RESEARCH Open Access
Func ion o RasGRP3 in he o ma ion and
p og ession o human b eas cance
Zsuzsanna Nagy
1
, Ilona Ko ács
2
, Miklós Tö ök
2
, DezsőTó h
3
, Gyö gy Ve eb
4
, K isz ina Buzás
5
, Is án Juhász
6
,
Pe e M Blumbe g
7
, Tamás Bí ó
1
and Gab iella Czi a
1*
Abs ac
In oduc ion: Ras guanine nucleo ide exchange ac o s (RasGEFs) media e he ac i a ion o he Ras signaling
pa hway ha is o e ac i a ed in many human cance s. The RasGRP3, an ac i a o o H-Ras and R-Ras p o ein exe s
oncogenic e ec s and he o e exp ession o he p o ein is obse ed in nume ous malignan cance ypes. He e, we
in es iga ed he pu a i e al e a ion o exp ession and po en ial unc ion o RasGRP3 in he o ma ion and p og ession
o human b eas cance .
Me hods: The RasGRP3 and phosphoRasGRP3 exp essions we e examined in human in asi e duc al adenoca cinoma
de i ed samples and cell lines (BT-474, JIMT-1, MCF7, SK-BR-3, MDA-MB-453, T-47D) bo h in mRNA (Q-PCR) and p o ein
(Wes e n blo ; immunohis ochemis y) le els. To explo e he biological unc ion o he p o ein, RasGRP3 knockdown
cul u es we e es ablished. To assess he ole o RasGRP3 in he iabili y o cells, annexin-V/PI s aining and Mi oP obe™
DilC1 (5) assay we e pe o med. To cla i y he unc ion o he p o ein in cell p oli e a ion and in he de elopmen o
chemo he apeu ic esis ance, CyQuan assay was pe o med. To obse e he RasGRP3 unc ion in umo o ma ion,
heSe e ecombinedimmunode iciency(SCID)mousemodelwasused.Toin es iga e he oleo hep o einin
Ras- ela ed signaling Q-PCR and Wes e n blo expe imen s we e pe o med.
Resul s: RasGRP3 exp ession was ele a ed in human b eas umo issue samples as well as in mul iple human
b eas cance cell lines. Down- egula ion o RasGRP3 exp ession in b eas cance cells dec eased cell p oli e a ion,
inducedapop osisinMCF7cells,andsensi izedT-47Dcells o he ac ion o d ugs Tamoxi en and as uzumab
(He cep in). Gene silencing o RasGRP3 educed umo o ma ion in mouse xenog a s as well. Inhibi ion o
RasGRP3 exp ession also educed Ak , ERK1/2 and es ogen ecep o alpha phospho yla ion downs eam om
IGF-I insulin like g ow h ac o -I (IGF-I) o epide mal g ow h ac o (EGF) s imula ion con i ming he unc ional ole
o RasGRP3 in he al e ed beha io o hese cells.
Conclusions: Taken oge he , ou esul s sugges ha he Ras ac i a o RasGRP3 may ha e a ole in he pa hological
beha io o b eas cance cells and may cons i u e a he apeu ic a ge o human b eas cance .
Keywo ds: Ras ac i a o , RasGRP3, Human b eas cance , Chemo he apeu ic esis ance, Tamoxi en, T as uzumab,
Tumo igenesis, EGF, IGF-I, Signaling pa hways
* Co espondence: [email p o ec ed]
1
DE-MTA “Lendüle ”Cellula Physiology Resea ch G oup, Depa men o
Physiology, Uni e si y o Deb ecen, Medical and Heal h Science Cen e ,
Resea ch Cen e o Molecula Medicine, Nagye dei k . 98, PO Box 22,
Deb ecen H-4032, Hunga y
Full lis o au ho in o ma ion is a ailable a he end o he a icle
© 2014 Nagy e al.; licensee BioMed Cen al L d. This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e
Commons A ibu ion License (h p://c ea i ecommons.o g/licenses/by/2.0), which pe mi s un es ic ed use, dis ibu ion, and
ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly c edi ed. The C ea i e Commons Public Domain
Dedica ion wai e (h p://c ea i ecommons.o g/publicdomain/ze o/1.0/) applies o he da a made a ailable in his a icle,
unless o he wise s a ed.
