The Olive Phenolic S-(-)-Oleocanthal as a Novel Intervention for Neuroendocrine Prostate Cancers: Therapeutic and Molecular Insights

Abstract

Background/Objectives. Prostate cancer (PCa) is among the leading causes of death from cancer in men. Frequent use of androgen receptor inhibitors induces PCa transdifferentiation, leading to poorly differentiated neuroendocrine PCa (NEPC). ROR2 is critical for NEPC pathogenesis by activating ASCL1, promoting lineage plasticity. Protein lysine methylation mediated by N-lysine methyltransferases SMYD2 and its downstream effector EZH2 upregulates the NEPC marker ASCL1 and enhances c-MET signaling, promoting PCa aggression. Epidemiological studies suggest a lower incidence of certain malignancies in Mediterranean populations due to their intake of an olive-phenolics-rich diet. Methods. Cell viability, gene knockdown, and immunoblotting were used for in vitro analyses. A nude mouse NEPC xenograft model evaluated the anti-tumor efficacy of purified and crude oleocanthal. Xenograft tumors were subjected to RNA-seq, qPCR, and Western blot analyses, with clinical validation performed using tissue microarrays. Results. A tissue microarray analysis showed that SMYD2 expression was significantly elevated in PCa tissues with higher IHS versus normal prostate tissue cores. The olive phenolic S-(-)-oleocanthal (OC) suppressed the de novo NEPC NCI-H660 cells proliferation. Male athymic nude mice xenografted with the NCI-H660-Luc cells were used to assess OC effects on de novo NEPC progression and recurrence. Male NSG mice transplanted with LuCaP 93 PDX tumor tissues generated a heterogeneous in vivo model used to assess OC effects against t-NEPC progression. Daily oral 10 mg/kg OC administration significantly suppressed the NCI-H660-Luc tumor progression and locoregional recurrence after primary tumor surgical excision. OC treatments effectively suppressed the progression of LuCaP 93 PDX tumors. OC-treated tumors revealed downregulation of ROR2, ASCL1, SMYD2, and EZH2, as well as activated c-MET levels versus the placebo control. RNA sequencing of the collected treated NEPC tumors showed that OC disrupted NEPC splicing, translation, growth factor signaling, and neuronal differentiation. Conclusions. This study's findings validate OC as a novel lead entity for NEPC management by targeting the ROR2-ASCL1-SMYD2-EZH2-c-MET axis.

Keywords: LuCaP 93 Patient-Derived Xenograft; RNA sequencing; ROR2-ASCL1-SMYD2-EZH2-c-MET axis; S–(–)–oleocanthal; de novo and treatment-induced neuroendocrine prostate cancer; recurrence.

Figure 1

Effect of SMYD2 knockdown on NCI-H660 cell viability: (A) Successful SMYD2 knockdown evidenced by comparing the Western blot of the parent cells versus SMYD2-KD NCI-H660 cell lysates. (B) Comparison of the viability of the parent NCI-H660 cells versus NCI-H660-KD cells in cell proliferation assay over 15 days. Vertical bars represent the normalized SMYD2 expression in NCI-H660-KD cells relative to parent control cells. Data are presented as mean ± SD (n = 3); Student’s t-test; * p < 0.05, ** p < 0.01. WT: Parent cells; KD: Knockdown.

Figure 2

Effects of OC treatments on NCI-H660 cell viability and SMYD2 expression level: (A) Antiproliferative effects of OC on the de novo NEPC NCI-H660 cells. Dose–response curve showing the inhibition of NCI-H660 cell proliferation at different OC concentrations. (B) Western blot analysis of SMYD2 expression in NCI-H660 cells treated with VC, 20 μM OC, and 40 μM OC. Representative Western blots showing the dose-dependent reduction in SMYD2 expression. Densitometric quantification of SMYD2 level was performed for all blots, with each experiment conducted in triplicate. The integrated optical density of SMYD2 bands was normalized to β-tubulin loading control. Bar graphs represent the mean relative protein expression of SMYD2 as a percentage of the vehicle-treated control (± SD, n = 3). Statistical significance was determined using unpaired t-tests, with * p < 0.05 and ** p < 0.01 indicating significant differences compared to the vehicle-treated control.

Figure 3

TMA expression of SMYD2 in PCa clinical tissues: (A) Comparison of SMYD2 expression in normal prostate versus PCa tissues expressed as immunohistochemical score (IHS). Unpaired t-tests calculated data significance at p < 0.0001. (B) Comparison of the SMYD2 expression IHS in normal prostate tissues (I) versus tissues collected from PCa patients with Gleason scores > 7 (II) at 400×.

Figure 4

Effects of OC on de novo NEPC NCI-H660-Luc tumor progression and locoregional recurrence, and t-NEPC LuCaP 93 PDX tumor progression: (A) Photographs comparing the collected OC versus placebo control-treated NCI-H660-Luc primary tumors after surgical excision. (B) NCI-H660-Luc tumor volume monitoring over the course of the study. (C) Comparison of OC versus placebo control-treated NCI-H660-Luc primary tumor weight. (D) Mean NCI-H660-Luc tumor-bearing nude mice body weight monitoring over the course of the study. (E) Whole-body-weight imaging of nude mice at the end of NCI-H660-Luc tumor recurrence experiment comparing OC versus placebo control-treatment effects on locoregional recurrence. (F) Photographs comparing the collected OC versus placebo control-treated LuCaP 93 PDX tumors in NSG at the end of the study after 7 dosing weeks. (G) Comparison of OC versus placebo control-treated LuCaP 93 PDX tumor volume over the course of the study. (H) Comparison of OC versus placebo control-treated LuCaP 93 PDX tumor weight at the end of the study. Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant.

