The effects of trastuzumab on HER2-mediated cell signaling in CHO cells expressing human HER2

Transcription

1 Mdi et l. BMC Cncer (2018) 18:238 RESEARCH ARTICLE Open Access The effects of trstuzum on HER2-medited cell signling in CHO cells expressing humn HER2 Hmid Mdi, Bk Nmi, Junfeng Tong, Gin Li nd Zhixing Wng * Astrct Bckground: Trgeted therpy with trstuzum hs ecome minsty for HER2-positive rest cncer without cler understnding of the mechnism of its ction. While mny mechnisms hve een suggested for the ction of trstuzum, most of them re not sustntited y experimentl dt. It hs een suggested tht trstuzum functions y inhiiting intrcellulr signling initited y HER2, however, the dt re very controversil. A mjor issue is the different cellulr ckground of vrious rest cncer cells lines used in these studies. Ech rest cncer cell line hs unique expression profile of vrious HER receptors, which could significntly ffect the effects of trstuzum. Methods: To overcome this prolem, in this reserch we dopted cell model tht llow us to specificlly exmine the effects of trstuzum on single HER receptor without the influence of other HER receptors. Three CHO cell lines stly expressing only humn EGFR (CHO-EGFR), HER2 (CHO-K6), or HER3 (CHO-HER3) were used. Vrious methods including cytotoxicity ssy, immunolotting, indirect immunofluorescence, cross linking, nd ntiody-dependent cellulr cytotoxicity (ADCC) were employed in this reserch. Results: We showed tht trstuzum did not ind EGFR nd HER3, nd thus did not ffect the homodimeriztion nd phosphoryltion of EGFR nd HER3. However, overexpression of HER2 in CHO cells, in the sence of other HER receptors, resulted in the homodimeriztion of HER2 nd the phosphoryltion of HER2 t ll mjor py residues. Trstuzum ound to HER2 specificlly nd with high ffinity. Trstuzum inhiited neither the homodimeriztion of HER2, nor the phosphoryltion of HER2 t most phosphotyrosine residues. Moreover, trstuzum did not inhiit the phosphoryltion of ERK nd AKT in CHO-K6 cells, nd did not inhiit the prolifertion of CHO-K6 cells. However, trstuzum induced strong ADCC in CHO-K6 cells. Conclusion: We concluded tht, in the sence of other HER receptors, trstuzum exerts its ntitumor ctivity through the induction of ADCC, rther thn the inhiition of HER2-homodimeriztion nd phosphoryltion. Keywords: HER receptors, EGFR, HER2, HER3, Trstuzum, Dimeriztion, Phosphoryltion, ADCC, CHO cells Bckground The HER fmily of receptor tyrosine kinses (RTKs) includes EGFR/HER1/ErB1, HER2/ErB2, HER3/ErB3, nd HER4/ErB4 [1, 2]. Except for HER4, the errnt ctivtion of HER receptor kinse ctivity contriutes to the tumorigenesis nd progression of rest cncer [3 11]. Overexpression of EGFR, HER2 nd HER3 occurs in * Correspondence: zhixing.wng@ulert.c Equl contriutors Deprtment of Medicl Genetics, nd Signl Trnsduction Reserch Group, Fculty of Medicine nd Dentistry, University of Alert, Edmonton, AB T6G 2H7, Cnd 30 40%, 20 30% nd ~ 20% of rest cncer cses, respectively [4, 11 16]. Trgeting HER2 hs proven to e n effective therpeutic strtegy for HER2-positive rest cncer [17, 18]. Since its pprovl y FDA in 1998, trstuzum, n ntiody ginst HER2, hs chnged the prdigm for the tretment of HER2-positive rest cncer [18, 19]. However, fter the initil success, cquired resistnce to trstuzum hs grdully developed, which posts chllenge tht needs to e overcome [18, 20, 21]. The ctivtion of HER receptors re induced y homoor hetero-dimeriztion [2, 22, 23]. Among HER receptors, The Author(s) Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4.0 Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Mdi et l. BMC Cncer (2018) 18:238 Pge 2 of 14 HER2 is n orphn receptor without direct lignd nd HER3 hs impired kinse ctivity. The heterodimeriztion mong vrious HER receptors is n importnt mechnism to ctivte ll HER receptors in response to lignd stimultion [2, 15, 24, 25]. The HER2 extrcellulr domin is lwys in the extended conformtion nd redy to e dimerized. Therefore, HER2 is the preferred heterodimeric prtner for other HER receptors [2, 26 28]. Overexpression of HER2 in cncers leds to the homodimeriztion nd the constitutive ctivtion of HER2 [15]. Ech HER receptor displys different inding ffinities for vrious downstrem signling proteins. While EGFR nd HER2 preferentilly ctivte the Rs-ERK pthwy leding to cell prolifertion HER3 preferentilly ctivtes the PI3K-AKT pthwy leding to cell survivl [15, 29]. The heterodimeriztion mong vrious HER receptors llows them to ply flexile nd complex roles in cell signling [2, 23 25, 29 39]. HER2 hs een therpeutic trget for treting rest cncer due to its overexpression in 20 30% of rest cncer ptients [6, 8, 11, 40]. Trstuzum is recominnt humnized monoclonl ntiody tht inds to the juxtmemrne region of HER2 [27, 41, 42]. Trstuzum is the first HER2-trgetted therpy pproved y FDA for metsttic rest cncer tretment. It showed strong ntitumor effects in oth mouse model nd HER2-positive rest cncer ptients [6, 8]. While mny mechnisms hve een proposed for the ntitumor ctivity of trstuzum, including oth extrcellulr nd intrcellulr ctions [6, 8, 43], the exct mechnisms re not known. The extrcellulr ction is through immune-medited response. When ound to the trget cells, the Fc portion of trstuzum will e recognized nd ttcked y Fc receptor on immune effector cells, principlly nturl-killer (NK) cells. In vitro, this process is clled ntiody-dependent cellulr cytotoxicity (ADCC). There re solid evidence to support ADCC s mjor mechnism for trstuzum ction [44 51]. On the other hnd, the dt regrding the intrcellulr mechnisms re either controversil t the eginning or chllenged y the recent dt [52]. Intrcellulr ction could e through the following mechnisms: inhiition of intrcellulr signl trnsduction, stimultion of HER2 internliztion nd degrdtion, inhiition of DNA repir, inhiition of proteolytic clevge of the HER2 extrcellulr domin, nd inhiition of ngiogenesis [6, 8, 43]. While mny recent pulictions clim tht erly studies support the role of trstuzum in inhiiting HER2 phosphoryltion [6, 52, 53], mny dt indicte tht trstuzum either hs no effect or stimultes HER2 phosphoryltion [52 56]. The dt regrding the effects of trstuzum on the dimeriztion of HER2, ctivtion of mjor signling pthwys including AKT nd ERK [6, 8, 43, 57, 58], nd HER2 endocytosis/downregultion [56, 59 61] re ll controversil. The dt regrding the role of trstuzum on DNA repir [62], proteolytic clevge of HER2 extrcellulr domin [63], nd ngiogenesis [64, 65] re very limited [6]. The most controversil mechnism regrding trstuzum function is its effect on the inhiition of HER2 ctivtion. A mjor reson ehind this controversy is the different cellulr ckground of vrious rest cncer cells lines used in those studies. Ech rest cncer cell line hs unique