Adenylyl Cyclase Type 8 Overexpression Impairs Phosphorylation-Dependent Orai1 Inactivation and Promotes Migration in MDA-MB-231 Breast Cancer Cells

Cance s 2019, 11, 1624; doi:10.3390/cance s11111624 www.mdpi.com/jou nal/cance s A icle Adenylyl Cyclase Type 8 O e exp ession Impai s Phospho yla ion-Dependen O ai1 Inac i a ion and P omo es Mig a ion in MDA-MB-231 B eas Cance Cells Jose Sanchez-Collado 1,† , Jose J. Lopez 1,†, * , Isaac Ja din 1 , Ped o J. Camello 2 , Debo a Falcon 3 , Se gio Regodon 1 , Gines M. Salido 1 , Ta ik Smani 3 and Juan A. Rosado 1, * 1 Depa men o Physiology, (Cellula Physiology Resea ch G oup), Ins i u e o Molecula Pa hology Bioma ke s, Uni e si y o Ex emadu a, 10003 Cace es, Spain 2 Depa men o Physiology, (Smoo h Muscle Physiology Resea ch G oup), Ins i u e o Molecula Pa hology Bioma ke s, Uni e si y o Ex emadu a, 10003 Cace es, Spain 3 Depa men o Medical Physiology and Biophysics, Ins i u e o Biomedicine o Se illa, 41013 Se illa, Spain † These au ho s con ibu ed equally o his wo k. * Co espondence: [email protected] (J.J.L.); [email p o ec ed] (J.A.R.); T el.: +34-927-257-100 (ex 51376) (J.A.R. and J.J.L.); Fax: +34-927-257-110 (J.A.R. and J.J.L.) Recei ed: 10 Oc obe 2019; Accep ed: 21 Oc obe 2019; Published: 23 Oc obe 2019 Abs ac : O ai1 plays a majo ole in s o e-ope a ed Ca 2+ en y (SOCE) in iple-nega i e b eas cance (TNBC) cells. This channel is inac i a ed ia di e en mechanisms, including p o ein kinase C (PKC) and p o ein kinase A (PKA)-dependen phospho yla ion a Se -27 and Se -30 o Se -34, espec i ely, which shapes he Ca 2+ esponses o agonis s. The Ca 2+ calmodulin-ac i a ed adenylyl cyclase ype 8 (AC8) was epo ed o in e ac di ec ly wi h O ai1, hus media ing a dynamic in e play be ween he Ca 2+ - and cyclic adenosine monophospha e (cAMP)-dependen signaling pa hways. He e, we show ha he b eas cance cell lines MCF7 and MDA-MB-231 exhibi enhanced exp ession o O ai1 and AC8 as compa ed o he non- umo al b eas epi helial MCF10A cell line. In hese cells, AC8 in e ac s wi h he O ai1α a ian in a manne ha is no egula ed by O ai1 phospho yla ion. AC8 knockdown in MDA-MB-231 cells, using wo di e en small in e e ing RNAs (siRNAs), a enua es hapsiga gin (TG)-induced Ca 2+ en y and also Ca 2+ in lux media ed by co-exp ession o O ai1 and he O ai1-ac i a ing small agmen (OASF) o STIM1 (s omal in e ac ion molecule-1). Con e sely, AC8 o e exp ession enhances SOCE, as well as Ca 2+ en y, in cells co-exp essing O ai1 and OASF. In MDA-MB-231 cells, we ound ha AC8 o e exp ession educes he O ai1 phosphose ine con en , hus sugges ing ha AC8 in e e es wi h O ai1 se ine phospho yla ion, which akes place a esidues loca ed in he AC8-binding si e. Consis en wi h his, he subse o O ai1 associa ed wi h AC8 in naï e MDA-MB-231 cells is no phospho yla ed in se ine esidues in con as o he AC8-independen O ai1 subse . AC8 exp ession knockdown a enua es mig a ion o MCF7 and MDA-MB-231 cells, while his maneu e has no e ec in he MCF10A cell line, which is likely a ibu ed o he low exp ession o AC8 in hese cells. We ound ha AC8 is equi ed o FAK ( ocal adhesion kinase) phospho yla ion in MDA-MB-231 cells, which migh explain i s ole in cell mig a ion. Finally, we ound ha AC8 is equi ed o TNBC cell p oli e a ion. These indings indica e ha o e exp ession o AC8 in b eas cance MDA-MB-231 cells impai s he phospho yla ion-dependen O ai1 inac i a ion, a mechanism ha migh suppo he enhanced abili y o hese cells o mig a e. Keywo ds: o ai1α; adenylyl cyclase 8; s o e-ope a ed calcium en y; b eas cance cells; mig a ion Cance s 2019, 11, 1624 2 o 24 1. In oduc ion B eas cance is one o he mos common malignancies in women wo ldwide. Among he di e en sub ypes, iple-nega i e b eas cance (TNBC) is mo e agg essi e and exhibi s a poo e p ognosis han o he ypes o b eas cance . Immunohis ochemically, TNBC is cha ac e ized by he lack o es ogen and p oges e one ecep o s o excess HER2 (human epide mal g ow h ac o ecep o 2) exp ession. Consequen ly, his ype o cance is esis an o ho monal he apies and chemicals ha a ge he HER2 ecep o [1]. S udies in TNBC cells e ealed ha Ca2+ signaling is emodeled and plays a key unc ional ole [2–5]. TNBC cells o e exp ess O ai1, which is esponsible o he ac i a ion o s o e-ope a ed Ca2+ en y (SOCE) [6]. O ai1 is a well-cha ac e ized egula o o he p oli e a ion and mig a ion o many TNBC cells, including he MDA-MB-231 cell line [7–9]. O ai1 is he po e- o ming subuni o he highly Ca2+-selec i e CRAC (Ca2+- elease ac i a ed Ca2+) channel, he bes cha ac e ized s o e-ope a ed channel [10–12]. The CRAC channel is ac i a ed by he endoplasmic e iculum Ca2+ senso STIM1 upon discha ge o he in acellula Ca2+ s o es, and he in lux o Ca2+ h ough he channel is modula ed by he egula ion o STIM1 by p o eins like SARAF (SOCE-associa ed egula o y ac o ) [13–15], as well as by CRAC channel inac i a ion. CRAC cu en s unde go Ca2+-dependen inac i a ion o p e en excessi e Ca2+ in lux. Two di e en mechanisms, e med as Ca2+-dependen inac i a ion (FCDI) ha occu s wi hin milliseconds [16] and slow Ca2+- dependen inac i a ion (SCDI) ha commences ens o seconds a e O ai1 ac i a ion [17], we e desc ibed, al hough he p ecise mechanism emains unclea . Two a ian s o O ai1, gene a ed by al e na i e ansla ion ini ia ion, we e ecen ly iden i ied [18], a long o m e med O ai1α o app oxima ely 33 kDa and a sho o m, O ai1β, lacking amino acids 1–63, o app oxima ely 23 kDa. O ai1α exhibi s a g ea e FCDI, hus sugges ing ha he N- e minal 63 amino acids migh play a ele an ole in his p ocess [11]. A ecen s udy epo ed di ec in e ac ion o O ai1 wi h he Ca2+ calmodulin-ac i a ed adenylyl cyclase ype 8 (AC8) [19]. Willoughby e al. speci ically iden i ied in e ac ion o he N- e minal egion o AC8 wi h esidues 26–34 o he O ai1 N- e minus [19]. This sequence, exclusi ely p esen in he O ai1α a ian , o e laps wi h h ee O ai1 phospho yla ion si es, Se -27, -30, and -34. Se -27 and -30 a e phospho yla ed by PKC in i o and in i o, leading o s ong inac i a ion o CRAC channel unc ion and SOCE [20]. On he o he hand, a ecen s udy demons a ed ha AC8 media es CRAC inac i a ion by phospho yla ion o O ai1 a Se -34 [21]. The unc ional in e ac ion be ween AC8 and O ai1 e eals a inely egula ed in e play be ween he cAMP and Ca2+ signaling pa hways. He e, we show ha TNBC MDA-MB-231 cells o e exp ess O ai1 and AC8, wi h p edominan o e exp ession o AC8 o e O ai1. In e ac ion o AC8 wi h O ai1 in e e es wi h phospho yla ion o he la e , p obably due o o e lapping o he phospho yla ion si es wi h he AC8-binding sequence o O ai1. In MDA-MB-231 cells, silencing o AC8 esul s in a enua ion o SOCE, while AC8 o e exp ession enhances Ca2+ in lux, hus sugges ing ha AC8 impai s he inac i a ion o O ai1. AC8 was also ound o be equi ed o b eas cance cell mig a ion, hus sugges ing ha AC8, by enhancing cAMP le els and/o Ca2+ in lux, plays an impo an unc ional ole in b eas cance cells. 