Nagy e al. Molecula Cance 2014, 13:96
h p://www.molecula -cance .com/con en /13/1/96
In oduc ion
The ac i a ion o Ras amily membe s is con eyed by
speci ic ups eam egula o s such as e.g. guanine nucleo-
ide exchange ac o s (RasGEF) [1]. Re lec ing hei signi i-
cance, gene ic loss o hese ac o s and hei unc ions was
shown o esul in simila e ec s o hose induced by he
loss o he Ras p o eins hemsel es [2,3]. In 1998, Ebinu
e al. ha e in oduced a new RasGEF called Ras Guanyl
nucleo ide Releasing Pep ide o RasGRP [4]. A unique ea-
u e o RasGRPs is he p esence o a C1 domain se ing
as a binding si e o he endogenous signaling molecule
diacylglyce ol (DAG) and i s exogenous coun e pa s, he
pho bol es e s. As RasGRPs a e egula ed di ec ly by bind-
ing DAG [5,6], hey ac as media o s o he a mul i ude
o ecep o -coupled mechanisms (induced e.g. by nume -
ous g ow h ac o s) ha ac i a e phospholipase C and he
ela ed signal ansduc ion machine ies [7,8]. RasGRP3 is
one o he ou membe s o RasGRP amily; i was shown
o ac i a e H-Ras and R-Ras [4,9].
The p esence and ac i i y o he Ras- ela ed subcellu-
la p ocesses and he induced biological esponses a e
one o he key de e minan s in malignan cell ans o m-
a ion and p og ession. E o s o iden i y he na u e and
ole o he Ras- ela ed in acellula signaliza ion pa h-
ways ha e ecen ly sugges ed ha no only he down-
s eam media o s bu also he ups eam egula o s o
Ras migh play a ole on hese mechanisms. In pe ec
ag eemen wi h his p oposal, membe s o RasGRP am-
ily a e sugges ed o unc ion as oncogenes in mul iple
cance s [10]. Indeed, RasGRP3 is highly exp essed in
human Bu ki ’s lymphoma, human p e-B-cell leukemia,
and na u al kille -like T-cell leukemia. O u he im-
po ance, we ha e ecen ly shown ha RasGRP3 is cen-
ally in ol ed in he egula ion o cell p oli e a ion,
su i al, mig a ion and umo o ma ion o human p os-
a e ca cinoma cells and se e al melanoma cell lines as
well as in he malignan ans o ma ion o human mela-
nocy es [11-13].
These indings which implica e p o-oncogenic e ec
o he p o ein and, u he mo e, he ac ha Ras was
ound is ch onically ac i a ed in b eas ca cinoma cells
ha lack mu a ed as [14], ha e mo i a ed us o assess
he exp ession and unc ion o RasGRP3 in b eas
de i ed in asi e duc al adenoca cinoma, a malignancy
cha ac e ized by a high endency o p oduce me a-
s a ic umo s [15]. In he p esen s udy, we p o ide
e idence ha exp essions o RasGRP3 and i s ac i e
o m (phosphoRasGRP3) a e highly ele a ed in human
b eas cance samples and ha i is also p esen in
a p ima y and se e al me as a ic human b eas cance
cell lines. Mo eo e , we also show ha RasGRP3 con ib-
u es o p oli e a ion, su i al, chemo he apeu ic esis ance
and in i o umo g ow h o b eas ca cinoma-de i ed
umo cell lines.
Resul s
RasGRP3 is ele a ed in human b eas de i ed duc al
adenoca cinoma
Fi s , using Q-PCR (Figu e 1A) and Wes e n blo
(Figu e 1B), we assessed he exp ession o he RasGRP3
p o ein and p esumably i s ac i e o m, phosphoRasGRP3,
in human b eas de i ed duc al adenoca cinoma (Tu)
as well as in no mal human b eas issue (Co) sam-
ples. We ound ha he le els o RasGRP3 and phos-
phoRasGRP3, albei exhibi ing ma ked in e -indi idual
a ia ions, we e signi ican ly highe in he umo sam-
ples compa ed o he con ols. In addi ion, we also
de e mined he cellula localiza ions o RasGRP3 and
phosphoRasGRP3 by immunohis ochemis y in sec ions
p epa ed om he diseased b eas issues. RasGRP3
was localized in he cy oplasm o he cells whe eas
he ac i e o m exhibi ed mos ly nuclea immuno e-
ac i i y (Figu e 1C). Human kidney de i ed samples
(9) se ed as posi i e con ols (PC) in immunohis o-
chemical expe imen s.
