calcium exchangers in tumorigenesis and tumor development of the upper gastrointestinal tract

Journal Pre-proof 2+ Plasma membrane Ca -permeable channels and sodium/calcium exchangers in tumorigenesis and tumor development of the upper gastrointestinal tract JianHong Ding, Zhe Jin, Xiaoxu Yang, Jun Lou, Weixi Shan, Yanxia Hu, Qian Du, Qiushi Liao, Jingyu Xu, Rui Xie PII:

S0304-3835(20)30040-9

DOI:

https://doi.org/10.1016/j.canlet.2020.01.026

Reference:

CAN 114668

To appear in:

Cancer Letters

Received Date: 20 November 2019 Revised Date:

30 December 2019

Accepted Date: 23 January 2020

Please cite this article as: J. Ding, Z. Jin, X. Yang, J. Lou, W. Shan, Y. Hu, Q. Du, Q. Liao, J. Xu, R. 2+ Xie, Plasma membrane Ca -permeable channels and sodium/calcium exchangers in tumorigenesis and tumor development of the upper gastrointestinal tract, Cancer Letters, https://doi.org/10.1016/ j.canlet.2020.01.026. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.

Abstract The upper gastrointestinal (GI) tumors are multifactorial diseases associated with a combination

of oncogenes

and

environmental

factors.

Currently,

surgery,

chemotherapy, radiotherapy and immunotherapy are relatively effective treatment options for the patients with these tumors. However, the asymptomatic phenotype of these tumors during the early stages poses as a significant limiting factor to diagnosis and often renders treatments ineffective. Therefore, new early diagnosis and effective therapy for upper GI tumors are urgently needed. Ca2+ is a pivotal intracellular second messenger and plays a crucial role in living cells by regulating several processes from cell division to death. The aberrant Ca2+ homeostasis is related to many human pathological conditions and diseases, including cancer, and thus the changes in the expression and function of plasma membrane Ca2+ permeable channels and sodium/calcium exchangers are frequently described in tumorigenesis and tumor development of the upper GI tract, including voltage-gated Ca2+ channels (VGCC), transient receptor potential (TRP) channels, store-operated channels (SOC) and Na+/Ca2+ exchanger (NCX). This review will summarize the current knowledge about plasma membrane Ca2+ permeable channels and sodium/calcium exchangers in the upper GI tumors and provide a synopsis of recent advancements on the role and involvement of these channels in upper GI tumors as well as a discussion of the possible strategies to target these channels and exchangers for diagnosis and therapy of the upper GI tumors.

1

Plasma membrane Ca2+-permeable channels and sodium/calcium exchangers in

2

tumorigenesis and tumor development of the upper gastrointestinal tract

3

JianHong Dinga,1, Zhe Jina,1, Xiaoxu Yanga, Jun Loua, Weixi Shana, Yanxia Hua, Qian Dua,

4

Qiushi Liaoa, Jingyu Xua,* , Rui Xiea,*

5

a

6

Guizhou, 563003, P.R. China

Department of Gastroenterology, Affiliated Hospital of Zunyi Medical University, Zunyi,

7 8

Running title: Calcium channels and transporters in upper gastrointestinal tumorogenesis

9

1Equal contribution

10

*Correspondence should be addressed to:

11

Prof. Rui Xie, MD, Ph.D, Department of Gastroenterology, Zunyi Medical University, China.

12

Email: [email protected]

13

Tel: +86 15120390626

14

Prof. Jingyu Xu, Ph.D., Department of Gastroenterology, Zunyi Medical University, China.

15

Email: [email protected]

16

Tel: +86 13765298886; Fax: +86 851 28609205

17

Abstract

18

The upper gastrointestinal (GI) tumors are multifactorial diseases associated with a combination

19

of oncogenes and environmental factors. Currently, surgery, chemotherapy, radiotherapy and

20

immunotherapy are relatively effective treatment options for the patients with these tumors.

21

However, the asymptomatic phenotype of these tumors during the early stages poses as a

22

significant limiting factor to diagnosis and often renders treatments ineffective. Therefore, new

23

early diagnosis and effective therapy for upper GI tumors are urgently needed. Ca2+ is a pivotal

24

intracellular second messenger and plays a crucial role in living cells by regulating several

25

processes from cell division to death. The aberrant Ca2+ homeostasis is related to many human

26

pathological conditions and diseases, including cancer, and thus the changes in the expression

27

and function of plasma membrane Ca2+ permeable channels and sodium/calcium exchangers are 1

28

frequently described in tumorigenesis and tumor development of the upper GI tract, including

29

voltage-gated Ca2+ channels (VGCC), transient receptor potential (TRP) channels, store-operated

30

channels (SOC) and Na+/Ca2+ exchanger (NCX). This review will summarize the current

31

knowledge about plasma membrane Ca2+ permeable channels and sodium/calcium exchangers in

32

the upper GI tumors and provide a synopsis of recent advancements on the role and involvement

33

of these channels in upper GI tumors as well as a discussion of the possible strategies to target

34

these channels and exchangers for diagnosis and therapy of the upper GI tumors.

