Our heartiest congratulations to Dr Wang Tingting for receiving the 2014 Avon Foundation-AACR International Travel Grant.

This award provides travel support to abstract presenters working in breast cancer or other female cancers travelling from countries where opportunities for scientific advancement are limited.

Title: Simvastatin induced apoptosis and suppressed proliferation of breast cancer through deactivating PI3K/Akt/mTOR pathway

Purpose: Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, an enzyme that catalyzes HMG-CoA conversion to mevalonate and enables subsequent cholesterol synthesis. Beyond its basic function in regulating HMG-CoA pathway, statins purportedly exert anti-tumoral effects on breast cancer by increasing apoptosis and decreasing proliferation. However, the biologic mechanisms for these actions are not fully elucidated. Prospective trials are warranted to clarify the impact of statins as an anti-cancer drug. The aims of this study were 1) to explore the effects of simvastatin on apoptosis, proliferation and commonly abrogated PI3K/Akt/mTOR pathway in a window-of-opportunity breast cancer trial; 2) to further confirm findings from the clinical trial by functional studies using breast cancer cell lines.

Experimental Design: A total of 16 female patients with newly diagnosed primary breast cancer were recruited into a preoperative window-of-opportunity trial. Patients received simvastatin for 10-28 days at a dose of 20mg daily before definitive breast cancer surgery. Pre- and post-treatment tumor biopsies were obtained for immunohistochemistry staining for cleaved caspase 3, Ki67, Pten, phospho-Akt and phospho-S6 ribosomal protein, and the changes between paired pre- and post-treatment tumors compared. For in vitro functional studies, apoptosis and proliferation assays were performed on breast cancer cell lines MCF7 and MDA-MB-231 48 hours after treatment with simvastatin. Using Western blots, the effects of simvastatin on apoptosis protein caspase 3, cell cycle proteins of c-myc, cyclin D1, p21 and p27, and of PI3K/Akt/mTOR pathway through phosphorylation of Akt, mTOR, p70S6K, S6 ribosomal protein and 4EBP1, were assessed.

Results: In the prospective clinical trial, short term simvastatin treatment led to significant increase in apoptosis in post-treatment tumors, as determined by positive cleaved caspase 3 (23.4±24.3 vs 8.9±7.4, p=0.002). Decreased trend for proliferation was also observed in post-treatment tumors (57.7±35.2 vs 74.6±59.9 for Ki67, p=0.245). Moreover, simvastatin treatment markedly suppressed PI3K/Akt/mTOR signalling pathway by activating Pten (66.1±65.2 vs 30.0±46.6, p=0.005) and by dephosphorylating Pten downstream effectors, Akt and S6 ribosomal protein (9.0±19.5 vs 93.0±87.4, p=0.002 for phospho-Akt; 63.7±51.6 vs 108.6±67.7, p=0.033 for phospho-S6 ribosomal protein). In MCF7 and MDA-MB-231 cells, simvastatin treatment similarly induced apoptosis and inhibited cell proliferation, with decrease in expression of c-myc and cyclin D1 and increase in cell cycle suppressor p21 and p27 at a dose dependent manner. Moreover, simvastatin treatment, at 20uM for MCF7 and 400nM for MDA-MB-231 cells, strongly suppressed sequential phosphorylation of PI3K/Akt/mTOR pathway cascades including Akt, mTOR, p70S6K, S6 ribosomal protein and 4EBP1.

Conclusion: In this study, prospectively treated clinical samples and in vitro functional studies concordantly showed simvastatin to promote apoptosis, suppress proliferation and dephosphorylate sequential signalling cascades of PI3K/Akt/mTOR pathway of breast cancer. These findings shed light on the biological and potential anti-tumor effects of simvastatin in breast cancer.

Authors:

Tingting Wang¹, Serena Seah¹, Ching-Wan Chan³, Mikael Hartman³, Janessa Thaw Dar², Philip Iau³, Boon-Cher Goh^{1, 2}, Soo-Chin Lee^{1, 2}

¹Cancer Science Institute, National University of Singapore, Singapore

²Department of Haematology and Oncology, National University Cancer Institute, National University Health System, Singapore, Singapore

³Department of Surgery, National University Cancer Institute, National University Health System, Singapore