Functional role of T-type calcium channels in tumour growth and progression: prospective in cancer therapy

Abstract

T-type Ca²⁺ channels represent a specific channel family overexpressed in different types of tumours. Their involvement in controlling the proliferation, angiogenesis and invasion of tumour cells, has been partially clarified. The article by Zhang et al. in this issue of BJP provides the first evidence of anti-tumoural effects of endostatin (ES) in U87 glioma cells. He demonstrated that ES or mibefradil (a L/T-type calcium channel blocker), reduces the proliferation and migration of U87 glioma cells in a T-type Ca²⁺ channel-dependent manner. However, the difference in the blocking effect of mibefradil on T-type calcium channel expression as compared with its ability to inhibit proliferation and migration, supports the idea of a broader T/L-type-independent effect of the mibefradil blocker. Overall, these findings provide new insights for the future development of a novel class of anti-T-type calcium channel blockers in the therapy of glioblastoma.

LINKED ARTICLE

This article is a commentary on Zhang et al., pp. 1247–1260 of this issue. To view this paper visit http://dx.doi.org/10.1111/j.1476-5381.2012.01852.x

Calcium plays a key role in intracellular signalling and controls many different cell processes such as proliferation, differentiation, growth, cell death and apoptosis. Thus, alterations in calcium signalling can cause defects in cell growth and invasion and are associated with certain types of cancer. A number of research groups have suggested a potential role for voltage-activated Ca²⁺ channels, in particular T-type, in the regulation of tumour growth and progression.

Molecular biology studies have expanded the repertoire of these Ca²⁺ channels revealing three main subfamilies of α₁ subunit called Ca_v1, Ca_v2 and Ca_v3. The third subfamily contains three members that are called T-types: Ca_v3.1 (α_1G), Ca_v3.2 (α_1H) and Ca_v3.3 (α_1I). The unique low voltage–dependent activation/inactivation and slow deactivation of T-type Ca²⁺ channels suggest that they play a direct role in regulating [Ca²⁺]_i, especially in non-excitable tissues, including some cancerous cells (Lory et al., 2006; Panner and Wurster, 2006). At low voltages, T-type Ca²⁺ channels produce the so-called ‘window current’ at appropriate values of membrane potential, that results in a sustained inward calcium current carried by the portion of channels that are not completely inactivated. This regulation of Ca²⁺ homeostasis allows T-type Ca²⁺ channels to control cell proliferation and differentiation. Therefore, loss of T-type Ca²⁺ channel control may lead to aberrant cell growth and tumour progression.

A role for these channels in tumour cell proliferation has been reported in breast, brain, colorectal, gastric, hepatic and prostate tumours, leukaemic cells, retinoblastoma cells and phaeochromocytoma cells (Lory et al., 2006; Panner and Wurster, 2006). Human breast adenocarcinoma MCF-7 cell line exhibits α_1G and α_1H T-type Ca²⁺ channel mRNA and T-current transiently (Taylor et al., 2008). Human prostate cancer epithelial cells (LNCap) have also been shown to display increased T-type Ca²⁺ channel (α_1H) current and mRNA. Finally, overexpression of T-type α₁-subunit genes, either α_1H alone or together with α_1G and α_1I, has been demonstrated in human oesophageal tumours, as compared with the normal counterpart that shows a lower α₁ expression. In accordance with the T-type Ca²⁺ channel expression, their blockade diminished the proliferation of oesophageal cancer cells through p53-dependent p21^CIP1 up-regulation (Lu et al., 2008).

The mechanisms underlying the in vivo anti-tumoural action of T-type Ca²⁺ channel antagonists are less well understood. In athymic nude mice implanted with MCF-7 breast cancer cells, injection of mibefradil (0.5 mg·100 µL⁻¹, twice a week) at tumour sites resulted in marked tumour degeneration and necrosis (Taylor et al., 2008). Furthermore, local intra-cerebral micro-infusion of endostatin (ES) improved treatment efficiency and survival in a xenograft orthotopic human glioblastoma multiforme (GBM) model.

The expression of T-type Ca²⁺ channels can vary depending on tumour stage. For instance, differentiation of epithelial prostate cancer cells into more aggressive neuroendocrine cells that express functional T-type Ca²⁺ triggers the release of growth factors stimulating the proliferation of neighbouring prostate cancer cells. Similarly, T-type Ca²⁺ channels mediate the release of growth factors in pheochromocytomas; in retinoblastoma cells the decreased proliferation was accompanied by a reduced expression of T-type channels mRNA and decreased T-currents.

In this issue of British Journal of Pharmacology, Zhang et al. (2012), by using ES, a proteolytic fragment of collagen XVIII, explored the functional role of T-type Ca²⁺ channels in U87 glioma cells and in HEK-293 and CHO heterologous cell expression systems.

