cance s
A icle
TRPC6 Channels A e Requi ed o P oli e a ion,
Mig a ion and In asion o B eas Cance Cell Lines
by Modula ion o O ai1 and O ai3 Su ace Exposu e
Isaac Ja din 1,†,* , Raquel Diez-Bello 1,†, Jose J. Lopez 1, Ped o C. Redondo 1,
Ginés M. Salido 1, Ta ik Smani 2and Juan A. Rosado 1,*
1Cellula Physiology Resea ch G oup, Depa men o Physiology, Ins i u e o Molecula Pa hology
Bioma ke s, Uni e si y o Ex emadu a, 10003 Cace es, Spain; [email p o ec ed] (R.D.-B.);
[email p o ec ed] (J.J.L.); [email p o ec ed] (P.C.R.); [email p o ec ed] (G.M.S.)
2Depa men o Medical Physiology and Biophysic, Ins i u e o Biomedicine o Se illa, 41013 Se illa, Spain;
[email p o ec ed]
*Co espondence: [email p o ec ed] (I.J.); [email p o ec ed] (J.A.R.);
Tel.: +34-927257100 (ex . 51376) (I.J. & J.A.R.);
Fax: +34-927257110 (I.J. & J.A.R.)
† These au ho s con ibu ed equally o his wo k.
Recei ed: 5 Augus 2018; Accep ed: 13 Sep embe 2018; Published: 14 Sep embe 2018
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Abs ac :
T ansien ecep o po en ial channels con ey signaling in o ma ion om a numbe o
s imuli o a wide a ie y o cellula unc ions, mainly by inducing changes in cy osolic Ca
2+
concen a ion. Di e en membe s o he TRPC, TRPM and TRPV sub amilies ha e been epo ed o
play a ole in umo igenesis. He e we show ha he es ogen ecep o posi i e and iple nega i e
b eas cance cell lines, MCF7 and MDA-MB-231, espec i ely, exhibi enhanced exp ession o he
TRPC6 channel as compa ed o he non- umo al MCF10A cell line.
In i o
TRPC6 knockdown using
shRNA impai ed MCF7 and MDA-MB-231 cell p oli e a ion, mig a ion and in asion de ec ed by
B dU inco po a ion, wound healing and Boyden chambe assays, espec i ely. Using RNAi-media ed
TRPC6 silencing as well as o e exp ession o he po e-dead dominan -nega i e TRPC6 mu an we
ha e ound ha TRPC6 plays a ele an ole in he ac i a ion o s o e-ope a ed Ca
2+
en y in he b eas
cance cell lines bu no in non- umo al b eas cells. Finally, we ha e ound ha TRPC6 in e ac s
wi h O ai1 and O ai3 in MCF7 and MDA-MB-231 cells and is equi ed o he ansloca ion o O ai1
and O ai3 o he plasma memb ane in MDA-MB-231 and MCF7 cells, espec i ely, upon Ca
2+
s o e
deple ion. These indings in oduce a no el mechanism o he modula ion o Ca
2+
in lux and he
de elopmen o di e en cance hallma ks in b eas cance cells.
Keywo ds: TRPC6; O ai1; O ai3; s o e-ope a ed calcium en y; MCF7; MDA-MB-231
1. In oduc ion
B eas cance is among he leading causes o cance dea h in women wo ldwide, accoun ing
o abou 25% o all diagnosed emale cance s [
1
]. B eas cance cells a e cha ac e ized by a high
p oli e a ion a e, esis ance o p og ammed cell dea h, and inc eased capabili y o mig a e and in ade
su ounding issues [
2
]. These hallma ks can de elop h ough di e en mechanisms ha lead o he
onse and p og ession o b eas cance , among hem he al e a ion in he PI3K pa hway [
3
], abno mal
ac i a ion o he MAPK signaling [4] o anomalous in acellula Ca2+ signaling [5].
Cy osolic ee-Ca
2+
concen a ion is a c ucial ac o o a a ie y o cellula p ocesses [
6
] and a
numbe o genes encoding ion channels ha e been ound among hose al e ed in cance cells [
7
]. The e
is a g owing body o e idence suppo ing he ele an ole o ion channels, and pa icula ly Ca
2+
Cance s 2018,10, 331; doi:10.3390/cance s10090331 www.mdpi.com/jou nal/cance s
Cance s 2018,10, 331 2 o 18
channels, in he mechanisms unde lying cell g ow h and p oli e a ion, mig a ion, apop osis esis ance
and angiogenesis in cance cells. Among he Ca
2+
channels in cance cells, O ai1,
he po e- o ming
subuni o he Ca
2+
elease-ac i a ed Ca
2+
(CRAC) channel [
8
,
9
], which is he bes cha ac e ized
s o e-ope a ed Ca
2+
channel, has been ound o be o e exp essed in he human cance cells in es iga ed,
including b eas cance [
10
], melanoma [
11
], clea cell enal ca cinoma [
12
] and non-small cell lung
ca cinoma [
13
], excep in p os a e cance cells, whose exp ession has been epo ed o be educed as
compa ed o no mal issue [
14
]. The in o ma ion conce ning he O ai1 homologs O ai2 and O ai3 is
a he sca ce bu O ai2 has been ound o be o e exp essed in pa a hy oid adenoma [
15
] and acu e
myeloid leukemia cells [
16
], while O ai3 is o e exp essed in es ogen ecep o -exp essing (ER
+
) b eas
cance cell lines [
17
] and in p os a e cance issue specimens ob ained om esec ion su ge ies as
compa ed o noncance ous issue [18].