35

Key words: Ion channel; Digestive system; Calcium signaling

36 37

Abbreviations

38

GI, gastrointestinal; VGCC, voltage-gated Ca2+ channels; TRP, transient receptor potential;

39

SOCC, store-operated Ca2+ channels; SOCE, store-operated Ca2+ entry; NCX, Na+/Ca2+

40

exchanger; [Ca2+]cyt, Cytosolic Ca2+ concentration; PMCAs, plasma membrane ATPases；ER，

41

endoplasmic reticulum；PM, Plasma membrane; STIM, stromal interaction molecule; LGC,

42

ligand-gated channels; SMOC, second messenger-operated channels; ASIC, acid-sensing ion

43

channels; MGC, mechano-gated channels; HVA, high-voltage-activated; LVA, low-voltage-

44

activated; TTCC, transient-type Ca2+ channels; CRAC, calcium release-activated calcium

45

channels; SICE, store-independent Ca2+ entry; ARC, arachidonate-regulated Ca2+; LRC,

46

leukotriene C4-regulated Ca2+; EC, esophageal cancer; ESCC, esophageal squamous cell

47

carcinoma; EA, esophageal adenocarcinoma; NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-

48

butanone; HCC, hepatocellular carcinoma; GC, gastric cancer; Hp, Helicobacter pylori; CaSR,

49

calcium sensing receptor; EMT, epithelial-to-mesenchymal transition; VPAC1, vasoactive

50

intestinal polypeptide receptor 1; LPS, Lipopolysaccharide. DSS, disease-specific survival; pT,

51

pathology tumor ； TGFβ ， transforming growth factor-β; PKCα, protein

52

GPCR,G Protein-Coupled Receptors ;COX-2,Cyclooxygenase-2; MNNG, N-methyl-N'-nitro-N-

53

nitrosoguanidine; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase; SPCA, Ca(2+)/Mn(2+)

54

ATPases

55

1.Introduction

56

Cytosolic Ca2+ concentration ([Ca2+]cyt) is essential for normal mammalian cell function, such as

57

gene transcription, proliferation, differentiation, migration, autophagy, metabolism, apoptosis, 2

kinase C alpha;

58

and angiogenesis[1]. [Ca2+]cyt is highly controlled by the fine regulation of ‘ON’ and ‘OFF’

59

mechanisms that ultimately generate Ca2+ signals with various amplitudes and frequencies. As

60

regarding the ON mechanisms, [Ca2+]cyt can either be delivered from extracellular space due to

61

the activity of Ca2+-permeable channels and Na+/Ca2+ exchanger (NCX) in plasma membrane

62

(PM), or occur as a result of a release from Ca2+-containing organelles (e.g. endoplasmic

63

reticulum)[1]. Living cells must maintain resting [Ca2+]cyt at low level (around 100 nM) to create

64

the wide dynamic range required for cellular Ca2+ signals. In order to maintain low resting Ca2+

65

concentration, cells remove Ca2+ using an energy-dependent mechanism, such as plasma

66

membrane ATPases (PMCAs), or NCX. Moreover Ca2+ is sequestered intracellularly into Ca2+-

67

containing organelles, primarily endoplasmic reticulum (ER), by means of mechanisms which

68

require either ATP hydrolysis (e.g. a sarco/endoplasmic reticulum Ca2+-ATPase pump), or a

69

favorable electrochemical gradient[2]. The increase in basal Ca2+ influx and remodeled Ca2+

70

signaling pathways may contribute to tumor progression by promoting proliferation, enhancing

71

invasiveness and conferring chemotherapeutic resistance[2, 3].

72

PM channels and sodium/calcium exchangers, [Ca2+]cyt buffers, and Ca2+-buffering organelles

73

regulate Ca2+ influx, storage, and extrusion to maintain [Ca2+]cyt. This finely tuned control of

74

[Ca2+]cyt is essential for differential modulation of various signaling pathways and [Ca2+]cyt-

75

regulated proteins involved in specific cellular processes. PM Ca2+-permeable channels support

76

Ca2+ to enter into the cytosol along its electrochemical gradient across the plasma membrane,

77

leading to an increase of [Ca2+]cyt. Various Ca2+-permeable channels and NCX are involved in

78

such transmembrane Ca2+ influx. Since Ca2+ is a ubiquitous second messenger involved in the

79

tuning of multiple fundamental cellular functions[1], it is not surprising that deregulated Ca2+

80

homeostasis has been observed in various disorders, including tumourigenesis[4, 5]. Since some

81

PM Ca2+ permeable channels and NCX referred to as oncogenic channels have been implicated

82

in tumorigenesis and tumor development of the upper GI tract, they may be used as potential

83

cancer biomarkers and therapeutic targets[6].

84

In this review, we will overview current observations supporting the aberrant expressions and

85

activities of some identified Ca2+-permeable channels and NCX in the upper GI tumors (Figure

86

1). Although Ca2+ disturbances in the PM could be compensated by internal Ca2+ stores activity

87

(such as PMCA and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) / Ca(2+)/Mn(2+)

3

88

ATPases (SPCA) expression), which is a new topic of other reviews, the current review focuses

89

on the role of aberrant Ca2+ movements across the plasma membrane in the upper GI tumors.