RasGRP3 is exp essed in human b eas de i ed duc al
adenoca cinoma cell lines and in ol ed in he egula ion
o g ow h o MCF7 and T-47D cells
We de e mined he exp ession o RasGRP3 in six di e en
human b eas duc al adenoca cinoma de i ed cell lines,
namely in BT-474, MDA-MB-453, MCF7, SK-BR-3,
T-47D and JIMT-1 cells ( he cha ac e is ics o he cell
lines and ele an e e ences a e shown in Addi ional
ile 1: Table S1). Quan i a i e PCR and Wes e n blo
analyses con i med he exp ession o RasGRP3 in all
cell lines wi h ba ely de ec able le els in BT-474 and
MDA-MB-453 cells (Figu e 2A). Fo compa ison, he
ela i ely high exp ession o RasGRP3 in he PC-3 p os-
a e adenoca cinoma de i ed cell line [12] se ed as a
posi i e con ol in bo h assays.
To explo e he unc ionali y o RasGRP3, namely he
ole o he p o ein in he egula ion o p oli e a ion, iabil-
i y, chemo he apeu ic esis ance and umo igenesis we
employed he shRNA-in e e ence echnique. Namely,
he cellula le els o he p o ein in MCF7 and T-47D
cells was supp essed by e o i al ec o s exp essing
shRNAs o RasGRP3 (shRasGRP3) o agains a non-
a ge ing sc ambled con ol (shSCR) o achie e a long
e m (s able) supp ession (sequences o he shRNA p obes
a e shown in Addi ional ile 2: Table S2). As de e mined
by con i ma o y Wes e n blo analysis, le els o RasGRP3
could be e ec i ely educed in bo h MCF7 and T-47D
cance cells whe eas in cells exp essing shSCR signi ican
modula ion o RasGRP3 exp ession was no obse ed
(Figu e 2B).
We assessed he possible al e a ions in cellula
unc ions o he T-47D and MCF7 de i ed “RasGRP3-
silenced”cells. As e ealed by g ow h cu e analysis
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using luo ime ic CyQUANT cellula p oli e a ion
assays om days 0–5 o cul u ing (Figu e 2C), down-
egula ion o RasGRP3 exp ession esul ed in a signi ican
supp ession o cell g ow h in bo h cell lines compa ed
o he p oli e a ion o cells bea ing he non- a ge ing
shSCR.
Figu e 1 RasGRP3 is exp essed in human b eas de i ed duc al adenoca cinoma. (A) Q-PCR and (B) Wes e n blo analysis o RasGRP3
and phosphoRasGRP3 exp essions in human no mal b eas (Con ols; n = 21) and b eas de i ed duc al adenoca cinoma (Tumo s; n = 33)
wi h a ious umo g ades (G1-G3) p esen ed acco ding o he molecula g ading o human b eas de i ed duc al adenoca cinoma (Luminal A; Luminal
B and HER-2). GAPDH was used as an in e nal con ol. The esul s a e ep esen a i e o h ee independen expe imen s o Q-PCR and wo independen
expe imen s o Wes e n blo . Values ep esen he mean ± SEM. (C) Rep esen a i e images o RasGRP3- and phosphoRasGRP3-speci ic immun eac i i y
wi h diaminobenzidine as a ch omogen (b own s aining) on human no mal and b eas de i ed duc al adenoca cinoma sec ions. Human kidney samples
we e included as a posi i e con ol (PC) [16]. Nuclei we e co-s ained by Maye ’s Hema oxylin (blue s aining). P eabso p ion nega i e con ol (NC) was
used. Images we e aken a magni ica ions 20x. The esul s a e ep esen a i e o ou independen expe imen s.
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RasGRP3 is in ol ed in he egula ion o su i al o MCF7
umo cells
Fu he mo e, as de e mined by low cy ome ic analysis
ollowing annexin-V/PI labeling, inhibi ion o RasGRP3
exp ession induced apop osis in MCF7 cells bu no in
he T-47D cell line (Figu e 3A). To u he suppo he
ole o RasGRP3 in he egula ion o apop osis a quan i a-
i e luo ime ic Mi oP obe™DilC1 (5) assay was pe -
o med. A educ ion in he mi ochond ial ansmemb ane
po en ial is one o he ea lies ma ke s o apop osis [16,17].
