metformin inhibits age related centrosome amplification in drosophila midgut stem cells through akt tor pathway

Inhibitory effect of metformin on age-related centrosome amplification in midgut ISCs.. Fourteen-day-old wild type flies without d–d” or with h–h” 5 mM metformin feeding for 6 days were tr

ARTICLE IN PRESS

G Model

MAD 10767 1–11

j o ur na l h o me pa g e :w w w e l s e v i e r c o m / l o c a t e / m e c h a g e d e v

Metformin inhibits age-related centrosome amplification in

Drosophila midgut stem cells through AKT/TOR pathway

Hyun-Jin Naa,1

Robert Arkingb, Mi-Ae Yooa,∗

a Department of Molecular Biology, Pusan National University, Busan 609-735, South Korea

Q3

b Department of Biological Sciences, Wayne State University, Detroit, MI 48202, USA

a r t i c l e i n f o

Article history:

Received 14 January 2015

Received in revised form 23 April 2015

Accepted 6 May 2015

Available online xxx

Keywords:

Drosophila intestinal stem cells (ISCs)

Metformin

Centrosome amplification

AKT/TOR pathway

DNA damage

a b s t r a c t

Wedelineatedthemechanismregulatingtheinhibitionofcentrosomeamplificationbymetforminin Drosophilaintestinalstemcells(ISCs).Age-relatedchangesintissue-residentstemcellsmaybeclosely associatedwithtissueagingandage-relateddiseases,suchascancer.Centrosomeamplificationisa hallmarkofcancers.OurrecentworkshowedthatDrosophilaISCsareanexcellentmodelforstemcell studiesevaluatingage-relatedincreaseincentrosomeamplification.Here,weshowedthatmetformin,a recognizedanti-cancerdrug,inhibitsage-andoxidativestress-inducedcentrosomeamplificationinISCs Furthermore,werevealedthatthiseffectismediatedviadown-regulationofAKT/targetofrapamycin (TOR)activity,suggestingthatmetforminpreventscentrosomeamplification byinhibitingtheTOR signallingpathway.Additionally,AKT/TORsignallinghyperactivationandmetformintreatment indi-catedastrongcorrelationbetweenDNAdamageaccumulationandcentrosomeamplificationinISCs, suggestingthatDNAdamagemightmediatecentrosomeamplification.Ourstudyrevealsthebeneficial andprotectiveeffectsofmetforminoncentrosomeamplificationviaAKT/TORsignallingmodulation.We identifiedanewtargetfortheinhibitionofage-andoxidativestress-inducedcentrosomeamplification

WeproposethattheDrosophilaISCsmaybeanexcellentmodelsystemforinvivostudiesevaluatingthe effectsofanti-cancerdrugsontissue-residentstemcellaging

©2015Z.PublishedbyElsevierIrelandLtd.ThisisanopenaccessarticleundertheCCBY-NC-ND

license(http://creativecommons.org/licenses/by-nc-nd/4.0/)

1 Introduction

Metformin,abiguanidedrug,isclinicallyapproved-and

well-toleratedforthetreatmentoftype2diabetes,andisofinterest

forcancerpreventionandtherapy(AljadaandMousa,2011;Baur

etal.,2010;Landmanetal.,2009).Thedirectmoleculartargetof

Abbreviations: 4E-BP, eukaryotic translation initiation factor 4E-binding

pro-tein; 8-oxo-dG, 8-oxo-2-deoxyguanosine; AMPK, 5AMP-activated protein kinase;

EBs, enteroblasts; ECs, enterocytes; EEs, enteroendocrine cells; EGFR, epidermal

growth factor receptor; IGF1R, insulin-like growth factor-1 receptor; InR, insulin

receptor; ISCs, intestinal stem cells; PBST, phosphate-buffered saline with 0.1%

Triton X-100; PH3, phospho-histone H3; PQ, paraquat; PTEN, phosphatase and

tensin homolog; Raptor, regulatory-associated protein of mTOR; Rheb, Ras homolog

enriched in brain; ROS, reactive oxygen species; S6K, ribosomal protein S6 kinase;

JAK/STAT, Janus kinase/signal transducers and activators of transcription; JNK, c-Jun

N-terminal kinase; AKT, protein kinase B; mTOR, mammalian target of rapamycin.

∗ Corresponding author at: Department of Molecular Biology, College of

Q4

Natural Science, Pusan National University, Busan 609-735, South Korea.

Tel.: +82 51 510 2278; fax: +82 51 513 9258.

E-mail address: mayoo@pusan.ac.kr (M.-A Yoo).

