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Breast Research and Treatment (2019) 173:11–21 https://doi.org/10.1007/s10549-018-4970-0

REVIEW

T-Type voltage gated channels: a target in ?

Anamika Bhargava1 · Sumit Saha1

Received: 24 July 2018 / Accepted: 15 September 2018 / Published online: 21 September 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract Purpose The purpose of this review article is to discuss the potential of T-type voltage gated calcium channels (VGCCs) as targets in breast cancer. Summary Breast cancer, attributable to the different molecular subtypes, has a crucial need for therapeutic strategies to counter the . VGCCs play an important role in regulating cytosolic free calcium levels which regulate cellular processes like tumorigenesis and cancer progression. In the last decade, T-type VGCCs have been investigated in breast cancer proliferation. blockers, in general, have shown an anti-proliferative and cytotoxic effects. T-type VGCC antagonists have shown growth inhibition owing to the inhibition of Ca­ V3.2 isoform. Ca­ V3.1 isoform has been indicated as a tumour-suppressor candidate and is reported to support anti-proliferative and apoptotic activity in breast cancer. The distribution of T-type VGCC isoforms in different breast cancer molecular subtypes is diverse and needs to be further investigated. The role of T-type VGCCs in breast cancer migration, and more importantly in epithelial to mesen- chymal transition (EMT) is yet to be elucidated. In addition, interlaced , using a combination of and T-type VGCC blockers, presents a promising therapeutic approach for breast cancer but more validation and clinical trials are needed. Also, for investigating the potential of T-type VGCC blocker therapy, there is a need for isoform-specific /antagonists to define and discover roles of T-type VGCC specific isoforms. Conclusion Our article provides a review of the role of T-type VGCCs in breast cancer and also discusses future of the research in this area so that it can be ascertained whether there is any potential of T-type VGCCs as drug targets in breast cancer.

Keywords Breast cancer · Voltage gated calcium channel · T-type VGCCs · Calcium influx

Introduction channels are mostly -gated ion channels that open in response to a ligand and mediate cation flux [3–5]. Two main 2+ 2 + Changes in the cytosolic free Ca­ concentration [Ca­ i ] rep- classes of calcium selective channels are known, namely resent an important signalling mechanism, which integrates store-operated calcium channels and voltage gated calcium other signal-transduction cascades and controls a variety channels (VGCCs) [6, 7]. Store-operated calcium channels of cellular processes [1]. Although there are many types of like Orai mediate calcium influx in response to the deple- calcium permeable ion channels like IP3 (Inositol Triphos- tion of intracellular calcium stores. VGCCs include L-type, phate) and RYR (Ryanodine Receptor) isoforms N-type, T-type, R-type and P/Q-type. These ion channels expressed in the intracellular organelles [2], plasma mem- consist of different subunits, although it is the α1 subu- brane has its own share of calcium permeable ion channels nit of 190–250 kDa that forms the calcium selective pore which we can classify into calcium selective and calcium [8]. The α1 subunit also determines the type of VGCC [9]. non-selective channels (Fig. 1). Calcium non-selective ion The that encode the α1 subunit include CACNA1S ­(CaV1.1), CACNA1C ­(CaV1.2), CACNA1D ­(CaV1.3) and CACNA1F (Ca­ V1.4) for L-type calcium channels; CACNA1A * Anamika Bhargava ­(CaV2.1), CACNA1B ­(CaV2.2) and CACNA1E ­(CaV2.3) for [email protected] P-/Q-type, N-type and R-type calcium channels respectively; CACNA1G CACNA1H CAC- 1 Biology Lab, Department of Biotechnology, and (Ca­ V3.1), (Ca­ V3.2) and Indian Institute of Technology Hyderabad (IITH), Kandi, NA1I ­(CaV3.3) for T-type calcium channel isoforms [10]. Telangana 502285, India