Mi oP obe DilC
1
(5) is a mi ochond ial memb ane po en-
ial sensi i e dye which accumula es in he mi ochond ia in
cells wi h ac i e memb ane po en ial. The s aining in ensi y
dec eases in cells wi h dis up mi ochond ial memb ane
po en ial. We ound ha RasGRP3 silencing signi ican ly
dec eased mi ochond ial memb ane po en ial (Figu e 3B),
in MCF7 cells, while no signi ican change was obse ed in
T-47D cells.
RasGRP3 exp ession con ibu es o esis ance o
Tamoxi en and He cep in in he MCF7 and T-47D b eas
cance umo cells
We also in es iga ed he sensi i i ies o “RasGRP3-silenced”
MCF7 and T-47D cells o amoxi en (Tamoxi en) used in
Figu e 2 RasGRP3 is exp essed in human b eas de i ed duc al adenoca cinoma cell lines and ep esses cell p oli e a ion o bo h MCF7
and T-47D de i ed cells. (A) Q-PCR and Wes e n blo analyses o RasGRP3 exp ession in mul iple b eas de i ed duc al adenoca cinoma cell lines.
PC-3 cells we e included as a posi i e con ol [12]. GAPDH was used as an in e nal con ol. The esul s a e ep esen a i e o h ee independen
expe imen s. Values ep esen he mean ± SEM. Exp ession o RasGRP3 was inhibi ed using a speci ic shRNA (shRasGRP3). A non- a ge ing sc ambled
shRNA (shSCR) was used as a con ol. (B) Con i ma ion o he ex en o supp ession o RasGRP3 exp ession was de e mined by Wes e n blo ing.
GAPDH was used as an in e nal con ol. P indica es he numbe o subcul u ing o he RasGRP3 silenced cells. Resul s a e ep esen a i e o 2
independen expe imen s. (C) The p oli e a ion o T-47D and MCF7 de i ed cells was de e mined using he CyQuan GR cell p oli e a ion assay, wi h
alues no malized o he le els o non- ans ec ed con ol cells. CyQuan assay was conduc ed e e y 24 hou s. Values ep esen he mean ± SEM o 2
independen expe imen s.
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endoc ine he apy and o he chemo he apeu ic d ug
as uzumab (He cep in) [18,19]. Down- egula ion o
RasGRP3 inc eased he sensi i i y o T-47D cells o he
g ow h-inhibi o y ac ions o bo h amoxi en and as u-
zumab; i.e. lowe concen a ion induced compa able
e ec s o hose ound wi h highe concen a ions on
cells exp essing he con ol shSCR (Figu e 4A). In e -
es ingly, supp ession o RasGRP3 le els did no a ec
hesensi i i yo heMCF7cells o heac ionso he
d ugs (Figu e 4B).
Figu e 3 Down- egula ion o RasGRP3 exp ession induces apop osis in MCF7 de i ed cells. (A) Rep esen a i e images o MCF7 and T-47D
de i ed cells s ained wi h Annexin-V and PI and analyzed by low cy ome y. Da a we e e alua ed wi h FSC Exp ess so we and p esen ed in he
pe cen age o ga ed cells. Values ep esen he mean ± SEM o h ee independen expe imen s. (B) Assessmen o mi ochond ial memb ane po en ials
o MCF7 and T-47D de i ed cells by luo ime ic DilC1 (5) assay wi h alues no malized o he le els o non- ans ec ed con ol cells. Values ep esen he
mean ± SEM o ou independen expe imen s.
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Down egula ion o RasGRP3 supp essed xenog a
umo o ma ion
To assess u he mo e he ole o RasGRP3 in in i o
umo o ma ion, we employed he SCID mouse xeno-
g a model in which umo s we e induced by MCF7
and T-47D cells exp essing ei he shRasGRP3 o shSCR.
In bo h cell lines, down- egula ion o RasGRP3 esul ed in
a ma ked educ ion in umo g ow h (Addi ional ile 3:
Figu e S1 and Addi ional ile 4: Figu e S2) as measu ed by
weigh o excised umo s in compa ison o hose induced
by he shSCR-exp essing cells (Figu e 5A). These di e en-
ial ea u es o he a ious cells on umo igenesis we e also
p o en by immunohis ochemical analysis o he exp es-
sion o he p oli e a ion ma ke Ki67. In umo s induced
by RasGRP3-silenced MCF7 cells he numbe o Ki67
posi i e cell was signi ican ly less han in he con ol
shSCR-exp essing ones (Figu e 5B and Addi ional ile 3:
Figu e S1 and Addi ional ile 4: Figu e S2). In e es ingly,
despi e he signi ican ly less umo size, s a is ical analysis
did no e eal di e ences in he Ki67 posi i e cell numbe
in he case o he T-47D cells.