90

2. Plasma membrane Ca2+-permeable channels

91

The Ca2+-permeable channels on PM are passive structures that pass Ca2+ down to its

92

electrochemical gradient. When open, extracellular Ca2+ enters through these channels to the

93

cytoplasm. Although PM Ca2+-permeable channels consist of several subclasses, in this review

94

we will focus on the following three major ones reported previously in the upper GI tumors:

95

voltage-gated calcium channels (VGCC)[7] store-operated Ca2+ channels (SOCC)[8] transient

96

receptor potential (TRP) channels[9]. VGCC (or Cav channels) are responsible for Ca2+ entry

97

mostly in the excitable cells during membrane depolarization. The SOCC is the major Ca2+ entry

98

mechanism in non-excitable cells, also termed store-operated Ca2+ entry (SOCE)[8]. This

99

channel opens when ER Ca2+ store is depleted in order to provide SOCE necessary for store

100

refilling as well as for signaling purposes. [8, 10]. Many of the TRP-channel family members

101

have their endogenous and exogenous chemical ligands as well[9].The TRP-channel family

102

participates in the sensing of endogenous and exogenous stimuli of various modalities, such as

103

temperature, osmolarity, membrane stretch, pH, and second messengers, etc[9]. There are also

104

several other PM Ca2+-permeable channel subclasses, such as ligand-gated channels (LGC),

105

second messenger-operated channels (SMOC), acid-sensing ion channels (ASIC), and mechano-

106

gated channels (MGC). However, there is no information in the literatures on their involvements

107

in the upper GI tumors.

108

2.1. VGCC

109

The VGCC are classified into high-voltage-activated (HVA) channels, including Cav1.1–1.4 and

110

2.1-2.3 and low-voltage-activated (LVA) channels consisting of Cav3.1-3.3, known as T

111

(“transient”)-type Ca2+ channels (TTCC)[7] .Cav1.1–1.4, known as L-type VGCC, contain main

112

pore-forming α1 subunits, α1S, α1C, α1D and α1F, respectively, whereas Cav2.1, Cav2.2 and

113

Cav2.3, known as P/Q-type, N-type and R-type VGCCs, contain α1A, α1B and α1E, respectively.

114

TTCC (Cav3.1, Cav3.2 and Cav3.3) contain α1G, α1H and α1I, respectively. Each α1 subunit of

115

Cav1 and Cav2, but not Cav3, forms molecular complexes with auxiliary subunits, β, α2δ and γ,

116

which regulate the cell surface expression and function of the channels[11]. Although VGCC

117

have important roles in excitable cells, such as neurons and cardiomyocytes, they are also

118

expressed in non-excitable cells, such as epithelial cells of GI tract. In the last decade, they have 4

119

also been implicated in cancer cells where they have been found to either promote or inhibit

120

proliferation depending on the type of cancer and the type of ion channel isoform expressed[12].

121

VGCC, especially TTCC, have been reported as promising target candidates in cancer therapy as

122

they have been reported to be upregulated in many types of cancer types[13, 14].

123

2.2. SOCC: This is another important PM Ca2+ permeable channel in non-excitable cells.

124

Depletion of Ca2+ from the ER triggers the oligomerization of STIM1, and its redistribution to

125

ER-plasma membrane (ER-PM) junctions where Orai1 accumulates in the plasma membrane.

126

This mechanism known as store-operated calcium entry (SOCE), STIM1 proteins are

127

redistributed upon Ca2+ store depletion and subsequently interact with Orai1 proteins on the

128

plasma membrane, leading to activation of Ca2+ influx. Orai1-related isoforms include Orai2 and

129

Orai3, which form highly selective Ca2+ channels[15] .The STIM1 and STIM2 proteins act as

130

Ca2+ sensors upon ER- Ca2+ store depletion to be able to interact with and activate Orai channels.

131

However, STIM1 is also located in the plasma membrane to play a different role as the store-

132

independent Ca2+ entry (SICE)[16]. Along with Orai1, Orai3 contributes to store-independent

133

Ca2+ channels, such as the arachidonate-regulated Ca2+ (ARC) channels [17] and leukotriene C4-

134

regulated Ca2+ (LRC) channels[18].

135

2.3. TRP (Transient Receptor Potential) channels:

136

There are at least 20 channels encoded by TRP genes in mammals. They are divided into seven

137

main subfamilies[19, 20]: the TRPC (“canonical”) family, the TRPV (“vanilloid”) family, the

138

TRPM (“melastatin”) family, the TRPP (“polycystin”) family, the TRPML (“mucolipin”) family,

139

the TRPA (“ankyrin”) family, and the TRPN (“nompC”) family. All functionally characterized

140

TRP channels are cationic channels permeable to Ca2+, with the exceptions of TRPM4 and

141

TRPM5, which are only permeable to monovalent cations. These channels are activated by a

142

wide range of stimuli, including binding of intra- and extracellular messengers, changes in

143

temperature, chemical agents, mechanical stimuli, and osmotic stress[21] . In some cellular

144

models, TRPC channels also participate in Ca2+ entry activated by store depletion along with

145

Orai1[22]. Like Orai proteins, members of the TRP family can be activated/mobilized by a

146

variety of extracellular signals, leading to changes in the Ca2+ concentration in spatially restricted

147

micro/nanodomains underneath the plasma membrane that support various Ca2+-dependent

148

intracellular pathways.

149 5

150

3. Plasma membrane Na+/Ca2+ exchanger

151

Na+/Ca2+ exchanger (NCX) has been recognized as an important pathway for Ca2+ efflux across

152

the plasma membrane, mainly when [Ca2+]cyt is relatively high[23]. Three different protein

153

isoforms (NCX1, NCX2, and NCX3) were described and their respective genes (SLC8A1,

154

SLC8A2, and SLC8A3) were cloned[24-26] Variations of these isoforms generated by

155

alternative splicing were also described[27]. NCX1 has a broad expression in multiple organs,

156

whereas NCX2 is found only in the brain and NCX3 in both brain and skeletal muscle[28, 29].