2. Resul s 2.1. Exp ession and In e ac ion o O ai1α and AC8 in Non-Tumo al and B eas Cance Cell Lines Consis en wi h p e ious s udies [3,6], Wes e n blo analysis o whole-cell lysa es om he non- umo al b eas epi helial MCF10A cell line and he es ogen ecep o posi i e (ER+) and TNBC cell lines MCF7 and MDA-MB-231, espec i ely, wi h a speci ic an i-human O ai1 an ibody e ealed a low exp ession o O ai1 in MCF10A cells and a signi ican ly highe exp ession o his p o ein in b eas cance cells (Figu e 1a,b; p < 0.05; n = 6). The inc eased exp ession o O ai1 in he b eas cance cell lines is consis en wi h he high exp ession o his p o ein in cance ous issue [22]. As shown in Figu e 1c,d, Wes e n blo analysis o whole-cell lysa es om MCF10A, MCF7, and MDA-MB-231 cells wi h a speci ic an i-AC8 an ibody e ealed ha his p o ein is sca cely exp essed in he non- umo al cell line, while i is highly exp essed in MCF7 and MDA-MB-231 b eas cance cells. The O ai1 and AC8 exp ession no malized o he β-ac in con en indica es ha O ai1 exp ession was 371 ± 12 and 393 ± Cance s 2019, 11, 1624 3 o 24 22% o ha in MCF10A cells in MCF7 and MDA-MB-231 cells, espec i ely, while he AC8 exp ession was 611 ± 75 and 621 ± 98% o ha in MCF10A cells in MCF7 and MDA-MB-231 cells, espec i ely; he e o e, he quan i a i e analysis indica ed ha AC8 o e exp ession in b eas cance cells is signi ican ly g ea e han ha o O ai1. P e ious s udies e ealed a unc ional ela ionship be ween O ai1 and AC8 [19,21]; hence, we nex explo ed he in e ac ion be ween bo h p o eins in he non- umo al and umo al b eas cell lines by co-immunop ecipi a ion o cell lysa es wi h an i-O ai1 an ibody, ollowed by Wes e n blo ing wi h an i-AC8 an ibody. The expe imen s we e pe o med in es ing cells as his in e ac ion was p e iously shown o be cons i u i e [19]. Ou esul s indica ed ha , while a de ec able in e ac ion was app ecia ed in non- umo al cells, he co-immunop ecipi a ion be ween O ai1 and AC8 was signi ican ly g ea e in MCF7 and MDA-MB-231 cells (Figu e 1e, ; p < 0.05; n = 6). Figu e 1. Exp ession and in e ac ion o O ai1 a ian s wi h Ca2+ calmodulin-ac i a ed adenylyl cyclase ype 8 (AC8) in non- umo al and b eas cance cell lines. (a–d) Non- umo al b eas epi helial MCF10A and b eas cance MCF7 and MDA-MB-231 cells we e lysed and subjec ed o Wes e n blo ing wi h an i-O ai1 (a) o an i-AC8 (c) an ibody, ollowed by ep obing wi h an i-β-ac in an ibody o p o ein loading con ol (b and d). The box-and-whiske plo s (o box plo s) ep esen O ai1 (b) o AC8 (d) exp ession no malized o he β-ac in con en . Molecula masses indica ed on he Cance s 2019, 11, 1624 4 o 24 igh we e de e mined using molecula -mass ma ke s un in he same gel; * p < 0.05 compa ed o he exp ession in MCF10A cells. (e) MCF10A, MCF7, and MDA-MB-231 cells we e lysed, and whole-cell lysa es we e immunop ecipi a ed (IP) wi h an i-O ai1 an ibody. Immunop ecipi a es we e subjec ed o 10% SDS-PAGE and subsequen Wes e n blo ing wi h speci ic an i-AC8 an ibody, as indica ed. Memb anes we e ep obed wi h he an ibody used o immunop ecipi a ion o p o ein loading con ol. The panels show esul s om one expe imen ep esen a i e o i e o he s. Molecula masses indica ed on he igh we e de e mined using molecula -mass ma ke s un in he same gel. ( ) The box plo ep esen s he quan i ica ion o AC8–O ai1 in e ac ion in es ing cells. Resul s a e p esen ed as a bi a y op ical densi y uni s, and exp essed no malized o he O ai1 exp ession. (g) MCF10A, MCF7, and MDA-MB-231 cells we e lysed, and whole-cell lysa es we e ea ed wi h N-glycosidase F (PNGaseF) and esol ed by 10% SDS-PAGE. The blo s we e p obed wi h an i-O ai1 an ibody and an i-β-ac in an ibody o 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. (h) The box plo ep esen s O ai1α o O ai1β exp ession no malized o he β-ac in con en . (i) The box plo ep esen s he O ai1α/O ai1β exp ession a io in he h ee cell lines in es iga ed; * p < 0.05 compa ed o he exp ession in MCF10A cells, $ p < 0.05 compa ed o he exp ession in MCF7 cells. (j) MDA-MB-231 cells we e ea ed wi h hapsiga gin (TG; 1 µM) o 1 min o le un ea ed (C), as indica ed, and lysed, and whole-cell lysa es we e immunop ecipi a ed (IP) wi h an i-AC8 an ibody o subjec ed o Wes e n blo ing wi h an i-O ai1 an ibody (WCL). Immunop ecipi a es we e ea ed wi h PNGaseF and hen subjec ed o 10% SDS- PAGE and subsequen Wes e n blo ing wi h speci ic an i-O ai1 an ibody, as indica ed. Memb anes we e ep obed wi h he an ibody used o immunop ecipi a ion o p o ein loading con ol. The panels show esul s om one expe imen ep esen a i e o i e o he s. Molecula masses indica ed on he igh we e de e mined using molecula -mass ma ke s un in he same gel. AC8 was epo ed o bind o an N- e minal sequence o O ai1 loca ed be ween amino acids 26 and 34, which con ains h ee se ines (27, 30, and 34) [23]. This sequence is only p esen in he mammalian-speci ic ull-leng h O ai1α a ian and is absen in he sho O ai1 a ian , O ai1β [18]; hus, AC8 was epo ed o in e ac solely wi h O ai1α [21]. We assessed he exp ession o O ai1α and O ai1β in he h ee b eas de i ed cell lines. The na i e O ai1 a ian exp ession was analyzed by Wes e n blo ing a e p o ein deglycosyla ion wi h PNGaseF. As shown in Figu e 1g, wo dis inc bands wi h lowe molecula weigh han glycosyla ed O ai1 we e de ec ed, co esponding o O ai1α and O ai1β. Ou esul s indica ed ha bo h O ai1 a ian s we e highly exp essed in he b eas cance MCF7 and MDA-MB-231 cell lines as compa ed o non- umo al MCF10A cells (Figu e 1h; p < 0.05; n = 6). Fu he mo e, we