Tumo -speci ic e ec o V-ATPase inhibi ion
1
Di e en ial egula ion o AMP-ac i a ed p o ein kinase in heal hy and cance cells
explains why V-ATPase inhibi ion selec i ely kills cance cells
Ka in Ba el1, Rol Mülle 2, Ka in on Schwa zenbe g1
1Depa men o Pha macy, Pha maceu ical Biology, Ludwig-Maximilians-Uni e si y o Munich,
Munich, Ge many.
2Helmhol z Cen e o Pha maceu ical Resea ch Saa land, Helmhol z Cen e o In ec ion Resea ch and
Depa men o Pha macy a Saa land Uni e si y, Saa land Uni e si y Campus, Saa b ücken, Ge many.
Running i le: Tumo -speci ic e ec o V-ATPase inhibi ion
To whom co espondence may be add essed: Ka in on Schwa zenbe g, Depa men o Pha macy,
Pha maceu ical Biology, LMU Munich, Bu enand s asse 5-13, 81377 Munich, Ge many; Tel.: 0049-
89-218077165; Fax: 0049-89-218077170; E-mail: ka in. on.schwa [email p o ec ed]
Keywo ds: V-ATPase, AMPK, umo me abolism, ROS, apop osis, cance , a chazolid, umo
supp esso , glucose s a a ion, pH homeos asis
Abs ac
The cellula ene gy senso AMP-
ac i a ed p o ein kinase (AMPK) is a
me abolic hub egula ing a ious pa hways
in ol ed in umo me abolism. He e, we epo
ha acuola H+-ATPase (V-ATPase)
inhibi ion di e en ially a ec s egula ion o
AMPK in umo and non- umo cells and ha
his di e en ial egula ion con ibu es o he
selec i i y o V-ATPase inhibi o s o umo
cells. In non-malignan cells, he V-ATPase
inhibi o a chazolid inc eased phospho yla ion
and lysosomal localiza ion o AMPK. We
no ed ha AMPK localiza ion has a p o-
su i al ole, as AMPK silencing dec eased
cellula g ow h a es. In con as , in cance
cells, we ound ha AMPK is cons i u i ely
ac i e and ha a chazolid does no a ec i s
phospho yla ion and localiza ion. Mo eo e ,
V-ATPase–independen AMPK induc ion in
he umo cells p o ec ed hem om
a chazolid-induced cy o oxici y, u he
unde lining he ole o AMPK as a p o-
su i al media o . These obse a ions indica e
ha AMPK egula ion is uncoupled om V-
ATPase ac i i y in cance cells and ha his
makes hem mo e suscep ible o cell dea h
induc ion by V-ATPase inhibi o s. In bo h
umo and heal hy cells, V-ATPase inhibi ion
induced a dis inc me abolic egula o y
cascade downs eam o AMPK, a ec ing ATP
and NADPH le els, glucose up ake, and
eac i e oxygen species (ROS) p oduc ion.
We could a ibu e he p o-su i al e ec s o
AMPK’s abili y o main ain edox
homeos asis by inhibi ing ROS p oduc ion and
main aining NADPH le els. In summa y, he
esul s o ou wo k indica e ha V-ATPase
inhibi ion has di e en ial e ec s on AMPK-
media ed me abolic egula ion in cance and
heal hy cells and explain he umo -speci ic
cy o oxici y o V-ATPase inhibi ion.
Al eady in he 1920ies O o Wa bu g
disco e ed ha umo cells show an al e ed
me abolism by using glycolysis as he main
ene gy sou ce e en in he p esence o oxygen,
named ae obic glycolysis o he Wa bu g
e ec . Since hen many modi ica ions in
oncogenes and umo supp esso s like
HIF1alpha, Ak , Ras o p53 could be di ec ly
connec ed o egula e he exp ession and
ac i i y o impo an componen s o umo
me abolism, p esen ing umo me abolism as
one hallma k o cance (1). Bu despi e
ongoing esea ch o iden i y he di e en
aspec s o cance me abolism, he me abolic
al e a ions ha a e c i ical o umo
p og ession emain la gely unknown.
Recen ly, he highly conse ed ene gy senso
AMP ac i a ed p o ein kinase (AMPK) came
in o ocus as me abolic hub showing o
egula e many di e en pa hways in ol ed in
umo me abolism. I belongs o a amily o
se ine/ h eonine kinases and consis s o a
ca aly ic α subuni and egula o y β and γ
subuni s (2). AMPK is ac i a ed by a a ie y
o me abolic o oncogenic s ess condi ions
h p://www.jbc.o g/cgi/doi/10.1074/jbc.RA119.010243The la es e sion is a
JBC Pape s in P ess. Published on Oc obe 11, 2019 as Manusc ip RA119.010243
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Tumo -speci ic e ec o V-ATPase inhibi ion
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like nu ien s a a ion o hypoxia and di ec s
he cell owa ds me abolic changes ha
p oduce ATP and es o e ene gy homeos asis
(3). By sensing he AMP:ATP a io i can
inc ease ca abolic p ocesses ha gene a e ATP
like a y acid oxida ion and glycolysis and
inhibi anabolic p ocesses ha consume ATP
such as p o ein and lipid syn hesis. Based on
he inding ha LKB1, he ups eam kinase
ac i a ing AMPK, is equen ly inac i a ed in
umo cells AMPK has been his o ically
p oposed as a umo supp esso . This
assump ion was con i med by showing ha
gene ic loss o AMPK accele a es umo
g ow h o an expe imen al model o
lymphangioma and by he ac ha
pha macological ac i a ion o AMPK leads o
g ow h inhibi ion o se e al umo cell lines
(4,5). Howe e , accumula ing e idence shows
ha AMPK has a dual ole in cance and can
ha e a p o- umo igenic ole as shown o
di e en umo s, especially unde s ess
condi ions like glucose dep i a ion o
oxida i e s ess (6,7).
