(-)-Oleocanthal Prevents Breast Cancer Locoregional Recurrence After Primary Tumor Surgical Excision and Neoadjuvant Targeted Therapy in Orthotopic Nude Mouse Models

Abstract

Breast cancer (BC) recurrence represents a challenge for survivors who have had their primary tumors surgically excised, and/or have completed radiation, neoadjuvant, or adjuvant therapeutic regimens. Current BC treatments mostly lack the ability to reduce the risk of disease recurrence. About 70% of BC patients will subsequently suffer disease relapse, manifesting as local, regional, or distant tumor recurrence, which clearly underscores the urgent need to discover novel recurrence inhibitors. (-)-Oleocanthal (OC) is a natural phenolic, found so far exclusively in extra-virgin olive oil (EVOO). OC exerts documented bioactivities against diverse cancer types, inflammation, and neurodegenerative diseases. Herein we report the novel activity of daily oral treatment with OC (10 mg/kg) in preventing BC locoregional recurrence in a nude mouse xenograft model generated by orthotopic inoculation with BT-474 cells as a luminal type B model. We further report inhibition of tumor recurrence by OC after completion of a lapatinib neoadjuvant regimen. However, in a recurrence model of triple-negative breast cancer (TNBC), OC treatment (10 mg/kg) did not effectively prevent tumor recurrence, but rather, was seen to significantly reduce the growth of recurrent tumors as compared to vehicle control-treated animals. Inhibition of tumor recurrence was associated with significant serum level reductions of the human BC recurrence marker CA 15-3 at the study end in animals treated with OC. OC treatment upregulated the expression of the epithelial marker E-cadherin and downregulated the levels of the mesenchymal marker vimentin in recurrent tumors vs. untreated control animals. OC treatment also reduced the activation of MET and HER2 receptors, as indicated by reduced phosphorylation levels of these proteins in recurrent tumors vs. controls. Collectively, the results of our studies provide the first evidence for suppression of BC tumor recurrence by oral OC treatment in an animal model for such recurrence, and furthermore, highlight favorable prospects for this natural product to emerge as a first-in-class BC recurrence inhibitor.

Keywords: HER2; MET; breast cancer; extra-virgin olive oil; lapatinib; neoadjuvant; oleocanthal; recurrence; surgical excision.

Figure 1

Oleocanthal treatment inhibited recurrence of BT-474 tumors in a xenograft orthotopic nude mice model. (A) Overview of the experimental design. (B) Incidence of BC recurrence in each experimental group. (C) Mean weight comparison (treated vs. control) for recurring tumors at the end of the experiment. Error bars indicate SD. *** p < 0.001 value represents the statistical significance as compared to vehicle-treated control group. (D) Mean volume comparison (treated vs. control) for recurring tumors at the end of the experiment. Error bars indicate SD. (E) Representative recurrent BT-474 breast tumors for each experimental group. (F) Mean tumor volumes for primary and recurrence phases over the treatment period. Data points represent the mean tumor volume, error bars indicate SD, for each experimental group. (G) Body weights (mean ± SD) of animals in each group during the primary and recurrence phases of the experiment.

Figure 2

Oleocanthal reduced growth of recurrent MDA-MB-231 tumors in a xenograft orthotopic nude mice model. (A) Overview of the experimental design. (B) Incidence of TNBC recurrence in each experimental group. (C) Mean weights (treated vs. control) for recurring tumors at the end of the experiment. Error bars indicate SD. * p < 0.05 for statistical significance comparing to vehicle-treated control group. (D) Mean volume comparison (treated vs. control) for recurring tumors at the end of the experiment. Error bars indicate SD. ** p < 0.01 for statistical significance. (E) Representative recurrent MDA-MD-231 breast tumors for each experimental group. (F) Mean tumor volumes for primary and recurrence phases over the treatment period. Points represent the mean tumor volume, error bars the SD, for each experimental group. (G) Body weight monitoring data over the experiment course. Points represent the mean body weight for animals in each group at each weighing. Error bars indicate SD.

Figure 3

Oleocanthal inhibits recurrence of BT-474 tumors in a xenograft orthotopic nude mouse model after neoadjuvant LP treatment. (A) Overview of the experiment layout. (B) Incidence of tumor recurrence in each experimental group. (C) Mean tumor weight comparison (treated vs. control) at the end of the experiment. Error bars indicate SD. * p < 0.05 when comparing treated to vehicle control groups. (D) Mean tumor volume at the end of experiment for tumor recurrence, comparing vehicle control (LP→VC) and OC-treated (LP→OC) groups. Error bars indicate SD. ** p < 0.01. (E) Representative recurrence tumors from each experimental group. (F) Mean tumor volumes for primary and recurrence phases over the treatment period. Points represent the mean tumor volume, error bars indicate SD for each experimental group. (G) Mean body weights of animals in each group during the primary and recurrence phases of the experiment. Error bars indicate SD.

Figure 4

Effect of oleocanthal treatment on E-cadherin, vimentin, total and active MET and HER2 RTKs in BC recurrence models. Top panels include representative Western blots for the indicated markers, and bottom panels represent the results of densitometric analysis performed on all blots. (A) Expression of the indicated markers in recurrent tumors from the BT-474 recurrence model animals in the different experimental groups, resected at the end of each experimental treatment protocol, (B) for tumors resected from MDA-MB-231 recurrence model animals, (C) for tumors resected from BT-474 recurrence model animals after neoadjuvant LP. Scanning densitometry was obtained for all blots, carried out in triplicates, and the integrated optical density of each band was normalized with the corresponding density found for β-tubulin in the same blot, results shown in the bar graphs under their respective Western blot images. Vertical bars in the graph indicate the normalized integrated optical density of bands visualized in each lane. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to respective vehicle-treated control group.