expression profile of vrious HER receptors, which could significntly ffect the effects of trstuzum. To overcome this prolem, in this reserch we dopted cell model tht llow us to specificlly exmine the effects of trstuzum on single HER receptor without the influence of other HER receptors. We im to conclusively determine if trstuzum specificlly inds only to HER2, nd locks HER2 homodimeriztion nd ctivtion. To chieve our ojective, we dopted CHO cell model. Besides the prentl CHO cells tht do not express ny detectle HER receptors, three stle CHO cell lines tht stly express only single HER receptor including EGFR (CHO-EGFR), HER2 (CHO-K6), nd HER3 (CHO-ErB3) were employed in this reserch. We demonstrte tht overexpression of HER2 in CHO cells resulted in the homodimeriztion of HER2 nd the phosphoryltion of HER2 t ll mjor py residues. Trstuzum ound to HER2 specificlly nd with high ffinity. Trstuzum neither inhiited the homodimeriztion of HER2, nor inhiited the phosphoryltion of HER2 t most phosphotyrosine residues. Moreover, trstuzum did not inhiit the phosphoryltion of ERK nd AKT in CHO-K6 cells, nd did not inhiit the prolifertion of CHO-K6 cells. However, trstuzum induced strong ADCC in CHO cells overexpressing HER2. Methods Cell culture nd tretment All cells were cultured t 37 C with 5% CO2 tmosphere. Chinese Hmster Ovry (CHO) cell ws purchsed from ATCC (ATCC CCL-61, Mnsss, VA). CHO cell stly expressing humn EGFR (CHO-EGFR) ws previously generted [66]. CHO cell stly expressing HER2 (CHO- K6) [67], nd HER3 (CHO-HER3) [68] were gifts from other ls. Dulecco s modified Egle s medium (DMEM) contining 10% FBS were used for cell culture. The cells were mintined t 37 C in 5% CO 2 tmosphere. To mintin the selection pressure G418 (500 mg/ml) were dded to the culture medium. For tretment, cells were strved overnight t DMEM contining 1% FBS nd then treted in this strvtion medium. EGF, trstuzum, norml IgG, CP , or vinoreline ws dded t indicted concentrtion for indicted time periods. Chemicls nd ntiodies CP HER2 inhiitor ws purchsed from Selleckchem (Houston, TX, USA). Lptini nd isotype

3 Mdi et l. BMC Cncer (2018) 18:238 Pge 3 of 14 control humn IgG were purchsed from Sigm-Aldrich (St. Louis, MO, USA). Trstuzum ws purchsed from Roche (Bsel, Switzerlnd). Mouse monoclonl nti-humn HER2 (9G6) nd (A-2), nti-humn EGFR (A-10), nd nti-humn HER3 (RTJ.2) ntiodies were purchsed from Snt Cruz Biotechnology Inc. (Dlls, TX, USA). Rit polyclonl nti-humn phospho-her2 Y-1005, Y-1112, Y-1127, Y-1139, Y-1196, nd Y-1248 ntiodies were purchsed from FroggBio (Toronto, ON, Cnd). All other chemicls were purchsed from Sigm-Aldrich. Cell prolifertion ssy y MTT Cell prolifertion ws determined y MTT ssy Vyrnt MTT Cell Prolifertion Assy Kit from Invitrogen (Grnd Islnd, NY). The detiled protocol of the ssy ws descried previously [69]. The cells were treted with vrious gents including EGF, trstuzum, norml IgG, CP , or vinoreline for 24 or 48 h. Ech vlue is the verge of t lest three independent experiments. Cell lystes nd immunolotting The lysis of the cells nd the immunolotting were descried previously [66]. Cross-linking ssy Cross-linking ssy ws employed to determine the dimeriztion of the receptors. CHO cells were cultured to suconfluency in 60 mm dishes. Following the tretment with EGF, trstuzum, nd norml humn IgG of indicted concentrtions for 1 h t 37 C, the cells were collected nd suspended in 0.2~ 0.5 ml PBS. BS3 [is(sulfosuccinimidyl)suerte] ws then dded to finl concentrtion of 1.0~ 2.5 mm. The cross-linking rection ws conducted on ice for 2 h. To stop the rection the quench solution (1 M Tris, ph 7.5, 1:100 dilution) ws dded nd incuted for 15 min on ice. The finl concentrtion of the quench solution ws 10 mm. Afterwrds, the cells were lysed with 1% NP-40 on ice for 1 h. The dimeriztion ws nlyzed y SDS-PAGE followed y immunolotting. A gel of 5% ws run to etter seprte the dimers from the monomers. Immunofluorescence stining ssy Cells were cultured on the immunofluorescence slides 48 h efore tretment strts. After tretment period, the slides were rinsed in tris-uffered sline (TBS; 6% tris, 8.8% NCl, 85.2% dh2o, PH = 7.6) nd the cells were fixed y cold methnol for 4 min. Blocking ws done with incution of slides in 1% BSA in TBS for n hour. The slides were then treted with 1 μg/ml indicted primry ntiodies in TBS for 1 h. Following rinsing in TBS for three times, the slides were incuted with 1 μg/ml FITC- nd/or TRITC-conjugted secondry ntiody in TBS for n hour in the drk. Therefter, the slides were wshed completely in TBS nd incuted in 1 μg/ml DAPI in TBS for 5 min t room temperture in the drk. The slides were oserved using DeltVision fluorescence microscopy system (Applied Precision Inc., Mississug, ON, Cnd). Antiody-dependent cellulr cytotoxicity (ADCC) ADCC of trstuzum in CHO cells expressing HER2 or EGFR ws determined y using Promeg ADCC Biossy kit ccording to Mnufcturer s instruction. Cultured cells were plted t the density of 15,000 cells per well in complete culture medium overnight efore iossy. On the dy of iossy, the series of concentrtions of trstuzum were dded to the cells, followed y ddition of ADCC Biossy Effector Cells. The E:T rtio ws 5:1. After 6 h of induction t 37 C, Bio-Glo Luciferse Assy Regent ws dded nd luminescence ws determined. Results Stle CHO cell lines expressing EGFR, HER2 nd HER3 HER2 heterodimerizes with EGFR nd HER3 in response to lignd stimultion [2, 15, 25]. HER2 lso homodimerizes nd ctivtes in cells with over-expressed HER2 [15, 70, 71]. Most HER2-positive rest cncer cells lso express either EGFR, HER3 or oth, which mkes it difficult to explin the oserved effects of trstuzum. Thus, to understnd the effects of trstuzum on HER2-medited cell signling in rest cncer cells, we pln first to study the effects of trstuzum in CHO cells tht selectively express single HER receptor. The results from these CHO cells will unmiguously define the role of trstuzum on HER2-medited cell signling under vrious expression profiles of HER receptors. Thus, these dt could e used to ccurtely interpret oservtion in rest cncer cells. We hve estlished CHO cell lines stly expressing EGFR (CHO-EGFR) [66]. CHO cells expressing HER2 (CHO-K6) or HER3 (CHO-HER3) were otined from other ls [67, 68]. Prentl CHO cells is used s control. We confirmed the expression of HER receptors in these cell lines y immunolotting nd immunofluorescence. As shown in Fig. 1, CHO-K6 cells expressed high level of HER2. CHO-EGFR cells expressed high level of EGFR. CHO- HER3 cells expressed high level of HER3 nd the prentl CHO cells did not express detectle HER2, EGFR nd HER3. Binding of trstuzum to HER receptors While trstuzum is n ntiody to HER2, it is possile tht it my wekly interct with EGFR nd HER3 due the sequence homology mong these receptors. Thus, we next exmined the inding of trstuzum to HER2, EGFR nd HER3. We showed y immunofluorescence