RasGRP3 is in ol ed in g ow h ac o -induced Ak , ERK1/2
and es ogen ecep o ac i a ion
G ow h ac o s such as insulin like g ow h ac o -I (IGF-I)
and epide mal g ow h ac o (EGF) ep esen impo an
signaling molecules in b eas cance [20,21]. In he inal
s age o ou expe imen s, we he e o e e alua ed he ole
o RasGRP3 in modula ing he IGF-I and EGF induced
ac i a ion o he Ras signaling pa hway in bo h MCF7
and T-47D cells. Cells we e ea ed wi h IGF-I and EGF
(Figu e 6A) as indica ed, and he ac i a ion o he possibly
mos impo an downs eam molecules ela ed o Ras,
ERK 1/2 and Ak kinases we e examined by Wes e n blo .
We con i med ha in bo h cell lines he down- egula ion
o RasGRP3 educed he IGF-I and EGF-induced ERK 1/2
and Ak phospho yla ion o he basal le el (Figu e 6A
and B). Consis en wi h he cen al ole o Ras signaling
pa hway in he ac i a ion o es ogen ecep o alpha
(ERα), dec easing he le el o pERK 1/2 and pAk led o
a educ ioninERαphospho yla ion (Figu e 6A).
Acco ding o ou esul s MCF7 cells we e mo e sen-
si i e o he g ow h ac o s han T-47D cells and he
Figu e 4 Down- egula ion o RasGRP3 inc eases he sensi i i y o amoxi en and as uzumab o he T-47D cell line. The indica ed MCF7
and T-47D de i ed cell lines we e seeded a a densi y o 1 x 10
4
cells/ well and ea ed wi h amoxi en (A) and as uzumab (B) ( amoxi en: MCF7
cells: 0–200 μM; T-47D cells: 0–100 μM; as uzumab: bo h MCF7 and T-47D cells: 0–10 μM). A e 72 hou s o incuba ion cell p oli e a ion was
de e mined using he CyQuan GR cell p oli e a ion assay. The esul s we e no malized o he le els o un ea ed cells. Values ep esen he
mean ± SEM o 2 independen expe imen s.
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onse o phospho ila ion e en s was di e en , wi h MCF7
de i ed “RasGRP3-silenced”cells appea ing o espond
o g ow h ac o s induced Ak and ERK1/2 down-
egula ion ea lie . In case o IGF-I applica ion (Addi ional
ile 5: Figu e S3) in MCF7 cells, pAKT was a ec ed in
5–30 min, while pAKT is a ec ed only a 30–40 min in
T-47D cells. A ma ked educ ion in ERK ½ ac i a ion
can be obse ed in bo h cells a 5 and 10 min, which is
s ill p esen a 20 and 30 mins in T-47D cells. A de-
c ease in ERαphospho ila ion can be no iced in MCF7
cells ea lie , bu he down- egula ion is mos a ec ed in
T-47D cells. In case o EGF ea men (Addi ional ile 6:
Figu e S4) he RasGRP3 media ed down- egula ion o Ak
(5–40 min) and ERK ½ (5–20 min) ac i a ion was mo e
e ec i e in MCF7 cells compa ed o T-47D, in which pAk
is a ec ed only a 30 min and he e is no signi ican
change in ERK ½ ac i a ion. Howe e T-47D cells show a
signi ican dec ease in ERαac i a ion (20–30 min) com-
pa ed o MCF7 cells, in which pERαis a ec ed only a
20 min. Impo an ly, down egula ion o RasGRP3 had
no measu able e ec on basal Ak , ERK1/2 and ERα
phospho yla ion and on he o al Ak , ERK1/2 and ERα
exp ession le els.