ound ha he exp ession o O ai1α was signi ican ly g ea e in MDA-MB-231 cells han in MCF7 cells (Figu e 1h; p < 0.05) while no di e ences we e de ec ed in he O ai1β exp ession among he cance cell lines in es iga ed. The la e migh explain he g ea e O ai1α/O ai1β exp ession a io in MDA-MB-231 cells as compa ed o MCF7 cells (Figu e 1i; p < 0.05; n = 6). The analysis o he exp ession a io be ween he O ai1 a ian s indica es a g ea e exp ession o O ai1β in all he cell ypes in es iga ed and, in e es ingly, a g ea e O ai1α/O ai1β exp ession a io in MCF7 and MDA-MB-231 b eas cance cells han in non- umo al MCF10A cells (Figu e 1i; p < 0.05; n = 6). We u he explo ed he in e ac ion o AC8 and O ai1α in MDA-MB-231 cells by co- immunop ecipi a ion o cell lysa es wi h an i-AC8 an ibody, ollowed by ea men o he immunop ecipi a es wi h PNGaseF and Wes e n blo ing wi h an i-O ai1 an ibody. As shown in Figu e 1j, ou esul s indica e ha O ai1α, bu no O ai1β, co-immunop ecipi a es wi h AC8 in es ing condi ions, con i ming p e ious esul s [19,21]. This in e ac ion was no modi ied by ea men o 1 min wi h he sa co/endoplasmic e iculum Ca2+ ATPase (SERCA) inhibi o hapsiga gin (TG; 1 µM), which is in ag eemen wi h a p e ious s udy by Willoughby and cowo ke s sugges ing a cons i u i e in e ac ion be ween bo h p o eins [19]. 2.2. The In e ac ion be ween O ai1 and AC8 Is Pa ially Dependen on Ca2+ In lux and O ai1 Phospho yla ion a Se -27 and -30 In o de o explo e he mechanism egula ing he O ai1–AC8 in e ac ion, we es ed he possible Ca2+ dependency o his e en . To add ess his issue, we assessed he ole o Ca2+ eleased om he Cance s 2019, 11, 1624 5 o 24 in acellula s o es and Ca2+ en y h ough O ai1 in he O ai1–AC8 co-immunop ecipi a ion. Hence, we es ed O ai1–AC8 in e ac ion in es ing cells o in cells ea ed wi h TG, o induce ne Ca2+ elease om in acellula Ca2+ s o es, suspended ei he in he p esence o 1 mM ex acellula Ca2+ o in a Ca2+- ee medium. When indica ed, cells we e loaded wi h dime hyl BAPTA, and suspended ei he in a Ca2+- ee medium, o p e en ises in cy osolic Ca2+ concen a ion ([Ca2+]c) induced by bo h Ca2+ elease and en y, o in he p esence o 1 mM ex e nal Ca2+, o p e en ises in [Ca2+]c due o Ca2+ elease bu allowing ises in Ca2+ concen a ion in he icini y o he O ai1 channels. Ou esul s indica ed ha , in he p esence o ex acellula Ca2+, he e was a de ec able O ai1– AC8 associa ion, which was una ec ed by BAPTA loading o ea men wi h TG (Figu e 2a,b; n = 6), as p e iously epo ed [19]. Figu e 2c–e depic a de ec able inc ease in he Ca2+ concen a ion in he O ai1 icini y in cells no loaded wi h BAPTA, as well as, wi h less in ensi y bu s ill signi ican , in BAPTA-loaded cells as de ec ed wi h G-GECO1.2-O ai1 [24] (p < 0.05; n = 6), and simila esul s we e ob ained using he nea plasma memb ane Ca2+ indica o u a-FFP18 ( he ini ial slopes o he inc ease in u a-FFP18 luo escence a io we e 1.0299 ± 0.1325 and 0.3793 ± 0.0205 in con ol and BAPTA- loaded cells, espec i ely, and he maximal u a-2 luo escence a ios we e 0.20 ± 0.01 and 0.11 ± 0.01 in con ol and BAPTA-loaded cells, espec i ely, Figu e S1, Supplemen a y Ma e ials), which demons a e ha de ec able ises in Ca2+ concen a ion in he O ai1 mic odomain due o Ca2+ in lux ia O ai1 we e s ill de ec able in BAPTA-loaded cells. Cell loading wi h dime hyl BAPTA was wi hou a signi ican e ec on he es ing u a-FFP18 luo escence a io ( he es ing a ios we e 0.60 ± 0.03 and 0.61 ± 0.01 in con ol and BAPTA-loaded cells, espec i ely). When Ca2+ en y was no allowed, ea men wi h TG esul ed in a educ ion in he O ai1–AC8 in e ac ion, a esponse ha was main ained when he ise in [Ca2+]c due o Ca2+ e lux om he s o es was p e en ed by BAPTA loading (Figu e 2a,b; n = 6). These indings sugges ha Ca2+ s o e deple ion i sel plays an inhibi o y ole in he O ai1–AC8 associa ion ha was o e come by Ca2+ in lux ia O ai1 channels. The e o e, upon agonis s imula ion, he O ai1–AC8 in e ac ion is s ongly dependen on Ca2+ in lux h ough he channel. O ai1 is phospho yla ed by PKC a esidues Se -27 and Se -30, an e en ha nega i ely egula es O ai1 unc ion [20]. As bo h se ine esidues a e loca ed wi hin he O ai1 AC8-binding egion, we explo ed whe he phospho yla ion a Se -27 and Se -30 al e s O ai1–AC8 in e ac ion. To in es iga e his issue, cells we e ans ec ed wi h yellow luo escen p o ein (YFP)-O ai1, he non- phospho yla able O ai1S27A/S30A mu an , o he phosphomime ic O ai1S27D/S30D mu an , o hey we e mock- ea ed, and he O ai1–AC8 in e ac ion was analyzed by co-immunop ecipi a ion om cell lysa es. Figu e 3, bo om panel, depic s ha exp ession o YFP-O ai1 p oduced a band o he p edic ed size (app oxima ely 60 kDa). Fu he mo e, exp ession o he O ai1S27A/S30A and O ai1S27D/S30D mu an s yielded se e al bands, one a he size o he na i e O ai1 and o he small size bands which migh be a ibu ed o O ai1 wi hou pos - ansla ional modi ica ions. As shown in Figu e 3, ou esul s indica ed ha AC8 was able o co-immunop ecipi a e wi h YFP-O ai1, as well as wi h he O ai1S27A/S30A and O ai1S27D/S30D mu an s. The e o e, hese indings indica e ha phospho yla ion o O ai1 a Se -27 and Se -30 is unlikely o in e e e wi h O ai1 binding o AC8. Cance s 2019, 11, 1624 6 o 24 Figu e 2. Role o Ca2+ mobiliza ion in he O ai1–AC8 in e ac ion. (a) MDA-MB-231 cells we e loaded wi h dime hyl BAPTA o le un ea ed, as indica ed, and hen suspended in a medium con aining 1 mM Ca2+ o in a Ca2+- ee medium (100 µM EGTA added). Cells we e ea ed wi h 1 µM TG o he ehicle, as indica ed, and lysed 1 min la e . Whole-cell lysa es we e immunop ecipi a ed (IP) wi h an i-O ai1 an ibody. Immunop ecipi a es we e subjec ed o 10% SDS-PAGE and subsequen Wes e n blo ing wi h speci ic an i-AC8 an ibody, as indica ed. Memb anes we e ep obed wi h he an ibody used o immunop ecipi a ion o p o ein loading con ol. The panels show esul s om one expe imen ep esen a i e o i e o he s. Molecula masses indica ed on he igh we e de e mined using molecula -mass ma ke s un in he same gel. (b) The box plo ep esen s he quan i ica ion o AC8–O ai1 in e ac ion in es ing and TG- ea ed cells. Resul s a e no malized o he O ai1 exp ession; * p < 0.05 compa ed o he co esponding con ol (un ea ed cells). (c) Cells we e ans ec ed wi