4 Mdi et l. BMC Cncer (2018) 18:238 Pge 4 of 14 Fig. 1 The expression of HER receptors in CHO cells stly trnsfected with single HER receptor including CHO-EGFR, CHO-K6, nd CHO-HER3. Immunolotting. The lystes of vrious CHO cells were seprted y gel electrophoresis nd immunolotted with ntiodies to HER receptors s indicted. The prent CHO cells (CHO) were used s control. Immunofluorescence. Vrious CHO cells were fixed nd stined with ntiodies to HER receptors s indicted. The expression of HER receptor ws reveled y FITC-conjugted secondry ntiody (Green). Cell nuclei were counterstined with DAPI. Size r: 10 μm tht trstuzum only ound to HER2, ut not EGFR nd HER3 (Fig. 2). As shown in Fig. 2, t the dosge rnging from 0.1 μg/ml to 10 μg/ml, trstuzum showed strong inding to HER2 in CHO-K6 cells. HER2 ws loclized to the plsm memrne (PM) in CHO-K6 cells under ll conditions s expected. Trstuzum ws lso locted to PM, co-loclizing with HER2, which indictes the inding of trstuzum to HER2 (Fig. 2). PM locliztion of trstuzum ws incresed with the incresed dosge. We lso determined the time course of trstuzum inding to HER2 in CHO-K6 cells. As shown in Fig. 2, t 5 min following trstuzum ddition, trstuzum hd lredy een well loclized to the PM, indicting rpid inding etween trstuzum nd HER2. Longer incution only slightly incresed the PM locliztion of trstuzum. However, even t the high dosge of 10 μg/ml, no inding of trstuzum to EGFR nd HER3 ws detectle in CHO-EGFR nd CHO-HER3 cells, respectively (Fig. 2c & d). These results indicte tht trstuzum inds to HER2 specificlly with high ffinity. The effects of trstuzum on the homodimeriztion of HER2 So fr, the reports re controversil regrding the effects of trstuzum on the dimeriztion or HER2. Here we exmined the effects of trstuzum on the homodimeriztion of HER2 y crosslinking nd immunolotting (Fig. 3). As shown in Fig. 3, overexpression of HER2 y itself resulted in high level of HER2 homodimeriztion. Clerly trstuzum did not lock the homodimeriztion of HER2. Interestingly, with the increse of the dosge from 0.1 μg/ml to 10 μg/ml, trstuzum slightly induced the dimeriztion of HER2 (Fig. 3). The induction of homodimeriztion of HER2 y trstuzum ws even more visile in the time course experiments (Fig. 3). The effects of trstuzum on the phosphoryltion of HER2 Activted HER2 phosphoryltes multiple tyrosine (Y) residues t its C-terminus. We hve exmined the phosphoryltion of following six tyrosine residues including Y1005, Y1112, Y1127, Y1139, Y1196, nd Y1248 (Figs. 4, 5 nd 6). As shown y immunolotting, for the control cells treted with norml IgG, HER2 ws well phosphorylted in ll of the py residues exmined (Fig. 4 & ). The phosphoryltion is likely due to the homodimeriztion induced y the overexpression of HER2. Tretment with trstuzum t the dosge rnging from 0.1 μg/ml to 10 μg/ml did not significntly lter the phosphoryltion levels of most phosphotyrosine residues including Y1005, Y1127, Y1196, nd Y1248. However, trstuzum prtilly inhiited the phosphoryltion of Y1139. Similr to norml IgG, EGF did not hve ny effects on the phosphoryltion of ll the py residues of HER2, which is not surprising s HER2 does not ind to EGF (Fig. 4 & ). These results were confirmed y time course experiments (Fig. 4c). Tretment from 15 min up to 2 h, did not chnge the phosphoryltion levels of ll py residues except for py1139 tht is prtilly inhiited (Fig. 4c). As controls, we hve exmined the effects of trstuzum on EGFR phosphoryltion in CHO-EGFR cells. As shown in Fig. 5, EGFR ws not phosphorylted in CHO-EGFR cells, nd ddition of EGF stimulted the phosphoryltion of EGFR. Tretment with trstuzum ws not le to inhiit EGF-induced EGFR phosphoryltion. Moreover, trstuzum y itself did not hve ny detectle effects on EGFR phosphoryltion in CHO- EGFR cells. We lso exmine the effects of chemicl inhiitor of HER2, CP on HER2 phosphoryltion in CHO-K6 cells. As shown in Fig. 5, vrious concentrtions of CP rnging from 1 to 100 μm significntly lock the phosphoryltion of HER2 t Y1005,

5 Mdi et l. BMC Cncer (2018) 18:238 Pge 5 of 14 c d Fig. 2 Binding of trstuzum to HER receptors in CHO-K6, CHO-EGFR nd CHO-HER3 cells s reveled y immunofluorescence. nd The inding of trstuzum to HER2 in CHO-K6 cells. CHO cells were treted with trstuzum t vrious concentrtions for 1 h () or t vrious time period t 10 μg/ml () s indicted. The memrne locliztion (inding) of trstuzum ws reveled y TRITC-conjugted donkey nti-humn IgG. The locliztion of HER2 ws reveled y rit nti-her2 ntiody followed y FITC-conjugted donkey nti-rit IgG. The cell nuclei were counter stined with DAPI. Yellow indicted the co-locliztion of trstuzum nd HER2. c The inding of trstuzum to EGFR in CHO-EGFR cells. The memrne locliztion (inding) of trstuzum ws reveled y TRITC-conjugted donkey nti-humn IgG. The locliztion of EGFR ws reveled y rit nti-egfr ntiody followed y FITC-conjugted donkey nti-rit IgG. The cell nuclei were counter stined with DAPI. d The inding of trstuzum to HER3 in CHO-HER3 cells. The memrne locliztion (inding) of trstuzum ws reveled y TRITC-conjugted donkey nti-humn IgG. The locliztion of HER3 ws reveled y rit nti-her3 ntiody followed y FITC-conjugted donkey nti-rit IgG. The cell nuclei were counter stined with DAPI. Size r: 10 μm which is in strk contrst from trstuzum s shown in Fig. 4. Furthermore, CP lso significntly inhiited the phosphoryltion of HER2 py1139, however, trstuzum only prtilly inhiited py1139. These results indicted tht trstuzum hs little, if ny, inhiitory effects on HER2 ctivtion/ phosphoryltion. We further exmine the effects of trstuzum on the phosphoryltion of HER2 y indirect immunofluorescence (Fig. 6). CHO-K6 cells either treted with trstuzum or control IgG were doule stined for oth trstuzum (TRITC, red) nd phosphor-her2 (FITC, green). Antiodies specific to six HER2 py residues including Y1005, Y1112, Y1127, Y1139, Y1196, nd Y1248 were used to determine the effects of trstuzum on HER2 phosphoryltion. As shown in Fig. 6, HER2 ws well phosphorylted on ll of these six py residues in the sence of trstuzum, indicting the utophosphoryltion due to overexpression. Tretment with trstuzum t the concentrtion rnging from 0.1 μg/ml to 10 μg/ml hd no effects on the phosphoryltion levels of these HER2 py residues including Y1005, Y1112, Y1127, Y1196, nd Y1248 (Fig. 6,, c & e). However, for py1139, trstuzum t 1 10 μg/ml showed some inhiitory effect (Fig. 6d). Together, our results indicted tht overexpression of HER2 resulted in strong HER2 phosphoryltion in ll its py residues studied here. Addition of trstuzum, in generl, did not inhiit the phosphoryltion of HER2.