1 These authors contributed equally to this article.

metforminismitochondrialrespiratory complex1(Owenetal., 2000).InhibitionofthisproteincomplexdecreasesATP produc-tion,whichincreasestheAMP/ATPratioandleadstosubsequent activationofAMP-activatedproteinkinase(AMPK),aninhibitorof mammaliantargetofrapamycin(mTOR)(Owenetal.,2000).Many studies supportfor theanti-tumor effects ofmetformin (Algire

et al., 2010; Berstein, 2012; Kato et al., 2012; Landman et al., 2009; Saito etal., 2013).Metforminhasbeenshown toinhibit humancancercellsproliferation, andit canbeusedtoprevent and treat a varietyof cancers (Alimova et al., 2009;Ashinuma

etal.,2012;Cantrelletal.,2010).Metforminblocksarethe produc-tionofendogenousreactiveoxygenspecies(ROS)(Halickaetal., 2011), and it inhibits pro-inflammatory responses and nuclear factorkappaBin humanvascularwallcells(Isodaetal.,2006) However,themolecularmechanismsunderlyingtheanti-tumor effectsofmetforminremainunclear

The centrosome is the major microtubule-organizing center and playsanimportant inkey cellularprocesses,includingcell division, cell migration, and cell polarity(Bettencourt-Dias and Glover,2007).Centrosomeaberrations,suchascentrosome ampli-fication,leadtogenomicinstability(Raoetal.,2009).Centrosome

http://dx.doi.org/10.1016/j.mad.2015.05.004

0047-6374/© 2015 Z Published by Elsevier Ireland Ltd This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ).

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Fig 1. Inhibitory effect of metformin on age-related centrosome amplification in midgut ISCs (For interpretation of the references to color in this figure legend, the reader

Q7

is referred to the web version of this article.)

(A) Guts from 14-day-old wild type flies (a–a” and e–e”), 45-day-old wild type flies (b–b” and f-f”), and 14-day-old Cat n1 mutant flies (c–c” and g–g”) without (a–c”) or with (e–g”) 5 mM metformin feeding for 7 days were stained with anti-␥-tubulin (red), anti-PH3 (green), and DAPI (blue) Fourteen-day-old wild type flies without (d–d”) or with (h–h”) 5 mM metformin feeding for 6 days were treated with 10 mM PQ in standard media for 20 h, after which their guts were stained with anti-␥-tubulin (red), anti-PH3 (green), and DAPI (blue) a’, b’, c’, d’, e’, f’, g’, and h’ indicates enlarged PH3 staining images a”, b”, c”, d”, e”, f”, g”, and h” indicates enlarged images of ␥-tubulin staining images Original magnification is 400× (B) The number of PH3-positive cells was counted in whole guts from 14-day-old wild type, 45-day-old wild type, 14-day-old Cat n1 mutant, and 14-day-old PQ-treated wild type flies, with (black bars) or without (gray bars) metformin feeding for 7 days N is the number of observed guts and n is the number of observed PH3-positive cells (C) The frequency of supernumerary centrosome (>2) per mitotic ISC in 14-day-old wild type, 45-day-old wild type, 14-day-old Cat n1 mutant, and 14-day-old PQ-treated wild type flies with (black bars) or without (gray bars) metformin feeding for 7 days guts The centrosome numbers were counted in mitotic ISCs (PH3-positive cell) in the midgut N is the number of observed guts and n is the number of observed ␥-tubulin-positive cells (D) The frequency of mitotic ISCs with supernumerary centrosome per gut in 14-day-old wild type, 45-day-old wild type, 14-day-old Cat n1 mutant, and 14-day-old PQ-treated wild type flies with (black bars)

or without (gray bars) metformin feeding for 7 days guts N is the number of observed guts The error bar represents standard error p-values were calculated using Student’s t-test.

(D’Assoro et al., 2002; Nigg, 2002; Pihan et al., 2003)

Centro-someamplificationisinvolvedintheinitialstagesoftumorigenesis

(Leonard et al., 2013;Nakajima et al.,2004; Nam etal., 2010) CentrosomeamplificationiscausedbyDNAdamage(Nigg,2002;

Xuet al.,1999).Interestingly,DNAdamage incurredduringG2 phase leadstogreatercentrosome amplificationthanG1 phase

49

50

51

52

53 54 55 56

ARTICLE IN PRESS

G Model

MAD 10767 1–11

H.-J Na et al / Mechanisms of Ageing and Development xxx (2015) xxx–xxx 3

Fig 2.Inhibitory effect of metformin on centrosome amplification in ISCs of midgut with ISC/EB-specific activation of AKT/TOR signaling pathway (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(A) Guts from 9-day-old esg ts > GFP (a and i), esg ts > GFP + InR (b and j), esg ts > GFP + PTEN RNAi (c and k), esg ts > GFP+AKT (d and l), esg ts > GFP + Rheb (e and m), esg ts > GFP + Raptor (f and n), esg ts > GFP + d4EBP RNAi (g and o), and esg ts > GFP + S6K STDE (h and p) flies without (a–h) or with (i–p) 5 mM metformin feeding for 7 days were stained with anti-␥-tubulin (red), anti-PH3 (green), and DAPI (blue) Original magnification is 400× (B) The number of PH3-positive cells was counted in whole guts of 9-day-old esg ts > GFP,

amplifica-tioncanswitchasymmetricdivision ofstemcells tosymmetric

division,leadingtopolyploidcellsandhyperplasiainDrosophila

(Bastoetal.,2008).Therefore,centrosomeamplificationinadult

stemcells,particularlythoseinhighturnovertissues,couldaffect

tissuehomeostasisandregeneration

TheAKT/TORpathwayisacentralsignalingpathway

regulat-inglifespan(HaigisandYankner,2010).TheTORpathwaycontrols

cellproliferation,survival,andmetabolism(Kapuriaetal.,2012;