In e es ingly, i was ecen ly disco e ed ha
he acuola H+-ATPase (V-ATPase) is
needed o ac i a ion o AMPK du ing glucose
s a a ion by o ming a complex wi h he
sca olding p o ein AXIN1 and LKB1 (8). The
V-ATPase is a highly conse ed mul isubuni
p o on pump loca ed a endolysosomal
memb anes o mos euka yo ic cells. I is
esponsible o main aining pH homeos asis
and is essen ial o in acellula a icking and
ecep o ecycling (9). I has been in oduced
as a p omising an i- umo a ge as inhibi ion
o he V-ATPase by se e al d ugs like
concanamycin o he myxobac e ial
compound a chazolid leads o apop osis
induc ion, inhibi ion o mig a ion and in asion
in a a ie y o umo cells in i o and in i o
wi hou a ec ing non-malignan cells (10,11).
Fu he mo e, i was also e ealed la ely ha
he V-ATPase has addi ional unc ions besides
jus egula ing pH and endocy osis. Apa om
being in ol ed in AMPK egula ion i was
shown o play a ole in mTOR-media ed amino
acid sensing and leads o an induc ion o
glycolysis (12,13), e ealing o play a ole in
me abolism.
The impo an ole o V-ATPase o AMPK
homeos asis and he con o e sial discussion
o AMPK ac i i y in umo con ex p omp ed
us o in es iga e he connec ion o V-ATPase
inhibi ion and AMPK ac i a ion in umo cell
su i al in de ail.
In his s udy we used a chazolid as a highly
po en ool o speci ically block he V-ATPase
and ound a di e en ial e ec on AMPK
ac i a ion in umo and non- umo cells which
esul s in di e en me abolic egula ion and
sensi i i y o apop osis induc ion. In non-
umo cells, AMPK is mos ly inac i e,
ea men wi h a chazolid howe e , lead o a
p o ound ac i a ion o AMPK ha ing a
p o ec i e e ec agains oxida i e s ess
induced by he d ug. Tumo cells on he
con a y, showed a cons i u i e ac i a ion o
AMPK i espec i e o a chazolid ea men ,
ye V-ATPase independen ac i a ion o
AMPK also p o ec ed om apop osis
induc ion. We p opose ha AMPK egula ion
in umo cells is uncoupled om V-ATPase
unc ion dep i ing hem om AMPK-
media ed p o ec ion and ende s hem mo e
sensi i e o cy o oxici y induced by V-ATPase
inhibi o s. Hence, dis inc AMPK egula ion
in cance and non-malignan cells accoun s o
he umo cell speci ici y o V-ATPase
inhibi o s.
Resul s
V-ATPase inhibi ion ac i a es AMPK in non-
umo cells
To es whe he inhibi ion o V-
ATPase leads o AMPK ac i a ion we ea ed
di e en umo (MDA-MB-231, MCF7, T24,
HUH7) and non- umo cells (HEK293,
MCF10A, HMLE) wi h a chazolid and
analyzed phospho yla ion o AMPK on
Th 172. We ound ha all umo cells ha e
cons i u i ely ac i a ed AMPK and ha
a chazolid had no e ec on he ac i a ion le el
(Fig. 1A and S1A). As onishingly howe e , V-
ATPase inhibi ion lead o a p o ound
ac i a ion o AMPK in all used non- umo
cells, which ypically showed a low basal
AMPK ac i a ion (Fig. 1B and S1B). To es
whe he his e ec is speci ic o V-ATPase
inhibi ion o a gene al s ess induced e ec we
s a ed he cells o glucose and subsequen ly
analyzed AMPK ac i a ion. As shown in
Figu es 1C and S1C, he e is an ac i a ion o
AMPK in all es ed cell lines and no di e ence
be ween umo and non- umo cells. These
esul s sugges ha he di e en ial e ec seen
on AMPK phospho yla ion is no induced by a
gene al s ess esponse bu speci ic o V-
ATPase inhibi ion. To u he con i m
a chazolid-induced AMPK ac i a ion we
analyzed phospho yla ion o ace yl CoA
ca boxylase (ACC). As a downs eam a ge o
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Tumo -speci ic e ec o V-ATPase inhibi ion
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AMPK ACC is a good ma ke o AMPK
ac i i y. He e we could show he same e ec
han wi h AMPK – an ac i a ion in non- umo
HEK293 cells and no change in MDA-MB-
231 cells (Fig. S2). To make su e ha V-
ATPase is inhibi ed in all cell lines o he same
ex end we used a pH sensi i e lyso acke and
could show a simila e ec by a chazolid
ea men (Fig. S3). To u he analyze AMPK
s a us in umo and non- umo issue we used
pa ien de i ed b eas - umo samples and
no mal b eas issue om he same pa ien s,
espec i ely, and analyzed ac i i y o AMPK
ia Wes e n Blo . In e es ingly, we ound ha
umo cells showed an inc eased le el o o al
AMPK and also a sligh ly highe le el o
phospho yla ed AMPK (Fig. 1D and S1D),
indica ing ha AMPK ac i a ion is bene icial
o cance cells.