6 Mdi et l. BMC Cncer (2018) 18:238 Pge 6 of 14 c d Fig. 3 The effects of trstuzum on HER2 homodimeriztion in CHO-K6 cells s reveled y crosslinking. Following trstuzum tretment s indicted, CHO-K6 cells were treted with BS 3 nd the homodimeriztion of HER2 ws reveled y immunolotting s descried in Mterils nd Methods. CHO-K6 cells were treted with trstuzum t vrious concentrtions rnging from μg/ml for 1 h. Quntifiction of the dt in (). c CHO-K6 cells were treted with 10 μg/ml trstuzum for vrious time period s indicted. Cells treted with norml humn IgG were used s control. d Quntifiction of the dt in (). The level of HER2 homodimeriztion were quntitted y densitometry nd expressed s the rtio of dimer/totl HER2. Ech vlue is the verge of t lest three experiments nd the error r is stndrd error. **: p < 0.01, ***: p < The only possile exception is tht trstuzum t higher dosge (1 10 ng/ml) slightly reduced the phosphoryltion of HER2 t py1139. The effects of trstuzum on the ctivtion of ERK nd AKT We finlly exmined the ctivtion of ERK nd AKT. The ERK nd AKT ctivtion ws mesured y their phosphoryltion. As shown in Fig. 7, the ERK phosphoryltion level is higher in CHO-K6 cells thn the control CHO cells, which suggests tht overexpression of HER2 incresed ERK ctivtion. However, we did not oserve the increse in AKT phosphoryltion, which is not surprising s HER2 homodimer hs very limited effects on the ctivtion of PI3K-AKT pthwy. We next exmined the effects of trstuzum on the phosphoryltion of ERK nd AKT in CHO-K6 cells. As shown in Fig. 7, tretment with trstuzum did not lock the phosphoryltion of ERK nd AKT. Trstuzum-induced ADCC Aove results suggests tht trstuzum did not inhiit HER2 dimeriztion nd phosphoryltion. Thus, it is interesting to find out if trstuzum cn induce ADCC in cells overexpressing HER2. Trstuzum-induced ADCC in CHO-K6 nd CHO-EGFR cells ws determined y using Promeg ADCC Biossy kit. As shown in Fig. 8, trstuzum induced very strong ADCC in CHO-K6 cells, ut not in CHO-EGFR cells. The effects of trstuzum on the prolifertion of CHO-K6 cells We next determined if trstuzum inhiits the prolifertion of cells with overexpressed HER2. MTT cell

7 Mdi et l. BMC Cncer (2018) 18:238 Pge 7 of 14 c Fig. 4 The effects of trstuzum on HER2 phosphoryltion in CHO-K6 cells y immunolotting. CHO-K6 cells were treted with trstuzum t vrious concentrtions for 1 h. The phosphoryltion of HER2 t Y1005, Y1127, Y1139, Y1196, nd Y1248 were then exmined y immunolotting s descried in Mterils nd Methods. Cells treted with norml humn IgG or EGF were used s controls. Quntifiction of the results in (). The phosphoryltion level of ech HER2 py residue ws normlized ginst the expression level of tuulin. Ech vlue is the verge of t lest three experiments nd the error r is stndrd error. **: p < c Time course experiments. CHO-K6 cells were treted with trstuzum (10 μg/ml) for vrious time s indicted. The phosphoryltion of vrious HER2 py residues were exmined y immunolotting prolifertion kit ws used to ssess the prolifertion of vrious CHO cells including CHO prentl cells, CHO- EGFR, CHO-K6, nd CHO-HER3 cells. Non-treted Cells were used s negtive controls, nd the cells treted with vinoreline (n nticncer drug) were used s positive controls. We first determined if overexpression of HER2 in CHO cells stimultes cell prolifertion y compring CHO-K6 cells with the prentl CHO cells. As shown in Fig. 9, the prolifertion rte of CHO-K6 cells is much higher thn tht of CHO cells, which indictes tht overexpression of HER2 stimultes cell prolifertion. We next exmined the effects of trstuzum on cell prolifertion. It is not surprising tht tretment with trstuzum for either 24 or 48 h hd no effects on the prolifertion of CHO, CHO-EGFR nd CHO-HER3 cells s these cell did not express HER2 (Fig. 9-d). Interestingly, even for CHO-K6 cells tht overexpressed HER2, trstuzum t high dosge did not hve ny effect on their prolifertion (Fig. 9e). However, vinoreline significntly inhiited the prolifertion of ll these CHO cells following 24 or 48 h incution (Fig. 9-e). Moreover, HER2 kinse inhiitors including Lptini nd CP significntly inhiited the prolifertion of CHO-K6 cells when t high dosge (Fig. 9f). Our dt indicted tht trstuzum did not inhiited the prolifertion of CHO-K6 cells tht overexpressed HER2. Discussion The most controversil mechnism regrding trstuzum function is its effect on the inhiition of HER2 ctivtion. A mjor reson ehind this controversy is the different cellulr ckground of vrious rest cncer cells lines used in those studies. Ech rest cncer cell line hs unique expression profile of vrious HER receptors, which could significntly ffect the effects of trstuzum due to the heterodimeriztion mong HER receptors. In this reserch we dopted CHO cell model. Besides the prentl CHO cells tht do not express ny detectle HER receptors, three stle CHO

8 Mdi et l. BMC Cncer (2018) 18:238 Pge 8 of 14 p-egfr (1068) EGFR Tuulin IgG EGF Trs 10 µg/ml ng/ml 50 ng/ml µg/ml 0.1 µg/ml 0.5 µg/ml 1 µg/ml 5 µg/ml 10 µg/ml Fig. 5 Control experiments to show the effects of trstuzum on EGFR phosphoryltion in CHO-EGFR cells nd the effects of CP on HER2 phosphoryltion in CHO-K6 cells. The effects of trstuzum on EGFR phosphoryltion or EGF-induced EGFR phosphoryltion in CHO-EGFR cells. Cells were treted with EGF nd/or trstuzum t vrious concentrtions s indicted. The phosphoryltion of EGFR ws determined y immunolotting with ntiody to EGFR py1068. The effects of chemicl inhiitor of HER2, CP on HER2 phosphoryltion in CHO-K6 cells. Cells were treted with CP t vrious concentrtions s indicted for 1 h. The phosphoryltion of HER2 ws exmined y immunolotting with ntiodies ginst HER2 py1005 nd py1139 cell lines tht stly express only single HER receptor including EGFR (CHO-EGFR), HER2 (CHO-K6), nd HER3 (CHO-ErB3) were employed in this reserch. Our cell model system voided the interference of other HER receptors, nd is very suitle to study the effects of trstuzum on the homodimeriztion of HER2 nd the phosphoryltion of HER2 homodimers. We im to conclusively determine if trstuzum specificlly inds only to HER2, nd locks HER2 homodimeriztion nd ctivtion. We showed tht trstuzum only ound to HER2 specificlly nd with high ffinity. Trstuzum did not ind to EGFR nd HER3 even t high dosge (10 ng/ml) (Fig. 2). Most HER2-positive rest cncer cells lso express EGFR nd HER3, our finding suggest tht ny trstuzum effects on these