On he o he hand, ansien ecep o po en ial (TRP) channels, especially ce ain membe s
o he TRPC, TRPM and TRPV sub amilies, ha e also been epo ed o play a ele an ole in he
p og ession o di e en ypes o cance . Among hem, TRPV6 is o e exp essed in a numbe o
cance cell ypes and pa icipa es in he p og ession o p os a e cance [
19
], acqui ing i s oncogenic
po en ial ia O ai1/TRPC1-dependen ansloca ion o he plasma memb ane [
20
]. TRPM8 egula es
he mo ili y o a a ie y o cance cells including o al squamous ca cinoma, lung cance o p os a e
cance cells [
21
], whe e i s plasma memb ane localiza ion and umo igenic po en ial a e egula ed by
TRP channel-associa ed ac o s [
22
]. S udies conce ning TRPC sub amily membe s ha e mainly ocused
on TRPC1, whose in ol emen in umo igenesis a ies depending on he s age and ype o cance
conside ed [
21
,
23
]. TRPC6 has been epo ed o play a ele an ole in he p oli e a ion o gas ic [
24
],
p os a e [
25
], esophageal squamous cell ca cinoma [
26
] and hepa ome cells [
27
]. Fu he mo e, TRPC6
is equi ed o mig a ion and in asion o hepa ocellula ca cinoma cells [
28
]. TRPC6 channels ha e
been shown o be o e exp essed in human b eas duc al adenoca cinoma compa ed o non- umo al
issue [
29
,
30
] and bo h, TRPC3 and TRPC6, ha e been epo ed o be signi ican ly up- egula ed in
b eas cance biopsies compa ed o no mal issue [
31
]; howe e , he molecula basis o he unc ional
ole o TRPC6 in b eas cance cells and i s in ol emen in he cance hallma ks emains unclea .
He e we show ha TRPC6 is equi ed o p oli e a ion, mig a ion and in asion o he ER
+
cell
line MCF7 and he iple nega i e MDA-MB-231 cell line. Silencing TRPC6 p o ein exp ession, as well
as o e exp ession o a po e-dead dominan -nega i e TRPC6 mu an has e ealed ha TRPC6 plays an
impo an ole in he ac i a ion o s o e-ope a ed Ca
2+
en y (SOCE) in bo h MCF7 and MDA-MB-231
cell lines, which is likely media ed by he ole o TRPC6 in he ansloca ion o he plasma memb ane
o O ai3 o O ai1, espec i ely, in he cell lines in es iga ed.
2. Resul s
2.1. TRPC6 Is O e exp essed in MCF7 and MDA-MB-231 B eas Cance Cell Lines and is Requi ed o B eas
Cance Cell P oli e a ion, Mig a ion and In asion
Consis en wi h he p e ious s udy by Ayda and cowo ke s [
31
], Wes e n blo analysis o whole
cell lysa es om he non- umo al b eas MCF10A cell line, he ER
+
and iple nega i e b eas cance
cell lines MCF7 and MDA-MB-231, espec i ely, wi h a speci ic an i-human TRPC6 an ibody e ealed
ha he exp ession o his p o ein is ela i ely low in he non- umo al cell line (Figu e 1). Fu he mo e,
TRPC6 exp ession in he MCF7 and MDA-MB-231 cell lines is signi ican ly g ea e (app oxima ely
350 and 460%, espec i ely) han in non- umo al cells. TRPC6 exp ession in he di e en cell lines,
no malized o he
β
-ac in con en and exp essed as pe cen age o he exp ession le el in MCF10A,
is shown in Figu e 1(ba g aphs; n = 6).
We ha e u he explo ed he in ol emen o TRPC6 in he abili y o MCF10A, MCF7 and
MDA-MB-231 o p oli e a e. To add ess his issue, cells ans ec ed wi h shTRPC6 o shRNA con ol
ec o (shRNAc ), we e subjec ed o he B dU cell p oli e a ion assay.
Cance s 2018,10, 331 3 o 18
Cance s 2018, 10, 331 3 o 18
Figu e 1. Cellula exp ession o TRPC6 in non- umo al and b eas cance cell lines. MCF10A, MCF7
and MDA-MB-231 cells we e lysed and subjec ed o Wes e n blo ing wi h an i-TRPC6 an ibody,
ollowed by ep obing wi h an i-β-ac in an ibody o p o ein loading con ol. Ba g aphs ep esen
TRPC6 exp ession no malized o he β-ac in con en and exp essed as pe cen age o he TRPC6
exp ession in non- umo al MCF10A cells. Molecula masses indica ed on he igh we e de e mined
using molecula -mass ma ke s un in he same gel. * p < 0.05 compa ed o TRPC6 exp ession in
MCF10A cells.
As shown in Figu e 2a, cell ans ec ion wi h shTRPC6 signi ican ly a enua ed TRPC6
exp ession in MCF10A, MCF7 and MDA-MB-231 cells (p < 0.05; n = 6). Nex , we explo ed he e ec
o ans ec ion wi h shTRPC6 in cell p oli e a ion in he h ee cell lines. Fo y-eigh hou s a e
ans ec ion ( ime = 0h), as well as 24, 48 and 72h la e , cell p oli e a ion was assessed. As expec ed,
he shTRPC6 was wi hou e ec in MCF10A p oli e a ion, which is consis en wi h he low na i e
TRPC6 exp ession and indica es a lack o e ec o shTRPC6 in cell p oli e a ion in his cell line (Figu e
2b; n = 6). In e es ingly, silencing TRPC6 p o ein exp ession signi ican ly a enua ed MCF7 and MDA-
MB-231 cell p oli e a ion a all he imes in es iga ed as compa ed o cells ans ec ed wi h shRNAc
(Figu e 2b; p < 0.05; n = 4). The e o e, ou obse a ions e eal ha TRPC6 is essen ial o ER+ and
iple nega i e b eas cance cell p oli e a ion.