In ol emen o V-ATPase inhibi ion in
AMPK ac i a ion
I is known ha V-ATPase in e ac s
wi h he sca olding p o ein AXIN1 in non-
umo cells s a ed o glucose a he lysosome
and ha his in e ac ion is equi ed o ac i a e
AMPK (8). The e o e, we es ed i a chazolid
leads o an in e ac ion o he V-ATPase and
AXIN1 in umo cells. We ound ha
ea men wi h a chazolid leads o an inc eased
in e ac ion o AXIN1 and he V-ATPase in he
non- umo cell line HEK293 bu no in he
b eas cance cell line MDA-MB-231 (Fig.
2A). A simila e ec could be shown o
colocaliza ion o AXIN1 and lysosomes (Fig.
S4). Fu he mo e, using lysosomal
ac iona ion we could show ha AMPK is
inc easingly loca ed a he lysosome in
a chazolid ea ed HEK293 cells bu no in
MDA-MB-231 cells (Fig. 2B). Fo AMPK
ac i a ion i is essen ial ha also LKB1 is
loca ed a he lysosome. We inally ound ha
a chazolid leads o a ans e o LKB1 o he
lysosome in HEK293 cells in con as o umo
cells whe e no di e ence be ween ea ed and
non- ea ed cells could be obse ed, as shown
by con ocal mic oscopy (Fig. 2C). These
esul s u he sugges ha he e is a di e ence
in AMPK ac i a ion by V-ATPase inhibi ion
in umo and non- umo cells.
V-ATPase inhibi ion al e s AMPK ela ed
me abolic pa ame e s
AMPK is ac i a ed due o an inc eased
a io o AMP:ATP, he e o e we analyzed he
e ec o V-ATPase inhibi ion on ATP con en .
We ound ha umo cells we e only sligh ly
a ec ed bu he non- umo cell lines HEK293
and MCF10A showed a signi ican educ ion
o ATP le el a e a chazolid ea men as
shown by a luminescen assay (Fig 3A).
Fu he mo e, classical AMPK ac i a ion is
induced by an inc eased le el o AMP.
The e o e we analyzed changes in AMP by
HPLC (Fig. S5). In e es ingly we could no
ind a clea inc ease nei he in non- umo
HEK293 cells, no in MDA-MB-231 cells –
which migh indica e a di e en ac i a ion
mode by V-ATPase inhibi ion. Bu we ound
an inc eased glucose up ake and a sligh bu
no signi ican up egula ion o Glucose
ecep o 1 (GLUT1) in HEK293 cells – which
can be a consequence o AMPK ac i a ion. In
con as , he umo cell lines showed only a
sligh e ec (MDA-MB-231) o e en a
dec ease (MCF7) in glucose up ake (Fig. 3B,
C) showing a dis inc me abolic egula ion o
umo and non- umo cells by V-ATPase
inhibi ion.
AMPK ac i a ion p o ec s non- umo cells
om a chazolid induced cy o oxici y
As AMPK ac i a ion is qui e
con o e sially discussed as ei he umo
supp esso o oncogene we analyzed i
a chazolid induced AMPK phospho yla ion is
p o-su i al o p o-apop o ic. The e o e, we
silenced AMPK in HEK293 cells (Fig. 4B)
and ea ed he cells wi h a chazolid. We ound
ha HEK293 cells wi h silenced AMPK
showed a dec eased g ow h a e a e
a chazolid ea men compa ed o con ol cells
(Fig. 4A) which sugges s a p o ec i e ole o
AMPK ac i i y.
I AMPK ac i a ion p o ec s cells om
a chazolid induced cy o oxici y, hen u he
inducing AMPK in umo cells should
dec ease a chazolid induced apop osis
induc ion. The e o e, we ea ed MDA-MB-
231 cells wi h he AMP analogue AICAR in
combina ion wi h a chazolid which inc eased
phospho yla ion o AMPK (Fig. 4C and S6A).
In e es ingly we ound ha apop osis
induc ion was signi ican ly educed by
combining a chazolid wi h AICAR compa ed
o a chazolid ea ed cells alone (Fig 4D). This
was accompanied by a dec eased clea age o
PARP-1 con i ming a dec eased apop osis
induc ion and a p o ec i e ole o AMPK
(Fig. 4E and S6B). To con i m ha his e ec
is eally AMPK dependen we epea ed he
expe imen wi h a silenced AMPKα1 and
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ound ha AICAR does no p o ec MDA-
MB-231 cells om a chazolid induced
apop osis con i ming he p o ec i e ole o
AMPK (Fig 4F and S6C).
P o-su i al ole o AMPK esul s om
di e en s ess esponse and main aining
edox homeos asis
As one known p o-su i al ole o
AMPK is he con ol o edox homeos asis by
p e en ing ROS accumula ion and
main aining NADPH le els, we analyzed i
a chazolid ea men leads o a dis u bance o
edox homeos asis. As shown in Fig. 5A, V-
ATPase inhibi ion leads o an induc ion o
ROS in umo cells bu a he o a dec ease o
ROS in non- umo cells. Fu he mo e,
a chazolid lead o an inc ease o
NADPH/NADP a io in HEK293 cells while
MDA-MB-231 cells showed a dec eased a io
(Fig. 5B) sugges ing a dis u bed edox balance
in umo cells. Ac i a ion o AMPK keeps he
edox balance by dec easing a y acid
syn hesis (7). The e o e, we analyzed he
ansc ip ion o FASN and SCD1 – wo majo
playe s in he a y acid syn hesis pa hway – in
umo and non- umo cells. In e es ingly we
ound ha a chazolid ea men lead o a s ong
up egula ion o bo h enzymes in umo cells
bu no in non- umo cells (Fig. 5C). To
connec hese e ec s wi h he p o-su i al
e ec o AMPK we ea ed MDA-MB-231
cells wi h he ROS sca enge Ti on and
analyzed apop osis induc ion (Fig 5D). We
ound ha combina ion o a chazolid wi h
Ti on dec eased apop osis induc ion. Fu he
suppo ing his hypo hesis is ha he
combina ion o AICAR and a chazolid
dec eases ROS p oduc ion in MDA-MB-231
cells (Fig. 5E). Finally, silencing o AMPK in
HEK293 cells leads o an up egula ion o ROS
a e a chazolid ea men con i ming ha
AMPK ac i a ion is esponsible o he
p o ec ion o ROS induced apop osis ia V-
ATPase inhibi ion (Fig. 5F).
