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Voltage-gated sodium channels (VGSCs) are the target for many therapies. Variation in membrane potential occurs throughout the cell cycle, yet little attention has been devoted to VGSCs and Na+,K+-ATPase in the cell cycle. We hypothesized that in addition to doubling DNA and cell membrane in anticipation of cell division, there should be a doubling of VGSCs and Na+,K+-ATPase compared to non-dividing cells. We tested this hypothesis in eight immortalized cell lines by correlating immunocytofluorescent labeling of VGSCs or Na+,K+-ATPase, with propidium iodide or DAPI fluorescence using flow cytometry. Cell surface expression of VGSCs during phases S through M was double that seen during phases G0 - G1. By contrast, Na+,K+-ATPase expression increased only 1.5-fold. The increases were independent of baseline expression of channels or pumps. The variation in VGSC and Na+,K+-ATPase expression has implications for both our understanding of sodium's role in controlling the cell cycle and variability of treatments targeted at these components of the Na+ handling system.

Changes in membrane potential dramatically impact the progression of cancer, yet little is known as to how ion transport contributes to disease progression. Voltage-gated sodium channels (VGSCs) and Na,K-ATPases are particularly attractive targets as both are upregulated in many cancers and have been correlated specifically with invasiveness in vitro and metastasis and in vivo. However, their precise upstream regulators and downstream effectors remain unclear. Additionally, it has not been determined whether these transporters are co-regulated in cancer, although they display overlap in their downstream effectors and have complementary activity on sodium ions. Such a system has, however, been described in neuronal models of pain, wherein inflammatory injury leads to an upregulation in VGSC expression and a subsequent proportional increase in Na,K-ATPase. We sought to describe a similar phenomenon in cancer cells, which could indicate that this neuronal response to injury may be hijacked as a driver of metastatic development. To elucidate the impact of inflammatory mediators on these ion transporters in cancer cells, we assessed the effects of TNFα and PGE2 on VGSCs and Na,K-ATPases in the human breast epithelial or cancer cells MCF-10a, MCF-7, and MDA-MB-231. RT-qPCR revealed increases in RNA expression for the clinically relevant VGSC subunit Nav1.5. Similar RNA increases were observed in the α1 and α3 subunits of Na,K-ATPase, although in all cases the magnitude and time point of maximal effect differed by cell line. Then a 1hr challenge of TNFα on MDA-MB-231 cells was used to examine whether the targets were independently regulated by inflammatory mediators, co-regulated via the transport of sodium ions, or co-regulated by the expression of one protein influencing the expression of the other protein. Transfection with siRNA against either the α1 or α3 subunit of Na,K-ATPase did not inhibit the inflammatory-mediated increase in RNA for Nav1.5. However, transfection with anti-Nav1.5 siRNA inhibited the upregulation of both Na,K-ATPase subunits. TNFα challenge following pre-incubation of MDA-MB-231 cells in a sodium-free medium completely inhibited the inflammatory-mediated upregulation of Nav1.5, Na,K-ATPase α1 or α3 RNA. Co-incubation of TNFα with the VGSC blocking drug protoxin I inhibited the inflammatory-mediated increase in Na,K-ATPase α1 or α3 RNA, but co-incubation with the Na,K-ATPase blocking drug ouabain did not inhibit the increase in Nav1.5 RNA and indeed may have potentiated the increase. Together these results are evidence that VGSCs and Na,K-ATPase are co-regulated by inflammatory mediators in metastatic breast cancer, that sodium is essential to this mechanism, and that the presence and ion-transporting function of VGSCs is necessary for the inflammatory-mediated effect on Na,K-ATPase but not vice versa. Citation Format: Steven D. Scahill, Harry J. Gould, Dennis Paul. Inflammatory co-regulation of voltage-gated sodium channels and Na,K-ATPases in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 276.

Voltage-gated sodium channels (VGSCs) are upregulated in aggressive carcinomas, making these channels a target for novel clinical therapies. Targeted Osmotic Lysis (TOL) is a novel cancer therapy that combines pharmacologically blocking Na+,K+, ATPase (sodium pumps) while stimulating VGSCs with a pulsed electric field. The consequent increase in intracellular Na+ causes an osmotic lysis of the cells that overexpress VGSCs. Noncancerous cells, with fewer VGSCs do not lyse. We hypothesized that variability in efficacy of TOL is due to VGSC expression that varies across the cell cycle. We assessed cell surface expression of VGSCs and Na+,K+-ATPase during phases of the cell cycle in which there are single copies of DNA compared with phases in which DNA has doubled. For this, DNA was labeled with either propidium iodide or DAPI and VGSCs were labeled with a pan-specific VGSC antibody while Na+,K+-ATPases were labelled with a pan-specific Na+,K+-ATPase antibody in eight immortalized cancer and non-cancer cell lines. With flow cytometry we showed that VGSC expression doubled during phases of cell division (S-M phases) while Na+,K+-ATPase expression increased by 1.5-fold. To further elucidate the role of VGSCs and Na+,K+-ATPases throughout the cell cycle, S-Trityl-L-cysteine (STLC) was used to suspend the cell cycle in M phase. We hypothesized that treating cancer cells with TOL in the S-M phases would lead to increased cell death, compared to cells that were not suspended in M phase by STLC (G0-G1 phases). Cells were either treated with STLC or media without drug for 24 hours, then exposed to TOL treatment. The efficacy of the treatment was measured using a MTT assay revealing that cell death was greater in STLC-treated cells than control cells. By using cell cycle inhibitors, we may increase the efficacy of TOL for treating advanced carcinomas that overexpress VGSCs. Citation Format: Samantha Edenfield, Harry J. Gould, Dennis Paul. Efficacy of targeted osmotic lysis is increased by cell cycle inhibition in M Phase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2312.

