Voltage-gated Na+ channels (VGSCs) are heteromeric protein complexes containing pore-forming α subunits and smaller, non-pore-forming β subunits. The β subunits are multifunctional channel modulators and are members of the immunoglobulin superfamily of cell adhesion molecules (CAMs). VGSCs are classically expressed in electrically excitable cells, e.g. neurons, where they mediate action potential firing, neurite outgrowth and migration during development. An increasing body of evidence indicates that VGSCs are also expressed in metastatic cells from a number of cancers, including breast cancer. The Nav1.5 α subunit (encoded by SCN5A) is expressed in cancer cell lines, where it enhances migration and invasion. SCN5A is up-regulated in tumor samples in several published datasets, associating with recurrence, metastasis, and reduced overall survival. We have previously shown that the VGSC-blocking antiepileptic drug phenytoin inhibits the migration and invasion of metastatic MDA-MB-231 cells in vitro. In addition, we have recently shown that the VGSC β1 subunit enhances breast tumor growth and metastasis in a xenograft model of breast cancer. The purpose of the present study was to establish whether or not VGSCs might be a viable therapeutic target by testing the effect of phenytoin on tumor growth and metastasis in vivo. We found that Nav1.5 expression was retained on MDA-MB-231 cells in orthotopic xenografts in immune-deficient mice. Mice were treated with phenytoin (60 mg/kg) or vehicle by daily intraperitoneal injection for 3 weeks, starting 1 week following implantation of tumor cells. Plasma phenytoin concentration was measured at the end of the experiment by liquid chromatography-mass spectrometry with single reaction monitoring (LC-SRM-MS) using metaxalone as an internal standard. Phenytoin significantly reduced tumor growth, detected by in vivo bioluminescent imaging and caliper measurements, without affecting animal weight. Phenytoin reduced the density of Ki67-positive cycling cells in the primary tumors, but did not affect the density of apoptotic cells expressing activated caspase-3, or the density of CD31-positive vessel structures. Phenytoin also reduced local invasion and the density of MMP9-positive cells within primary tumors. Finally, phenytoin significantly reduced metastasis to the liver, lungs and spleen, detected by bioluminescent imaging and GFP immunohistochemistry. This is the first study showing that phenytoin reduces tumor growth and metastasis in vivo. Together, our data support the hypothesis that VGSCs are up-regulated in breast cancer, favoring an invasive phenotype, and may thus be promising targets for therapeutic intervention. We propose that pharmacologically targeting VGSCs should be further studied as a potentially novel, cost-effective, anti-cancer therapy. Repurposing FDA-approved oral VGSC-blocking antiepileptic or antiarrhythmic drugs, e.g. phenytoin, may improve patient outcomes in the adjuvant setting.

Citation Format: William J Brackenbury, Michaela Nelson, Ming Yang, Adam A Dowle, Jerry R Thomas. Targeting voltage-gated sodium channels with the antiepileptic drug phenytoin inhibits breast tumor growth and metastasis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-03-10.