157

Again, the first described role of NCX was to extrude Ca2+ from cells (forward mode), although

158

in some situations the exchanger may also promote Ca2+ influx (reverse mode). Functional

159

studies have shown that NCX transports three Na+ to one Ca2+ in the opposite direction

160

(antiporter)[30].The asymmetry in the charge movement across the membrane results in an

161

electrogenic transport[31]. Although NCX has been extensively studied in cerebral and cardiac

162

diseases and the therapeutic potential of its inhibitors in brain and heart injuries, the literature

163

about the molecular and functional aspects of NCX in cancer is scarce. In addition, NCX was

164

reported to be involved in esophageal cancer, but it has not been explored in normal stomach and

165

not mention to gastric cancer.

166

4. Plasma membrane Ca2+- permeable channels and Na+/Ca2+ exchangers in esophageal

167

cancer

168

4.1. Esophageal cancer (EC): Although EC is the eighth most common cancer and the sixth

169

leading cause of human cancer death worldwide[32], it ranks as the third most common and

170

fourth most common cause of cancer-related death in China[33].There are two major types of EC:

171

esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EA)[34]

172

Gastroesophageal reflux disease and Barrett’s esophagus are major risk factors for EA, whereas

173

ESCC is strongly related to environmental factors, such as smoking, alcohol consumption, and

174

zinc deficiency [35] . The main treatment options for EC include surgery, chemotherapy and

175

radiotherapy, but its current prognosis is poor. According to the National Cancer Institute, over

176

the last decades, the 5-year survival rate of patients with EC was still under 20%[36] These poor

177

outcomes have led to extensive research regarding the mechanisms of the carcinogenesis and

178

effective chemoprevention for EC. Hence, there is an urgent need for therapies that improve

179

survival and optimize palliative care for patients with EC.

180

4.2. Role of Plasma membrane Ca2+- permeable channels and NCX in EC 6

.

181

4.2.1 VGCC: Among the different subfamily of VGCC implicated in cancer, TTCC have been

182

found to be linked to tumor initiation and progression[37]. In particular, TTCC trigger cancer

183

cell proliferation via the mitogen-activated kinase pathway[38], while TTCC inhibition induces

184

cancer cell apoptosis[39].Since pharmacological TTCC inhibitors are under early phase of

185

development in several tumor types in clinical trials[40], modulation of TTCC may be a

186

promising strategy for cancer treatment. Lu F et al reported that mRNAs for three TTCC α1-

187

subunits (α1G, α1H, and α1I) were detected in 17 of ESCC cell lines. Cell proliferation was

188

reduced by TTCC blocker mibefradil in TE8 cells expressing mRNA for TTCC α1-subunits and

189

exhibiting T-type current. Silencing the α1G-gene that encodes functional TTCC in TE8 cells

190

also significantly decreased TE8 cell proliferation[41] . These findings suggest a functional role

191

of TTCC in EC and inhibition of TTCC reduces cell proliferation of EC.

192

4.2.2 TRP channels:

193

In human ESCC, the TRP channels mediates Ca2+ entry to activate Ca2+ signaling–dependent

194

pathways (such as mitogen-activated protein kinase (MAPK), which in turn rebuilt the cell

195

processes such as proliferation, apoptosis and migration[42].

196

TRPC regulate the cell cycle, specifically the G2/M phase transition, and are essential for glioma

197

progression[43]. TRPC6 channels were found to be abundant in human ESCC and are essential

198

for cell proliferation. Inhibition of TRPC6 channels in ESCC cells suppressed their proliferation

199

and induced G2/M phase arrest[44, 45]. Stratified analysis according to the pathological stage

200

revealed its discernibility on disease-specific survival (DSS). Liberati S et al also reported that

201

high expression of TRPV2 was observed more frequently in ESCC patients with advanced

202

pathology tumor (pT) stage, lymph node metastasis and advanced pathological stage[46].

203

Consistently, both mRNA and protein expression of TRPV1, TRPV2, and TRPV4 were

204

upregulated in ESCC cell lines, but not in nontumor esophageal squamous cell lines[47].

205

Immunohistochemical staining showed that TRPM7 expression was an independent prognostic

206

factor of good postoperative survival of patients with ESCC[48]. TRPV2 is involved in the

207

maintenance of cancer stem cells, and the TRPV2 channel inhibitor tranilast has potential to

208

become a targeted therapeutic agent against ESCC[49]. TRPV6 is downregulated in ESCC and is

209

predictive of survival of ESCC patients[50]. Suppression of TRPM7 expression with TRPM7-

210

siRNA was found to increase the proliferation, migration and invasion of ESCC cells, suggesting

211

that TRPM7 might serve as a cancer suppressor of ESCC[48]. Therefore, different 7

TRP

212

channels play different roles in EC formation and progression, but the underlying molecular

213

mechanisms need further investigation.