Nex , we assessed he ele ance o TRPC6 in he abili y o hese cell lines o mig a e. MCF10A,
MCF7 and MDA-MB-231 cells we e subjec ed o he well-es ablished wound healing assay. Cells
we e seeded, sc a ched, and cul u ed in medium supplemen ed wi h 1% se um o p e en u he
cell g ow h. Mig a ion o cells was quan i a ed as desc ibed in Ma e ials and Me hods. To explo e
he ole o TRPC6 in cell mig a ion MCF10A, MCF7 and MDA-MB-231 cells we e ans ec ed wi h
shTRPC6 o con ol plasmid and cell mig a ion was e alua ed. As shown in Figu e 3a, MCF10A,
MCF7 and MDA-MB-231 cells ans ec ed wi h shRNAc signi ican ly educed he wound size
du ing he i s 48 h (p < 0.05; n = 3). TRPC6 exp ession silencing did no a ec he abili y o MCF10A
o mig a e (Figu e 3a; n = 3), which is consis en wi h he low exp ession o TRPC6 in his cell line.
In e es ingly, silencing TRPC6 exp ession signi ican ly a enua ed MCF7 and MDA-MB-231
mig a ion as compa ed o cells ans ec ed wi h shRNAc (Figu e 3a; p < 0.05; n = 3), which indica es
ha TRPC6 plays an impo an ole in MCF7 and MDA-MB-231 cell mig a ion.
We ha e u he in es iga ed he ole o TRPC6 in in i o in asion analysed using he answell
mig a ion assay. A e ans ec ion wi h shRNAc , a signi ican amoun o MCF7 and MDA-MB-231
cells, especially he la e , passed ac oss he answell inse (Figu e 3b). We e en ound a la ge
numbe o MDA-MB-231 cells adhe ed o he su ace o he lowe chambe (Figu e 3b, bo om panel).
By con as , we we e unable o de ec MCF10A cells in he unde su ace o he answell inse [32].
In e es ingly, as depic ed in Figu e 3b, a lesse numbe o MCF7 and MDA-MB-231 cells we e able o
mig a e o he unde su ace o he answell inse upon TRPC6 exp ession silencing as compa ed o
cells ea ed wi h con ol shRNA (p < 0.05; n = 5). Consis en ly, he numbe o in asi e MDA-MB-231
cells a ached o he su ace o he lowe chambe was clea ly educed a e ans ec ion wi h
shTRPC6 (Figu e 3b, bo om panel).
Figu e 1.
Cellula exp ession o TRPC6 in non- umo al and b eas cance cell lines. MCF10A, MCF7
and MDA-MB-231 cells we e lysed and subjec ed o Wes e n blo ing wi h an i-TRPC6 an ibody,
ollowed by ep obing wi h an i-
β
-ac in an ibody o p o ein loading con ol. Ba g aphs ep esen
TRPC6 exp ession no malized o he
β
-ac in con en and exp essed as pe cen age o he TRPC6
exp ession in non- umo al MCF10A cells. Molecula masses indica ed on he igh we e de e mined
using molecula -mass ma ke s un in he same gel. * p< 0.05 compa ed o TRPC6 exp ession in
MCF10A cells.
As shown in Figu e 2a, cell ans ec ion wi h shTRPC6 signi ican ly a enua ed TRPC6 exp ession
in MCF10A, MCF7 and MDA-MB-231 cells (p< 0.05; n = 6). Nex , we explo ed he e ec o ans ec ion
wi h shTRPC6 in cell p oli e a ion in he h ee cell lines. Fo y-eigh hou s a e ans ec ion (
ime = 0 h
),
as well as 24, 48 and 72 h la e , cell p oli e a ion was assessed. As expec ed, he shTRPC6 was wi hou
e ec in MCF10A p oli e a ion, which is consis en wi h he low na i e TRPC6 exp ession and indica es
a lack o e ec o shTRPC6 in cell p oli e a ion in his cell line (Figu e 2b; n = 6). In e es ingly, silencing
TRPC6 p o ein exp ession signi ican ly a enua ed MCF7 and MDA-MB-231 cell p oli e a ion a all
he imes in es iga ed as compa ed o cells ans ec ed wi h shRNAc (Figu e 2b; p< 0.05; n = 4).
The e o e, ou obse a ions e eal ha TRPC6 is essen ial o ER
+
and iple nega i e b eas cance
cell p oli e a ion.
Nex , we assessed he ele ance o TRPC6 in he abili y o hese cell lines o mig a e. MCF10A,
MCF7 and MDA-MB-231 cells we e subjec ed o he well-es ablished wound healing assay. Cells
we e seeded, sc a ched, and cul u ed in medium supplemen ed wi h 1% se um o p e en u he cell
g ow h. Mig a ion o cells was quan i a ed as desc ibed in Ma e ials and Me hods. To explo e he ole
o TRPC6 in cell mig a ion MCF10A, MCF7 and MDA-MB-231 cells we e ans ec ed wi h shTRPC6
o con ol plasmid and cell mig a ion was e alua ed. As shown in Figu e 3a, MCF10A, MCF7 and
MDA-MB-231 cells ans ec ed wi h shRNAc signi ican ly educed he wound size du ing he i s
48 h (p< 0.05; n = 3). TRPC6 exp ession silencing did no a ec he abili y o MCF10A o mig a e
(Figu e 3a; n = 3), which is consis en wi h he low exp ession o TRPC6 in his cell line. In e es ingly,
silencing TRPC6 exp ession signi ican ly a enua ed MCF7 and MDA-MB-231 mig a ion as compa ed
o cells ans ec ed wi h shRNAc (Figu e 3a; p< 0.05; n = 3), which indica es ha TRPC6 plays an
impo an ole in MCF7 and MDA-MB-231 cell mig a ion.
We ha e u he in es iga ed he ole o TRPC6 in
in i o
in asion analysed using he answell
mig a ion assay. A e ans ec ion wi h shRNAc , a signi ican amoun o MCF7 and MDA-MB-231
cells, especially he la e , passed ac oss he answell inse (Figu e 3b). We e en ound a la ge
numbe o MDA-MB-231 cells adhe ed o he su ace o he lowe chambe (Figu e 3b, bo om panel).