cells must e initited through the interction etween trstuzum nd HER2. We next exmined the effects of trstuzum on HER2 dimeriztion. HER2 is n orphn receptor nd does not hve lignd. However, HER2 re heterodimerized with EGFR in response to EGF stimultion nd heterodimerized with HER3 in response to HRG [1]. HER2 is lso homodimerized when overexpressed in cells. CHO-K6 cells only expresses single HER receptor HER2, not EGFR, HER3 or HER4. Thus our results re regrding the effects of trstuzum on the homodimeriztion of HER2. We showed tht in CHO-K6 cells HER2 ws mostly dimerized, likely due to the overexpression (Fig. 3). This is not surprising. As reveled y crystl structures of the HER2 extrcellulr region, HER2 dopts n extended configurtion, which resemles the configurtion of EGFR seen in ech molecule of n EGFR dimer. Thus, ErB2 possesses constitutive, or lignd independent, ctivted conformtion, which llows the HER2 homodimeriztion when overexpressed [1, 27, 72]. We lso showed tht trstuzum did not lock the homodimeriztion of HER2 (Fig. 3). While it is originlly proposed tht trstuzum cts to lock HER2 dimeriztion, so fr, no reserch hs een done to determine the effects of trstuzum on the homodimeriztion of HER2. Given the fct tht trstuzum inds to the juxtmemrne region of HER2 [27], which is not essentil for HER2 dimeriztion, our results re not surprising. Wht surprising is tht our dt suggest tht trstuzum t high dosge ctully enhnced the homodimeriztion of HER2 (Fig. 3). While we re not certin how trstuzum stimultes the homodimeriztion of HER2, it is possile tht it functions through HER2 trnsmemrne domin. Mny dt support the role of HER2 trnsmemrne domin in HER2 dimeriztion nd ctivtion [27]. Prts of juxtmemrne region hs lso een implicted in HER2 dimeriztion nd ctivtion [73 75]. As trstuzum inds to the extrcellulr juxtmemrne region of HER2, it will likely ffect the function of HER2 trnsmemrne domin nd juxtmemrne region in terms of HER2 dimeriztion. It is possile tht somehow the specific effects of trstuzum enhnced the interction etween two HER2 trnsmemrne domins nd thus incresed HER2 homodimeriztion s we oserved here. It hs een elieved tht trstuzum functions to inhiit HER2 ctivtion/phosphoryltion nd HER2- medited cell signling [6, 52, 53]. However, our dt indicted tht trstuzum only hd very limited effects on HER2 phosphoryltion. Among six py residues exmined in this reserch, HER2 hd no effects on the phosphoryltion of py1005, py1112, py1027, py1196, nd py1248 (Figs. 5 nd 6). While HER2 decresed the phosphoryltion of py1139, which is much weker inhiition when compred with CP (Figs. 5, 6 nd 7). In generl, this is consistent with our oservtion regrding the role of trstuzum in HER2 dimeriztion. Trstuzum did not lock HER2 dimeriztion, thus it did not lock HER2 phosphoryltion. It is not cler how the effects of HER2 trnsmemrne domin on HER2 dimeriztion ffect the phosphoryltion of HER2. Some reserch indicted the presence of n lterntive dimeriztion mode of

9 Mdi et l. BMC Cncer (2018) 18:238 Pge 9 of 14 c d e f Fig. 6 The effects of trstuzum on HER2 phosphoryltion in CHO-K6 cells y immunofluorescence. CHO-K6 cells were treted with trstuzum t concentrtions rnging from μg/ml for 1 h. The phosphoryltion of HER2 t Y1005 (), Y1112 (), Y1127 (c), Y1139 (d), Y1196 (e), nd Y1248 (f) were then exmined y immunofluorescence s descried in Methods. The locliztion of trstuzum ws reveled y TRITC-conjugted donkey nti-humn IgG. The locliztion of HER2 ws reveled y rit nti-phosphoher2 ntiody followed y FITC-conjugted donkey nti-rit IgG. The cell nuclei were counter stined with DAPI. Yellow indicted the co-locliztion of trstuzum nd phosphoher2. The cells treted with norml humn IgG (10 μg/ml) were used s negtive controls. Size r: 10 μm HER2. In this mode, HER2 dimeriztion is medited y oth trnsmemrne domin nd the cytoplsmic juxtmemrne region of HER2. Such dimeriztion mode exert inhiiting effects on the HER2 kinse ctivity [73 75]. Thus, in theory, the enhnced dimeriztion through the interction of trnsmemrne domin nd the juxtmemrne region could result in the inhiition of certin HER2 phosphoryltion including py1139. Recently, some reserches with vrious rest cncer cell lines hve shown tht trstuzum did not significntly lter HER2 phosphoryltion [53 56, 76]. Moreover, there is one reserch shows the enhnced phosphoryltion of py1248 in response to trstuzum [52]. Our results suggest tht trstuzum hs, if ny, limited effects on HER2-medited intrcellulr signling. Indeed, when we exmined the effects of trstuzum on the phosphoryltion of ERK nd AKT, we showed tht trstuzum did not lock the phosphoryltion of oth ERK nd AKT in CHO-K6 cells (Fig. 7). Together, our dt indicte tht trstuzum did not significntly

10 Mdi et l. BMC Cncer (2018) 18:238 Pge 10 of 14 Fig. 7 The effects of trstuzum on the phosphoryltion of ERK nd AKT in CHO-K6 cells. The phosphoryltion of ERK nd AKT ws reveled y immunolotting s descried in Mterils nd Methods. The phosphoryltion of ERK nd AKT in CHO prentl cells nd in CHO-K6 cells. The effects of trstuzum on the phosphoryltion of ERK nd AKT in CHO-K6 cells. Cells were treted with trstuzum with indicted concentrtions for 7 h. Cells treted with norml humn IgG ws used s negtive control nd cells treted with CP (CP) ws used s positive control lter HER2 ctivtion nd HER2 medited intrcellulr signling in the sence of other HER receptors. However, we need to e cutious to pply these findings to rest cncer cells. CHO cell is derived from hmster ovry, thus the expressed humn HER2 my not e coupled well with downstrem signling cscdes. We then exmined if trstuzum induces ADCC in CHO-K6 cell. We showed tht trstuzum indeed induces strong ADCC in CHO-K6 cells (Fig. 8). This is specificlly due to the expression of HER2 in CHO-K6 cells s there is no ADCC oserved in CHO-EGFR cells (Fig. 8). The role of trstuzum in the induction of ADCC in HER2-positive rest cncer cells hve een consistently well supported y mny reserches [44 51]. Our results confirmed the role of trstuzum in the induction of ADCC in simple ut specific cell setting. Fig. 8 Trstuzum-induced ADCC in CHO-K6 nd CHO-EGFR cells. Trstuzum-induced ADCC ws exmined in oth CHO-K6 nd CHO-EGFR cells y using Promeg ADCC Biossy kit s descried in Methods We lso showed tht trstuzum did not ffect cell prolifertion in CHO-K6 cells (Fig. 9). Some reports indicted tht trstuzum hd little effect on prolifertion