In vitro cancer research predominantly utilizes established cell lines of a chosen cancer type as phenotypic representations of various disease states, whether they be non-cancerous, non-invasive, highly metastatic, or derived from distinct cellular origins within an organ or tissue. While such methodology is useful to study cancer cells from diverse disease states, when one considers the tremendous complexity of intertumoral heterogeneity, it becomes problematic to use cell lines derived from distinct individuals with diverse malignancies to study the progression of tumorigenesis in a single cancer type. To address this need, a novel murine model was developed, composed of six cell lines isolated from progressively advanced tumors all derived from a single parental line (Ray et al., Exp. Cell Res. 340, 1-11). Each cell line should represent a snapshot of a particular stage of this cancer’s development, with the entire model providing a platform to study tumorigenic changes incurred as the transformed cells progressed from a localized tumor to an aggressively malignant growth. However, isolating primary cell lines can be an inefficient process, and a successfully isolated line may not accurately represent the dominant neoplastic cell type from a tumor. For that reason, it is necessary to characterize the cell lines from this model to confirm that the observed in vitro behavior of the cells matches the expected phenotype of the tissue from which it was derived. To this end, we performed an in vitro characterization of each cell line in the model. The three lines derived from distant metastatic tumors displayed significantly higher rates of two dimensional motility and invasive capacity as compared to the parental cell line, the line derived from a primary tumor or from a local metastasis. The distant metastatic lines also displayed elevated rates of extracellular acidification, an indication of increased rates of aerobic glycolysis. These measures demonstrated convincingly that the behavior of the cells in culture mirrored the expected phenotype of the tissue from which they were derived, thus validating the utility of the model. We then performed a comprehensive proteomic analysis via orbitrap tandem mass spectrometry, yielding expression data on over 3,000 proteins across all cell lines. This analysis yielded expected results that further validated the model, such as showing an increasing trend of expression of cancer-associated proteins like transcription factor AP-1 and decreasing trends of expression of tumor suppressors like protein NDRG1. However, it also revealed striking changes in protein expression in targets not currently implicated in tumorigenesis, thus providing novel targets for further examination. In total, this study validated a novel murine cell model as a valuable tool in the study of tumorigenesis, while providing a robust proteomic data set to guide future research into cancerous progression. Citation Format: Steven D. Scahill, Kelly Jean Sherman, Jessie Guidry, Harry J. Gould, Dennis Paul. In vitro characterization of a novel murine model of cancerous progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1684.

Targeted osmotic lysis (TOL), the concurrent stimulation of voltage-gated sodium channels (VGSCs) and blockade of Na+ pumps, kills up to 100% of highly malignant, human and murine breast cancer cells in vitro and slows the growth of murine breast cancer homografts and increases survival of the hosts in vivo when compared to homografts treated with negative controls. In this study we compared the efficacy of TOL and paclitaxel, a current standard chemotherapeutic agent for treating advanced murine breast cancer, as a positive control. For this, BALB/c mice with 4T1 homografts were treated with TOL alone, paclitaxel alone or with a combination regimen of paclitaxel and TOL. For TOL, mice received 8 hourly doses (3 mg/kg) of digoxin on 2 successive days. Upon reaching steady-state levels (dose 5) the mice were exposed to 4 X 30 minutes of pulsed electric field stimulation (18 V/m, 10 ms pulses, 15 interstimulus intervals) hourly for a total of 2 hours of stimulation. Alternatively, mice received paclitaxel (20 mg/kg) by i.p. injection. Mice that were treated concurrently with TOL and paclitaxel received paclitaxel 2 days prior to treatment with TOL. Groups of mice that received digoxin alone, stimulation alone, paclitaxel + digoxin, paclitaxel + stimulation or vehicle alone served as negative controls. Initially, the homografts were measured daily and then every other day with a digital caliper until criteria for humane endpoint euthanasia were met. The mean time to meeting criteria for humane endpoint euthanasia in mice treated with paclitaxel alone (2 days) was identical to that observed in the negative controls. Mice treated with TOL alone, on average, met criteria at post-treatment day 5, whereas administration of paclitaxel two days before TOL reduced the effectiveness of TOL, decreasing mean survival to day 3 post-treatment. Immunohistochemical evidence indicated that paclitaxel’s effect on tubulin may impede the transport and insertion of channels into the cell membrane, thereby reducing the channel density and the effectiveness of TOL. By contrast, the administration of TOL either before or after treatment with paclitaxel improved host survival. Normal tissues were unaffected. These results are evidence that TOL is equal to or better than paclitaxel for increasing survival of murine hosts with advanced breast cancer when TOL is administered before or between paclitaxel dosing cycles. Sequential use of TOL may well improve survival, but simultaneous administration of paclitaxel and TOL will likely reduce TOL’s adjunctive benefit. Because of the conserved nature of the sodium channel/sodium pump mechanism, the comparable efficacy to TOL to a currently approved treatment and the favorable adverse effect profile, we submit that TOL warrants further study as a promising option for treating individuals with advanced neoplastic disease. Citation Format: Harry J. Gould, Kelly J. Sherman, Dennis Paul. Efficacy of targeted osmotic lysis using pulsed electric field stimulation compared to paclitaxel for treating murine, triple-negative breast carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1071.