By con as
, we we e unable o de ec MCF10A cells in he unde su ace o he answell inse [
32
].
In e es ingly, as depic ed in Figu e 3b, a lesse numbe o MCF7 and MDA-MB-231 cells we e able o
mig a e o he unde su ace o he answell inse upon TRPC6 exp ession silencing as compa ed o
cells ea ed wi h con ol shRNA (p< 0.05; n = 5). Consis en ly, he numbe o in asi e MDA-MB-231
cells a ached o he su ace o he lowe chambe was clea ly educed a e ans ec ion wi h shTRPC6
(Figu e 3b, bo om panel).
Cance s 2018,10, 331 4 o 18
Cance s 2018, 10, 331 4 o 18
Figu e 2. TRPC6 exp ession is equi ed o MCF7 and MDA-MB-231 cell p oli e a ion. (a) MCF10A,
MCF7 and MDA-MB-231 cells we e ans ec ed wi h shTRPC6 o shRNA con ol ec o (shRNAc ),
as indica ed. A e 48h cells we e lysed and subjec ed o Wes e n blo ing wi h an i-TRPC6 an ibody,
ollowed by ep obing wi h an i-β-ac in an ibody o p o ein loading con ol. Molecula masses
indica ed on he igh we e de e mined using molecula -mass ma ke s un in he same gel. (b)
MCF10A, MCF7 and MDA-MB-231 cells we e ans ec ed wi h shTRPC6 o sc amble plasmid and 48
h la e cell p oli e a ion was assessed o a u he 24, 48 and 72 h using he B dU cell p oli e a ion
assay ki , as desc ibed in he Ma e ial and Me hods. Ba g aphs ep esen cell p oli e a ion 0, 24, 48
and 72 h a e cell ans ec ion, p esen ed as B dU up ake a e. * p < 0.05 compa ed o he
co esponding con ol (cells ans ec ed wi h shRNAc ).
Figu e 3. Con .
Figu e 2.
TRPC6 exp ession is equi ed o MCF7 and MDA-MB-231 cell p oli e a ion. (
a
) MCF10A,
MCF7 and MDA-MB-231 cells we e ans ec ed wi h shTRPC6 o shRNA con ol ec o (shRNAc ),
as indica ed. A e 48h cells we e lysed and subjec ed o Wes e n blo ing wi h an i-TRPC6 an ibody,
ollowed by ep obing wi h an i-
β
-ac in an ibody o p o ein loading con ol. Molecula masses
indica ed on he igh we e de e mined using molecula -mass ma ke s un in he same gel.
(b) MCF10A
,
MCF7 and MDA-MB-231 cells we e ans ec ed wi h shTRPC6 o sc amble plasmid and 48 h la e
cell p oli e a ion was assessed o a u he 24, 48 and 72 h using he B dU cell p oli e a ion assay ki ,
as desc ibed
in he Ma e ial and Me hods. Ba g aphs ep esen cell p oli e a ion 0, 24, 48 and 72 h
a e cell ans ec ion, p esen ed as B dU up ake a e. * p< 0.05 compa ed o he co esponding con ol
(cells ans ec ed wi h shRNAc ).
Cance s 2018, 10, 331 4 o 18
Figu e 2. TRPC6 exp ession is equi ed o MCF7 and MDA-MB-231 cell p oli e a ion. (a) MCF10A,
MCF7 and MDA-MB-231 cells we e ans ec ed wi h shTRPC6 o shRNA con ol ec o (shRNAc ),
as indica ed. A e 48h cells we e lysed and subjec ed o Wes e n blo ing wi h an i-TRPC6 an ibody,
ollowed by ep obing wi h an i-β-ac in an ibody o p o ein loading con ol. Molecula masses
indica ed on he igh we e de e mined using molecula -mass ma ke s un in he same gel. (b)
MCF10A, MCF7 and MDA-MB-231 cells we e ans ec ed wi h shTRPC6 o sc amble plasmid and 48
h la e cell p oli e a ion was assessed o a u he 24, 48 and 72 h using he B dU cell p oli e a ion
assay ki , as desc ibed in he Ma e ial and Me hods. Ba g aphs ep esen cell p oli e a ion 0, 24, 48
and 72 h a e cell ans ec ion, p esen ed as B dU up ake a e. * p < 0.05 compa ed o he
co esponding con ol (cells ans ec ed wi h shRNAc ).
Figu e 3. Con .
Figu e 3. Con .
Cance s 2018,10, 331 5 o 18
Cance s 2018, 10, 331 5 o 18
Figu e 3. Role o TRPC6 in b eas cance cell mig a ion and in asion. MCF10A, MCF7 and MDA-MB-
231 cells we e ans ec ed wi h shTRPC6 o con ol shRNAc . Fo y-eigh hou s a e ans ec ion
cells we e subjec ed o wound healing assay (a) o answell mig a ion assay (b) as desc ibed in
Me hods. (a) Images we e acqui ed a 0 and 48 h om he beginning o he assay. The do ed lines
de ine he a eas lacking cells. The ba g aphs ep esen he wound size, in mic ome e s, a he
di e en condi ions, exp essed as he mean ± SEM o h ee independen expe imen s. * p < 0.05
compa ed o he ime = 0 h. § p < 0.05 compa ed o he co esponding ime in shRNAc ans ec ed
cells. (b) Images show he s ained cells as ob ained om he answell mig a ion assay subjec ed o
he di e en expe imen al condi ions. The ba g aphs ep esen he pe cen age o cell in asion as
compa ed o MDA-MB-231 cells ans ec ed wi h shRNAc , exp essed as he mean ± SEM o i e
independen expe imen s. * p < 0.05 compa ed o he co esponding shRNAc ans ec ed cells.
Bo om panels show ep esen a i e pic u es o he in asi e cells adhe ed o he bo om o he lowe
chambe .
Figu e 3.