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Fig. 1 Calcium influx chan- nels on the plasma membrane. Calcium selective channels comprise of Orai and ­CaV family. Calcium non-selective channels comprise of P2X, 5-HT3 receptor, TRP channels, NMDA, AMPA and kainate receptors. NMDA, AMPA and kainate receptors are predomi- nantly found in

Although VGCCs have important roles in the excitability of ­CaV1 (L-type) and ­CaV2 (P/Q, N and R-type) channels but excitable cells, such as those in the central they are only ∼ 25% identical in the amino acid sequence [9]. and cardiovascular tissue, they are also expressed in non- Currents through T-type α1 subunit are similar to currents excitable types where they perform a variety of cellular through native T-type VGCCs, suggesting that the β, α2δ and functions. In the last decade, they have also been implicated γ subunits may have alternative roles [17]. The alternative in cancer cells where they have been found to both promote roles of auxiliary subunits have recently been reviewed by or inhibit proliferation depending on the type of cancer and Hofmann et al. [18] which also includes essential physi- the type of ion channel isoform expressed [11]. ological processes. VGCCs have been reported as promising target can- T-type VGCCs perform various functions such as atrial didates in cancer therapy [12]. Among VGCCs, T-type pacemaking, low-threshold exocytosis, neuronal firing, G1/S VGCCs are being suggested as a promising target as they checkpoint regulation, cell proliferation, atrioventricular have been reported to be upregulated in many cancer types. regulation etc. [20–25]. T-type VGCCs are therefore impli- For detailed information on the role of T-type VGCCs in cated in several pathologies including autism, , cancer growth and progression, please see reviews [13–15]. and cancer. Because of the extreme importance of Our review is focussed on the role of T-type VGCCs in T-type VGCCs and their abundant expression in the nervous breast cancer and future opportunities therein to validate system, a major that involves the T-type VGCC as a them as potential drug targets in breast cancer. In this review drug target is absence epilepsy. Experiments on the GAERS we have covered (1) The data indicating the role of T-type (Genetic Absence Epilepsy Rat of Strasbourg) mice showed VGCCs in breast cancer and (2) The scope of research in this that there was a marked increase in the activity of Ca­ V3.2 area to elucidate the direct/ indirect role of T-type VGCCs in isoform of T-type VGCCs which triggers the burst firing breast cancer regulation. We have reviewed the involvement of thalamic neurons in epilepsy seizure [26]. The Ca­ V3.2 of T-type VGCCs in breast cancer among the vast number of isoform of T-type VGCCs is also reported to be upregu- due to the following reasons: (1) Breast cancer has lated in visceral and [27] in animal models, a huge mortality rate, (2) Due to varied molecular subtypes, where ­CaV3.2 contributes to the perception of pain and is prognosis and therapy is a challenge in breast cancer and (3) regarded as a viable drug target. The evidence of involve- Reports regarding the role of T-type VGCCs in breast cancer ment of T-type VGCCs in the manifestation of such are insufficient, inconclusive and non-specific to come up as cardiovascular diseases, renal injury and different human with definitive . cancers such as breast, colon, , leukaemia, ovarian and is supported by their over/re-expression in T‑type voltage gated calcium channels cancerous and diseased tissue as compared to the normal tissue [24, 28–34]. Various studies have also indicated their Originally, Hagiwara and colleagues described two inward supportive role in cancer proliferation, survival and cell- currents, labelled I and II, in starfish eggs which were both cycle regulation in cancer progression. However, a clear calcium influx currents [16]. Today, “I” and “II” are widely casual or consequential relationship of T-type VGCCs with recognized as high-voltage activated (HVA) and low-volt- the various above said diseases is not well established. age activated (LVA) channels respectively. T-type VGCCs belong to the family of LVA channels [17]. Three isoforms Breast of the T-type ­(CaV3) VGCC α1 subunit have been discovered by cDNA cloning and sequencing (Table 1). These calcium Breast cancer is emerging as the most common female channel subunits have the same molecular organization as cancer worldwide which, according to the World Cancer

