Blocking channels to metastasis : Targeting sodium transport in breast cancer

The development of therapies that can suppress invasion and prevent metastasis, 'anti-metastatic drugs', is an important area of unmet therapeutic need. The new results of a recent open-label, multicentre randomised trial published in J Clin Oncol showed a significant disease-free survival (DFS) benefit for breast cancer patients receiving presurgical, peritumoral injection of lidocaine, an amide local anaesthetic, which blocks voltage-gated sodium channels (VGSCs). VGSCs are expressed on electrically excitable cells, including neurons and cardiomyocytes, where they sustain rapid membrane depolarisation during action potential firing. As a result of this key biophysical function, VGSCs are important drug targets for excitability-related disorders, including epilepsy, neuropathic pain, affective disorders and cardiac arrhythmia. A growing body of preclinical evidence highlights VGSCs as key protagonists in regulating altered sodium influx in breast cancer cells, thus driving invasion and metastasis. Furthermore, prescription of certain VGSC-inhibiting medications has been associated with reduced cancer incidence and improved survival in several observational studies. Thus, VGSC-inhibiting drugs already in clinical use may be ideal candidates for repurposing as possible anti-metastatic therapies. While these results are promising, further work is required to establish whether other VGSC inhibitors may afford superior metastasis suppression. Finally, increasing preclinical evidence suggests that several other ion channels are also key drivers of cancer hallmarks; thus, there are undoubtedly further opportunities to harness ion transport inhibition that should also be explored

Dual roles of voltage-gated sodium channels in development and cancer

Voltage-gated Na(+) channels (VGSCs) are heteromeric protein complexes containing pore-forming α subunits together with non-pore-forming β subunits. There are nine α subunits, Na(v)1.1-Na(v)1.9, and four β subunits, β1-β4. The β subunits are multifunctional, modulating channel activity, cell surface expression, and are members of the immunoglobulin superfamily of cell adhesion molecules. VGSCs are classically responsible for action potential initiation and conduction in electrically excitable cells, including neurons and muscle cells. In addition, through the β1 subunit, VGSCs regulate neurite outgrowth and pathfinding in the developing central nervous system. Reciprocal signalling through Na(v)1.6 and β1 collectively regulates Na(+) current, electrical excitability and neurite outgrowth in cerebellar granule neurons. Thus, α and β subunits may have diverse interacting roles dependent on cell/tissue type. VGSCs are also expressed in non-excitable cells, including cells derived from a number of types of cancer. In cancer cells, VGSC α and β subunits regulate cellular morphology, migration, invasion and metastasis. VGSC expression associates with poor prognosis in several studies. It is hypothesised that VGSCs are up-regulated in metastatic tumours, favouring an invasive phenotype. Thus, VGSCs may have utility as prognostic markers, and/or as novel therapeutic targets for reducing/preventing metastatic disease burden. VGSCs appear to regulate a number of key cellular processes, both during normal postnatal development of the CNS and during cancer metastasis, by a combination of conducting (i.e. via Na(+) current) and non-conducting mechanisms

Nav1.5 regulates breast tumor growth and metastatic dissemination in vivo

Voltage-gated Na(+) channels (VGSCs) mediate action potential firing and regulate adhesion and migration in excitable cells. VGSCs are also expressed in cancer cells. In metastatic breast cancer (BCa) cells, the Na(v)1.5 α subunit potentiates migration and invasion. In addition, the VGSC-inhibiting antiepileptic drug phenytoin inhibits tumor growth and metastasis. However, the functional activity of Na(v)1.5 and its specific contribution to tumor progression in vivo has not been delineated. Here, we found that Na(v)1.5 is up-regulated at the protein level in BCa compared with matched normal breast tissue. Na(+) current, reversibly blocked by tetrodotoxin, was retained in cancer cells in tumor tissue slices, thus directly confirming functional VGSC activity in vivo. Stable down-regulation of Na(v)1.5 expression significantly reduced tumor growth, local invasion into surrounding tissue, and metastasis to liver, lungs and spleen in an orthotopic BCa model. Na(v)1.5 down-regulation had no effect on cell proliferation or angiogenesis within the in tumors, but increased apoptosis. In vitro, Na(v)1.5 down-regulation altered cell morphology and reduced CD44 expression, suggesting that VGSC activity may regulate cellular invasion via the CD44-src-cortactin signaling axis. We conclude that Na(v)1.5 is functionally active in cancer cells in breast tumors, enhancing growth and metastatic dissemination. These findings support the notion that compounds targeting Na(v)1.5 may be useful for reducing metastasis