Role o TRPC6 in b eas cance cell mig a ion and in asion. MCF10A, MCF7 and
MDA-MB-231 cells we e ans ec ed wi h shTRPC6 o con ol shRNAc . Fo y-eigh hou s a e
ans ec ion cells we e subjec ed o wound healing assay (
a
) o answell mig a ion assay (
b
) as
desc ibed in Me hods. (
a
) Images we e acqui ed a 0 and 48 h om he beginning o he assay.
The do ed
lines de ine he a eas lacking cells. The ba g aphs ep esen he wound size, in mic ome e s,
a he di e en condi ions, exp essed as he mean
±
SEM o h ee independen expe imen s. * p< 0.05
compa ed o he ime = 0 h. § p< 0.05 compa ed o he co esponding ime in shRNAc ans ec ed
cells. (
b
) Images show he s ained cells as ob ained om he answell mig a ion assay subjec ed o
he di e en expe imen al condi ions. The ba g aphs ep esen he pe cen age o cell in asion as
compa ed o MDA-MB-231 cells ans ec ed wi h shRNAc , exp essed as he mean
±
SEM o i e
independen expe imen s. * p< 0.05 compa ed o he co esponding shRNAc ans ec ed cells. Bo om
panels show ep esen a i e pic u es o he in asi e cells adhe ed o he bo om o he lowe chambe .

Cance s 2018,10, 331 6 o 18
We con i med he ole o TRPC6 in b eas cance cell mig a ion and p oli e a ion by exp essing
a po e-dead dominan -nega i e TRPC6 (TRPC6dn) mu an . As shown in Figu e 4a, exp ession o
he TRPC6dn mu an signi ican ly educed MCF7 and MDA-MB-231 mig a ion as compa ed o cells
ans ec ed wi h emp y ec o (p< 0.05; n = 3).
Cance s 2018, 10, 331 6 o 18
We con i med he ole o TRPC6 in b eas cance cell mig a ion and p oli e a ion by exp essing
a po e-dead dominan -nega i e TRPC6 (TRPC6dn) mu an . As shown in Figu e 4a, exp ession o he
TRPC6dn mu an signi ican ly educed MCF7 and MDA-MB-231 mig a ion as compa ed o cells
ans ec ed wi h emp y ec o (p < 0.05; n = 3).
Figu e 4. Exp ession o TRPC6dn mu an a enua es cell mig a ion and p oli e a ion in b eas cance
cells. (a) MCF7 and MDA-MB-231 cells we e ans ec ed wi h TRPC6dn exp ession plasmid o emp y
ec o (mock), as indica ed. Fo y-eigh hou s a e ans ec ion cells we e subjec ed o wound healing
assay as desc ibed in Me hods. Images we e acqui ed a 0 and 48 h om he beginning o he assay.
The do ed lines de ine he a eas lacking cells. The ba g aphs ep esen he wound size, in
mic ome e s, a he di e en condi ions, exp essed as he mean ± SEM o h ee independen
expe imen s. * p < 0.05 compa ed o he ime = 0h. § p < 0.05 compa ed o he co esponding ime in
mock- ea ed cells. (b) MCF7 and MDA-MB-231 cells we e ans ec ed wi h TRPC6dn exp ession
plasmid o emp y ec o (mock), as indica ed, and 48 h la e cell p oli e a ion was assessed o a
u he 24, 48 and 72 h using he B dU cell p oli e a ion assay ki , as desc ibed in he Ma e ial and
Me hods. Ba g aphs ep esen cell p oli e a ion 0, 24, 48 and 72 h a e cell ans ec ion, p esen ed as
B dU up ake a e. * p < 0.05 compa ed o he co esponding con ol (mock- ans ec ed cells).
Figu e 4.
Exp ession o TRPC6dn mu an a enua es cell mig a ion and p oli e a ion in b eas cance
cells. (
a
) MCF7 and MDA-MB-231 cells we e ans ec ed wi h TRPC6dn exp ession plasmid o emp y
ec o (mock), as indica ed. Fo y-eigh hou s a e ans ec ion cells we e subjec ed o wound healing
assay as desc ibed in Me hods. Images we e acqui ed a 0 and 48 h om he beginning o he assay.
The do ed
lines de ine he a eas lacking cells. The ba g aphs ep esen he wound size, in mic ome e s,
a he di e en condi ions, exp essed as he mean
±
SEM o h ee independen expe imen s.
*p< 0.05
compa ed o he ime = 0 h. § p< 0.05 compa ed o he co esponding ime in mock- ea ed cells.
(b) MCF7
and MDA-MB-231 cells we e ans ec ed wi h TRPC6dn exp ession plasmid o emp y ec o
(mock), as indica ed, and 48 h la e cell p oli e a ion was assessed o a u he 24, 48 and 72 h using
he B dU cell p oli e a ion assay ki , as desc ibed in he Ma e ial and Me hods. Ba g aphs ep esen
cell p oli e a ion 0, 24, 48 and 72 h a e cell ans ec ion, p esen ed as B dU up ake a e. * p< 0.05
compa ed o he co esponding con ol (mock- ans ec ed cells).
Cance s 2018,10, 331 7 o 18
Fu he mo e, exp ession o he TRPC6dn mu an signi ican ly a enua ed MCF7 and MDA-MB-231
cell p oli e a ion a all he imes in es iga ed as compa ed o cells ans ec ed wi h emp y ec o
(Figu e 4b; p< 0.05; n = 3). These indings con i m ha TRPC6 is equi ed o MCF7 and MDA-MB-231
b eas cance cells mig a ion and p oli e a ion.