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Table 1 T-Type voltage gated calcium channel properties. adapted from [17, 19]

T-Type VGCCs CaV3.1 CaV3.2 CaV3.3

Other names α13.1, α1G α13.2, α1H α13.3, α1I Molecular information Human: 2377aa, O43497, chr. Human: 2353aa, O95180, chr. Human: 2251aa, AAM67414, chr. 17q22, CACNA1G 16p13.3, CACNA1H 22q13.1, CACNA1I Ion selectivity Sr2+ > Ba2+ = Ca2+ Ba2+ = Ca2+ Ba2+ = Ca2+ Blockers Selective: NNC 55 0396 Selective: NNC 55 0396 Selective: NNC 55 0396 Non-selective: Nickel, , Non-selective: Nickel, Mibefradil, Non-selective: Nickel, Mibefradil Nimopidine and Nimopidine and anaesthetics Note CaV3.2 is more sensitive than ­CaV3.1 to block by nickel ­(IC50 = 12 µl) Functional assays Patch-clamp, calcium imaging Patch-clamp, calcium imaging Patch-clamp, calcium imaging Channel Distribution (in general) , Ovary, Placenta, Kidney, Liver, , Brain Brain, Heart Physiological function Thalamic oscillations, muscle Thalamic oscillations, muscle Thalamic oscillations, contraction contraction

T-type VGCCs can be divided into three subclasses: Ca­ V3.1, ­CaV3.2 and ­CaV3.3

Currently, the only major means of restricting mortal- Table 2 Breast cancer subtypes with their specific endocrine receptor ity in breast cancer is early screening [43]. This is because, markers. adapted from [39] although the success of surgical breast conservation in later Molecular subtype Molecular markers stages is based on tumour downsizing using radiotherapy and chemotherapy, there are still numerous limitations of 1. Luminal A ER+/PR+/HER2− − + − breast cancer therapy mainly due to molecular evolution and ER /PR /HER2 ER+/PR−/HER2− heterogeneity [44–52]. The rise of sub-clones during metas- 2. Luminal B ER+/PR+/HER2+/− tasis pose as one of the major cause of therapy-resistance ER+/PR−/HER2+/− and therefore in-depth characterization of the clones with + − +/− ER /PR /HER2 repeated biopsies are being pursued as ongoing research − − − 3. TNBC ER /PR /HER2 to tackle such issues. Besides the problem of molecular − − + 4. HER2 enriched ER /PR /HER2 heterogeneity, another major problem is the occurrence of Breast cancer can be categorized into molecular subtypes of Lumi- mutated ‘un-druggable’ targets. Keeping in mind the effects nal A, Luminal B, Triple Negative Breast Cancer (TNBC) and HER2 of tumour microenvironment [53] and changes in the intrin- enriched sic subtypes during metastasis [54], rigorous clinical studies are needed for a comprehensive idea of the transcriptome, proteome and epigenome interactions which may provide Research Fund International, represents a quarter of all opportunities for personalized cancer therapy and may pave cancers [35, 36]. Today, breast cancer is considered as a way for the discovery of novel drug targets [55]. group of diseases that can be distinguished by different molecular subtypes, clinical behaviours, risk factors and treatment responses [37]. These distinct molecular subtypes Role of T‑type voltage gated calcium of breast cancer have been identified using gene expression channels in breast cancer profiles [38]. A classification of distinct molecular subtypes in breast cancer has been accomplished using receptors as It has been lately well-recognized that the alterations in cal- biological markers which includes the presence or absence cium signalling are crucial for the sustenance of prolifera- of oestrogen receptor (ER+/ER−), progesterone receptor tion and migration in tumour progression. Amongst calcium (PR+/PR−) and human epidermal growth factor receptor influx pathways like TRP channels and store-operated cal- 2 (HER2+/HER2−) (Table 2). Detailed information on the cium influx, VGCCs have received less attention in breast subtype identification and classification, clinicopathological cancer previously [56]. This could be due to the fact that features, gene expression profiling and clinical management cancer cells are non-excitable cells and the presence of of the breast cancer heterogeneity has been reviewed before VGCCs in cancer cells was not much regarded. However, [39–42]. today it is widely accepted that VGCCs are functionally