214 215

4.2.3 SOCC: Zinc deficiency is known to be associated with high incidences of human EC and

216

leads to a highly proliferative hyperplastic condition in the upper GI tract. To examine if zinc

217

deficiency contributes to the progression of EC, Choi S et al found that zinc supplementation

218

significantly inhibits proliferation of ESCC cell lines and that the effect of zinc is reversible with

219

a specific Zn2+ chelator. Mechanistically, they elucidated that zinc may inhibit cell proliferation

220

of ESCC cells through Orai1-mediated [Ca2+]cyt oscillations, and revealed that Orai1-SOCE

221

signaling pathway is important in ESCC cells[51]. Indeed, high expression of Orai1 was found in

222

ESCC, which was associated with poor overall and recurrence-free survival. Human ESCC cells

223

exhibited strikingly hyperactive in [Ca2+]cyt oscillations, which were sensitive to Orai1 channel

224

blockers and to Orai1 silencing. Moreover, pharmacologic inhibition of Orai1 activity or

225

reduction of Orai1 expression suppressed proliferation and migration of ESCC in vitro and

226

slowed tumor formation and growth in in vivo xenografted mice. These findings imply Orai1 as

227

a novel biomarker for ESCC prognostic stratification and also highlight Orai1-mediated Ca2+

228

signaling pathway as a potential target for treatment of ESCC[52]. Recently, Cui C et al

229

evaluated the anticancer effect of a novel potent SOCC inhibitor, RP4010, and found that

230

treatment with RP4010 reduced [Ca2+]cyt oscillations in EC cells and cell proliferation, caused

231

cell cycle arrest at G0/G1 phase in vitro, decreased nuclear translocation of nuclear factor kappa

232

B (NF-κB) in vivo and in vitro, and finally inhibited tumor growth in vivo. Therefore, RP4010

233

has the therapeutic potential in EC patients via inhibition of SOCC-mediated Ca2+ signaling[53].

234

In addition, the patients with ESCC putatively have elevated levels of ORAI1, which correlates

235

with poor overall and recurrence-free survival[52]. These findings conclude that ORAI1-

236

mediated SOCC-Ca2+ signaling is involved in the formation and progression of EC and that

237

SOCC inhibitors have the therapeutic potential in EC therapy.

238

4.2.4 NCX: Although NCX and the therapeutic potential of its inhibitors have been extensively

239

studied in heart diseases, there are few studies about the molecular and functional aspects of

240

NCX in cancer. The literature about the molecular and functional aspects of NCX in cancer is

241

scarce. Also NCX1 was reported as promising biomarkers in EC. We also found that NCX1 8

242

expression was significantly higher in ESCC primary tissues compared to the noncancerous

243

tissues and was overexpressed in tumor samples from the smoking patients. Furthermore, NCX1

244

expression correlates with the smoking status of ESCC patients. Consistently, the expression of

245

NCX1 proteins was significantly higher in human ESCC cell lines compared to normal

246

esophageal epithelial cell line. Therefore, it might serve as a novel prognostic biomarker for

247

ESCC patients in early stage[54]. We revealed that 4-(methylnitrosamino)-1-(3-pyridyl)-1-

248

butanone (NNK), a tobacco-specific nitrosamine, potentiated the [Ca2+]cyt signaling via the

249

Ca2+entry mode of NCX. NNK dose-dependently promoted proliferation and migration of human

250

ESCC cells induced by NCX1 activation[54]. Therefore, NNK may activate the Ca2+ entry mode

251

of NCX1 in ESCC cells, leading to cell proliferation and migration, and NCX1 protein is a novel

252

potential target for ESCC therapy.

253

4.2.5 A coupling of PM Ca2+-permeable channels and NCX: a coupling of Ca2+-permeable

254

channels and NCX may simultaneously involve in tumorigenesis and development of human

255

cancer. Most Ca2+- permeable channels and NCX play multiple roles in the cancer-related

256

processes, such as tumor growth, invasion, or metastasis. Focusing on either single channel type

257

or NCX may not achieve the maximum effect. Therefore, in cancer therapy, different modulators

258

of Ca2+- permeable channels and NCX are needed to inhibit tumor progression to the maximum.

259

We previously reported that transforming growth factor-β (TGFβ) induces Ca2+ entry via both

260

TRPC1 and NCX1 to raise [Ca2+)]cyt in pancreatic cancer cells, which is essential for protein

261

kinase C alpha (PKCα) activation and subsequent tumour cell invasion. Our data suggest that

262

TRPC1 and NCX1 may be among the potential therapeutic targets for pancreatic cancer[55].

263

Recently, we investigated a coupling of TRPC6 and NCX1 in tumorigenesis and tumor

264

development of human hepatocellular carcinoma (HCC). We found that TGFβ-stimulated

265

[Ca2+]cyt signaling in HCC cells through the formation of a TRPC6/NCX1 molecular complex.

266

This TRPC6/NCX1/ Ca2+ signaling mediated TGFβ-induced migration, invasion, and

267

intrahepatic metastasis of human HCC cells in nude mice. Our data reveal the role of the

268

TRPC6/NCX1 molecular complex in HCC and in regulating TGFβ and Ca2+ signaling, and

269

implicate both TRPC6 and NCX1 as potential targets for HCC therapy in[56]. However, we do

270

not know yet if molecular complex of TRP and NCX also exist in tumorigenesis and

271

development of the upper GI tract, which needs further investigation.