2.2. Func ional Role o TRPC6 in SOCE in B eas Cance Cell Lines
As ou esul s indica e ha TRPC6 knockdown signi ican ly a enua es ele an ea u es o cance
cells, such as p oli e a ion, mig a ion and
in i o
in asion, we ha e explo ed he possible mechanism
unde lying he unc ional ole o TRPC6 in hese cells. SOCE has been epo ed o play an impo an
ole suppo ing se e al cance hallma ks [
16
,
33
,
34
]. Hence, we ha e e alua ed whe he TRPC6
plays a ole in he ac i a ion o SOCE in b eas cance cells by ans ec ing non- umo al MCF10A
and cance MCF7 and MDA-MB-231 cells wi h shTRPC6 o shRNAc , as con ol. As depic ed in
Figu e 5a–c,
in cells ans ec ed
wi h shRNAc suspended in a Ca
2+
- ee medium, ea men wi h
he SERCA inhibi o TG (1
µ
M) esul ed in a ansien inc ease in cy osolic ee-Ca
2+
concen a ion
due o Ca
2+
elease om he in acellula Ca
2+
s o es. Subsequen addi ion o CaCl
2
(1 mM) o he
ex acellula medium esul ed in a u he inc ease in cy osolic ee-Ca
2+
concen a ion indica i e
o SOCE. TG-induced Ca
2+
elease was simila in all he cell lines in es iga ed while Ca
2+
in lux
was signi ican ly g ea e in MDA-MB-231 cells (Figu e 5g,h; p< 0.05; n = 40 cells/day/3–5 days).
A enua ion o TRPC6 exp ession by cell ans ec ion wi h shTRPC6 signi ican ly inhibi ed SOCE in
MCF7 and MDA-MB-231 cells by 70%, wi hou ha ing any e ec on Ca
2+
elease om he in acellula
s o es (Figu e 5a–c,g–h; p< 0.05). T ans ec ion o MCF10A cells wi h shTRPC6 did no signi ican ly
al e TG-induced Ca
2+
elease o en y, which is consis en wi h he low TRPC6 exp ession a he
p o ein le el in hese cells. Al oge he hese indings indica e ha TRPC6 plays a ele an ole in
he ac i a ion o SOCE in MCF7 and MDA-MB-231 b eas cance cells while his p o ein has no a
de ec able ole in non- umo al MCF10A cells.
Cance s 2018, 10, 331 7 o 18
Fu he mo e, exp ession o he TRPC6dn mu an signi ican ly a enua ed MCF7 and MDA-MB-
231 cell p oli e a ion a all he imes in es iga ed as compa ed o cells ans ec ed wi h emp y ec o
(Figu e 4b; p < 0.05; n = 3). These indings con i m ha TRPC6 is equi ed o MCF7 and MDA-MB-
231 b eas cance cells mig a ion and p oli e a ion.
2.2. Func ional Role o TRPC6 in SOCE in B eas Cance Cell Lines
As ou esul s indica e ha TRPC6 knockdown signi ican ly a enua es ele an ea u es o
cance cells, such as p oli e a ion, mig a ion and in i o in asion, we ha e explo ed he possible
mechanism unde lying he unc ional ole o TRPC6 in hese cells. SOCE has been epo ed o play
an impo an ole suppo ing se e al cance hallma ks [16,33,34]. Hence, we ha e e alua ed whe he
TRPC6 plays a ole in he ac i a ion o SOCE in b eas cance cells by ans ec ing non- umo al
MCF10A and cance MCF7 and MDA-MB-231 cells wi h shTRPC6 o shRNAc , as con ol. As
depic ed in Figu e 5a–c, in cells ans ec ed wi h shRNAc suspended in a Ca2+- ee medium,
ea men wi h he SERCA inhibi o TG (1 µM) esul ed in a ansien inc ease in cy osolic ee-Ca2+
concen a ion due o Ca2+ elease om he in acellula Ca2+ s o es. Subsequen addi ion o CaCl2 (1
mM) o he ex acellula medium esul ed in a u he inc ease in cy osolic ee-Ca2+ concen a ion
indica i e o SOCE. TG-induced Ca2+ elease was simila in all he cell lines in es iga ed while Ca2+
in lux was signi ican ly g ea e in MDA-MB-231 cells (Figu e 5g,h; p < 0.05; n = 40 cells/day/3-5 days).
A enua ion o TRPC6 exp ession by cell ans ec ion wi h shTRPC6 signi ican ly inhibi ed SOCE in
MCF7 and MDA-MB-231 cells by 70%, wi hou ha ing any e ec on Ca2+ elease om he in acellula
s o es (Figu es 5a–c and 4g–h; p < 0.05). T ans ec ion o MCF10A cells wi h shTRPC6 did no
signi ican ly al e TG-induced Ca2+ elease o en y, which is consis en wi h he low TRPC6
exp ession a he p o ein le el in hese cells. Al oge he hese indings indica e ha TRPC6 plays a
ele an ole in he ac i a ion o SOCE in MCF7 and MDA-MB-231 b eas cance cells while his
p o ein has no a de ec able ole in non- umo al MCF10A cells.
Figu e 5. Con .
Figu e 5. Con .