h G- GECO1.2-O ai1. Fo y-eigh hou s la e , cells we e suspended in a Ca2+- ee medium and s imula ed wi h TG (1 µM) o 2 min, ollowed by addi ion o CaCl2 ( inal concen a ion 1 mM) o he medium o ini ia e Ca2+ en y. Images a e ep esen a i e o six independen expe imen s. (d–e) Ba g aphs ep esen he quan i ica ion o he G-GECO (g een gene ically encoded Ca2+ indica o o op ical imaging) luo escence in cells loaded wi h dime hyl BAPTA (BAPTA) o le un ea ed (con ol), as indica ed. Fluo escence was analyzed a es , 30 s a e he addi ion o 1 µM TG and 30 s a e he subsequen addi ion o CaCl2 ( inal concen a ion 1 mM). Cance s 2019, 11, 1624 7 o 24 Figu e 3. Role o O ai1 phospho yla ion a Se -27 and Se -30 in he O ai1–AC8 in e ac ion. MDA-MB- 231 cells we e ans ec ed wi h pEYPF-O ai1, o he O ai1S27A/S30A and O ai1S27D/S30D mu an s, o hey we e mock- ea ed, as indica ed. Cells we e hen ea ed wi h 1 µM TG o he ehicle (C) and lysed 1 min la e . Whole-cell lysa es we e immunop ecipi a ed (IP) wi h an i-AC8 an ibody. Immunop ecipi a es we e subjec ed o 10% SDS-PAGE and subsequen Wes e n blo ing wi h speci ic an i-O ai1 an ibody, as indica ed. Memb anes we e ep obed wi h he an i-AC8 an ibody o p o ein loading con ol. Al e na i ely, he cell lysa es we e subjec ed o 10% SDS-PAGE and subsequen Wes e n blo ing wi h an i-O ai1 an ibody. The panels show esul s om one expe imen ep esen a i e o i e o he s. Molecula masses indica ed on he igh we e de e mined using molecula -mass ma ke s un in he same gel. HC: hea y chain o he an ibody used o immunop ecipi a ion; na i e O ai1: pos - ansla ionally modi ied O ai1; O ai1: O ai1 wi hou pos - ansla ional modi ica ions. 2.3. Role o AC8 in he Ac i a ion o S o e-Ope a ed Ca2+ En y in B eas Cance MDA-MB-231 Cells SOCE in MDA-MB-231 cells was epo ed o be en i ely dependen on O ai1 unc ion [3,6]. Hence, we explo ed he unc ional ole o he in e ac ion be ween O ai1 and AC8 in hese cells. To assess he ole o AC8 in SOCE, MDA-MB-231 cells we e ans ec ed wi h wo di e en comme cial small in e e ing RNAs (siRNAs) o AC8 o sc amble plasmids o analyze hei e ec on TG-e oked Ca2+ mobiliza ion. As shown in Figu e 4a,b, ans ec ion wi h bo h plasmids a enua ed he AC8 exp ession by abou 50% in 48 h. As depic ed in Figu e 4c, in cells ans ec ed wi h sc amble plasmids 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 he u a-2 luo escence a io due o Ca2+ elease om he in acellula Ca2+ s o es. Cell s imula ion wi h TG in a medium con aining 1 mM Ca2+ esul ed in a g ea e and sus ained ise in he u a-2 luo escence a io as a esul o Ca2+ elease om he in acellula s o es and en y h ough plasma memb ane channels (Figu e 4d). Cell ans ec ion wi h he siAC8 plasmids was wi hou signi ican e ec on he es ing u a-2 luo escence a io (in he absence o ex acellula Ca2+, he es ing a ios we e 0.27 ± 0.01, 0.26 ± 0.01 and 0.28 ± 0.01 in cells ans ec ed wi h sc amble plasmid, siAC8#1 and siAC8#2, espec i ely, while, in he p esence o 1 mM ex acellula Ca2+, he es ing a ios we e 0.30 ± 0.02, 0.32 ± 0.01 and 0.33 ± 0.02 in cells ans ec ed wi h sc amble plasmid, siAC8#1 and siAC8#2, espec i ely). Fu he mo e, ans ec ion wi h he siAC8 plasmids did no modi y TG- induced Ca2+ elease (Figu e 4e,g,i). The ini ial peak u a-2 luo escence a ios we e 0.10 ± 0.01, 0.10 ± 0.01, and 0.09 ± 0.02 in cells ans ec ed wi h sc amble plasmid, siAC8#1, o siAC8#2, espec i ely, hus indica ing ha AC8 does no ha e a signi ican e ec on he abili y o MDA-MB-231 cells o accumula e Ca2+ in he in acellula s o es o on he Ca2+ leakage a e om he s o es. By con as , a enua ion o AC8 exp ession by ans ec ion o he siAC8 plasmids educed TG-e oked Ca2+ mobiliza ion in he p esence o 1 mM ex acellula Ca2+ (Figu e 4 ,h,i; he ini ial slopes o he inc ease in u a-2 luo escence a io we e 0.0065 ± 0.0004, 0.0036 ± 0.0003, and 0.0040 ± 0.0002 in cells Cance s 2019, 11, 1624 8 o 24 ans ec ed wi h sc amble plasmid, siAC8#1, o siAC8#2, espec i ely, while he ini ial peak u a-2 luo escence a ios we e 0.93 ± 0.02, 0.64 ± 0.03, and 0.65 ± 0.02, in cells ans ec ed wi h sc amble plasmid, siAC8#1, o siAC8#2, espec i ely). As TG-e oked Ca2+ elease was una ec ed by a enua ion o he AC8 exp ession, his e ec should be a ibu ed o a educ ion in TG-induced Ca2+ in lux. I we conside he a ea unde he cu e (AUC) o he en y o Ca2+ s imula ed by TG, co ec ed by sub ac ion o he esponse o TG in he absence o ex e nal Ca2+, ans ec ion o he siAC8#1 and siAC8#2 plasmids signi ican ly educed SOCE by 40 ± 2% and 31 ± 2%, espec i ely (Figu e 4j; p < 0.05). Figu e 4. AC8 is equi ed o ull s o e-ope a ed Ca2+ en y ac i a ion in MDA-MB-231 b eas cance cells. (a–b) MDA-MB-231 cells we e ans ec ed wi h wo di e en small in e e ing RNA (siRNA) AC8 plasmids (siAC8#1 and siAC8#2) o a sc amble plasmid (sc), as indica ed. Fo y-eigh hou s a e ans ec ion, cells we e lysed and subjec ed o Wes e n blo ing wi h an i-AC8 an ibody, ollowed by Cance s 2019, 11, 1624 9 o 24 ep obing wi h an i-β-ac in an ibody o p o ein loading con ol (a). Molecula masses indica ed on he igh we e de e mined using molecula -mass ma ke s un in he same gel. (b) The box plo ep esen s AC8 exp ession unde he di e en expe imen al p ocedu es no malized o he β-ac in con en . (c–j) MDA-MB-231 cells we e ans ec ed wi h siAC8#1, siAC8#2, o sc amble plasmids, 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) o wi h a medium con aining 1 mM CaCl2, as indica ed, and hen s imula ed wi h TG (1 µM). (i and j) Ba g aphs ep esen TG-induced Ca2+ elease (i) and mobiliza ion (j) in MDA-MB-231 cells ans ec ed wi h he indica ed plasmids. Da a a e exp essed as he AUC o means ± SEM (s anda d e o o he mean) o 40 cells/day/3–5 days and p esen ed as a pe cen age o con ol (cells ans ec ed wi h sc amble plasmid). (k and l) MDA-MB-231 cells we e ans ec ed wi h siAC8#1, siAC8#2, o sc amble plasmids, 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 1 µM TG, ollowed by addi ion o CaCl2 ( inal concen a ion 1 mM) o he medium o ini ia e Ca2+ in lux. (l) Ba g aphs ep esen TG-induced Ca2+ en y unde he di e en expe imen al condi ions. Da a a e exp essed as he AUC o means ± SEM o 40 cells/day/3–5 days and p esen ed as a pe cen age o con ol (cells ans ec ed wi h sc amble plasmid); * p < 0.05 as