Conflicting observations are available on the expression of α1 subunits of T-type Ca²⁺ channels Ca_v3.1 (α_1G), Ca_v3.2 (α_1H) and Ca_v3.3 (α_1I) in U87 glioma cells. Recently, Panner et al. (2005) have demonstrated a decrease in the expression of the α_1G and α_1H subunit associated with decreased proliferation. By contrast, using real-time PCR and electrophysiological methods, Lu et al., (2005) failed to detect T-type channel mRNA and T-currents. In the present paper, Zhang et al. (2012) confirmed Panner's data showing that U87 cells express all of the three α1 subunits of T-type Ca²⁺channels. By using transfected HEK293 or CHO cells, they found that only Ca_V3.1 and Ca_V3.2, but not Ca_V3.3 or Ca_V1.2 (L-type), channel currents were significantly inhibited by ES.

Zhang et al. (2012) showed that treatment with ES inhibited T-currents in U87 cells, whereas L-currents were not affected. Moreover, pretreatment of U87 glioma cells with the T-type Ca²⁺ channel blocker, mibefradil, or with the L-type blockers nifedipine or nimodipine, inhibited cell proliferation and migration. This inhibitory effect was associated with a hyperpolarizing shift in the voltage-dependence of inactivation. The authors also observed that inhibition of T-currents induced by ES was highly dependent on the inactivation state of the channel. ES-mediated hyperpolarization induced a shift of the steady-state inactivation curve (approximately −15 mV), whereas the activation of curve was not affected. Although it is unclear whether the hyperpolarizing shift of the steady-state inactivation curve would produce a significant modification in T-type ‘window current’, it is conceivable that it could depend on an increased number of channels remaining in the inactivated state after activation. Further studies are needed to address how making fewer T-type Ca²⁺ channels available for opening mechanistically, contributes to the inhibitory effect of ES on glioma cellular responses.

Mibefradil (Posicor) was the first mixed T/L channel blocker to be marketed for its ability to block T-currents. Unfortunately, it is metabolized by cytochromes P450 3A4 and 2D6, leading to drug–drug interactions. Mibefradil can exert non-specific anti-proliferative effects because it accumulates and is hydrolyzed inside the cells and the resulting metabolites can block T-type Ca²⁺ channels. Thus, to avoid these problems, in the present paper, data obtained with mibefredil were also evaluated by using NNC 55–0396, a mibefradil nonhydrolyzable analogue without L-type Ca²⁺ channel efficacy (Panner and Wurster, 2006).

ES or mibefradil inhibits fibronectin-induced migration of U87 glioma cells. Similarly, results were induced in fibrosarcoma cells, where mibefradil suppressed T-type Ca²⁺-mediated Ca²⁺ spikes, waves, cell motility and invasive properties (Huang et al., 2004). The magnitude of the inhibitory effect of mibefradil on the motility of U87 cells was higher than that induced by ES, suggesting that this blocker also shows broader T/L-type-independent effects.

The findings by Zhang et al. (2012) also demonstrate that the ES-mediated effects on the induced proliferation and migration of U87 glioma cells is not mediated by G-proteins or tyrosine-kinase signalling pathways, raise a crucial question as to the nature of the signalling pathways involved in ES inhibition of Ca²⁺ T-type channels and how they regulate Ca_V3.1/2 activity. T-channel activity can be modulated by hormones and neurotransmitters acting through signalling intermediates such as protein kinases A and C, calmodulin-dependent protein kinase II, tyrosine kinase, G-proteins and lipid derivatives such as arachidonic acid. Recent reports suggest a role for PKC and ERK pathways in T-type channel activation. Phorbol-12-myristate-13-acetate potently enhances, although to different extents, the current amplitude of Ca_v3.1, Ca_v3.2 and Ca_v3.3 channels, via PKC activation (Park et al., 2006). At present, it is not completely understood whether the PKC-mediated stimulation might depend on direct phosphorylation of Ca_v3 channels or represent an indirect consequence of phosphorylation of associated targeting, anchoring or signalling protein(s). Ciliary neurotrophic factor increases the expression and currents of T-type Ca²⁺ channels by triggering JAK/STAT and ERK signalling pathways (Trimarchi et al., 2009). Further studies are necessary to elucidate this issue.

An expansion of the list of ion channels implicated in cancer development is expected. It is difficult to ascribe tumour development to the malfunction of a single ion channel. For instance, regulation of K⁺ or TRP channels can affect the membrane potential, which in turn regulates the window currents mediated by T-type Ca²⁺ channels. However, as in many cases there are already known pharmacological modulators (blockers and activators) of ion channels, the identification of a single defective ion channel in a particular cancer could provide a ready-to-go therapeutic approach (Gray and Macdonald, 2006).

In conclusion, the study of Zhang and colleagues highlighted the therapeutic potential of ES via targeting T-type calcium channels for the treatment of human GBM. Recently, a sequential administration of a T-channel blocker to synchronize cells at the G₁/S checkpoint of the cell cycle before the administration of chemotherapy (Interlaced Therapy™), has been reported by Tau Therapeutics (LLC Charlottesville, USA). Thus, in GBM, the administration of mibefradil before temozolomide has been found to overcome the resistance of GBM patients to temozolomide and significantly increases their life-span. However, because T-type Ca²⁺ channels are normally expressed in the brain, heart and endocrine tissues of the human body, the potential side effects of the specific channel blockers must to be considered for therapeutic applications.

Glossary

ES

endostatin

GBM

glioblastoma multiforme