Discussion
This wo k p o ides e idence ha V-
ATPase inhibi ion by a chazolid leads o a
di e en ial me abolic egula ion in umo and
non- umo cells which esul s in an inc eased
sensi i i y o umo cells owa ds he
ea men . Ou majo indings a e ha V-
ATPase inhibi ion leads o AMPK ac i a ion
only in heal hy cells which p o ec s hem om
a chazolid-induced cy o oxici y. In umo
cells, as depic ed in Figu e 6, his p o ec ion is
missing which esul s in inc eased apop osis
induc ion caused by a dis inc e ec on AMPK
downs eam cascade including ATP, glucose
up ake, NADPH le el and ROS p oduc ion.
As hese e ec s could be ab oga ed by
ac i a ing AMPK in umo cells independen ly
o V-ATPase, a no el ole o AMPK in V-
ATPase inhibi ion induced cy o oxici y was
un a eled. This p o ides a no el in e es ing
insigh in o he egula ion o s ess esponses
in di e en cell ypes and a be e
unde s anding o he mechanism o ac ion o
V-ATPase inhibi ion ega ding me abolism.
One majo p oblem in cance he apy is o ind
compounds ha speci ically a ge cance cells
and lea e non-malignan cells una ec ed and
he eby minimizing oxic side e ec s and
make chemo he apy mo e bea able o cance
pa ien s. In ensi e esea ch has led o he
in oduc ion o se e al compounds ha a ge
e.g. g ow h ac o ecep o s ha a e
o e exp essed in umo s like ce uximab
(EGFR) (14), as uzumab (HER2) (15) o
ima inib which a ge s he bc -abl y osinkase,
a umo -speci ic mu a ion (16). Bu despi e he
success expe ienced wi h hese compounds
selec i i y o chemo he apeu ics is s ill a
majo challenge.
The V-ATPase is a p omising an i- umo
a ge and i s inhibi ion was shown o ha e a
p onounced e ec on umo cells, bu lea es
non- umo cells almos una ec ed as we and
o he s could al eady show (11,17). We could
e en in oduce mo e umo and non- umo
cells lines o p o e a clea di e ence in
sensi i i y owa ds V-ATPase inhibi ion in
umo e sus non- umo cells (Fig. S7). The
an i- umo e ec o V-ATPase inhibi ion was
mos ly con ibu ed o he dis u bance o
endocy o ic ecycling p ocesses and se e al
publica ions by ou g oup and o he s could
elucida e se e al unde lying mechanisms
(11,13,18,19). In ecen yea s howe e , i
became ob ious ha he V-ATPase also plays
a pi o al ole in nu ien sensing. Zoncu e al.
showed ha he V-ATPase is ine i able o
mTOR-media ed amino acid sensing a he
lysosome, by building a complex be ween
mTORC1, he V-ATPase and Ragula o (12).
In e es ingly, Zhang e al. ound ha he V-
ATPase is no only equi ed o mTOR
egula ion bu also o AMPK ac i a ion unde
glucose dep i a ion. Thei g oup imp essi ely
showed ha he V-ATPase-Ragula o complex
se es as a docking si e o LKB1-media ed
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AMPK ac i a ion wi h he aid o he
sca olding p o ein AXIN1, by o ming V-
ATPase-Ragula o /LKB1-AMPK complex a
he lysosome (8). Finally, McGui e e al.
showed ha glucose s a a ion leads o an
inc eased V-ATPase assembly which is
p eceded by AMPK ac i a ion (20). These
indings s eng hen he hypo hesis ha V-
ATPase is in ol ed in me abolic egula ion o
he cell.
Zhang e al showed ha an in ac V-ATPase is
needed o build he complex o
LKB1/AXIN1/AMPK and V-ATPase and ha
inhibi ing he V-ATPase wi h concanamycin
mimicks glucose s a a ion and he e o e
ac i a es AMPK in HEK293 cells (8). I was
also shown ha ano he V-ATPase inhibi o ,
ba ilomycin, can ac i a e AMPK in myoblas s
(21). Consis en wi h hei indings we could
show ha inhibi ing he V-ATPase unc ion
pha macologically wi h a chazolid lead o an
ac i a ion o AMPK in HEK293 and o he
non- umo cells, o an inc ease in he binding
o V-ATPase o AXIN1 and o an inc eased
lysosomal localiza ion o he AMPK-LKB1
complex. In e es ingly, in umo cells
a chazolid had no in luence on AMPK
phospho yla ion and binding o V-ATPase o
AXIN1 was a he dec eased a e a chazolid
ea men , hough all umo cells al eady
showed a phospho yla ed AMPK in con ols.
This inding is consis en wi h o he g oups
showing high cons i u i e AMPK ac i i y in
umo cells like p os a e cance me as asis o
glioblas oma umo s (22,23).
Di e en ial ac i a ion was no due o a s ess
esis ance o o he esis ance mechanisms in
umo cells as glucose dep i a ion lead o an
inc eased ac i a ion o AMPK in all cell lines.