compa ed o sc amble- ea ed cells. Simila esul s we e obse ed when we es ima ed TG-induced Ca2+ en y using he Ca2+ add- back p o ocol. As shown in Figu e 4k, ans ec ion o siAC8#1 o #2 plasmids signi ican ly a enua ed SOCE ( he ini ial peak u a-2 luo escence a ios upon addi ion o Ca2+ o TG- ea ed cells we e 1.68 ± 0.35, 0.90 ± 0.05, and 1.09 ± 0.04 in cells ans ec ed wi h sc amble plasmid, siAC8#1, o siAC8#2, espec i ely, and he ini ial slopes o he inc ease in u a-2 luo escence a io we e 0.0448 ± 0.0091, 0.0213 ± 0.0022, and 0.0207 ± 0.0029 in cells ans ec ed wi h sc amble plasmid, siAC8#1, o siAC8#2, espec i ely). In an a emp o asce ain mo e speci ically he ole o AC8 in Ca2+ in lux h ough O ai1, we analyzed he en y o Ca2+ in cells exp essing O ai1 and he O ai1-ac i a ing small agmen (OASF; amino acids 233–474) o STIM1. MDA-MB-231 cells we e ans ec ed wi h exp ession plasmids o pEYFP-O ai1 and pEYFP-OASF in combina ion wi h he siAC8 o sc amble plasmids. Exp ession o O ai1, OASF, o bo h in he absence o p esence o he siAC8 plasmids did no signi ican ly al e he es ing u a-2 luo escence a io ( es ing a ios we e 0.30 ± 0.01, 0.32 ± 0.01, 0.30 ± 0.01, 0.32 ± 0.01, 0.30 ± 0.01, and 0.31 ± 0.01 in cells ans ec ed wi h sc amble plasmid, O ai1, OASF, O ai1 + OASF, O ai1 + OASF + siAC8#1, and O ai1 + OASF + siAC8#2, espec i ely). As shown in Figu e 5, exp ession o O ai1 alone had a negligible e ec , i any, on he u a-2 luo escence a io, as p e iously epo ed [25]. On he o he hand, OASF exp ession signi ican ly enhanced Ca2+ in lux in hese cells, which exp essed a signi ican amoun o endogenous O ai1 (Figu e 5c). Co-exp ession o O ai1 and OASF esul ed in a obus ac i a ion o Ca2+ en y independen ly o Ca2+ s o e deple ion as compa ed o mock- ea ed cells, which did no display a signi ican cons i u i e Ca2+ en y. Ca2+ en y induced by co-exp ession o O ai1 and OASF was impai ed by AC8 silencing (Figu e 5e, ,g; p < 0.05), hus sugges ing ha AC8 di ec ly modula es Ca2+ en y h ough O ai1 in MDA-MB-231 cells. The ini ial peak u a-2 luo escence a ios we e 0.09 ± 0.01, 0.10 ± 0.01, 0.28 ± 0.02, 0.84 ± 0.07, 0.13 ± 0.01, and 0.13 ± 0.01 in cells ans ec ed wi h sc amble plasmid, O ai1, OASF, O ai1 + OASF, O ai1 + OASF + siAC8#1, and O ai1 + OASF + siAC8#2, espec i ely. The ole o AC8 in O ai1 channel unc ion in MDA-MB-231 cells was u he explo ed by es ing he e ec o AC8 o e exp ession on Ca2+ in lux in cells exp essing exogenous O ai1 and OASF. Cell ans ec ion wi h AC8 plasmid was wi hou signi ican e ec on he es ing u a-2 luo escence a io (in he absence o ex acellula Ca2+, he es ing a ios we e 0.34 ± 0.01, 0.36 ± 0.01, 0.32 ± 0.01, and 0.32 ± 0.01 in cells ans ec ed wi h O ai1 + OASF, O ai1 + OASF + AC8, emp y ec o (mock), and AC8 o e exp ession plasmid alone, espec i ely; Figu e 6). As shown in Figu e 6a, YFP-AC8 was e icien ly exp essed in MDA-MB-231 cells. In e es ingly, Ca2+ en y induced by co-exp ession o O ai1 and OASF, es ima ed as he AUC, was signi ican ly enhanced by 30% upon AC8 o e exp ession (Figu e 6b,c; p < 0.05). The ini ial peak u a-2 luo escence a ios we e 0.84 ± 0.02, 1.01 ± 0.03, 0.89 ± 0.04, and 1.11 ± 0.05 in cells ans ec ed wi h O ai1 + OASF, O ai1 + OASF + AC8, emp y ec o (mock), and AC8 o e exp ession plasmid alone, espec i ely, whe eby he wo la e ones Cance s 2019, 11, 1624 16 o 24 indica ed. Fo y-eigh hou s la e , cells we e lysed and subjec ed o 10% SDS-PAGE and Wes e n blo ing wi h an i-phospho-FAK (Y397) o an i-FAK speci ic an ibodies. Memb anes we e ep obed wi h an i-β-ac in an ibody o p o ein loading con ol. Blo s a e ep esen a i e o i e sepa a e expe imen s. The box plo ep esen s FAK y osine phospho yla ion p esen ed as he phospho- FAK/ o al FAK a io. 2.6. AC8 Is Requi ed o TNBC Cell P oli e a ion We u he explo ed he ole o AC8 in cell p oli e a ion in TNBC cells. Wes e n blo analysis o whole-cell lysa es om MCF10A and MCF7, as well as he TNBC cell lines MDA-MB-231, BT20, and Hs578T, wi h a speci ic an i-AC8 an ibody e ealed ha his p o ein is highly exp essed in MCF7 and TNBC cells (Figu e 11a). Nex , we explo ed he ole o AC8 in MDA-MB-231 and HS578T cells. As shown in Figu e 4a and 11c, cell ans ec ion wi h siAC8#1 and siAC8#2 signi ican ly a enua ed AC8 exp ession in MDA-MB-231 and Hs578T cells, espec i ely (p < 0.05; n = 4). Silencing AC8 p o ein exp ession signi ican ly a enua ed MDA-MB-231 and Hs578T cell p oli e a ion a all imes in es iga ed as compa ed o cells ans ec ed wi h sc amble plasmid (Figu e 11b,c; p < 0.05; n = 4). The e o e, ou obse a ions e eal ha AC8 plays an impo an ole in TNBC cell p oli e a ion. Figu e 11. Role o AC8 in iple-nega i e b eas cance (TNBC) cell p oli e a ion. (a) Non- umo al b eas epi helial MCF10A, luminal MCF7 b eas cance , and TNBC cells (MDA-MB-231, BT20, and Hs578T) we e lysed and subjec ed o Wes e n blo ing wi h an i-AC8 an ibody, ollowed by ep obing wi h an i-β-ac in an ibody o p o ein loading con ol. (b,c) MDA-MB-231 (b) and Hs578T (c) cells we e ans ec ed wi h siAC8#1, siAC8#2, o sc amble plasmid (Sc), as indica ed. Fo y-eigh hou s la e , cell p oli e a ion was assessed o a u he 24 and 48 h using he b omodeoxyu idine (B dU) cell p oli e a ion assay ki , as desc ibed in Sec ion 4. The box plo ep esen s cell p oli e a ion 0, 24, and 48 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 sc amble plasmid). (c, op panel) Hs578T cells we e lysed and subjec ed o 10% SDS-PAGE and Wes e n blo ing wi h an i-AC8 an ibody. Blo s a e ep esen a i e o h ee sepa a e expe imen s. 3. Discussion O ai1 was epo ed o play a majo ole in Ca2+ in lux in he TNBC cell line MDA-MB-231. SOCE, a majo mechanism o Ca2+ en y in his cell ype, was ound o be s ongly dependen on O ai1 [6], whose plasma memb ane exp ession is modula ed by TRPC6 channels [3]. Fu he mo e, O ai1 is in ol ed in s o e-independen Ca2+ in lux h ough a unc ional in e ac ion wi h K 10.1 po assium channels ha media e se um-induced mig a ion [9]. O ai1 Ca2+ channels play an impo an unc ional Cance s 2019, 11, 1624 17 o 24 ole in b eas cance cells suppo ing a numbe o cance hallma ks such as mig a ion, p oli e a ion, o su i al [33–35]. The O ai1 N- e minal in acellula egion con ains se e al phospho yla ion si es. Se -27 and -30 