Abstract 1673: mGluR1 as a potential therapeutic target in the treatment of triple negative breast cancer

Abstract

Abstract Introduction: Metabotropic glutamate receptors are expressed throughout the central nervous system where they initiate a host of signaling events that regulate neuron function. We have identified the presence of metabotropic glutamate receptor 1 (mGluR1) in breast cancer cells and demonstrated a role for these receptors in regulating the pro-proliferative phenotype of these cells, both in vitro and in vivo. In this study, we investigate the signaling mechanism(s) by which mGluR1 mediates this pro-proliferative effect, as well as investigating a potential role for mGluR1 in mediating anti-apoptotic signaling events in triple negative breast cancer. Methods: We studied the effect of mGluR1 expression on cell growth using MDA-MB-231 cells stably transduced with plasmids expressing shRNA against GRM1, the gene coding for mGluR1 protein. Cell growth was measured by MTT assay and a role for mGluR1 in mediating apoptosis was assessed by measuring PARP cleavage products and Annexin V staining, using Riluzole or BAY36-7620 (BAY), non-competitive inhibitors of mGluR1 activity. To examine potential signaling mechanisms mediating cell growth, MDA-MB-231 cells were grown in glutamate-free media and stimulated with the mGluR1 agonist, L-Quisqualic acid, in the presence or absence of BAY. PKC and phosphorylated Akt levels were examined by Western analysis. Since mGluR1 is known to protect nerve cells from oxidative stress and increased oxidative stress can result in p53-induced apoptosis, we measured the effect of Riluzole on p53 expression and oxidative stress levels in MDA-MB-231 cells. Oxidative stress was measured using immunofluorescent staining with carboxy-H2DCFDA and p53 by Western blot analysis. Results: MDA-MB-231 cells were transduced with Lentiviral constructs expressing shRNA against GRM1 and cell growth assessed by MTT assay. Growth of these cells was inhibited 50% by shRNA against GRM1 compared to non-silencing scrambled control. In addition, inhibition of mGluR1 activity by BAY in MDA-MB-231 cells prevented signaling through Akt which ultimately resulted in increased p53 expression and superoxide production and apoptosis. Incubation of MDA-MB-231 or BT549 cells with varying doses of Riluzole resulted in increased PARP cleavage by 24 hours. FACS analysis also showed a three- and ten-fold increase in Annexin V staining of MDA-MB-231 cells after 24 and 48 hr incubation with Riluzole, respectively. BAY also induced Annexin V staining of these cells by almost 3-fold at the 48 hr timepoint. Conclusion: Our results showing that inhibition of mGluR1 inhibits proliferation and increases apoptosis and oxidative stress implicate mGluR1, or one of its downstream signaling molecules, as potential new molecular targets for the treatment of breast cancer. Because it is an FDA-approved oral drug with low toxicity, Riluzole represents a promising approach in the treatment of triple negative breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1673. doi:10.1158/1538-7445.AM2011-1673