The e o e his e ec is speci ic o V-ATPase
inhibi ion and sugges s a di e en ial
egula ion o V-ATPase inhibi ion induced
s ess esponses in umo and non- umo cells.
In e es ingly, we did no ind an inc ease o
AMP, nei he in umo no non- umo cells.
This e ec migh suppo he special ole o
V-ATPase induced AMPK ac i a ion.
Rega ding his dis inc e ec we wan ed o
in es iga e i he ac i a ion o AMPK in non-
umo cells plays a ole o a chazolid-induced
cy o oxici y.
Looking a he ole o AMPK in umo con ex
makes clea ha AMPK egula ion is highly
complex and depends on he cell ype, he
se e i y and he kind o he s ess signal and i s
ole as umo supp esso o oncogene is
discussed con o e sially (24).
In he umo con ex AMPK has long been
seen as an an i- umo a ge which o igina ed
om he disco e y o he AMPK ups eam
kinase LKB1, he loss o which esul s in he
de elopmen o Peu z-Je ghe synd ome ha
comes along wi h an inc eased cance isk
(25). Fu he mo e, he use o - o en unspeci ic
- AMPK-ac i a o s, we e shown o inhibi
umo g ow h in a a ie y o s udies (26-28).
Ye , in ecen yea s i became ob ious ha
AMPK ac i a ion can e y well be
umo igenic and can e en be essen ial o
umo p og ession as shown o glioblas oma,
p os a e cance (23,29,30) o myc
o e exp essing umo s (31). In ha ega d,
mos da a showing a umo -supp essi e ole
o AMPK ely on pha macological s udies,
whe eas expe imen s using gene ic ools
mos ly suppo a p o-su i al ole o AMPK.
Se e al oncogenes like S c o Myc we e
shown o ac i a e AMPK (31,32) and silencing
o an AMPK subuni impai s he abili y o
cells o o m umo s in i o (33). In addi ion,
loss o he umo supp esso ollicullin also
ac i a es AMPK (34). All hese da a sugges a
con ex speci ic ole o AMPK ac i i y.
In iguingly, we ound ha silencing
AMPK1α in HEK293 cells ea ed wi h
a chazolid lead o a dec eased g ow h a e
compa ed o wild ype cells sugges ing a p o-
su i al unc ion o AMPK ac i a ion.
Mo eo e , inc easing AMPK ac i i y in
MDA-MB-231 b eas cance cells by he
AMPK ac i a o AICAR p o ec ed he cells
om a chazolid-induced cy o oxici y – an
e ec ha could be diminished by silencing
AMPK1α. Hence ou indings suppo a p o-
su i al ole o AMPK ac i a ion a e V-
ATPase inhibi ion. Impo an ly, ea ing non-
umo cells wi h a combina ion o a chazolid
and AICAR had no e ec on apop osis
induc ion (Fig. S8).
A e showing ha induc ion o AMPK by
a chazolid has a p osu i al e ec we wan ed
o elucida e he mechanism behind i . AMPK
leads o a a ie y o me abolic changes
acili a ing su i al in pe iods o poo
nu i ion o o he me abolic s esses. The bes
known p o-su i al unc ions o AMPK
ac i a ion a e au ophagy, mac omolecule
impo , edox homeos asis and enhanced
glycolysis (35-37). In ega d o glucose
me abolism, we ound an inc eased glucose
up ake and sligh ly highe GLUT1 exp ession
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Tumo -speci ic e ec o V-ATPase inhibi ion
6
selec i ely in non- umo cells and an
un a ying o e en dec eased glucose up ake in
umo cells which co ela es wi h he
di e en ial ac i a ion o AMPK upon V-
ATPase inhibi ion. This is in cohe ence wi h
he da a o o he s, showing an AMPK
dependen inc eased up ake o glucose and
glucose ecep o exp ession o p omo e b eas
cance g ow h (38) and p o ec ion om
oxida i e s ess (39). Conce ning edox
homeos asis, Jeon e al showed ha AMPK
ac i a ion p o ec s om ROS p oduc ion and
main ains NADPH le els cons an du ing
glucose dep i a ion leading o cell su i al (7)
– he e o e p o iding e idence ha AMPK
ac i a ion p omo es cell su i al du ing
ene gy s ess. Addi ionally AMPK was shown
o be esponsible o he an i-oxidan e ec s o
es e a ol (40) and essen ial o he edox
balance in leukemia cells whe e AMPK
inhibi ion esul s in educed leukemic
p og ession (41). Inc eased ROS le els ha e
been epo ed o be a ea u e o V-ATPase
inhibi ion in umo cells (42,43) which we
could con i m in his s udy o MDA-MB-231
and MCF-7 cells using a chazolid as V-
ATPase inhibi o . ROS p oduc ion in non-
umo cells on he o he hand was no a ec ed.
These di e ences came along wi h a dec eased
NADPH/NADP le el and syn hesis o wo
majo enzymes o lipid syn hesis selec i ely in
umo cells. Impo an ly he a chazolid
induced dis u bance in edox homeos asis was
o e come by AMPK ac i a ion in non- umo
cells, while umo cells ailed o ac i a e
AMPK upon V-ATPase inhibi ion. Simila
e ec s we e also epo ed o Myc-posi i e
melanoma whe e AMPK ac i a ion could
p o ec he cells om ROS induced apop osis
(44). We ound ha sca enging ROS by Ti on
dec eased a chazolid induced apop osis in
umo cells and combina ion wi h AICAR
dec eased ROS p oduc ion, whe eas AMPK
silencing esul ed in inc eased ROS
p oduc ion a e V-ATPase inhibi ion e en in
non- umo cells. This poin s o he impo ance
o he edox homeos asis main ained by
AMPK unde me abolic s ess which is
impo an o alle ia ing cy o oxic e ec s o
V-ATPase inhibi ion.