1 3 14 Breast Cancer Research and Treatment (2019) 173:11–21 expressed in non-excitable cells like immune cells, kid- T-type VGCCs are involved in cell survival as mibefradil ney cells, prostate cells, breast cells, pulmonary vascular is a relatively selective blocker of T-type VGCCs. Mibe- and epithelial [29, 57–60]. Early fradil- induced cell death was primarily due to necrosis as investigations indicated that use of calcium channel blockers compared to -induced cell death, which was due can increase the risk or occurrence of cancer however these to [72]. No isoform-specific results were reported findings are contradicted by the findings in the last decade, in the study by Bertolesi et al. In agreement with the study indicating a therapeutic role of calcium channel blockers. of Bertolesi et al., Gray et al. 2004 gave evidence of signifi- These findings have been discussed below. cant expression of ­CaV3.2 in breast lines (MDA- MB-231,361 and 435) and suggested the pro-proliferative Do calcium channel blockers induce the risk role of ­CaV3.2 by transfecting HEK293 cells with ­CaV3.2 of breast cancer? and using T-type antagonists mibefradil and TH-1177 [73]. In 2006, Asaga et al. identified and confirmed a significant In 1996, Pahor et al. reported a higher cancer incidence in expression of CACNA1H gene (coding for ­CaV3.2 T-type patients treated with calcium channel antagonists as com- VGCC) for the first time in MCF-7, MDA-MB-435 and pared with those not taking calcium channel antagonists T-47D cell lines using Restriction Landmark Genome Scan- [61]. A year later in 1997, Fitzpatrick AL et al. reported a ning (RLGS) [74]. RLGS is a genome analysis technique higher incidence of breast carcinoma upon the use of cal- for simultaneous and rapid visualizations of around hundred cium channel blockers and anti-hypertensive drugs in post- restriction sites using restriction combinations spe- menopausal women using time-dependent Cox proportional cific to DNA modifications. RLGS can be used to visualize hazards regression models [62]. Their study also reported methylation, amplification and deletion differences in the that sustained-release drugs led to comparatively less cancer genome of an organism [75]. CACNA1H gene was previ- incidence as compared to immediate-release drugs. How- ously found to be hypo-methylated in T-cell leukaemia com- ever, the authors could not provide any conclusive correla- pared to peripheral mononuclear cells, thus suggest- tion due to a limited number of cases in their study and ing its role as a possible regulated by epigenetic suggested an obvious need for further investigation to con- methylation [76]. This opened up a research front to reveal firm or refute their observations. Further, studies of larger detailed roles of Ca­ V3.2 T-type VGCCs in breast