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Oleuropein is a natural inhibitor of PAI-1-mediated proliferation in human ER-/PR- breast cancer cells

Abstract

Purpose

Elevated expression of PAI-1 has been widely linked with adverse outcomes in a variety of human cancers, such as breast, gastric and ovarian cancers, rendering PAI-1 a prognostic biomarker. As a result, several chemical inhibitors are currently being developed against PAI-1; however, the clinical setting where they might confer survival benefits has not yet been elucidated.

Methods

RNA sequencing data analysis from the TCGA/GTEx cancer portals (n = 3607 samples). In silico molecular docking analyses to predict functional macromolecule interactions. ER-/PR- (MDA-MB-231) and ER+/PR+ (MCF-7) breast cancer cell lines implemented to assess the effect of oleuropein as a natural inhibitor of PAI-1-mediated oncogenic proliferation.

Results

We show that high PAI-1 levels inversely correlate with ER and PR expressions in a wide panel of estrogen/progesterone-responsive human malignancies. By implementing an in silico molecular docking analysis, we identify oleuropein, a phenolic component of olive oil, as a potent PAI-1-binding molecule displaying increased affinity compared to the other olive oil constituents. We demonstrate that EVOO or oleuropein treatment alone may act as a natural PAI-1 inhibitor by incrementally destabilising PAI-1 levels selectively in ER-/PR- breast cancer cells, accompanied by downstream caspase activation and cell growth inhibition. In contrast, ER+/PR+ breast cancer cells, where PAI-1 expression is absent or low, do not adequately respond to treatment.

Conclusions

Our study demonstrates an inverse correlation between PAI-1 and ESR1/PGR levels, as well as overall patient survival in estrogen/progesterone-responsive human tumours. With a focus on breast cancer, our data identify oleuropein as a natural PAI-1 inhibitor and suggest that oleuropein-mediated PAI-1 destabilisation may confer clinical benefit only in ER-/PR- tumours.

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Acknowledgements

We would like to thank Yanni’s Olive Grove Company in Potidea Chalkidiki, Greece for providing the early harvest EVOO.

Funding

This research is co-financed by Greece and the European Union (European Social Fund- ESF) through the Operational Program «Human Resources Development, Education and Lifelong Learning» in the context of the project ‘Reinforcement of Postdoctoral Researchers - 2nd Cycle’ (grant number: 2019-050-0503-18066), implemented by the State Scholarships Foundation (ΙΚΥ).

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Authors

Contributions

EET performed experiments, analysed and interpreted data. G.G. performed the in silico data analysis. SNL and MPT assisted with experimental equipment. AAP contributed towards data interpretation and co-supervised the project. AP designed the study, performed experiments, analysed and interpreted data, wrote the manuscript and supervised the project.

Corresponding author

Correspondence to Angelos Papaspyropoulos.

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All authors declare no conflict of interest.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Supplementary Information

Below is the link to the electronic supplementary material.

10549_2020_6054_MOESM1_ESM.pptx

Supplementary Fig. 1. Densitometry providing quantification for Western blots. (a) Quantification of PAI-1 and CASP-8 levels based on β-actin expression for Fig. 3a. (b) Same as (a), for Fig. 3c. (c) Same as (a), for Fig. 3e. **P<0.01 and ***P<0.001, respectively, of Student’s t-test; n.s.: non-significant. Error bars indicate s.e.m. Data shown are representative of at least 3 independent experiments. Supplementary Fig. 2 PAI-1 and TP53 correlation in estrogen/progesterone-responsive tumours. (a) Correlation analysis between PAI-1 and TP53 levels expressed in transcripts per million (TPM) across 9 estrogen/progesterone-responsive human tumour types (BRCA, COAD, LUAD, LUSC, OV, PRAD, READ, UCEC, UCS; n indicates total number of tumour samples). RNA seq data were retrieved from the TCGA and GTEx databases and analysed using the GEPIA and GEPIA2 online tools. (b) Overall survival data of cancer patients in (a) based on TP53 expression levels. TP53 level changes do not confer a significant effect on survival (p=0.58). See also Fig 1. Data were retrieved from the TCGA and GTEx databases using the GEPIA online tool(pptx 335 kb)

10549_2020_6054_MOESM2_ESM.pptx

Supplementary Table 1 Characterisation of the phenolic and non-phenolic content of EVOO used in in vitro experiments (pptx 73 kb)

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Tzekaki, E.E., Geromichalos, G., Lavrentiadou, S.N. et al. Oleuropein is a natural inhibitor of PAI-1-mediated proliferation in human ER-/PR- breast cancer cells. Breast Cancer Res Treat 186, 305–316 (2021). https://doi.org/10.1007/s10549-020-06054-x

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Keywords

  • Plasminogen activator inhibitor-1 (PAI-1)
  • Extra virgin olive oil (EVOO)
  • Oleuropein
  • ER/PR-responsive human cancer
  • MDA-MB-231
  • MCF-7