Ionic modulation of immune checkpoint proteins

Despite extensive basic and clinical research on immune checkpoint regulatory pathways, little is known about the effects of the ionic tumour microenvironment on immune checkpoint expression and function. We screened effects of ion channel modulating compounds on indoleamine-2',3'-dioxygenase (IDO1) activity. Here, we describe a mechanistic link between Na+/K+ ATPase inhibition by cardiac glycosides and activity of IDO1, a well-characterized immune checkpoint. IDO1 catalyses the rate-limitig step of tryptophan catabolim and inhibits the immune response to the tumour by local depletion of tryptophan, an amino acid essential for anabolic functions in cancer and T cells

The sodium channel-blocking antiepileptic drug phenytoin inhibits breast tumour growth and metastasis.

Background Voltage-gated Na+ channels (VGSCs) are heteromeric protein complexes containing pore-forming ? subunits and smaller, non-pore-forming ? subunits. VGSCs are classically expressed in electrically excitable cells, e.g. neurons. VGSCs are also expressed in tumour cells, including breast cancer (BCa) cells, where they enhance cellular migration and invasion. However, despite extensive work defining in detail the molecular mechanisms underlying the expression of VGSCs and their pro-invasive role in cancer cells, there has been a notable lack of clinically relevant in vivo data exploring their value as potential therapeutic targets. Findings We have previously reported that the VGSC-blocking antiepileptic drug phenytoin inhibits the migration and invasion of metastatic MDA-MB-231 cells in vitro. The purpose of the present study was to establish whether VGSCs might be viable therapeutic targets by testing the effect of phenytoin on tumour growth and metastasis in vivo. We found that expression of Nav1.5, previously detected in MDA-MB-231 cells in vitro, was retained on cells in orthotopic xenografts. Treatment with phenytoin, at a dose equivalent to that used to treat epilepsy (60 mg/kg; daily), significantly reduced tumour growth, without affecting animal weight. Phenytoin also reduced cancer cell proliferation in vivo and invasion into surrounding mammary tissue. Finally, phenytoin significantly reduced metastasis to the liver, lungs and spleen. Conclusions This is the first study showing that phenytoin reduces breast tumour growth and metastasis in vivo. We propose that pharmacologically targeting VGSCs, by repurposing antiepileptic or antiarrhythmic drugs, should be further studied as a potentially novel anti-cancer therapy

Subcellular dynamics and functional activity of the cleaved intracellular domain of the Na+ channel β1 subunit

The voltage-gated Na(+) channel β1 subunit, encoded by SCN1B, regulates cell surface expression and gating of α subunits and participates in cell adhesion. β1 is cleaved by α/β and γ-secretases, releasing an extracellular domain and intracellular domain (ICD), respectively. Abnormal SCN1B expression/function is linked to pathologies including epilepsy, cardiac arrhythmia, and cancer. In this study, we sought to determine the effect of secretase cleavage on β1 function in breast cancer cells. Using a series of GFP-tagged β1 constructs, we show that β1-GFP is mainly retained intracellularly, particularly in the endoplasmic reticulum and endolysosomal pathway, and accumulates in the nucleus. Reduction in endosomal β1-GFP levels occurred following γ-secretase inhibition, implicating endosomes and/or the preceding plasma membrane as important sites for secretase processing. Using live-cell imaging, we also report β1ICD-GFP accumulation in the nucleus. Furthermore, β1-GFP and β1ICD-GFP both increased Na(+) current, whereas β1STOP-GFP, which lacks the ICD, did not, thus highlighting that the β1-ICD is necessary and sufficient to increase Na(+) current measured at the plasma membrane. Importantly, although the endogenous Na(+) current expressed in MDA-MB-231 cells is tetrodotoxin (TTX)-resistant (carried by Na(v)1.5), the Na(+) current increased by β1-GFP or β1ICD-GFP was TTX-sensitive. Finally, we found β1-GFP increased mRNA levels of the TTX-sensitive α subunits SCN1A/Na(v)1.1 and SCN9A/Na(v)1.7. Taken together, this work suggests that the β1-ICD is a critical regulator of α subunit function in cancer cells. Our data further highlight that γ-secretase may play a key role in regulating β1 function in breast cancer