cancer. cohort and some prospective trials have failed to conclude a Soon afterwards in 2008, Taylor et al. reported significant significant correlation between the use of calcium channel expression of both isoforms, ­CaV3.1 and ­CaV3.2 T-type antagonists and increased breast cancer incidence [63–69]. VGCCs in ER + and ER- breast cancer cell lines under non- Recently in 2016, Xuan Ye et al. published a meta-analy- confluent conditions and demonstrated that T-type VGCC sis on whether calcium channel blockers may increase the specific antagonists like NNC-55-036 andCa ­ V3.2 knock- risk of breast cancer. They concluded that long-term use of down have an anti-proliferative role rather than a cytotoxic calcium channel blockers may lead to a potential increased role in MCF-7 (ER+) and MDA-MB-231 (ER-) breast can- breast cancer risk [70]. In contrast, LE Wilson et al. found no cer cell lines in a dose-dependent manner [33]. Later, T-type significant increase in the breast cancer risk among women VGCC antagonists were reported to result in the blocking who had been using calcium channel blockers for 10 years of G1/S checkpoint of the , thus supporting their or more [71], thus aiding to the big question ‘Do calcium anti-proliferative role in breast cancer [77–79]. channel blockers increase breast cancer incidence?’. A major limitation in all these cohort studies is the lack of informa- Isoform‑specific role of T‑type voltage gated calcium tion on the type of calcium channel blockers used to predict channel in breast cancer cancer incidence. This limitation, therefore, makes the gen- eralized statistics too dilute to elucidate whether a long-term In the previous section, we have described evidence that dose of T-type VGCC blockers or any other selective chan- suggests a supportive role of T-type VGCCs in breast can- nel antagonist will affect breast cancer risk. cer proliferation. But, due to distinct kinetics and epigenetic regulation of different isoforms of T-type VGCCs, it is Do T‑type voltage gated channel blockers have imperative to define isoform-specific role of T-type VGCCs an anti‑proliferative role in breast cancer? in breast cancer. In literature, there have been divergent observations regarding the isoform-specific role of T-type Bertolesi GE et al. in 2002 reported the growth-inhibitory VGCCs in breast cancer. cytotoxic role of calcium channel antagonists pimozide and As early as in year 1999, Toyota et al. reported a methyl- mibefradil using MCF-7 cell line (ER+ epithelial cancer ation-induced suppression of ­CaV3.1 in breast cancers, thus cell line derived from breast adenocarcinoma). The study giving evidence of a candidate tumour-suppressor property, showed that mibefradil induced cell death, indicating that and observed that breast cancers have higher methylation

