Identifying potential biomarkers for breast cancer proliferation and angiogenesis

V. Petroni Magri¹, A. G. Fenech¹, M. T. Camilleri Podesta², G. Grech³. ¹Clinical Pharmacology and Therapeutics, University of Malta, Msida, Malta, ²Anatomy, University of Malta, Msida, Malta, ³Pathology, University of Malta, Msida, Malta.

Introduction: The phosphatidylinositol-4,5-bisphosphate 3-kinase pathway regulates proliferation and angiogenesis in normal and cancer cells^1,2. Mammalian target of rapamycin (mTOR) inhibitors (rapamycin) suppress hypoxia inducible factor 1, alpha subunit (HIF1A) translation³ and vascular endothelial growth factor (VEGF) production⁴. HIF1A overexpression occurs in various cancers, controlling angiogenesis and invasion². VEGF is a strong pro-angiogenic factor, resulting in cancer growth³. The aim of this research was to investigate rapamycin effects on breast cancer cells by analysing cell viability and RNA expression of pro-angiogenic factors.

Method: Human breast cancer cell lines: MDA-MB-468, MDA-MB-436, MDA-MB-231, Hs 578T, HCC1937, BT-20 (ER^-, PR^-, HER2^-); MCF7 (ER⁺, PR⁺, HER2^-); MDA-MB-453 (ER^-, PR^-, HER2⁺) were seeded for 24 hours and then treated with 0, 10, 25, 35, 50, 65, 75, 100ng/mL rapamycin. Rapamycin sensitivity was determined by MTT assays performed 24, 48 and 72 hours after treatment. Additionally, 24 hours after rapamycin addition, the cells were lysed to prepare RNA for transcript quantification using a Luminex^® bead-based 33-GenePlex assay.

Results: MDA-MB-436 and HCC1937 cells were observed to be rapamycin-insensitive, since cell viability loss, if any, was not maintained across the investigated rapamycin concentrations and time-points. MDA-MB-468, MDA-MB-231, Hs 578T, BT-20, MCF7 and MDA-MB-453 resulted to be rapamycin-sensitive. In rapamycin-sensitive cells rapamycin was considered to be most effective after 48 hours, with viability loss ranging from 31% to 48%. A statistically significant positive correlation of cell viability with HIF1A (rho=0.403, p=0.010) and VEGFA (rho=0.682, p=0.000) RNA levels was recorded for rapamycin-sensitive cells, but not for rapamycin-insensitive cells.

Conclusions: The effects of rapamycin on suppressing cell proliferation vary among different breast cancer cell types. A positive correlation of HIF1A and VEGFA expression levels with rapamycin-sensitive cell viability suggests that cell viability decrease might be a result of HIF1A and VEGFA RNA expression reduction, upon rapamycin addition. This may be of clinical benefit by predicting similar results in vivo. Knowledge of HIF1A and VEGFA levels may predict candidates who will respond successfully to mTOR inhibitors, which will reduce tumorigenic properties associated with overexpression of HIF1A and VEGFA. Hence, HIF1A and VEGFA expression may act as potential biomarkers in personalised breast cancer management.

References:

1. Satheesha S et al. (2011). Mol Cancer 10:19.

2. Karar J and Maity A (2011). Front Mol Neurosci 4:51.

3. Masoud GN and Li W (2015). Acta Pharm Sin B 5(5): 378-89.

4. Guda M et al. (2002). Nat Med 8(2): 128-135.