Abstract 4024: iNOS inhibition increases survival in triple negative breast cancer by targeting metastasis and epithelial-mesenchymal transition
Authors: Granados Sergio M. , Liu Yi , Dave Bhuvanesh , Landis Melissa D. , Gross Steven S. , Chang Jenny C. 
Published: May 2015

Triple negative breast cancer (TNBC) is an aggressive form of breast cancer with no effective targeted therapy. Inducible nitric oxide synthase (iNOS) is associated with poor survival in breast cancer patients by increasing tumor aggressiveness. This work aimed to investigate the potential of iNOS inhibitors as a targeted therapy for TNBC. We hypothesized that inhibition of endogenous iNOS would decrease TNBC aggressiveness by reducing metastasis through modulation of epithelial-mesenchymal transition (EMT)-inducing factors. We also propose an effective clinically relevant-targeted therapeutic regimen by re-purposing the iNOS inhibitor L-NMMA which has been extensively investigated in thousands of patients with cardiogenic shock.

Cell migration was determined in confluent MDA-MB-231 cells with a “wound healing assay” treated with different concentrations of the specific iNOS inhibitor 1400W (0, 0.0001, 0.001, 0.01, 0.1, 1, 2, 4 mM) in low serum conditions (1%) for 72h. EMT-inducing factors (Snail, Slug, Twist1, and Zeb1) were determined by Western blot in MDA-MB-231 cells. For in vivo studies, 3x106 MDA-MB-231 and SUM159 cells were injected in the right mammary fat pad of female SCID Beige mice (n=10/group). The clinically relevant dose regimen consisted on two cycles of docetaxel (20mg/kg, i.p, on day 0) twelve hours before being combined with L-NMMA (400mg/kg on day 1, and 200mg/kg for 4 additional days by oral gavage) and amlodipine on day 0 (10mg/kg, i.p, daily, for 6 days). Docetaxel alone, as well as saline (i.p) + sterile water (oral gavage) were used as controls.

Our results show that the specific iNOS inhibitor 1400W was able to diminish cell migration in vitro for all concentrations tested. Importantly, impact of iNOS inhibition on cell migration was further correlated with a dose-dependent decrease of the EMT transcription factors- Snail, Slug, and Twist1. Zeb1 was mainly affected at millimolar concentrations. In vivo, the combination of L-NMMA and docetaxel was able to decrease tumor growth in a MDA-MB-231 and SUM159 orthotopic models. Additionally, the combination with amlodipine did significantly prevent the L-NMMA-dependent increase of blood pressure. More importantly, this dose regimen also improved survival compared to docetaxel alone.

In conclusion, targeted therapy with iNOS inhibitor impacts cell migration and changes in epithelial-mesenchymal transition might influence metastatic events. Based on these findings, we have planned a phase Ib/II targeted therapeutic trial by re-purposing L-NMMA as an anti-cancer indication. Targeting TNBC with L-NMMA could significantly alter clinical practice for treatment of breast cancer in the near future.

Citation Format: Sergio M. Granados, Yi Liu, Bhuvanesh Dave, Melissa D. Landis, Steven S. Gross, Jenny C. Chang. iNOS inhibition increases survival in triple negative breast cancer by targeting metastasis and epithelial-mesenchymal transition. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4024. doi:10.1158/1538-7445.AM2014-4024