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in ­CaV3.1 gene as compared to normal mammary epithe- essential signalling pathways in breast cancer or mediate lial cells [34]. In 2002, Ohkubo et al. reported that ­CaV3.1 anti-cancer roles. The roles of T-type VGCCs known so far silencing in MCF-7 cells showed enhanced proliferation are summarized in Box 1. compared to ­CaV3.2 silencing which showed no significant effect [80]. The study also indicated that calcium influx Box 1: role of T‑type VGCCs in breast through ­CaV3.1 channel supported apoptosis and provided cancer another evidence of ­CaV3.1 being a candidate tumour- suppressor gene but suggested further investigation for the • Ca 3.1 oncogenic role of ­Ca 3.2 in breast cancer. More recently, V V Anti-proliferative; supports apoptosis. Ranzato et al. 2014 investigated the role of T-type VGCCs Possible tumour-suppressor gene candidate. in regulating calcium influx to mediate epigallocatechin • Ca 3.2 in MCF-7 cells [81]. Ranzato and colleagues V Pro-Proliferative. showed an increased Ca­ 3.2 activity for mediating epigal- V Possible oncogene candidate. locatechin cytotoxicity rather than ­Ca 3.1, contradicting V • Ca 3.3 earlier reports, where blocking of Ca­ 3.2 showed an anti- V V Pro-Proliferative. proliferative effect in breast cancer [33]. More importantly, Overexpressed in invasive breast carcinoma. this study indicates that although Ca­ V3.2 inhibition provides an anti-cancer therapy, Ca­ V3.2 calcium influx can also aid chemotherapeutic drugs against breast cancer. The third iso- The scope of T‑type voltage gated calcium channel research in breast cancer form of T-type VGCCs, ­CaV3.3, has been shown to modulate pressure-stimulated proliferation in MCF-7 cell line [82]. In this study, a higher concentration of nickel chloride was Despite numerous studies showing a role of T-type VGCCs required to inhibit external pressure exposed-proliferation in cell-cycle arrest [31, 84], induction of necrosis [72], apop- in the colon, prostate and breast cancer cell lines, hinting totic cell death [85, 86], reduced mTORC2/Akt signalling at ­CaV3.3 channels to be responsible for the proliferation in [87] and stimulation of p38 MAP activity [85] in MCF-7 cells [82]. Confocal imaging showed a major role different cancer types, there are a lot of gaps in the case of of ­CaV3.3 in mediating calcium influx required for the acti- breast cancer owing to disparate reports of T-type VGCC vation of PKCβ dependent cell proliferation pathway [82]. activity and expression in different breast cancer subtypes. A summary of all the studies regarding the role of T-type VGCCs in breast cancer has been provided in Table 3. EMT and metastasis Besides the diverse role of T-type VGCC isoforms in breast cancer, the distribution of these isoforms in differ- An aspect which is yet unexplored is the role of T-type ent molecular subtypes of breast cancer is not clear and VGCCs in epithelial to mesenchymal transition (EMT) by needs more investigations. Pera et al. in 2016 suggested regulating basal markers. A characteristic feature of the that Ca­ V3.2 may be a prognostic marker for luminal and tumour cells undergoing EMT is their capacity to evade HER2 breast cancer types as compared to the basal type. body’s immune responses directed against tumour cells, RT-qPCR results showed increased ­CaV3.2 levels in luminal- resist apoptosis and anti-cancer drugs [88, 89]. They also like breast cancer lines like MCF-7 and T47D than basal-like exhibit -like properties which allow the tumour breast cancer lines like MDA-MB-231 and MDA-MB-468 cells to act as a reservoir that replenishes and expands the [83]. Recently in 2017, Phan et al. in their statistical studies tumour cell population [90]. Cancer stem cells are impli- reported that CACNA1G, CACNA1H and CACNA1I have a cated in EMT, therapy-resistance and tumour relapse for aid- lower expression in breast cancer compared to other cancer ing cancer sustenance. For detailed reviews on cancer stem types. The analysis showed that CACNA1G is expressed cells, please refer to [91, 92]. EMT represents an important significantly more in invasive lobular breast cancer as com- stage in cancer prognosis as the tumour cells undergoing pared to mucinous breast cancer [12]. Of note, CACNA1I EMT also show therapeutic resistance. Given these attrib- was found to be in the top 4–7% of genes that are upregu- utes, EMT is a key hallmark of , and targeting lated in ductal and invasive breast carci- EMT pathways may be of prime importance in cancer treat- noma stroma [12]. ment. For a detailed review of EMT in cancer please refer to As described above, the reports of distribution and role [93, 94]. Therefore, a big question is if T-type VGCCs are of T-type VGCCs in breast cancer are divergent and con- involved in EMT. The answer is we don’t know yet. In 2000, tradictory. Therefore, there is a tremendous scope of fur- Bringmann and colleagues reported T-type VGCCs in radial ther research focussing on the other roles of T-type VGCCs glial stem cells and found that the T-type VGCC activity in breast cancer and more importantly how they regulate decreases during development [95]. Rodriguez-Gomez