Cance s 2018,10, 331 8 o 18
Cance s 2018, 10, 331 8 o 18
Figu e 5. TRPC6 is equi ed o s o e-ope a ed Ca2+ en y in b eas cance cell lines. (a–c) MCF10A,
MCF7 and MDA-MB-231 cells we e ans ec ed wi h shTRPC6 o sc amble plasmid (shRNAc ), as
indica ed. Fo y-eigh hou s a e ans ec ion, u a-2-loaded cells we e pe used wi h a Ca2+- ee
medium (100 µM EGTA added) and hen s imula ed wi h TG (1 µM) ollowed by ein oduc ion o
ex e nal Ca2+ ( inal concen a ion 1 mM) o ini ia e Ca2+ en y. Da a a e mean ± SEM o 40 cells/day/3-
5 days. (d– ) MCF7 and MDA-MB-231 cells we e ans ec ed wi h TRPC6dn mu an exp ession
plasmid o emp y ec o (mock), as indica ed. A e 48 h cells we e lysed and subjec ed o wes e n
blo ing wi h an i-TRPPC6 an ibody, ollowed by ep obing wi h an i-β-ac in an ibody o p o ein
loading con ol (d). Molecula masses indica ed on he igh we e de e mined using molecula -mass
ma ke s un in he same gel. (e and ) Fo y-eigh hou s a e ans ec ion, u a-2-loaded cells we e
pe used wi h a Ca2+- ee medium (100 µM EGTA added) and hen s imula ed wi h TG (1 µM)
ollowed by ein oduc ion o ex e nal Ca2+ ( inal concen a ion 1 mM) o ini ia e Ca2+ en y. Da a a e
mean ± SEM o 40 cells/day/3-5 days. Ba g aphs ep esen TG-induced Ca2+ elease (g) and en y (h)
in MCF10A, MCF7 and MDA-MB-231 cells un ea ed o ans ec ed wi h he indica ed plasmids. Da a
a e exp essed as mean ± SEM and p esen ed as pe cen age o con ol (MCF10A cells ea ed wi h
sc amble plasmid). * ep esen s p < 0.05 as compa ed o sc amble- ea ed MCF10A cells. § ep esen s
p < 0.05 as compa ed o he same cell line ans ec ed wi h shRNAc .
In o de o u he explo e whe he he obse ed e ec depends on ca ion en y h ough he
channel o i is a he associa ed o a mechanism in ol ing he exp ession o he p o ein i sel , we
o e exp essed he TRPC6dn mu an in MCF7 and MDA-MB-231 cells and looked o i s e ec on TG-
induced Ca2+ elease and en y. As shown in Figu e 5d, TRPC6dn was e icien ly exp essed in bo h
cell ypes. As depic ed in Figu es 5e–h, o e exp ession o TRPC6dn in MCF7 and MDA-MB-231 cells
signi ican ly educed TG-e oked Ca2+ en y o a simila ex en o ans ec ion o shTRPC6 (p < 0.05 as
compa ed o con ol; n = 40 cells/day/3–5 days), which indica es ha ca ion in lux h ough TRPC6
plays an impo an ole in SOCE in hese cells. O e exp ession o TRPC6dn also esul ed in a
signi ican dec ease in he abili y o MCF7 cells o accumula e Ca2+ in o TG-sensi i e s o es (Figu e
5e,g; p < 0.05; n = 40 cells/day/3–5 days), an e ec ha migh be a ibu ed o he inhibi ion o SOCE.
Figu e 5.
TRPC6 is equi ed o s o e-ope a ed Ca
2+
en y in b eas cance cell lines. (
a
–
c
) MCF10A,
MCF7 and MDA-MB-231 cells we e ans ec ed wi h shTRPC6 o sc amble plasmid (shRNAc ),
as indica ed
. Fo y-eigh hou s a e ans ec ion, u a-2-loaded cells we e pe used wi h a Ca
2+
- ee
medium (100
µ
M EGTA added) and hen s imula ed wi h TG (1
µ
M) ollowed by ein oduc ion
o ex e nal Ca
2+
( inal concen a ion 1 mM) o ini ia e Ca
2+
en y. Da a a e mean
±
SEM o
40 cells/day/3–5 days
. (
d
–
) MCF7 and MDA-MB-231 cells we e ans ec ed wi h TRPC6dn mu an
exp ession plasmid o emp y ec o (mock), as indica ed. A e 48 h cells we e lysed and subjec ed
o wes e n blo ing wi h an i-TRPPC6 an ibody, ollowed by ep obing wi h an i-
β
-ac in an ibody
o p o ein loading con ol (
d
). Molecula masses indica ed on he igh we e de e mined using
molecula -mass ma ke s un in he same gel. (
e
and
) Fo y-eigh hou s a e ans ec ion, u a-2-loaded
cells we e pe used wi h a Ca
2+
- ee medium (100
µ
M EGTA added) and hen s imula ed wi h TG
(1
µ
M) ollowed by ein oduc ion o ex e nal Ca
2+
( inal concen a ion 1 mM) o ini ia e Ca
2+
en y.
Da a a e mean
±
SEM o 40 cells/day/3–5 days. Ba g aphs ep esen TG-induced Ca
2+
elease (
g
)
and en y (
h
) in MCF10A, MCF7 and MDA-MB-231 cells un ea ed o ans ec ed wi h he indica ed
plasmids. Da a a e exp essed as mean
±
SEM and p esen ed as pe cen age o con ol (MCF10A cells
ea ed wi h sc amble plasmid). * ep esen s p< 0.05 as compa ed o sc amble- ea ed MCF10A cells.
§ ep esen s p< 0.05 as compa ed o he same cell line ans ec ed wi h shRNAc .
In o de o u he explo e whe he he obse ed e ec depends on ca ion en y h ough he
channel o i is a he associa ed o a mechanism in ol ing he exp ession o he p o ein i sel , we
o e exp essed he TRPC6dn mu an in MCF7 and MDA-MB-231 cells and looked o i s e ec on
TG-induced Ca
2+
elease and en y. As shown in Figu e 5d, TRPC6dn was e icien ly exp essed in bo h
cell ypes. As depic ed in Figu e 5e–h, o e exp ession o TRPC6dn in MCF7 and MDA-MB-231 cells
signi ican ly educed TG-e oked Ca
2+
en y o a simila ex en o ans ec ion o shTRPC6 (
p< 0.05
as
compa ed o con ol; n = 40 cells/day/3–5 days), which indica es ha ca ion in lux h ough TRPC6
plays an impo an ole in SOCE in hese cells. O e exp ession o TRPC6dn also esul ed in a signi ican
dec ease in he abili y o MCF7 cells o accumula e Ca
2+
in o TG-sensi i e s o es (Figu e 5e,g; p< 0.05;
n = 40 cells/day/3–5 days), an e ec ha migh be a ibu ed o he inhibi ion o SOCE.