We p opose ha V-ATPase inhibi ion by
a chazolid leads o a s ess esponse in non-
umo cells in which AMPK sus ains cellula
homeos asis like ene gy supply and edox
homeos asis. In umo cells howe e , V-
ATPase does no lead o an ac i a ion o
AMPK-media ed me abolic changes
supposedly due o a de ec i e egula ion o
AMPK which makes he cells mo e ulne able
o V-ATPase induced me abolic s ess (Fig. 6).
These esul s demons a e he signi icance o
he V-ATPase o umo me abolism, gi e a
i s insigh in o he mechanisms o umo -
speci ic e ec s o V-ATPase inhibi ion and
unde line he impo ance o unde s and
mechanis ic di e ences o umo and non-
umo cells.
Expe imen al p ocedu e
Cell Cul u e and Compounds
MDA-MD-231, HEK293, T24 and
MCF-7 cells we e ob ained om DSMZ
(Heidelbe g, Ge many) and MDA-MB-231
and HEK293 cells we e cul u ed in DMEM
medium supplemen ed wi h 10% FCS. MCF-
7 cells we e main ained in RPMI1640 medium
supplemen ed wi h 10% FCS, Insulin and 1%
non-essen ial amino acids. MCF10A cells
we e pu chased om ATCC and cul u ed in
DMEM-F12 supplemen ed wi h 5% ho se
se um, 100 mg/ml epide mal g ow h ac o , 10
mg/ml insulin, 1 mg/ml hyd oco isone, 1
mg/ml chole a oxin and 1%
penicillin/s ep a idin. HMLE cell we e a kind
gi o D . Ch is ina Scheel (Helmhol z Cen e
Munich) and cul u ed in MEGCM medium
ob ained om P omoCell (Heidelbe g,
Ge many).
S a a ion expe imen s we e pe o med in
glucose- ee DMEM supplemen ed wi h 10%
o dialyzed FCS.
A chazolid was p o ided by P o . Di k
Menche, Uni e si y o Bonn, AICAR was
pu chased om Toc is bioscience (B is ol,
UK).
Pa ien -de i ed b eas umo samples and
heal hy b eas issue samples we e a kind gi
om he non-p o i o ganiza ion PATH
biobank (Augsbu g, Ge many) (45). The
collec ion o samples was app o ed by he
local e hics commi ee in Bonn and all pa ien s
p o ided w i en in o med consen . The
sample collec ion was ca ied ou acco ding o
he decla a ion o Helsinki.
Immunoblo ing
Cells we e lysed in a bu e con aining
EDTA 2 mM, NaCl 137 mM, Glyce ol 10 %,
Na4P2O7 2 mM, T is-Base 20 mM, T i on X-
100 1 % and Na-Glyce olphospha e 20 mM a
pH 7,5. Fo immunop ecipi a ion cells we e
lysed and incuba ed wi h an an ibody agains
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Tumo -speci ic e ec o V-ATPase inhibi ion
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AXIN1 o e nigh . Subsequen ly he lysa e
was incuba ed wi h P o ein Aga ose A beads
(Sigma Ald ich, Tau ki chen, Ge many),
washed wi h lysa e bu e and be a-
me cap oe hanol con aining sample bu e was
added. Equal amoun s o p o ein we e
sepa a ed on a SDS-PAGE and ans e ed o
a ni ocellulose memb ane. The memb anes
we e blocked wi h 5% milkpowde and hen
p obed wi h p ima y an ibodies.
Fo loading con ol 2,2,2-T ichlo oe hanol
(TCE) was added o he polyac ylamide gel
be o e polyme iza ion. The yp ophans o
p o eins unde go an ul a iole ligh -induced
eac ion wi h TCE which p oduces ligh in he
isible ange so ha p o eins can be de ec ed
(46). In some cases ac in was used as loading
con ol.
Following an ibodies we e used: pAMPK
Th 172, AMPK, AMPK1alpha, pACC Se 79,
ACC, PARP1 (Cell Signaling Technology,
Dan e s, MS), GLUT1 (No us Biologicals),
ac in, ab7, AXIN1, V-ATPase D, (San a C uz
Bio echnology, Dallas, TX), HRP-goa -an i-
abbi (Bio-Rad, Munich, Ge many) and HRP-
goa -an i-mouse (San a C uz Bio echnology).
Isola ion o Lysosomes
Lysosomes we e isola ed as
p e iously desc ibed (47). B ie ly, 1,5 x 106
cells we e seeded in 10 cm dishes and we e
ea ed as indica ed. Subsequen ly medium
was emo ed and 250 µl homogeniza ion
bu e was added o he cells. The cell
suspension was ans e ed o a glas g inding
essel and homogenized using a po e
homogenize . Nex , he homogena e was
cen i uged 15 min a 14.000 g a 4°C. The
supe na an was ans e ed o a 2 ml
polyca bona e cen i uga ion ube and an equal
olume o 16 mM CaCl2 o p ecipi a e he
lysosomes was added. Then he homogena e
was cen i uged a 25.000 g o 15 min. Nex ,
supe na an is disca ded and he pelle
esuspended in one olume o washing bu e
and again cen i uged. Now he pelle
con aining lysosomes is esuspended in 20 µl
o Washing bu e and used o Wes e n blo
analysis.