1 3 16 Breast Cancer Research and Treatment (2019) 173:11–21 - 3.2 V Ca 3.1 knock 3.2 in support - V V Ca Ca 3.2 demethylation 3.2 demethylation V ­ Ca 3.3 channel was discovered was 3.3 channel V 3.1 was aberrantly methylated aberrantly methylated 3.1 was in breast 3.2 overexpressed 3.3 supported proliferation. V V V in breast cancer was reported cancer was in breast observed upon ­ observed and suppressed in most of the in most and suppressed cancer cell lines, thus indicating role a tumour suppressing cancer in HEK293 overexpression by cells of activation Pressure-mediated ­ Ca using RLGS in breast cancer in breast using RLGS cell lines and indicated possible candidature oncogene channel supported- channel epigallocate cancer in breast cytotoxicity chin ing proliferation was reported in was ing proliferation cancer breast down proliferation of cancer cells proliferation eration in breast cancer in breast eration A direct role of ­ role A direct Significance of interlaced therapy Significance of interlaced therapy Enhanced proliferation was was Enhanced proliferation Ca Confirmed Calcium influx ­ through Ca Pro-proliferative role investigated investigated role Pro-proliferative Ca T-type VGCC blockers inhibited blockers VGCC T-type - antagonists inhibited prolif T-type Conclusion 3.2: Pro-proliferative significant effect on 3.1: No V V proliferation Ca Pro-proliferative Pro-proliferative NA Ca Anti-proliferative Anti-proliferative NA Pro-proliferative Pro-proliferative Pro-proliferative - calcium influx (anti-pro T-type or Pro-proliferative) liferative - - 2 NiCl b laced therapy using NNC-55- laced therapy 0396 and ProTx-I epigallocatechin 55-036 and ­ specific ers derived from the chemical the from chemical ers derived structure of L-type blockers Isoform-specific siRNA and siRNA Isoform-specific inter siRNA, Isoform-specific and siRNA Isoform-specific NNC- siRNA, Isoform-specific NA and TH-1177 Mibefradil NA and isoform- NNC-55-0396 siRNA Various T-type VGCC block VGCC T-type Various Mibefradil and Pimozide Mibefradil - antagonist/chemothera T-type peutic drug used in the study, if any MDA-MB-231, MDA-MB-435, MDA-MB-435, MDA-MB-231, SKBr3, BT20, MDA-MB-474, CAMA1 MB-231, 361, 435 and T-47D MDA-MB-435 MDA-MB-231 MB-361 Breast cancer cell lines: MCF-7, Breast cancer cell line: MCF-7 Breast cancer cell lines: MDA- Breast cancer cell lines: MCF-7, Breast cancer cell lines: MCF-7, Breast cancer cell line: MCF-7 Breast cancer cell line: MCF-7 Breast cancer cell line: MCF-7 Breast Breast cancer cell line: MDA- Breast Breast cancer cell line: MCF-7 Breast Experimental system 3.1 3.1 3.1 3.2 3.2 and 3.1 3.2 3.2 3.3 a V V V V V V V V V Ca Ca ND ND Ca Ca Ca Ca T-type VGCC VGCC T-type if isoform, defined Ca Ca Ca not applicable not b Summary of the reported roles of T-type VGCCs in breast cancer in breast Summary VGCCs of the reported of T-type roles not not defined, Ranzato et al. 2014 [ 81 ] et al. Ranzato Bertolesi 2002 [ 72 ] GE et al. 2004 [ 77 ] McCalmont WF et al. Taylor et al. 2008 [ 33 ] et al. Taylor Basson et al. 2014 [ 82 ] Basson et al. Pottle J et al. 2013 [ 78 ] J et al. Pottle Gray et al. 2004 [ 73 ] et al. Gray 3 Table a Article Asaga et al. 2006 [ 74 ] et al. Asaga Ohkubo 2002 [ 80 ] et al. Toyota et al. 1999 [ 34 ] et al. Toyota