To alida e u he a ole o RasGRP3 in he modula-
ion o he ac i a ion o ERαwe examined he e ec o
RasGRP3 gene-silencing on he exp ession o se e al
ERα- egula ed genes [22], namely p oges e one ecep o
(PGR), ca hepsin D (CTSD), cy och ome C (CYCS) and
lo ic in (LOR) de e mined by Q-PCR. Excep lo ic in
we ound a signi ican educ ion in he exp ession o
hese genes in “RasGRP3-silenced”MCF7 and T-47D
cells (Figu e 6B). In case o T-47D cells a signi ican de-
c ease was obse ed in shSCR cells in hei cy och ome
C exp ession compa ed o he non- ea ed cells, bu in
shRasGRP3 cells his dec ease was mo e exp essi e.
RasGRP3 modula es he exp ession o IGF-I and EGF
ecep o s in MCF7 cells
To u he cla i y he ole o RasGRP3 on he ac i a ion
o he downs eam signaling molecules, he e ec o
RasGRP3 inhibi ion on he exp ession o ups eam IGF-I,
EGF, and Human Epide mal G ow h Fac o Recep o 2
(HER-2) ecep o s was in es iga ed (Figu e 7). Supp es-
sion o RasGRP3 le els dec eased he exp ession o bo h
IGF-I and EGF ecep o s in MCF7 cells, whe eas i had no
e ec on he ecep o exp ession o T-47D cells. RasGRP3
down- egula ion did no modi y he exp ession o HER-2
ecep o o he cells.
Discussion
Ac i a ion o Ras signaling has long been ecognized o
be impo an o umo igenesis and p og ession o can-
ce cells [23,24]. Mo eo e , i was also sugges ed ha
Ras can be “ch onically o e ac i a ed”by a ious up-
s eam signaling elemen s [15] such as e.g. RasGRPs [1].
P e ious s udies ha e clea ly iden i ied he exis ence
o RasGRP3 on human Bu ki ’s lymphoma, p e-B-cell
leukemia, na u al kille -like T-cell leukemia, me as a ic
p os a e cance and malignan melanoma [11-13]. Using
Q-PCR and issue mic oa ay i was p o ed ha RasGRP3
is o e exp essed in p os a e umo and melanoma sam-
ples. Howe e , o ou bes knowledge, we a e he i s
o desc ibe and quan i a i ely asses he exp ession o
RasGRP3 and i s ac i e o m on human b eas duc al
adenoca cinoma cells. Indeed, mu ually complemen a y
Q-PCR, Wes e n blo and immunohis ochemical analyses
e ealed ha he exp ession o RasGRP3 and he ac i e
phosphoRasGRP3 a e ele a ed in nume ous human b eas
umo samples as well as in mul iple b eas de i ed duc al
adenoca cinoma cell lines. We ha e also p esen ed ha
he ela i e le el o RasGRP3 and phosphoRasGRP3
Figu e 5 Supp ession o RasGRP3 exp ession inhibi s xenog a umo g ow h o bo h MCF7 and T-47D cells. SCID mice we e injec ed
subcu aneously wi h 4x10
6
cells o he indica ed MCF7 and T-47D de i ed cells (n = 4 mice o each g oup). The animals we e sac i iced 12 weeks
a e injec ion, and he umo s we e excised, weigh ed (A) and p ocessed o immunohys ochemical analysis. Values ep esen he mean ± SEM o
ou independen expe imen s. (B) Digi alized images o Ki67-speci ic immun eac i i y (Addi ional ile 3: Figu e S1 and Addi onal ile 4: Figu e S2)
ob ained on sec ions p epa ed om umo s de eloped by MCF7 and T-47D de i ed cells we e analysed using Image J image analysis so we . In
e e y each sec ion he numbe o cells we e coun ed a 5 andomly placed, equal a eas o in e es (AOI) and he a e age o Ki67 imunnoposi i e
cells o he 5 AOI was de ined. Ra ios: in T-47D shSCR n = 1213/1098; in T-47D shRasGRP3 n = 1216/1096; in MCF7 shSCR n = 1268/757; in MCF7
shRasGRP3 n = 1112/553. The numbe s ep esen o al cell numbe /Ki67 posi i e cells a ios. Values ep esen he mean ± SEM.