T ans ec ion
Cells we e ans ec ed wi h
GenaxxoFec (Genaxxon Bioscience,
Müns e , Ge many) acco ding o he
manu ac u e ’s ins uc ions. B ie ly,
AMPKα1 was silenced using ON-TARGET-
Plus SMARTpool siRNA om Dha macon
(Schwe e, Ge many) and non- a ge ing
siRNA as a con ol and we e ea ed as
indica ed 48 h a e ans ec ion.
Immunocy ochemis y
Cells we e seeded on µ-slides 8-well
ibidiT ea (IBIDI, Ma ins ied, Ge many) and
we e ea ed as indica ed. Cells we e ixed and
s ained as desc ibed be o e (48) and analyzed
wi h a Zeiss LSM 510 Me a con ocal
mic oscope (Jena, Ge many). Following
an ibodies we e used: LKB1 (Cell Signaling
Technology, Dance s, MS), Lamp1
(De elopmen al S udies Hyb idoma Bank
Iowa, IA) AlexaFluo 488-goa -an i- abbi ,
AlexaFluo 543-goa -an i-mouse (In i ogen,
Wal ham, MA).
Apop osis Assay
Subdiploid DNA con en was
de e mined acco ding o Nicole i e al (49).
B ie ly, cells we e ea ed as indica ed,
ha es ed, pe meabilized wi h Na iumci a e
con aining T i on X-100, s ained wi h 25
µg/ml p opidiumiodide and analyzed by low
cy ome y (Beck on Dickinson, Heidelbe g,
Ge many). Subdiploid cells le o he G1-peak
we e conside ed as apop o ic.
Cy o oxici y assay
Cells we e seeded in a 96 well pla e,
ea ed as indica ed and g ow h inhibi ion was
analyzed wi h he CellTi e Blue assay
(P omega, Madison, WI) acco ding o
manu ac u e ’s ins uc ions.
qRT-PCR analysis
To al mRNA was isola ed om cell
cul u e samples using Qiagen RNeasy Mini
Ki (Qiagen, Hilden, Ge many) acco ding o
he manu ac u e ’s ins uc ions. Fo cDNA
syn hesis, he High Capaci y cDNA Re e se
T ansc ip ion Ki (Applied Biosys ems, Fos e
Ci y, CA) was used. qRT-PCR was pe o med
wi h he AB 7300 RealTime PCR sys em, he
TaqMan Gene Exp ession Mas e Mix
(Applied Biosys ems) and he SYBR G een
PCR Mas e Mix (Applied Biosys ems)
acco ding o he manu ac u e 's ins uc ions.
All designed p ime s we e pu chased om
Me abion (Ma ins ied, Ge many).
Glucose up ake
Cells we e ea ed as indica ed.
Subsequen ly cells we e incuba ed wi h 100
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Tumo -speci ic e ec o V-ATPase inhibi ion
8
µM 2-NBDG (2-[N-(7-Ni obenz-2-Oxa-1,3-
Diazol-4-yl)Amino]-2-Deoxy-D-Glucose)
(In i ogen, Wal ham, MA) in HANKS bu e
o 30 min a 37°C. Then cells we e ha es ed
and changes in glucose up ake we e measu ed
by low cy ome y.
ROS Measu emen
Cells we e ea ed as indica ed,
ha es ed and incuba ed wi h 10 µM o 2′,7′-
dichlo o luo escein diace a e (Sigma Ald ich,
Tau ki chen, Ge many) o 30 min a 37°C.
A e a washing s ep wi h PBS, eac i e
oxygen species p oduc ion was analyzed by
low cy ome y.
ATP
Cells we e seeded in a 96-well pla e
and ea ed as indica ed. ATP le el was
de e mined using he CellTi e -Glo assay
(P omega) acco ding o he manu ac u e ’s
ins uc ion and measu ed in a luminome e
(Be hold Technologies, Bad Wildbad,
Ge many) acco ding o manu ac u e ’s
ins uc ions.
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Tumo -speci ic e ec o V-ATPase inhibi ion
9
Acknowledgemen : We would like o hank Be nade e G ohs and Ri a Soche o hei excellen wo k
and D . Simone Mose o doing HPLC analysis. This wo k was suppo ed by he DFG g an FOR 1406
SCHW 1781/1-1.
Con lic o in e es : The au ho s decla e ha hey ha e no con lic s o in e es wi h he con en s o
his a icle
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a Helmhol z Zen um ue In ek ions o schung - BIBLIOTHEK- on No embe 14, 2019h p://www.jbc.o g/Downloaded om

a Helmhol z Zen um ue In ek ions o schung - BIBLIOTHEK- on No embe 14, 2019h p://www.jbc.o g/Downloaded om
a Helmhol z Zen um ue In ek ions o schung - BIBLIOTHEK- on No embe 14, 2019h p://www.jbc.o g/Downloaded om
a Helmhol z Zen um ue In ek ions o schung - BIBLIOTHEK- on No embe 14, 2019h p://www.jbc.o g/Downloaded om
Ka in Ba el, Rol Mülle and Ka in on Schwa zenbe g
explains why V-ATPase inhibi ion selec i ely kills cance cells
Di e en ial egula ion o AMP-ac i a ed p o ein kinase in heal hy and cance cells
published online Oc obe 11, 2019J. Biol. Chem.