Cance s 2018,10, 331 9 o 18
2.3. TRPC6 Exp ession Is Requi ed o Plasma Memb ane Localiza ion o O ai1 and O ai3 in B eas
Cance Cells
B eas cance MCF7 and MDA-MB-231 cells ha e been epo ed o exp ess bo h O ai1 and O ai3
channels. Howe e , he ela i e exp ession le el and unc ion di e s om ER
+
MCF7 cells o iple
nega i e MDA-MB-231 cells [
35
]. While SOCE in MDA-MB-231 cells en i ely depends on O ai1, MCF7
SOCE is mainly media ed by O ai3, whose exp ession, egula ed by ER
α
[
17
], is p edominan o e
ha o O ai1 [
35
]. Ou esul s con i m ha O ai1 is o e exp essed in he b eas cance cell lines and
ha O ai3 exp ession is signi ican ly enhanced in MCF7 (Figu e 6a; p< 0.05; n = 6), as p e iously
epo ed [
35
]. In o de o explo e he mechanism unde lying he sensi i i y o SOCE o TRPC6
exp ession and unc ion we ha e i s in es iga ed he in e ac ion o TRPC6 wi h O ai1 and O ai3 by
co-immunop ecipi a ion om MCF7 and MDA-MB-231 cell lysa es. Res ing and TG- ea ed cells we e
used o his s udy o de e mine whe he Ca
2+
s o e deple ion plays any ole in he possible in e ac ion
be ween TRPC6 and he O ai p o eins in es iga ed. As shown in Figu e 6b,c, immunop ecipi a ion
o cell lysa es wi h an i-TRPC6 an ibody ollowed by Wes e n blo ing wi h an i-O ai1 o an i-O ai3
an ibody e eals ha TRPC6 in e ac s wi h bo h p o eins in es ing cells. In e es ingly, ou esul s
sugges ha in MCF7 cells he in e ac ion o TRPC6 wi h O ai3 is appa en ly g ea e han wi h O ai1,
and, con e sely, in MDA-MB-231 cells, TRPC6 seems o in e ac p edominan ly wi h O ai1 o e O ai3
(Figu e 6b,c; n = 6). Al hough hese appa en di e ences migh be a ibu ed o he use o wo di e en
an ibodies, i we look a he associa ion o TRPC6 wi h O ai1, whose exp ession we and o he s ha e
ound o be simila in MCF7 and MDA-MB-231 cells [
35
] (Figu e 6a), and we no malize he da a
wi h he amoun o TRPC6 pulled down, ou esul s indica e ha mo e O ai1 is bound o TRPC6 in
MDA-MB-231 cells (p< 0.05; n = 6). In addi ion, we ha e ound ha he in e ac ion o TRPC6 wi h
O ai1 and O ai3 is no al e ed by ea men wi h 1
µ
M TG o 3 min (Figu e 6b,c), which, as depic ed
in Figu e 5, is able o induce signi ican s o e deple ion.
Cance s 2018, 10, 331 9 o 18
2.3. TRPC6 Exp ession Is Requi ed o Plasma Memb ane Localiza ion o O ai1 and O ai3 in B eas Cance
Cells
B eas cance MCF7 and MDA-MB-231 cells ha e been epo ed o exp ess bo h O ai1 and O ai3
channels. Howe e , he ela i e exp ession le el and unc ion di e s om ER+ MCF7 cells o iple
nega i e MDA-MB-231 cells [35]. While SOCE in MDA-MB-231 cells en i ely depends on O ai1,
MCF7 SOCE is mainly media ed by O ai3, whose exp ession, egula ed by ER [17], is p edominan
o e ha o O ai1 [35]. Ou esul s con i m ha O ai1 is o e exp essed in he b eas cance cell lines
and ha O ai3 exp ession is signi ican ly enhanced in MCF7 (Figu e 6a; p < 0.05; n = 6), as p e iously
epo ed [35]. In o de o explo e he mechanism unde lying he sensi i i y o SOCE o TRPC6
exp ession and unc ion we ha e i s in es iga ed he in e ac ion o TRPC6 wi h O ai1 and O ai3 by
co-immunop ecipi a ion om MCF7 and MDA-MB-231 cell lysa es. Res ing and TG- ea ed cells
we e used o his s udy o de e mine whe he Ca2+ s o e deple ion plays any ole in he possible
in e ac ion be ween TRPC6 and he O ai p o eins in es iga ed. As shown in Figu e 6b,c,
immunop ecipi a ion o cell lysa es wi h an i-TRPC6 an ibody ollowed by Wes e n blo ing wi h
an i-O ai1 o an i-O ai3 an ibody e eals ha TRPC6 in e ac s wi h bo h p o eins in es ing cells.
In e es ingly, ou esul s sugges ha in MCF7 cells he in e ac ion o TRPC6 wi h O ai3 is appa en ly
g ea e han wi h O ai1, and, con e sely, in MDA-MB-231 cells, TRPC6 seems o in e ac
p edominan ly wi h O ai1 o e O ai3 (Figu e 6b,c; n = 6). Al hough hese appa en di e ences migh
be a ibu ed o he use o wo di e en an ibodies, i we look a he associa ion o TRPC6 wi h O ai1,
whose exp ession we and o he s ha e ound o be simila in MCF7 and MDA-MB-231 cells [35]
(Figu e 6a), and we no malize he da a wi h he amoun o TRPC6 pulled down, ou esul s indica e
ha mo e O ai1 is bound o TRPC6 in MDA-MB-231 cells (p < 0.05; n = 6). In addi ion, we ha e ound
ha he in e ac ion o TRPC6 wi h O ai1 and O ai3 is no al e ed by ea men wi h 1 µM TG o 3
min (Figu e 6b,c), which, as depic ed in Figu e 5, is able o induce signi ican s o e deple ion.
Figu e 6. Con .
Figu e 6. Con .
Cance s 2018,10, 331 16 o 18
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