mice after 5 weeks of treatment. One-way ANOVA followed by Tukey’s post-test was used to compare all the groups to control group, * p < 0.05, ** p < 0.01. CTR was significantly different from any treatment or association and UniPR1331 was significantly different from DTX, CPT, and their associations (data not reported in the graph for simplicity). All the groups passed Shapiro–Wilk normality test.