a e he main phospho yla ion si es a ge ed by PKCβ1 [20], while Se -34 was ound o be a a ge o p o ein kinase G (PKG) [36] and, mo e ecen ly, Zhang and cowo ke s e ealed ha Se -34 is also a PKA phospho yla ion si e [21]. Phospho yla ion o O ai1 a any o hese se ine esidues was demons a ed o esul in O ai1 channel inac i a ion and educ ion o Ca 2+ in lux. These h ee se ines a e loca ed in he O ai1 AC8-binding sequence (amino acids 26–34). He e, we show ha AC8 binding o O ai1 a enua es O ai1 se ine phospho yla ion in MDA-MB-231 cells, leading o enhanced Ca 2+ en y h ough he channel. This s a emen is based on di e en obse a ions. Fi s o all, we ound ha AC8 and he O ai1α and O ai1β a ian s a e o e exp essed in b eas cance MDA-MB-231 and MCF7 cells as compa ed o non- umo al b eas epi helial cells. Secondly, O ai1α, bu no O ai1β lacking he N- e minal 63 amino acids, cons i u i ely in e ac s wi h AC8. Thi dly, while he subse o O ai1 no associa ed wi h AC8 is phospho yla ed in se ine esidues bo h unde es ing condi ions and upon s imula ion wi h he SERCA inhibi o TG, he AC8-associa ed O ai1 subse is no se ine- phospho yla ed, hus sugges ing ha AC8 in e ac ion migh in e e e wi h O ai1 phospho yla ion a Se -27, -30, and -34. Fou hly, AC8 o e exp ession impai s O ai1 se ine phospho yla ion. Las ly, AC8 exp ession silencing signi ican ly educes TG-induced Ca 2+ en y, as well as Ca 2+ in lux media ed by co-exp ession o O ai1 wi h he STIM1 OASF egion in hese cells; con e sely, AC8 o e exp ession leads o enhanced Ca 2+ in lux in cells co-exp essing O ai1 and OASF, as well as TG-e oked Ca 2+ in lux. A ecen epo by Zhang and cowo ke s showed an elegan unc ional in e ac ion be ween O ai1 and AC8, whe e AC8 ac i a ion media es an O ai1 inac i a ion mechanism d i en by local cAMP and PKA ac i a ion ha , in u n, phospho yla es O ai1 a Se -34 [21]. He e, we show ha binding o AC8 o O ai1, a he sequence be ween amino acids 26 and 34, impai s phospho yla ion o O ai1 a Se -27, Se -30, and Se -34, he O ai1 phospho yla ion si es cha ac e ized a p esen [20,21,36]. The e o e, acco ding o p e ious esul s [21], Ca 2+ in lux h ough he O ai1 subse associa ed wi h AC8 should be in ol ed in he inac i a ion o he AC8-independen O ai1 channels. In naï e cells, wi h a no mal AC8 exp ession, AC8 is expec ed o p omo e inac i a ion o a la ge subse o O ai1 channels; howe e , in b eas cance MDA-MB-231 cells, which p edominan ly o e exp ess AC8 o e O ai1, he AC8/O ai1 s oichiome y is shi ed in a o o AC8 and, subsequen ly, he subse o AC8- independen O ai1 is expec ed o be educed, and he AC8-induced O ai1 inac i a ion is, hus, impai ed (Figu e 12). Figu e 12. Ca oon summa izing he impai men o phospho yla ion-dependen O ai1 inac i a ion by AC8 o e exp ession. In non- umo al b eas epi helial cells, Ca 2+ in lux ia O ai1 ac i a es AC8, which, in u n, esul s in he ac i a ion o PKA, leading o inac i a ion o a la ge subse o AC8- Cance s 2019, 11, 1624 18 o 24 independen O ai1 channels. In b eas cance MDA-MB-231 cells, he g ea e AC8 o e exp ession modi ies he AC8/O ai1 s oichiome y, hus p e en ing phospho yla ion-dependen O ai1 inac i a ion. We u he explo ed he unc ional ele ance o he AC8–O ai1 in e ac ion in MDA-MB-231 cells. In b eas cance cells, O ai1 was epo ed o play an impo an ole in mig a ion [37,38] and he p esen s udy p o ides e idence o a ole o AC8 suppo ing b eas cance cell mig a ion. Silencing AC8 exp ession a enua es mig a ion o es ing and agonis s imula ed MDA-MB-231 cells, while he same expe imen al maneu e was wi hou e ec in he non- umo al b eas epi helial MCF10A cell line, an obse a ion ha is likely a ibu ed o he low AC8 exp ession in his cell line. The ole o AC8 in cell mig a ion is no speci ic o TNBC cells, as simila esul s we e obse ed in he luminal b eas cance MCF7 cell line, which exhibi s some common ea u es wi h he MDA-MB-231 cell line, such as AC8 and O ai1 o e exp ession. While he ole o AC8 in cell mig a ion migh be a ibu ed o impai men o O ai1 inac i a ion and, hus, he suppo o Ca2+ in lux, we canno ule ou he possibili y ha cAMP gene a ion by AC8 also plays a ole in b eas cance cell mig a ion, as PKA ac i a ion in MDA-MB-231 and MCF7 cells by he cAMP analogue 8-b omo-cAMP pe se enhances cell mig a ion and, con e sely, pha macological PKA inhibi ion a enua es i . Finally, we in es iga ed he mechanism unde lying he ole o AC8 in MDA-MB-231 cell mig a ion. Focal adhesion u no e is essen ial o cell mig a ion, and he cy osolic y osine kinase FAK plays a cen al ole in he dynamics o ocal adhesions [39]. Hence, we assessed whe he he ole o AC8 in cell mig a ion migh be a ibu ed o he egula ion o FAK phospho yla ion a Ty -397, which is used as an indica o o FAK ac i a ion [40,41]. Ou esul s indica e ha FAK phospho yla ion a Ty -397 is s ongly dependen on AC8 exp ession, hus sugges ing ha FAK ac i a ion migh unde lie he pa icipa ion o AC8 in MDA-MB-231 cell mig a ion. We u he obse ed ha AC8 plays a ele an ole in TNBC cell p oli e a ion, ano he cellula unc ion egula ed by SOCE in b eas cance cells [3]. 4. Ma e ials and Me hods 4.1. Reagen s Fu a-2 ace oxyme hyl es e ( u a-2/AM) was om Molecula P obes (Leiden, The Ne he lands). TG, abbi polyclonal an i-O ai1 an ibody (ca alog numbe O8264, epi ope: amino acids 288–301 o human O ai1), abbi polyclonal an i-β-ac in an ibody (ca alog numbe A2066, epi ope: amino acids 365–375 o human β-ac in), KT5720, 8-b omoadenosine 3’-5’-cyclic monophospha e sodium, BAPTA (1,2-Bis(2-aminophenoxy)e hane-N,N,N′,N′- e aace ic acid e akis(ace oxyme hyl es e )), EGTA (e hylene glycol-bis(2-aminoe hyle he )-N,N,N′,N′- e aace ic acid), HEPES (4-(2- Hyd oxye hyl)pipe azine-1-e hanesul onic acid), EDTA (e hylenedini ilo e aace ic acid) and bo ine se um albumin (BSA) we e om Sigma (S Louis, MO, USA). Rabbi monoclonal an i-FAK an ibody (ca alog numbe ab40794, epi ope: wi hin amino acids 700–800 o human FAK) and abbi monoclonal an i-FAK (phospho Y-397) an ibody (ca alog numbe ab81298) we e om Abcam (Camb idge, UK). Ho se adish pe oxidase-conjuga ed goa an i-mouse immunoglobulin G (IgG) an ibody and goa an i- abbi IgG an ibody we e om Jackson labo a o ies (Wes G o e, PA, USA). Clean-Blo ™ IP De ec ion Reagen , ca bamylcholine chlo ide (ca bachol), abbi polyclonal an i- adenyla e cyclase 8 an ibody (ca alog numbe PA5-72589, epi ope: amino acids 946–972 o human adenyla e cyclase 8), Supe Signal® Wes Du a ex ended du a ion subs a e eagen , Silence ® Adenylyl cyclase 8 p e-designed siRNAs (Ids#: 119586 and 119587), and Silence ® Selec Nega i e Con ol siRNA we e om The moFishe Scien i ic (Wal ham, MA, USA). PNGase F om Elizabe hkingia mi icola was om P omega Co po a ion (Madison, WI, USA) Dha maFECT kb ans ec ion eagen was om Dha macon Inc (La aye e, CO, USA). P o ein A aga ose was om Me ck-Millipo e (Bu ling on, MA, USA). Plasmids used we e kindly p o ided by Ch is oph Romanin (YFP-O ai1 and YFP-OASF; Uni e si y o Linz, Linz, Aus ia), Michelle Halls (YFP- adenylyl cyclase 8; Monash Uni e si y, Aus alia), and Agus in Gue e o (Flag-O ai1-S27A/S30A and Cance s 2019, 11, 1624 19 o 24 Flag-O ai1-S27D/S30D; CINVESTAV, Mexico). G-GECO1.2-O ai1 was a gi om Michael Cahalan (Addgene plasmid #73562; h p://n2 .ne /addgene:73562; Resea ch Resou ce Iden i ie : Addgene_73562). Fu a-FFP18/AM was om San a C uz Bio echnology (Dallas, TX, USA). All o he eagen s we e o analy ical g ade. 4.2. Cell Cul u e and T ans ec ion The MCF10A cell line was p o ided by D . Po ie -Ca e eau (Uni e si é F ançois Rabelais Tou s, F ance). MCF7 and MDA-MB-231 cell lines we e ob ained om Ame ican Type Cul u e Collec ion (Manassas, VA, USA). Hs578T cells we e p o ided by D . Beni ez (CNIO, Mad id, Spain). Cells we e cul u ed up o 20–25 passages a 37 °C wi h 5% CO2 in Dulbecco's Modi ied Eagle Medium (DMEM)- F12 (MCF10A) o DMEM (MCF7 and MDA-MB-231), supplemen ed wi h 10% ( / ) ho se o e al bo ine se um, espec i ely, and 100 U/mL penicillin and s ep omycin, as desc ibed p e iously [3]. Fo Wes e n blo ing and immunop ecipi a ion assays, cells (2 × 106) we e pla ed in 75-cm2 lasks and cul u ed o 48–72 h, while, o calcium imaging, wound healing assay, and con ocal de e mina ion o G-GECO1.2 luo escence assays, cells (2 × 105 o 2.5 × 105) we e seeded in a 35-mm six-well mul idish. MDA-MB-231 cells we e ans ec ed wi h exp ession plasmids o YFP-O ai1, YFP-AC8, YFP- OASF, Flag-O ai1-S27A/S30A, and Flag-O ai1-S27D/S30D o sc amble plasmid using Dha maFECT kb ans ec ion eagen . Plasmids we e used a 1 µg/mL. AC8 siRNAs and nega i e con ol siRNA we e also ans ec ed in o cell using Dha maFECT kb ans ec ion eagen . siRNAs we e used o silencing expe imen s a 1 µg/mL. 4.3. Measu emen o Cy osolic F ee-Calcium Concen a ion ([Ca2+]c) Cells we e loaded wi h u a-2 by incuba ion wi h 2 µM u a 2/AM o 30 min a 37 °C as desc ibed p e iously [42]. Co e slips wi h cul u ed cells we e moun ed on a pe usion chambe and placed on he s age o an epi luo escence in e ed mic oscope (Nikon Eclipse Ti2, Ams e dam, The Ne he lands) wi h an image acquisi ion and analysis sys em o ideomic oscopy (NIS-Elemen s Imaging So wa e, Nikon, Ams e dam, The Ne he lands). Cells we e con inuously supe used a oom empe a u e wi h HEPES-bu e ed saline (HBS) con aining (in mM) 125 NaCl, 5 KCl, 1 MgCl2, 5 glucose, and 25 HEPES, pH 7.4, supplemen ed wi h 0.1% (w/ ) BSA. Cells we e examined a 40× magni ica ion (Nikon CFI S FLUOR 40× Oil, Ams e dam, The Ne he lands) and we e al e na i ely exci ed wi h ligh om a xenon lamp passed h ough a high-speed monoch oma o Op oscan ELE 450 (Cai n Resea ch; Fa e sham, UK) a 340/380 nm. Fluo escence emission a 505 nm was de ec ed using a cooled digi al sCMOS came a Zyla 4.2 (Ando ; Bel as , UK) and eco ded using NIS-Elemen s AR so wa e (Nikon; Tokyo, Japan). Fluo escence a io (F340/F380) was calcula ed pixel by pixel, and he da a we e p esen ed as ΔF340/F380, as p e iously desc ibed [13,43,44]. TG-e oked Ca2+ elease and Ca2+ mobiliza ion, as well as cons i u i e Ca2+ in lux in cells co-exp essing YFP-O ai1 and YFP-OASF, we e es ima ed as he a ea unde he cu e (AUC) measu ed as he in eg al o he ise in u a-2 luo escence a io o 2.5 min a e he addi ion o TG in he absence o p esence o ex acellula Ca2+, espec i ely, aking a sample e e y second. To compa e he a e o inc ease in u a-2 luo escence be ween di e en ea men s we used he cons an o he exponen ial inc ease. T aces we e i ed o he equa ion: y = A(1 − e−K1T ) e−K2T, whe e K1 is he cons an o he exponen ial inc ease. 4.4. Measu emen o Nea -Plasma-Memb ane F ee-Calcium Concen a ion Cells we e incuba ed wi h 5 µM u a-FFP18/AM o 2 h a 37 °C as desc ibed p e iously [45]. Co e slips wi h cul u ed cells we e moun ed on a pe usion chambe and placed on he s age o an epi luo escence in e ed mic oscope (Nikon Eclipse Ti2, Ams e dam, The Ne he lands) wi h an image acquisi ion and analysis sys em o ideomic oscopy (NIS-Elemen s Imaging So wa e, Nikon). Cells we e con inuously supe used wi h HBS supplemen ed wi h 0.1% (w/ ) BSA a oom empe a u e and we e examined a 40× magni ica ion (Nikon CFI S FLUOR 40× Oil, Ams e dam, The Ne he lands). Cells we e al e na i ely exci ed wi h ligh om a xenon lamp passed h ough a high- Cance s 2019, 11, 1624 20 o 24 speed monoch oma o Op oscan ELE 450 (Cai n Resea ch; Fa e sham, UK) a 335 and 364 nm, and luo escence emission, a 490 and 502 nm, espec i ely, was de ec ed using a cooled digi al sCMOS came a Zyla 4.2 (Ando ; Bel as , UK) and eco ded using NIS-Elemen s AR so wa e (Nikon, Tokyo, Japan). Fluo escence a io (F335/F364) was calcula ed pixel by pixel, and he da a we e p esen ed as ΔF335/F364. TG-e oked Ca2+ en y was measu ed as he in eg al o he ise in u a-FFP18 luo escence a io o 3 min a e he addi ion o ex acellula Ca2+ aking a sample e e y second (AUC). To compa e he a e o inc ease in u a-FFP18 luo escence be ween di e en ea men s, aces we e i ed o he equa ion men ioned in Sec ion 4.3. 