Introduction: At a global level breast cancer is the second most common cancer, and the most frequent in women.
Statins are 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) inhibitors. HMG-CoA reductase is a rate-limiting enzyme in the mevalonate pathway, making it important in the pathology of cancer. The serine/threonine kinase, mammalian target of rapamycin (mTOR) regulates cell survival, proliferation and growth via translational initiation control, and this pathway is deregulated in many types of cancer which are associated with phosphatidylinositol-3-kinase (PI3K)/Akt signaling activation. HMG-CoA reductase expression is dependent on eukaryotic translation initiation factor 4E (eIF4E), and requires high translation initiation efficiency for polysome recruitment. eIF4E availability is reduced by mTOR inhibitors such as rapamycin and metformin, leading to a reduction in HMG-CoA reductase expression.
Objectives: This research aims to study the proposed combinatory treatments of rapamycin or metformin with HMG-CoA reductase inhibitors, in order to identify breast cancer subtypes sensitive to the proposed drug combinations. The specific mechanisms of action will also be studied.
Methodology: The breast cancer cell lines of Hs587T, MCF-7 and MDA-MB-468 were seeded in 96-well plates. Following 24 hours, triplicate wells of each cell line were treated with 0, 2, 4, 6, 8, 10, 12, and 14mM metformin. MTT assays were carried out 24, 48, and 72 hours post treatment. Successively, similar procedures for each cell line were carried out using 0, 10, 25, 35, 50, 65, 75, 100ng/mL rapamycin. For every cell line the time point and the dose at which a suppression of cell viability (not reaching IC50) occurred for 3 consecutive doses, were defined as the sensitization time point (Ts) and the sensitization dose (Ds) respectively.
Ongoing research: The same cell lines are currently being pre-treated with Ds of metformin and rapamycin separately, and at time Ts simvastatin is added at a range of µM concentrations. Following these conditions MTT assays are being carried out, and compared to control experiments using simvastatin alone. Annexin V FITC / propidium iodide flow cytometry based apoptosis assays, as well as Western Blot analysis for the quantification of HDJ2, HMG-CoA reductase, and eukaryotic translation initiation factor 4E binding protein 1 proteins are being planned to be carried out at a later stage.
Results: Time Ts and dose Ds for MCF-7 cells treated with rapamycin were found to be 72 hours and 12.5ng/mL respectively. MCF-7 cells were found to exhibit sensitivity to metformin that was too high to be considered for further combinatory treatment with simvastatin, since IC50 was reached post 48 hours of treatment with 10mM. Substantial sensitivity of all breast cancer cell lines studied was also observed for rapamycin or metformin treatment alone. In addition, previous work published by our group shows that the breast cancer cell lines MDA-MB-436 and BT-20 are sensitive to rapamycin.
Conclusion: Cell viability assays have shown that response to mTOR modulators is time, cell type and dose dependent. This project will determine optimum conditions for the combinatory treatment described above. The targeting of these two specific mechanisms converging onto a common target is novel, and expected to result in a higher efficacy, as well as decreased unwanted effects in an in vivo situation due to the lower individual doses used. Drug combinations would thus be superior to using either drug alone.
Citation Format: Vanessa Petroni, Marie Therese Camilleri Podesta, Anthony George Fenech, Godfrey Grech. Identification of novel drug combinations to target molecular pathways involved in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Targeting the PI3K-mTOR Network in Cancer; Sep 14-17, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(7 Suppl):Abstract nr B22.
- ©2015 American Association for Cancer Research.