272 9

273

5. Plasma membrane Ca2+- permeable channels in gastric cancer

274

5.1. Gastric cancer (GC): GC is one of the most common cancers in the world and ranks second

275

in overall cancer-related deaths[57, 58] .High incidence regions include Asia, Eastern Europe

276

and Middle and South America[59]. Several major factors are known to increase the risk of

277

developing GC, such as infection by Helicobacter pylori (Hp) and Epstein-Barr virus, tobacco

278

use, high salt intake, and obesity. The Lauren classification is the most commonly used

279

classification system for GC[60, 61], which distinguishes GC into two main classes: intestinal

280

and diffuse[62]. The intestinal type tumors often occur in the antrum and incisura portions of the

281

stomach and consist mainly of well differentiated cells that have a slow growth rate and the

282

tendency to form glands. The incidence of this type of GC is often related to environmental

283

factors and occurs predominantly in men and elderly individuals. In contrast, the diffuse type

284

involves the entire stomach and it is made up of poorly differentiated cells lacking intercellular

285

adhesions that tend to scatter throughout the stomach, which explains the increased incidence of

286

metastasis and poor prognosis associated with this type of GC. Furthermore, the diffuse type is

287

typically correlated with genetic abnormalities and is more frequently diagnosed in females and

288

young patients[61]. Common therapeutic options for GC include surgery, radiotherapy, and

289

chemotherapy[63, 64].Studies have shown that patients with advanced stage GC have a poor

290

prognosis with a 5-year survival rate which is less than 30%[65]. Recently, advances in the

291

biology and molecular profiling of GC have resulted in targeted treatments and better survival

292

rates in selecting patients with advanced GC. However, the development of more highly

293

effective and selective targeted agents is still an important issue for the appropriate management

294

of advanced GC and more research is needed to further improve outcome.

295 296

5.2. Role of Plasma membrane Ca2+- permeable channels in GC

297

5.2.1 VGCC: as early as 1990, Tatsuta M first reported that caerulein, a Ca2+ mobilizer,

298

enhanced N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) -induced GC in rats, which could be

299

attenuated by calcium channel blockers verapamil and MgCl2. These findings indicate that

300

VGCC may play an important role in caerulein enhancement of GC[66]. Later on, Toyota M at

301

al. detected aberrant methylation of CACNA1G in GC patients and suggested that inactivation of

302

CACNA1G may play a role in GC development by modulating Ca2+ signaling to potentially

303

affect cell proliferation and apoptosis of GC[67].Furthermore, WANAJO A et al found that 10

304

exogenous expression of CACNA2D3 strongly inhibited GC cell growth and adhesion in HEK-

305

293T and NUGC4 cells, and inverse effects were seen by CACNA2D3 siRNA treatment in the

306

CACNA2D3-positive cell lines, indicating that CACNA2D3 may have tumor suppressive

307

functions[68]. Also, WANAJO A et al reported that among 4 voltage-dependent 2δ subunit

308

genes of the VGCC, loss of CACNA2D3 expression through aberrant promoter

309

hypermethylation may contribute to GC [68]. Toyota M at al. reported that aberrant methylation

310

of CACNA1G, a gene encoding a TTCC, was detected in 25% of GC patients, suggesting as a

311

diagnostic biomarker[67]. Fornaro L et al found that expression of all CACNA genes of TTCC in

312

GC was associated with overall survival among stage I-IV patients, suggesting that CACNA

313

gene expression is linked to tumour prognosis. However, CACNA-1H was the single best

314

predictor of clinical outcome, showing significant association with overall survival in all stage I-

315

IV patients, with high expression associated with poorer survival outcomes[40]. Although

316

VGCC and TTCC are promising diagnostic and prognosis biomarker of GC, their roles in GC

317

and the underlying molecular mechanisms need further investigation.

318 319

5.2.2 TRP channels:

320

An increasing number of reports have shown that TRP channels are involved in tumor

321

progression of GC. Cai et al. demonstrated that TRPC6 is functionally expressed in GC cell lines

322

and that pharmacological inhibition of this channel significantly reduced cell growth through

323

G2/M cell cycle arrest. These in vitro results were complemented with in vivo studies showing

324

that inhibition of TRPC6 suppressed GC growth in nude mice[69]. In addition to its proliferative

325

role, TRPC6 was also found as a key player in GC epithelial-to-mesenchymal transition

326

(EMT)[70], the most common process involved in cancer metastasis[71] . Mechanistically, it has

327

been suggested that TRPC6 operates by modulating Ras/Raf/ERK1/2 signaling. TRPM2 was

328

functionally expressed in GC cells and that its inhibition reduced cell bioenergetics, suppressed

329

cell invasion, and decreased cell survival via a JNK-dependent and mTOR-independent

330

autophagy pathway. The loss of TRPM2 led to a decrease in tumor growth in vivo using a

331

mouse model[72, 73]. Almasi et al. later confirmed that TRPM2 expression levels were

332

correlated with poor patient survival, particularly in those with late/advanced stages. Inhibition

333

of TRPM2 also promoted the effectiveness of chemotherapy drugs, suggesting that TRPM2

334

inhibition in conjunction with known chemotherapeutics could represent an efficacious strategy 11

335

for GC treatment[72]. Similarly, TRPM7 plays an influential role in the growth and survival of

336

GC cells. It can also depress cell apoptosis and is likely to be a potential target for

337

pharmacological therapy of GC[74, 75]. Among the members of the TRPV family, TRPV2,

338

TRPV4 and TRPV6 have been shown to play a prominent role in GC. Both mRNA and protein

339

expression levels of TRPV2 were found increasing according to tumor stage, and were

340

associated with poor prognosis in the Lauren’s intestinal type GC. Therefore, TRPV2 might be

341

used as a prognostic biomarker for GC[76]. Moreover, we reported that TRPV4 was upregulated

342

in GC cells and its activation evoked a large outwarded rectifying current leading to a marked

343

elevation in [Ca2+]cyt. Additional studies have also uncovered that the function of TRPV4 in GC

344

cells is facilitated through the activation of G Protein-Coupled Receptors (GPCR), the calcium

345

sensing receptor (CaSR)- and vasoactive intestinal polypeptide receptor 1 (VPAC1)[77, 78].