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JA et al. 2012 also reported mouse embryonic stem cells using interlaced therapy for breast cancer, there is a major to express T-type VGCCs where the expression of T-type scope of research to come up with better adjuvant and neo- VGCCs supported cell proliferation [96]. A recent report adjuvant chemotherapeutic approaches alongside T-type by Jacquemet et al. showed a role of L-type VGCCs in reg- VGCC antagonists. ulating filopodia stability in breast cancer and pancreatic Early reports of non-specific calcium channel block- cancer and thus suggested its possible role in cell invasion ers showing better prognosis in breast cancer indicated an and metastasis. In their experiments, they found that T-type associated role of T-type VGCCs [62, 65, 72], but, only VGCC has no role in regulating filopodia number [97]. Yet, after T-type VGCC isoform-specific siRNA-knockdown, there is no demonstrated direct association between VGCCs the anti-cancer or oncogenic role of T-type VGCC could and cell migration in breast cancers. In 2012, Hirooka et al. be established. The search for isoform-specific agonists and found that T-type VGCC was significantly overexpressed antagonists for T-type VGCCs is also one of the major chal- in undifferentiated compared to the differ- lenges in the field of T-type VGCCs as targets in cancer entiated cells, suggesting an association with a malignant therapy [108]. These agonists/antagonists may also be useful potential of a tumour [74, 98]. This is yet to be investigated in other pathological conditions where T-type VGCCs are in case of breast cancer. There are reports indicating epige- implicated [26, 27, 109]. Furthermore, epigenetic modifica- netic modifications in T-type VGCCs such as methylation tion of ­CaV3.1 playing a role in apoptosis and proliferation of CACNA2D3 mRNA to support metastatic phenotype in inhibition also suggests that novel T-type VGCC specific breast cancer [99]. epigenetic modulators could be targeted to combat pathol- Along with the foresaid reports, considering the expres- ogy in breast cancer. Drug repurposing can be done through sion of T-type VGCCs in undifferentiated retinoblastoma, strategies to improve selectivity, potency and lead- we may hypothesize a role of T-type VGCCs in propagating ing to better specificity and optimum action on tumour stemness in cancer cells and perhaps even their involvement cells, thus having therapeutic benefits [54, 81, 110–112]. in the development of cancer stem cells [98, 100]. The scope of T-type VGCCs in breast cancer has been sum- This suggests that there is more to be discovered regard- marized in Box 2. ing the role of T-type VGCCs in regulating migration path- ways in breast cancer. The function of T-type VGCCs may Box 2: scope of T‑type VGCCs in breast extend to roles in invasion, and colonization cancer rather than being restricted to regulating proliferation as intracellular calcium levels and calcium influx are crucial • Stemness regulation and metastatic potential. for all of these processes. • Interlaced therapy. Interlaced therapy and drug repurposing • Isoform-specific T-type VGCC /antagonist. • Novel T-type VGCC isoform-specific physiological roles. T-type VGCC antagonists have shown reduced proliferation, in vitro and in vivo, in many cancer types like breast, T-cell, prostate [77, 101], ovarian [28] and [102]. Conclusion Reports also show that the anti-proliferative role of T-type VGCC antagonists is due to the inhibition of mTORC2/ Akt In conclusion, the question: ‘Is T-type VGCC an important signalling and regulation of the G1/S cell-cycle checkpoint, target in breast cancer?’, can be hinted with a positive reply, which sensitizes cancer cells to radiotherapy and chemo- however, there is no definitive answer to it yet, as a plethora therapy [87, 102–105]. This property has been exploited in of research awaits to recognize the causal/consequential interlaced therapy which was introduced by Tau Therapeu- association of T-type VGCCs with breast cancer. tics for repurposing drugs like mibefradil (a drug designed Funding to be a hypertensive drug, as T-type VGCC blocker) for This work was supported by grants to AB: BioCARe, DBT, [TM] India, (BT/BioCARe/01/9701/2013-14), seed grant, IITH (SG/IITH/ anti-cancer therapy. Tau’s Interlaced Therapy is based F145/2016-17/SG-27), ECR SERB-DST (ECR/2017/000242) and on the concept that using a T-type calcium , MHRD, India fellowship to SH. the population of metabolically vulnerable cancer cells in S-phase can be increased which sensitize cells to cytotoxic Compliance with ethical standards radiotherapy and chemotherapy [106]. Interlaced Therapy using T-type channel antagonists has been a success with Conflict of interest The authors declare having no financial competing cytotoxic using (TMZ) for interest. [107], paclitaxel for breast cancer [78] and carbo- Ethical approval This article does not contain any studies with human platin for [28]. As there is just one report of participants or animals performed by any of the authors.

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