Figure 5

Molecular effects of OC on NCI-H660-Luc and LuCaP 93 PDX tumors. Tumor lysates were prepared by pooling tissues from three randomly selected mouse tumors (out of five per group) for each analysis. Western blot analysis showing the effects of OC treatment compared to placebo on (A) ROR2 and ASCL1 expression levels in NCI-H660-Luc tumors and (B) ROR2 and ASCL1 expression levels in LuCaP 93 PDX tumors. (C) Western blot comparison of OC treatment effects on SMYD2, EZH2, c-MET, p-c-MET Tyr1234/1235, p-c-MET Tyr1349, and p-c-MET Tyr1356 expression levels in NCI-H660 primary tumors versus the placebo control. Densitometric analysis quantified protein levels, with all blots performed in triplicate. The integrated optical density of each band was normalized to β-tubulin loading control. Bar graphs next to the Western blot images display the normalized optical density for each protein. Data are expressed as mean ± SEM (n = 3). *** p < 0.001, **** p < 0.0001 versus placebo control analyzed by one-way ANOVA. (D) qPCR analysis indicated a significant reduction in mRNA expression levels of SMYD2, EZH2, and total c-MET in OC-treated NCI-H660 primary tumors compared to placebo controls. Data are presented as mean ± SEM (n = 3). *** p <0.001 versus placebo control, analyzed by unpaired t-tests. (E) Western blot comparison of OC treatment effects on SMYD2, EZH2, c-MET, p-c-MET Tyr1234/1235, p-c-MET Tyr1349, and Tyr1356 expression levels in LuCaP 93 PDX tumors versus placebo controls. Densitometric analysis of the blots was performed in triplicate. The integrated optical density of each band was normalized to β-tubulin loading control. The bar graphs next to the blots represent the normalized optical density for each protein. Data are presented as mean ± SEM (n = 3). Statistical significance is indicated by *** p < 0.001, **** p <0.0001, analyzed by one-way ANOVA. (F) qPCR analysis revealed significant reduction in mRNA expression levels of SMYD2, EZH2, and increase in total c-MET in OC-treated LuCaP 93 PDX tumors versus placebo control. Data are expressed as mean ± SEM (n = 3). Statistical significance indicated by *** p <0.001; ns is non-significant, analyzed by unpaired t-test.

Figure 6

GO enrichment and PPI network analysis of downregulated DEGs in NCI-H660 and LuCaP 93 PDX tumors following OC treatments: (A) Dot plot illustrates the proportion of significantly enriched downregulated DEGs associated with distinct biological processes in NCI-H660 tumors. (B) Cnet plot visualizing the interaction network of downregulated DEGs mapped to the topmost five significantly enriched biological processes in NCI-H660 tumors. (C) Dot plot depicting the ratio of significantly enriched downregulated DEGs in LuCaP 93 PDX tumors, highlighting the affected biological pathways. (D) Cnet plot showing the network connectivity of downregulated DEGs linked to topmost five enriched biological processes in LuCaP 93 PDX tumors. DEGs identified using log2FC > 1.5 or log2FC < −1.5 with an adjusted p-value < 0.05. (E) STRING network analysis of downregulated DEGs in NCI-H660 tumors after OC treatments revealed 53 nodes and 18 edges, with a PPI enrichment p-value of 0.000227 (log2FC < −5, adjusted p-value < 0.05). (F) STRING analysis of downregulated DEGs in LuCaP 93 PDX tumors in response to OC treatments identified 80 nodes and 20 edges, with a PPI enrichment p-value of 0.0207 (log2FC < −3, adjusted p-value < 0.05).

Figure 6

GO enrichment and PPI network analysis of downregulated DEGs in NCI-H660 and LuCaP 93 PDX tumors following OC treatments: (A) Dot plot illustrates the proportion of significantly enriched downregulated DEGs associated with distinct biological processes in NCI-H660 tumors. (B) Cnet plot visualizing the interaction network of downregulated DEGs mapped to the topmost five significantly enriched biological processes in NCI-H660 tumors. (C) Dot plot depicting the ratio of significantly enriched downregulated DEGs in LuCaP 93 PDX tumors, highlighting the affected biological pathways. (D) Cnet plot showing the network connectivity of downregulated DEGs linked to topmost five enriched biological processes in LuCaP 93 PDX tumors. DEGs identified using log2FC > 1.5 or log2FC < −1.5 with an adjusted p-value < 0.05. (E) STRING network analysis of downregulated DEGs in NCI-H660 tumors after OC treatments revealed 53 nodes and 18 edges, with a PPI enrichment p-value of 0.000227 (log2FC < −5, adjusted p-value < 0.05). (F) STRING analysis of downregulated DEGs in LuCaP 93 PDX tumors in response to OC treatments identified 80 nodes and 20 edges, with a PPI enrichment p-value of 0.0207 (log2FC < −3, adjusted p-value < 0.05).