346

Furthermore, pharmacological inhibition and/or genetic depletion of TRPV4 expression

347

abolished CaSR and VPAC1-mediated GC cell proliferation and invasion, as well as tumor

348

growth and metastasis. Mechanistically, GPCR/TRPV4 promoted GC survival through Ca2+/β-

349

catenin pathway[77, 78]. Consistently, Mihara H. et al reported that Hp infection-dependent

350

DNA methylation suppressed TRPV4 expression in human gastric epithelia, suggesting that

351

TRPV4 methylation may be involved in Hp-associated gastric disease [79]. Finally, Chow et al.

352

found that TRPV6 expression was upregulated in GC cells, and further discovered that TRPV6

353

mediated GC cell death via the Ca2+/p53/JNK pathway [80]. Therefore, over past decades several

354

TRP channels have been identified to play important promoting roles in the development and

355

progress of GC, and inhibition of TRP channel may suppress the progress of GC development.

356

5.2.3 SOCC: Liu B et al reported that STIM1 silencing in GC cells significantly inhibited cell

357

proliferation by arresting the cell cycle at the G0/G1 phase, and increasing apoptotic rate of GC

358

cells. STIM1 knock down also reduced the migration and invasion of GC cells, suggesting

359

STIM1 is crucial for the proliferation and invasion of GC cells[81]. We reported that nucleotides

360

activated P2Y6 receptors to suppress GC growth through a novel SOCC/ Ca2+/β-catenin-

361

mediated anti-proliferation of GC cells, suggesting the potential therapeutic role of nucleotides-

362

mediated SOCC in GC[82]. Lipopolysaccharide (LPS) is a major component of the outer

363

membrane of gram-negative bacteria, such as Hp. Wong JH et al found that LPS mediated

364

gastric inflammation through activation of the SOCC, initiation of downstream NF-κB signaling,

365

and phosphorylation of ERK1/2. Phosphorylated ERK1/2 promotes the nuclear translocation of 12

366

NF-κB, and eventually elevates the expression level of Cyclooxygenase-2 (COX-2), a major

367

inflammatory gene[83].. Finally, Xia J et al reported Orai1 and STIM1 expressions were higher

368

in GC tissues, which was associated with more advanced disease, more frequent recurrence, and

369

higher mortality rates in GC patients. The disease-free survival rates of Stage I–III patients and

370

the overall survival rates of Stage IV patients were significantly worse when the tumors had high

371

Orai1 and/or STIM1 expressions. Orai1/STIM1 knockdown lowered the proliferation,

372

metabolism, migration, and invasion of GC cell lines. Consistently, Orai1/STIM1 knockdown

373

significantly reduced tumor growth and metastasis in athymic mice. Also, Orai1/STIM1

374

knockdown changed the markers of the cell cycle and EMT. These studies confirm the critical

375

roles of Orai1/STIM1in the tumorigenesis and development of GC[84]. Together, those studies

376

demonstrate that inhibition of SOCC may become a novel prevention strategy and a therapeutic

377

intervention for GC.

378 379

6. Conclusion

380

Over the last decades, studies have demonstrated the involvement of plasma membrane Ca2+-

381

permeable channels and NCX in various types of cancers, so that they have been regarded as

382

oncochannels [85] . These oncochannels are being verified in the upper GI tumor, and are likely

383

promising diagnostic biomarkers and therapeutic targets for EC and GC in clinic[85]. However,

384

this field of the upper GI cancer research is still in its infancy compared with classical oncology.

385

First, some experimental results are mainly based on transcript expression but not protein levels,

386

and at the end we do not know if the Ca2+ homeostasis of the cells is changed. Second, not all

387

aspects of oncogenic channel functions are fully understood for different types of cancer and not

388

all results obtained in the in vitro experimentation and even in vivo animal modeling can be

389

easily transferred to human cancer, which requires validation in human subjects. Third, there is a

390

need for isoform-specific agonists and antagonists to define and discover roles of the specific

391

isoforms of Ca2+-permeable channels and NCX. Although a combination of chemotherapy drugs

392

and Ca2+-permeable channels and NCX blockers present a promising therapeutic approach for

393

upper GI tumors, more validation and clinical trials are needed. It is necessary to establish

394

whether actual mutations can underlie oncogenic properties of the channels in certain types of

395

cancer; and if so what impact on channel function they may have. This will provide further solid

396

arguments to regard cancer hallmarks and maybe even certain cancer types as a whole as 13

397

oncochannelopathy. Future work should focus on characterization of their roles in tumor

398

development (primary tumor and/or metastatic development) and on their clinical relevance. It is

399

of interest to modulate the activity of plasma membrane Ca2+- permeable channels and NCX

400

expressed in upper GI cancer cells, in a sufficient manner to disrupt Ca2+ homeostasis selectively

401

in cancer cells. To this, extensive characterization of the structure, localization, expression

402

levels, and functioning is required in order to allow the development of highly specific and

403

potent modulators to pharmacologically target these proteins.

404 405

Consent for publication

406

We have obtained consents to publish this paper from all the participants of this study.

407 408

Availability of supporting data

409

Not applicable.

410 411

Competing interests

412

The authors declare that they have no competing interests.