4.5. Immunop ecipi a ion and Wes e n Blo ing The immunop ecipi a ion and Wes e n blo ing we e pe o med as desc ibed p e iously [46]. B ie ly, 500-µL aliquo s o cell suspension (4 × 106 cell/mL) we e lysed wi h an equal olume o ice- cold 2× NP-40 bu e , pH 8, con aining 274 mM NaCl, 40 mM T is, 4 mM EDTA, 20% glyce ol, 2% nonide P-40, 2 mM Na3VO4, and comple e EDTA- ee p o ease inhibi o able s. Aliquo s o cell lysa es (1 mL) we e immunop ecipi a ed by incuba ion wi h 2 µg o an i-O ai1 o an i-AC8 an ibody and 25 µL o p o ein A aga ose o e nigh a 4 °C on a ocking pla o m. The so elu ion o p o eins bound o he p o ein A aga ose was pe o med acco ding o he p o ocol desc ibed by An obus and Bo ne [47]. The immunop ecipi a es we e esol ed by 10% SDS-PAGE, and sepa a ed p o eins we e elec opho e ically ans e ed on o ni ocellulose memb anes o subsequen p obing. Blo s we e incuba ed o e nigh wi h 10% (w/ ) BSA in T is-bu e ed saline wi h 0.1% Tween-20 (TBST) o block esidual p o ein binding si es. Immunode ec ion o O ai1 and β-ac in was achie ed by incuba ion o 1 h wi h an i-O ai1 an ibody dilu ed 1:500 in TBST o 1 h wi h an i-β-ac in an ibody dilu ed 1:2000 in TBST, espec i ely. AC8, p-FAK, and FAK we e achie ed by incuba ion o e nigh wi h an i-AC8, an i-p-FAK (phospho Y-397) and an i-FAK an ibody dilu ed 1:500 in TBST, espec i ely. The p ima y an ibody was emo ed, and blo s we e washed six imes o 5 min each wi h TBST. To de ec he p ima y an ibody, blo s we e incuba ed o 1 h wi h ho se adish pe oxidase-conjuga ed goa an i- mouse IgG an ibody, ho se adish pe oxidase-conjuga ed goa an i- abbi IgG an ibody dilu ed 1:10000 in TBST, o Clean-Blo ™ IP De ec ion Reagen dilu ed 1:250 in TBST, and hen exposed o enhanced chemiluminiscence eagen s o 5 min. The densi y o bands was measu ed using a C-DiGi Chemiluminescen Wes e n Blo Scanne (LI-COR Biosciences, Lincoln, NE, USA). Da a we e no malized o he amoun o p o ein eco e ed by he an ibody used o he immunop ecipi a ion. 4.6. Wound Healing Assay The wound healing assay was pe o med as desc ibed p e iously [3]. MDA-MB-231 cells we e seeded in a 35-mm six-well mul idish o ob ain con luence a e 24 h. Nex , cells we e cul u ed in medium supplemen ed wi h 1% se um, and a wound was c ea ed using a s e ile 200-µL plas ic pipe e ip. Pho og aphs we e aken immedia ely o a he imes indica ed using an in e ed mic oscope Nikon Eclipse TS100 (Tokyo, Japan). Mig a ion o cells was quan i a ed using Fiji ImageJ (NIH; Be hesda, MD, USA). 4.7. Con ocal De e mina ion o G-GEC01.2 Fluo escence G-GECO1.2-O ai1 ans ec ed MDA-MB-231 cells we e seeded on co e slips and moun ed on a pe usion chambe and placed on he s age o an epi luo escence in e ed mic oscope Nikon Eclipse Ti (Tokio, Japan) wi h an image acquisi ion and analysis sys em o ideomic oscopy NIS-Elemen s Imaging So wa e (Nikon, Ams e dam, The Ne he lands). Cells we e con inuously supe used wi h HBS supplemen ed wi h 0.1% (w/ ) BSA a oom empe a u e. Cells we e examined a 60× magni ica ion and exci ed using a con ocal lase -scanning sys em (Melles-G io , IDEX Heal h & Science, Walling o d, CT, USA) a 488 nm. Fluo escence emission a 515 nm was de ec ed and eco ded using NIS-Elemen s AR so wa e (Nikon). GECO luo escence was de e mined (a) be o e he addi ion o TG ( es ing) in he absence o ex acellula Ca2+ (100 µM EGTA added), (b) 30 s a e he addi ion o 1 µM TG in he absence o ex acellula Ca2+, and (c) 30 s a e he addi ion o 1 mM Cance s 2019, 11, 1624 21 o 24 CaCl2 o he ex acellula medium. Images we e analyzed using ImageJ so wa e (NIH, Be hesda, MD, USA). 4.8. De e mina ion o Cell P oli e a ion To de e mine cell p oli e a ion, cells we e seeded a a concen a ion o 5 × 103/well in o 96-well pla es and, a e 0, 24, and 48 h, cell p oli e a ion was assessed using a speci ic cell p oli e a ion assay ki based on he measu emen o B dU inco po a ion du ing DNA syn hesis acco ding o he manu ac u e ’s ins uc ions (BioVision, Milpi as, CA, USA). Abso bance in samples was measu ed using a pla e eade (Epoch, Bio ek, Swindon, UK) a 450 nm, p esen ed as a bi a y uni s. 4.9. S a is ical Analysis Analysis o s a is ical signi icance was pe o med using he K uskal–Wallis es combined wi h Dunn´s pos hoc es (o one-way analysis o a iance combined wi h Tukey pos hoc es o he analysis o Ca2+ de e mina ions) (G aphPad P ism Windows 5.04, San Diego, CA, USA). Fo compa ison be ween wo g oups, he Mann–Whi ney U es was used. A p- alue <0.05 was conside ed o be s a is ically signi ican . 5. Conclusions In conclusion, ou esul s p o ide s ong e idence o he impai men o phospho yla ion- dependen O ai1 inac i a ion by AC8 o e exp ession in MDA-MB-231 cells, a mechanism ha enhances Ca2+ in lux, b eas cance cell mig a ion (by suppo ing FAK ac i a ion), and cell p oli e a ion. These indings sugges ha AC8 migh be a good candida e o he de elopmen o an i- umo al s a egies in b eas cance . Supplemen a y Ma e ials: The ollowing a e a ailable online a www.mdpi.com/xxx/s1: Figu e S1: Ca2+ mobiliza ion nea he plasma memb ane in MDA-MB-231 b eas cance cells; Figu e S2: Role o he cAMP–PKA pa hway in MCF7 cell mig a ion; Figu e S3: Role o AC8 in MCF7 cell mig a ion; Figu e S4: Role o AC8 in MCF10A cell mig a ion. Au ho Con ibu ions: Concep ualiza ion, J.J.L., T.S., and J.A.R.; unding acquisi ion, G.M.S., T.S., and J.A.R.; in es iga ion, J.S.-C., J.J.L., I.J., S.R., P.J.C., D.F., T.S., and J.A.R.; supe ision, J.A.R.; w i ing—o iginal d a , J.A.R.; w i ing— e iew and edi ing, S.R., G.M.S., and T.S. All au ho s e iewed and app o ed he manusc ip . Funding: This wo k was suppo ed by MINECO (G an s BFU2016-74932-C2-1-P and BFU2016-74932-C2-2-P) and Jun a de Ex emadu a-FEDER (Fondo Eu opeo de Desa ollo Regional G an s IB16046 and GR18061). J.J.L. and I.J. a e suppo ed by a con ac om Jun a de Ex emadu a (TA18011 and TA18054, espec i ely). J.S.-C. is suppo ed by a con ac om Minis y o Science, Inno a ion, and Uni e si ies, Spain. Acknowledgmen s: We a e g a e ul o Agus in Gue e o (Cen e o Resea ch and Ad anced S udies o he Na ional Poly echnic Ins i u e, Mexico), Ch is oph Romanin (Uni e si y o Linz, Aus ia), and Michelle Halls (Monash Uni e si y, Aus alia) o p o iding plasmids. We a e also g a e ul o Ja ie Beni ez (Spanish Na ional Cance Resea ch (CNIO), Spain) o p o iding he Hs578T cells. The BT-20 human b eas ca cinoma cell line was gene ously p o ided by Abela do López-Ri as and Ca men Palacios (Cell Dea h and Signaling Resea ch G oup, CABIMER Se ille, Spain). We hank Sand a Al a ado o echnical assis ance. Con lic s o In e es : The au ho s decla e no con lic s o in e es . Re e ences 1. Ramadan, W.S.; Vazhappilly, C.G.; Saleh, E.M.; Menon, V.; AlAzawi, A.M.; El-Se a i, A.T.; Mansou , W.; El-Awady, R. 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