Nagy e al. Molecula Cance 2014, 13:96 Page 7 o 17
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Figu e 6 E ec s o down- egula ion o RasGRP3 on he Ras signaling pa hway I. RasGRP3 is in ol ed in IGF-I and EGF (A) dependen Ak , ERK
and ERαphospho ila ion. RasGRP3 knockdown cell lines c ea ed om MCF7 and T-47D cells we e ea ed wi h o wi hou IGF-I (100 pg/ml) and EGF
(100 pg/ml) as indica ed. Ak and phospho yla ed Ak , ERK and phospho yla ed ERK, ERαand phospho yla ed ERαwe e de ec ed by immunoblo ing
o cell lysa es. Le els o o al Ak , ERK and ERαwe e used as con ol. All esul s we e ep esen a i e o 2 independen expe imen s. (B) Q-PCR analyses
o ERα- egula ed genes, namely p oges e one ecep o (PGR), ca hepsin D (CTSD), cy och ome C (CYCS) and lo ic in (LOR). GAPDH was used
as an in e nal con ol. The esul s a e ep esen a i e o h ee independen expe imen s. Values ep esen he mean ± SEM. In case o T-47D cells
lo ic in exp ession *indica es signi ican di e ence compa ed o non- ans ec ed con ol cells, while
#
indica es signi ican di e ence compa ed o
sc ambled con ol cells.
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exp essions in he umo issues was ma kedly highe
compa ed o he no mal issues. We also p esen ed he
exp ession o RasGRP3 in b eas cance de i ed cell lines.
O u he impo ance, we a e he i s o show ha he
subcellula localiza ion pa e n o RasGRP3 and i s ac-
i e o m a e ma kedly di e en ; i.e., RasGRP3 is local-
ized mos ly in he cy oplasm whe eas phosphoRasGRP3
showed p ominen nuclea s aining in he b eas cance
cells. RasGRP3 is a high-a ini y a ge o he second mes-
senge diacylglyce ol (DAG), i s analogues and pho bol-
es e s (PMA) egula ing hei ansloca ion and exchange
ac o ac i i y [5,6]. As p e iously shown RasGRP3 local-
ized mainly in he cy osol [6]. 1,2-dioc anoyl-sn-glyce ol
(DOG), a memb ane-pe meable analogue o DAG and
pho bol es e PMA also induced RasGRP3 edis ibu ion
o he pe inuclea egion and, o a lesse ex en , o he
plasma memb ane [6]. The localiza ion o he plasma
memb ane is ele an o he ac i a ion o Ras [25]. The e
a e se e al examples o RasGEFs o Ras ha a e ec ui ed
o he plasma memb ane wi h ac i a ion, such as SOS and
RasGRF2 [26,27]. Localiza ion o RasGRP3 in he nucleus
has no been epo ed be o e, and i s signi icance is
puzzling. Howe e , o ms o H-Ras and Rap1, which
a e bo h po en ial a ge s o RasGRP3, ha e been ound
in he nuclei o N-ni osodie hylamine-induced li e
umo cells and squamous cell ca cinoma lines, espec -
i ely [28,29]. In ac , subcellula edis ibu ion in e-
sponse o DAG o i s analogs is one o he hallma ks
o ac i a ion o RasGRPs, he in e ac ion o he p o ein
wi h speci ic in acellula p o eins can also pa icipa e
in he ansloca ion e en s. The ole o an adap o p o-
ein RACK has been shown in he case o a well known
DAG ecep o p o ein kinase C [30]. Dynein ligh chain
1 (DLC1), a componen o he cy oplasmic dynein com-
plex is a no el RasGRP3-in e ac ing p o ein pa icipa ing
in he subcellula localiza ion o RasGRP3 [31]. DLC1 is
no only in ol ed in cy oskele on-media ed mo ili y and
in acellula anspo e en s [32] bu was also epo ed
esponsible o egula e he nuclea localiza ion o ce ain
p o eins, namely p53 and b ain-en iched p o ein PMES-2
Figu e 7 E ec s o down- egula ion o RasGRP3 on he Ras signaling pa hway II. IGF1R, EGFR and HER-2 exp essions we e de ec ed by Q-PCR
and immunoblo ing o RasGRP3 knockdown cell lines c ea ed om MCF7 and T-47D cells. GAPDH (Q-PCR) and βac in (Wes e n Blo ) we e used as
in e nal con ols. The esul s a e ep esen a i e o h ee (Q-PCR) o wo (Wes e n blo ) independen expe imen s. Values ep esen he mean ± SEM.
Nagy e al. Molecula Cance 2014, 13:96 Page 9 o 17
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doi:10.1186/1476-4598-13-96
Ci e his a icle as: Nagy e al.:Func ion o RasGRP3 in he o ma ion
and p og ession o human b eas cance . Molecula Cance 2014 13:96.
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