413

Funding

414

This study was supported by research grants the National Natural Science Foundation of China

415

(No. 81970541 to JY; No. 811660412 to XR)

416 417

Authors' contributions

418

J.H.D and Z.J wrote the manuscript. X.X.Y, J.L W.X.S, Y.X.H, Q.D, and Q.S.L collect the

419

literature. R.X. primarily revised and finalized manuscript J.Y.X revised the manuscript for

420

clarity and style. All authors read and approved the final manuscript. All authors read and

421

approved the final manuscript.

422 423

References: 14

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656 657 658 659 660 661 662 663 664 665 19

666

Fig 1. Diagram showing the ion channels and transporters directly implicated in Ca2+

667

homeostasis in the cancer cells of the upper gastrointestinal tract.

668

Influx of Ca2+ is primarily mediated by VGCC (voltage-gated Ca2+ channels), transient receptor

669

potential channels (TRP), sodium-calcium exchanger (NCX) and store-operated Orai channels

670

that are activated by STIM1 protein. Efflux of Ca2+ is achieved by PM Ca2+ ATPase (PMCA).

671

Release of Ca2+ from the ER/SR is mediated through IP3 (IP3R). The reuptake of Ca2+ into the

672

ER/SR is primarily mediated by sarcoplasmic/ER Ca2+ ATPase (SERCA)

673

20

Table 1. PM Ca2+-permeable channels and NCX in esophageal cancer Channels Function Mechanisms

References

VGCC Expert Rev Antican Ther. 2013; TTCC

Promoter

Trigger ESCC cell proliferation 13: 589 Silence α1G-gene decreases ESCC cell proliferation

Cell Calcium 2008; 43:49

FEBS Open Bio 2019; 9:206

TRP TRPV1

Promoter

Upregulation of both mRNA&proteins

TRPV2

Promoter

Upregulation of both mRNA&proteins

Cur Protein and Pept Sci, 2014; 15:732 Overexpression is associated with poor prognosis

Med Oncol 2014: 31:17

Maintenance of cancer stem cells

J Gastroenterol 2017

TRPV4

Promoter

Upregulation of both mRNA&proteins

FEBS Open Bio 2019; 9:206

TRPM7

Suppressor

Suppress proliferation, migration and invasion

Anticancer Res 2917: 37: 1161

TRPC6

Promoter

Overexpression is associated with poor prognosis

Med Oncol 2013: 30:607

Promote cell proliferation and cell cycle

Gut 2009;58:1443

Zinc inhibits cell proliferation via Orai1/Ca2+

FASEB J. 2018: 32:1

Overexpression is associated with poor prognosis

Oncotarget 2014:15:3455

Promotes proliferation and cell cycle via Ca2+/NF-kB

Can Lett 2018; 432:169

STIM1/Orai1 Orai1

Promoter

NNK promotes proliferation and migration via NCX

Promoter

Oncotarget 2016;27:63816 NCX1/Ca2+ signaling

1

Table 2. PM Ca2+-permeable channels in gastric cancer Channels Function Mechanisms

References

VGCC CACNA2D3

Suppressor

Inhibition of growth and adhesion

Gastroenterology 2008; 135:580

CACNA1G

Promoter

Aberrant methylation

Caner Res.1999: 59, 4535

CACNA1H

Promoter

Association with overall survival

PLoS One 2017;12:e0182818

TRPV2

Promoter

Over-expression/Poor prognosis

J. Clin. Med. 2019; 8: 662

TRPV4

Promoter

CaSR/TRPV4/Ca2+

Cancer Res. 2017; 77:6499

Promoter

VIPC1/TRPV4/Ca2+

Oncogene 2019; 38:3946

TRP

TRPV4

methylation

/Hp-induced

Promoter

Helicobacter. 2017;22 dyspepsia

TRPV6

Suppressor

CAP-induced apoptosis

BBA 2007;1773:565

TRPM2

Promoter

JNK/Autophage/Mito funct

JBC. 2018 ;293:3637

TRPM5

Promoter

pHe/Ca2+/MMP-9-mediated metastasis

Oncotarget 2017; 8:78312

TRPM7

Promoter

Mg2+/Zn2+ involved

Integr Med Res. 2016;5:124

TRPC1/3

Promoter

TGFβ1-induced EMT via Ras/Raf1/ERK

Cell Biol Int. 2018;42:975

TRPC6

Promoter

14a blocks TRPC6 to kill GC cells

Cancer Lett. 2018;432:47

Promoter

TGFβ1-induced EMT via Ras/Raf1/ERK

Cell Biol Int. 2018;42:975

Promoter

G2/M phase arrest

Int J Cancer 2009;125:2281

STIM/Orai

Promoter

Upregulates MACC1

Cancer Lett. 2016;381:31

STIM/Orai

Promoter

SOCE facilitates LPS/NFkB/COX-2

Sci Rep. 2017; 16;12813

STIM1

Promoter

Proliferation&invasion of SGC7901

Mol Med Rep. 2015;12:3047

STIM1/Orai1

2

Highlights Voltage-gated Ca2+ channels (VGCC), transient receptor potential (TRP) channels,

store-operated

(NCX) involve

in

the

channels

(SOC)

tumorigenesis

and

and

Na+/Ca2+ exchanger

development

of

the

upper

gastrointestinal tract. Ca2+ permeable channels and sodium/calcium exchangers present abnormal expression and activity in upper gastrointestinal tumors. Calcium channels can be used as a promising diagnostic biomarkers and therapeutic targets for esophageal cancer and gastric cancer.