nd survivl [58, 77]. However, other reports indicted tht trstuzum inhiited ErB2 ctivtion, nd decresed the ctivtion of ERK nd PI3K-AKT pthwys, which leds to reduced cell prolifertion [57]. Given tht trstuzum hs little effects on the phosphoryltion of HER2, it is likely tht trstuzum hs no effects on HER2-medited cell signling leding to cell prolifertion. Although trstuzum induces ADCC in CHO-K6 cell, under the culture conditions used for the MTT ssy, no effector cells were present nd no ADCC response re expected. It is interesting to note tht our ove finding re different from the oservtion y Ghosh [78]. Ghosh et l. reported tht trstuzum inhiited HER2 homodimer-medited ERK phosphoryltion nd cell growth. The difference could e due to the different model system used in these two studies. The HER2 receptor used in the reserch y Ghosh et l. is fused with FKBP, nd the receptor homodimeriztion is induced y chemicl linker AP1510 tht dimerizes the receptor intrcellulrly through the fused FKBP. It should e noted tht while we oserved strong inhiition of HER2 phosphoryltion y CP t 1 μm (Fig. 5). We only oserved the inhiition of CHO-K6 cell prolifertion t much higher CP concentrtion (Fig. 9f). We re not sure wht cuse this discrepncy, however, there re severl possile explntions. Firstly, t 1 μm, CP my not completely inhiit Her2 phosphoryltion, we cn see wek phosphoryltion of Y1139 in Fig. 5. There could e wek phosphoryltion of HER2 t other py residues tht were not exmined. A wek HER2 phosphoryltion my still e

11 Mdi et l. BMC Cncer (2018) 18:238 Pge 11 of 14 c d e f Fig. 9 The effects of trstuzum on the prolifertion of CHO, CHO-EGFR, CHO-K6, nd CHO-HER3 cells. The cell prolifertion ws exmined y MTT ssy s descried in Mterils nd Methods. The effects of HER2 overexpression on the prolifertion of CHO cells. Cell prolifertion of oth CHO prentl cells nd CHO-K6 cells ws exmined. -e The effects of trstuzum on the prolifertion of CHO, CHO-EGFR, CHO-K6 nd CHO-HER3 cells. Cell were treted with vrious concentrtion of trstuzum s indicted for 24 nd 48 h. Non treted cells were used s negtive control nd the cells treted with vinoreline (VR) were used s positive controls. CHO cells. c CHO-HER3 cells. d CHO-EGFR cells. e CHO-K6 cells. f The effects of other HER2 inhiitors on the prolifertion of CHO-K6 cells. CHO-K6 cells were treted with HER2 kinse inhiitors lptini (Lp) nd CP of indicted concentrtion. Ech vlue is the verge of t lest three experiments nd the error r is stndrd error. ***: p < ****: p < sufficient to support cell growth. Secondly, there could e the existence of kinse-independent effects of HER2 receptors. There re mny reports supporting the existence of kinse-independent cell signling of vrious receptor tyrosine kinses including EGFR nd Insulin receptor [79 82]. Conclusion Together, in this reserch we dopted cell model tht llow us to specificlly exmine the effects of trstuzum on single HER receptor without the influence of other HER receptors. Three CHO cell lines stly expressing only humn EGFR, HER2, or HER3 were used. These model system llow us to specificlly exmine the effects of trstuzum on the homodimeriztion of HER2 nd the phosphoryltion of HER2 homodimers. We demonstrte tht overexpression of HER2 in CHO cells results in the homodimeriztion of HER2 nd the phosphoryltion of HER2 t ll mjor py residues. Trstuzum inds to HER2 specificlly nd with high ffinity. Trstuzum does not inhiit the homodimeriztion of HER2. Trstuzum does not inhiit the phosphoryltion of HER2 t most phosphotyrosine residues. We lso oserved tht trstuzum neither inhiits the phosphoryltion of the downstrem signling proteins including ERK nd AKT, nor inhiits the prolifertion of CHO cells overexpressing HER2. However, cution is needed to pply these findings to rest cncer cells. However, trstuzum induces strong ADCC in CHO cell overexpressing HER2. We concluded tht trstuzum exerts its ntitumor ctivity through the induction of ADCC, rther thn the inhiition of HER2-medited cell signling in the sence of other HER receptors.

12 Mdi et l. BMC Cncer (2018) 18:238 Pge 12 of 14 Arevitions ADCC: Antiody-dependent cellulr cytotoxicity; CHO cells: Chinese Hmster Ovry cells; EGF: Epiderml growth fctor; EGFR: Epiderml growth fctor receptor; ERK: Extrcellulr signl-regulted kinses; FDA: Food nd Drug Administrtion; HER2: Humn epiderml growth fctor receptor 2; HER3: Humn epiderml growth fctor receptor 3; HER4: Humn epiderml growth fctor receptor 4; MTT: (3-(4,5-dimethylthizol-2yl)-2,5-diphenyltetrzolium romide; NK cells: Nturl-killer cells; PM: The plsm memrne; RTK: Receptor tyrosine kinse Acknowledgements We thnk Drs. Hitt nd Buchholz for providing us with CHO-HER3 nd CHO-K6 cell lines. Funding Our reserch ws supported in prt y grnts from the Cndin Brest Cncer Foundtion (CBCF) to ZW nd Cndin Institutes of Helth Reserch (CIHR) to ZW. BN is supported y Grdute Studentship from Women nd Children s Helth Reserch Institute (WCHRI). No specific funding ws received for this study. Avilility of dt nd mterils In ddition to the dt reported in this mnuscript, ll the primry dt will e ville upon request. All the mteril we generted for this reserch will e ville to other reserchers upon request. Authors contriutions All uthors hve red nd pproved the mnuscript. HM: Prticipting in the design of the project, performnce of the experiments including cell culture, cytotoxicity ssy, crosslinking, nd immunolotting, nd prticipting in the dt nlysis nd mnuscript writing. BN: Prticipting in the design of the project, performnce of the experiments including cell culture nd immunofluorescence, nd prticipting in the dt nlysis nd mnuscript writing. JT: Performing experiments including cell culture nd ADCC ssy, nd prticipting in dt nlysis. GL: Performing experiments including cell culture, cytotoxicity ssy, nd immunolotting, nd prticipting in dt nlysis. ZW: Prticipting in the design of the project, nd prticipting in dt nlysis nd the writing of the mnuscript. Ethics pprovl nd consent to prticipte The CHO Cells used in this reserch is purchsed from ATCC. The CHO-EGFR, CHO-HER2 nd CHO-HER3 cells were derived from CHO cells s descried in Methods section. Ethics pprovl nd consent to prticipte is not required. Consent for puliction Not pplicle. Competing interests All uthors declre tht they hve no competing interests. Pulisher s Note Springer Nture remins neutrl with regrd to jurisdictionl clims in pulished mps nd institutionl ffilitions. Received: 6 June 2017 Accepted: 19 Ferury 2018 References 1. Yrden Y, Sliwkowski MX. Untngling the ErB signlling network. NtRevMolCell Biol. 2001;2(2): Citri A, Yrden Y. EGF-ERBB signlling: towrds the systems level. NtRevMolCell Biol. 2006;7(7): Peles E, Yrden Y. Neu nd its lignds: from n oncogene to neurl fctors. BioEssys : news nd reviews in moleculr, cellulr nd developmentl iology. 