10.1074/jbc.RA119.010243Access he mos upda ed e sion o his a icle a doi:
Ale s:
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o choose om all o JBC's e-mail ale sClick he e
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Supplemen al Ma e ial and Me hods
Cell lines, an ibodies
Panc3.027 panc ea ic cance cells we e ob ained om ATCC and cul i a ed in RPMI1640
media supplemen ed wi h 10% FCS and 10 mg/ml insulin. HepaRGTM cells we e ob ained om The mo
Fishe Scien i ic. Cells we e pla ed and main ained in Williams’ medium E supplemen ed wi h
Glu aMAXTM and HepaRG™ Thaw, Pla e, & Gene al Pu pose Medium Supplemen (Thaw, Pla e, &
Gene al Pu pose Wo king Medium) pu chased om The mo Fishe Scien i ic. Hepa ocellula
ca cinoma cell line HepG2 was pu chased om Ge man Resea ch Cen e o Biological Ma e ial
(DSMZ) and cul u ed in DMEM, 10% FCS.
pACC Se 79 and ACC an ibodies we e om Cell Signaling Technology, Dan e s, MA.
Lyso acke s aining
Se e al umo and non- umo cells we e seeded in IBIDI slides and ea ed wi h a chazolid o
4h. Subsequen ly a pH sensi i e Lyso acke (In i ogen, Wal ham, MA) was added o he wells and
cells we e measu ed a e an incuba ion ime o 30 min a he con ocal mic oscope. Acidic lysosomes
show a ed s aining whe eas alkaline lysosomes show no s aining. Pic u es ep esen one pic u e ou o
h ee expe imen s.
De e mina ion o AMP
2 x 106 cells we e seeded in 10 cm dishes and ea ed wi h a chazolid o 20 hou s. Subsequen ly
hey we e sc aped o and washed wice wi h PBS. The pelle was esuspended in 6 % pe chlo ic acid
and incuba ed o 20 min. A e ho oughly o exing he lysa e was cen i uged o 15 min a 14 000
pm. The supe na an was neu alized wi h 2M KOH and again cen i uged. The supe na an was used
o HPLC measu emen . AMP was quan i ied using e e se-phased HPLC (Agilen 1260 In ini y II LC
sys em wi h a 1260 In ini y Degasse , a 1100 Se ies qua e na y pump and 1100 Se ies diode a ay
de ec o ) and sepa a ed on a C18 column (20EC-C18 4 µm 46 x 150 mm a 15°C p o ec ed by
Phenomenex ODS 4 x 3 mm i.d. p e-column) o 10 min a a low- a e o 0,8 ml/min. The mobile-phase
was i luo ace ic acid.

1.5
1
0.5
0
co
a ch 10nM
co a ch
pAMPK/AMPK ( old induc ion)
MDA-MB-231
MCF-7
T24
pAMPK/AMPK ( old induc ion)
co a ch
HEK293
co
a ch 10nM
MCF10A
HMLE
4
3
2
1
0
*
**
pAMPK/AMPK ( old induc ion)
4
3
2
1
0
MDA-MB-231
MCF7
HEK293
co -gluc
*
*
*
2
1.5
1
0.5
0N T
pAMPK/AMPK ( old induc ion)
*
B
C D
A
Figu e S1: Quan i ica ion o AMPK Wes e nblo s. The umo cell lines MCF-7, MDA-MB-231 (MDA)
and T24 (A) and he non- umo cell lines HEK293, MCF10A and HMLE (B) we e ea ed wi h 10 nM
a chazolid (a ch) o 24 hou s and phospho yla ion o AMPK a Th 172 and o al AMPK p o ein we e
analyzed by Wes e n Blo . (C) HEK293, MCF-7 and MDA-MB-231 we e incuba ed in glucose- ee media
o 24 hou s and phospho yla ion o AMPK was analyzed. (D) Phospo yla ion o AMPK was analyzed in
pa ien -de i ed b eas cance issue and no mal b eas issue om he same pa ien . Quan i ica ion ep esen s
h ee di e en expe imen s. *p<0.05, s uden s - es .
HEK293 MDA
pACC
(Se 79)
ACC
LC
- + - + a ch
300 kDa -
200 kDa -
300 kDa -
200 kDa -
2
1.5
1
0.5
0
co a ch
2
1.5
1
0.5
0
pACC old induc ion
pACC/ACC
pACC/LC
pACC/ACC
pACC/LC
co a ch
pACC old induc ion
Figu e S2: A chazolid leads o phospho yla ion o ACC. HEK293 non- umo and MDA-MB-231 umo
cell lines we e ea ed wi h 10 nM a chazolid o 24 h. Phospho yla ion o ACC a Se 79 was analyzed by
wes e n blo . Quan i ica ion was done o pACC e sus ACC and pACC e sus loading con ol (LC) which
showed simila esul s.
HEK293 MDA
70 kDa -
50 kDa -
HEK293
HMLE
HUH7
MCF7
MDA-MB-231
T24
Co A ch 10nM
Hoechs
Lyso acke
Figu e S3: A chazolid leads o alkaliza ion o lysosomes. Di e en umo and non- umo cell lines we e
ea ed wi h 10 nM a chazolid o 24 h. Subsequen ly cells we e s ained wi h a pH sensi i e Lyso acke and
analyzed by con ocal mic oscopy. The scale ba is 25 µm, apa om MCF-7 (10 µm).
Lamp1
AXIN1 me ge
co
co
a ch
a ch
HEK293
MDA
Figu e S4: Colocaliza ion o AXIN1 and Lamp1 is shown a e a chazolid ea men in HEK293 cells
and cons i u i ely exis en in MDA-MB-231 cells. Cololocaliza ion o Lamp1 and AXIN1 was de e mined
in HEK293 and MDA-MB231 cells a e ea men wi h 10 nM a chazolid o 24 h by con ocal s aining. One
ep esen a i e image ou o h ee independen expe imen s is shown.