1993;15(12): Rjkumr T, Gullick WJ. The type I growth fctor receptors in humn rest cncer. Brest Cncer ResTret. 1994;29(1): Hynes NE, McDonld G. ErB receptors nd signling pthwys in cncer. CurrOpinCell Biol. 2009;21(2): Spector NL, Blckwell KL. Understnding the mechnisms ehind trstuzum therpy for humn epiderml growth fctor receptor 2-positive rest cncer. JClinOncol. 2009;27(34): Ci Z, Zhng H, Liu J, Berezov A, Murli R, Wng Q, Greene MI. Trgeting erb receptors. Semin Cell Dev Biol. 2010;21(9): Fiszmn GL, Jsnis MA. Moleculr mechnisms of Trstuzum resistnce in HER2 overexpressing rest cncer. IntJBrest Cncer. 2011;2011: Mhipl A, Kothri N, Gupt S. Epiderml growth fctor receptor inhiitors: coming of ge. Cncer control : journl of the Moffitt Cncer Center. 2014;21(1): Lluch A, Eroles P, Perez-Fidlgo JA. Emerging EGFR ntgonists for rest cncer. Expert opinion on emerging drugs. 2014;19(2): Rimwi MF, Schiff R, Osorne CK. Trgeting HER2 for the tretment of rest cncer. Annu Rev Med. 2015;66: Slmon DJ, Clrk GM, Wong SG, Levin WJ, Ullrich A, McGuire WL. Humn rest cncer: correltion of relpse nd survivl with mplifiction of the HER-2/neu oncogene. Science (New York, NY). 1987;235(4785): Slmon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE, Levin WJ, Sturt SG, Udove J, Ullrich A. Studies of the HER-2/neu proto-oncogene in humn rest nd ovrin cncer. Science (New York, NY). 1989;244(4905): Gullick WJ. The c-erb3/her3 receptor in humn cncer. Cncer Surv. 1996;27: Hynes NE, Lne HA. ERBB receptors nd cncer: the complexity of trgeted inhiitors. NtRevCncer. 2005;5(5): Cpdevil J, Elez E, Mcrull T, Rmos FJ, Ruiz-Echrri M, Ternero J. Anti-epiderml growth fctor receptor monoclonl ntiodies in cncer tretment. Cncer Tret Rev. 2009;35(4): Ross JS, Slodkowsk EA, Symmns WF, Puszti L, Rvdin PM, Hortogyi GN. The HER-2 receptor nd rest cncer: ten yers of trgeted nti-her-2 therpy nd personlized medicine. Oncologist. 2009;14(4): De Mttos-Arrud L, Cortes J. Use of pertuzum for the tretment of HER2-positive metsttic rest cncer. Adv Ther. 2013;30(7): Dwood S, Broglio K, Buzdr AU, Hortogyi GN, Giordno SH. Prognosis of women with metsttic rest cncer y HER2 sttus nd trstuzum tretment: n institutionl-sed review. Journl of clinicl oncology : officil journl of the Americn Society of Clinicl Oncology. 2010;28(1): Nht R, Yu D, Hung MC, Hortogyi GN, Estev FJ. Mechnisms of disese: understnding resistnce to HER2-trgeted therpy in humn rest cncer. Nt Clin Prct Oncol. 2006;3(5): Arteg CL, Sliwkowski MX, Osorne CK, Perez EA, Puglisi F, Ginni L. Tretment of HER2-positive rest cncer: current sttus nd future perspectives. Nt Rev Clin Oncol. 2012;9(1): Heldin CH. Dimeriztion of cell surfce receptors in signl trnsduction. Cell. 1995;80(2): Mrmor MD, Skri KB, Yrden Y. Signl trnsduction nd oncogenesis y ErB/HER receptors. IntJRditOncolBiolPhys. 2004;58(3): Crrwy KL 3rd, Cntley LC. A neu cquintnce for erb3 nd erb4: role for receptor heterodimeriztion in growth signling. Cell. 1994;78(1): Pinks-Krmrski R, Soussn L, Wtermn H, Levkowitz G, Alroy I, Klpper L, Lvi S, Seger R, Rtzkin BJ, Sel M, et l. Diversifiction of Neu differentition fctor nd epiderml growth fctor signling y comintoril receptor interctions. EMBO J. 1996;15(10): Tzhr E, Wtermn H, Chen X, Levkowitz G, Krungrn D, Lvi S, Rtzkin BJ, Yrden Y. A hierrchicl network of interreceptor interctions determines signl trnsduction y Neu differentition fctor/neuregulin nd epiderml growth fctor. MolCell Biol. 1996;16(10): Cho HS, Mson K, Rmyr KX, Stnley AM, Gelli SB, Denney DW Jr, Lehy DJ. Structure of the extrcellulr region of HER2 lone nd in complex with the Herceptin f. Nture. 2003;421(6924): Grrett TP, McKern NM, Lou M, Ellemn TC, Adms TE, Lovrecz GO, Kofler M, Jorissen RN, Nice EC, Burgess AW, et l. The crystl structure of truncted ErB2 ectodomin revels n ctive conformtion, poised to interct with other ErB receptors. MolCell. 2003;11(2): Burgess AW. EGFR fmily: structure physiology signlling nd therpeutic trgets. Growth fctors (Chur, Switzerlnd). 2008;26(5): Erp HS, Dwson TL, Li X, Yu H. Heterodimeriztion nd functionl interction etween EGF receptor fmily memers: new signling prdigm with implictions for rest cncer reserch. Brest Cncer ResTret. 1995;35(1):

13 Mdi et l. BMC Cncer (2018) 18:238 Pge 13 of Wllsch C1, Weiss FU, Niederfellner G, Jlll B, Issing W, Ullrich A. Heregulin-dependent regultion of HER2/neu oncogenic signling y heterodimeriztion with HER3. EMBO J. 1995;14(17): Krungrn D, Tzhr E, Beerli RR, Chen X, Grus-Port D, Rtzkin BJ, Seger R, Hynes NE, Yrden Y. ErB-2 is common uxiliry suunit of NDF nd EGF receptors: implictions for rest cncer. EMBO J. 1996;15(2): Gmett DC, Person G, Cerione RA, Friederg I. Secondry dimeriztion etween memers of the epiderml growth fctor receptor fmily. JBiolChem. 1997;272(18): Grus-Port D, Beerli RR, Dly JM, Hynes NE. ErB-2, the preferred heterodimeriztion prtner of ll ErB receptors, is meditor of lterl signling. EMBO J. 1997;16(7): Alroy I, Yrden Y. The ErB signling network in emryogenesis nd oncogenesis: signl diversifiction through comintoril lignd-receptor interctions. FEBS Lett. 1997;410(1): Wtermn H, Sni I, Geiger B, Yrden Y. Alterntive intrcellulr routing of ErB receptors my determine signling potency. JBiolChem. 1998; 273(22): Wtermn H, Levkowitz G, Alroy I, Yrden Y. The RING finger of c-cl medites desensitiztion of the epiderml growth fctor receptor [in process cittion]. JBiolChem. 1999;274(32): Lenferink AE, Pinks-Krmrski R, vn de Poll ML, vn Vugt MJ, Klpper LN, Tzhr E, Wtermn H, Sel M, vn Zoelen EJ, Yrden Y. Differentil endocytic routing of homo- nd hetero-dimeric ErB tyrosine kinses confers signling superiority to receptor heterodimers. EMBO J. 1998;17(12): Worthylke R, Opresko LK, Wiley HS. ErB-2 mplifiction inhiits down-regultion nd induces constitutive ctivtion of oth ErB-2 nd epiderml growth fctor receptors. JBiolChem. 1999;274(13): Ahmed S, Smi A, Xing J. HER2-directed therpy: current tretment options for HER2-positive rest cncer. Tokyo: Brest cncer; Fendly BM, Kotts C, Vetterlein D, Lewis GD, Winget M, Crver ME, Wtson SR, Srup J, Sks S, Ullrich A, et l. The extrcellulr domin of HER2/neu is potentil immunogen for ctive specific immunotherpy of rest cncer. JBiolResponse Mod. 1990;9(5): Lewis GD, Figri I, Fendly B, Wong WL, Crter P, Gormn C, Sheprd HM. Differentil responses of humn tumor cell lines to nti-p185her2 monoclonl ntiodies. Cncer ImmunolImmunother. 1993;37(4): Nuti M, Bellti F, Visconti V, Npoletno C, Domenici L, Cccett J, Zizzri IG, Ruscito I, Rhimi H, Benedetti-Pnici P, et l. Immune effects of trstuzum. J Cncer. 2011;2: Clynes RA, Towers TL, Prest LG, Rvetch JV. Inhiitory fc receptors modulte in vivo cytotoxicity ginst tumor trgets. Nt Med. 2000;6(4): Arnould L, Gelly M, Penult-Llorc F, Benoit L, Bonnetin F, Migeon C, Cret V, Fermeux V, Bertheu P, Grnier J, et l. Trstuzum-sed tretment of HER2-positive rest cncer: n ntiody-dependent cellulr cytotoxicity mechnism? Br J Cncer. 2006;94(2): Vrchett S, Gielli N, Oliviero B, Nrdini E, Gennri R, Gtti G, Silv LS, Villni L, Tgliue E, Menrd S, et l. Elements relted to heterogeneity of ntiody-dependent cell cytotoxicity in ptients under trstuzum therpy for primry operle rest cncer overexpressing Her2. Cncer Res. 2007;67(24): Kute T, Stehle JR Jr, Ornelles D, Wlker N, Delono O, Vughn JP. Understnding key ssy prmeters tht ffect mesurements of trstuzum-medited ADCC ginst Her2 positive rest cncer cells. Oncoimmunology. 2012;1(6): Petricevic B, Lengle J, Singer J, Schet M, Fzeks J, Steger G, Brtsch R, Jensen-Jrolim E, Bergmnn M. Trstuzum medites ntiody-dependent cell-medited cytotoxicity nd phgocytosis to the sme extent in oth djuvnt nd metsttic HER2/neu rest cncer ptients. J Trnsl Med. 2013;11: Duong MN, Cleret A, Mter EL, Chett K, Mthe D, Vlsesi-Wittmnn S, Clemenceu B, Dumontet C. Adipose cells promote resistnce of rest cncer cells to trstuzum-medited ntiody-dependent cellulr cytotoxicity. Brest cncer reserch : BCR. 2015;17: Shi Y, Fn X, Deng H, Brezski RJ, Rycyzyn M, Jordn RE, Strohl WR, Zou Q, Zhng N, An Z. Trstuzum triggers phgocytic killing of high HER2 cncer cells in vitro nd in vivo y interction with Fcgmm receptors on mcrophges. Journl of immunology (Bltimore, Md : 1950). 2015; 194(9): Scltriti M, Verm C, Guzmn M, Jimenez J, Prr JL, Pedersen K, Smith DJ, Lndolfi S. Rmon y Cjl S, Arris J, et l. Lptini, HER2 tyrosine kinse inhiitor, induces stiliztion nd ccumultion of HER2 nd potentites trstuzum-dependent cell cytotoxicity. Oncogene. 2009;28(6): Dokmnovic M, Wu Y, Shen Y, Chen J, Hirsch DS, Wu WJ. Trstuzuminduced recruitment of Csk-homologous kinse (CHK) to ErB2 receptor is ssocited with ErB2-Y1248 phosphoryltion nd ErB2 degrdtion to medite cell growth inhiition. Cncer iology & therpy. 2014;15(8): Gijsen M, King P, Perer T, Prker PJ, Hrris AL, Lrijni B, Kong A. HER2 phosphoryltion is mintined y PKB negtive feedck loop in response to nti-her2 herceptin in rest cncer. PLoS Biol. 2010;8(12):e Agus DB, Akit RW, Fox WD, Lewis GD, Higgins B, Piscne PI, Lofgren JA, Tindell C, Evns DP, Miese K, et l. Trgeting lignd-ctivted ErB2 signling inhiits rest nd prostte tumor growth. Cncer Cell. 2002;2(2): Nht R, Hung MC, Estev FJ. The HER-2-trgeting ntiodies trstuzum nd pertuzum synergisticlly inhiit the survivl of rest cncer cells. Cncer Res. 2004;64(7): Longv KE, Pedersen NM, Hsleks C, Stng E, Mdshus IH. Herceptininduced inhiition of ErB2 signling involves reduced phosphoryltion of Akt ut not endocytic down-regultion of ErB2. IntJCncer. 2005;116(3): Ykes FM, Chinrtnl W, Ritter CA, King W, Seelig S, Arteg CL. Herceptin-induced inhiition of phosphtidylinositol-3 kinse nd Akt is required for ntiody-medited effects on p27, cyclin D1, nd ntitumor ction. Cncer Res. 2002;62(14): Xi W, Bisi J, Strum J, Liu L, Crrick K, Grhm KM, Treece AL, Hrdwicke MA, Dush M, Lio Q, et l. Regultion of survivin y ErB2 signling: therpeutic implictions for ErB2-overexpressing rest cncers. Cncer Res. 2006;66(3): Cuello M, Ettenerg SA, Clrk AS, Kene MM, Posner RH, Nu MM, Dennis PA, Lipkowitz S. Down-regultion of the erb-2 receptor y trstuzum (herceptin) enhnces tumor necrosis fctor-relted poptosis-inducing lignd-medited poptosis in rest nd ovrin cncer cell lines tht overexpress erb-2. Cncer Res. 2001;61(12): Austin CD, De Mziere AM, Piscne PI, vn Dijk SM, Eigenrot C, Sliwkowski MX, Klumpermn J, Scheller RH. Endocytosis nd sorting of ErB2 nd the site of ction of cncer therpeutics trstuzum nd geldnmycin. MolBiolCell. 2004;15(12): Vlreg G, Montemurro F, Srotto I, Petrelli A, Ruini P, Tcchetti C, Agliett M, Comoglio PM, Giordno S. TGFlph expression impirs Trstuzum-induced HER2 downregultion. Oncogene. 2005;24(18): Pietrs RJ, Pegrm MD, Finn RS, Mnevl DA, Slmon DJ. Remission of humn rest cncer xenogrfts on therpy with humnized monoclonl ntiody to HER-2 receptor nd DNA-rective drugs. Oncogene. 1998;17(17): Molin MA, Codony-Servt J, Alnell J, Rojo F, Arris J, Bselg J. Trstuzum (herceptin), humnized nti-her2 receptor monoclonl ntiody, inhiits sl nd ctivted Her2 ectodomin clevge in rest cncer cells. Cncer Res. 2001;61(12): Izumi Y, Xu L, di Tomso E, Fukumur D, Jin RK. Tumour iology: herceptin cts s n nti-ngiogenic cocktil. Nture. 2002;416(6878): Wen XF, Yng G, Mo W, Thornton A, Liu J, Bst RC Jr, Le XF. HER2 signling modultes the equilirium etween pro- nd ntingiogenic fctors vi distinct pthwys: implictions for HER2-trgeted ntiody therpy. Oncogene. 2006;25(52): Wng Q, Zhu F, Wng Z. Identifiction of EGF receptor C-terminl sequences nd di-leucine motif 1010 LL 1011 s essentil in EGF receptor endocytosis. ExpCell Res. 2007;313: Munch RC, Muhlech MD, Schser T, Kneissl S, Jost C, Pluckthun A, Cichutek K, Buchholz CJ. DARPins: n efficient trgeting domin for lentivirl vectors. Moleculr therpy : the journl of the Americn Society of Gene Therpy. 2011;19(4): Mendrol JM, Berger MB, King MC, Lemmon MA. The single trnsmemrne domins of ErB receptors self-ssocite in cell memrnes. J Biol Chem. 2002;277(7): Wng RC, Chen X, Prissenti AM, Joy AA, Tuszynski J, Brindley DN, Wng Z. Sensitivity of docetxel-resistnt MCF-7 rest cncer cells to microtuuledestilizing gents including vinc lkloids nd colchicine-site inding gents. PLoS One. 2017;12(8):e Ngy P, Jenei A, Kirsch AK, Szollosi J, Dmjnovich S, Jovin TM. Activtiondependent clustering of the erb2 receptor tyrosine kinse detected y scnning ner-field opticl microscopy. J Cell Sci. 1999;112(Pt 11):