LaplanteandSabatini,2012).HyperactivationofAKT/TORsignaling

isobservedinmanyhumancancers(Alliouacheneetal.,2008)

Fur-thermore,TORsignaling-relatedfactors,includinginsulinreceptor

(InR),rashomologenrichedinbrain(Rheb),phosphataseandtensin

homolog(PTEN),regulatory-associatedproteinofmTOR(Raptor),

ribosomalproteinS6kinase(S6K),andtheeukaryotictranslation

initiationfactor4E-bindingprotein(4E-BP),arealsoimplicatedin

tumorigenesis(Alliouacheneetal.,2008).Inparticular,thelossof

PTENisassociatedwithcentrosomeabnormalities(Leonardetal.,

2013;Levineetal.,1998;Nametal.,2010).Constitutiveactivation

ofAKThasalsobeenshowntocausecentrosomeabnormalitiesand

amplification(Leonardet al.,2013;Nametal.,2010).However,

theeffectofmetforminoncentrosome abnormalities

amplifica-tionand themodulationof molecularpathwayshavenot been

reported

Adultstemcellsmaintaintissuehomeostasisandrepairduring

adulthoodthroughtheirabilitytoself-renewandproduce

differen-tiatedcells(Amcheslavskyetal.,2011).Age-relatedchangesinstem

cellsareassociatedwithtissueagingandage-relateddisease(Liu

andRando,2011).Therefore,understandingage-relatedchanges

instemcellsmayprovidemechanisticinsightsintotissue

homeo-stasisandstem cell-deriveddiseases,includingcancer.Directed

modulationof stemcellaging couldresultin thepreventionof

age-relateddiseasesandthuspromotehealthyaging

TheDrosophilamidgutisanexcellentmodelsystemtostudy

adult stem cells (Andriatsilavo et al., 2013; Jiang and Edgar,

2011).Theintestineisasensitiveorganandisexposedto

vari-ousenvironmentalstressorsanddamageduringreplication.The

Drosophilaintestinal stemcells (ISCs)undergoself-renewaland

producetwomajortypesofcells,enterocytes(ECs)and

enteroen-docrinecells(EEs),fromenteroblasts(EBs)(MicchelliandPerrimon,

2006;OhlsteinandSpradling,2006,2007).ISCsareonlymitotic

cells(MicchelliandPerrimon,2006;OhlsteinandSpradling,2006,

2007),andseveralconservedsignalingpathwaysregulate

prolifer-ationofISCs,suchastheepidermalgrowthfactorreceptor(EGFR),

WntJanuskinase(JAK)/signaltransducerandactivatorof

transcrip-tion(STAT),c-JunN-terminalkinase(JNK),Hippo,PVR/PVF2and

InR/TORpathways(Beebeetal.,2009;Biteauetal.,2008;Biteau

etal.,2010;Choietal.,2008;JiangandEdgar,2009;Jiangetal.,

2011;Jiangetal.,2009;O’Brienetal.,2011;Renetal.,2010;Xu

etal.,2011)

Previous studies have reported age-associated increases in ISChyperproliferationandmis-differentiation,leadingto intesti-nal hyperplasia (Biteau et al., 2008; Choi et al., 2008; Park

et al., 2009) Recently, we reported that foci of the DNA dou-blestrandbreakmarkerDrosophilaorthologof␥H2AX(␥H2AvD) and theoxidative DNA damage marker8-oxo-dG, accumulated with age and in response to oxidative stress in ISCs (Park

et al., 2012) We also reported that centrosome amplification

is increased in ISCs that were aged or exposed to oxidative stress(Park etal.,2014).Basedonthesedata,Drosophila midgut ISCs may be a suitable model to evaluate the effects of anti-cancer drugs on tissue-resident stem cell aging in vivo We recentlyreportedthatmetformininhibitsage-related accumula-tionofDNAdamageinDrosophilaISCsandhyperplasia(Naetal., 2013)

Inthepresentstudy,weinvestigatedtheeffectsofmetformin

oncentrosomeamplificationinDrosophilaISCsandthemechanism

ofitsanti-cancereffect.Ourresultsshowedthatmetformin pre-ventsage-andoxidativestress-inducedcentrosomeamplification

inDrosophilamidgutISCs,whichiscorrelatedwiththeinhibition

ofDNAdamageaccumulationviadown-regulationoftheAKT/TOR signalingpathway

2.1 Flystock Fly stocks were maintained at 25◦C on standard food under a ∼12h/12h light/dark cycle Food consisted of 79.2% water, 1% agar, 7% cornmeal, 2% yeast, 10% sucrose, 0.3% bokinin, and 0.5% propionic acid To avoid larval over-population, 50–60 adult flies per vial were transferred to new food vials every 2–3 days Oregon-R (OR) was used

as wild type Catn1/TM3 (#4014), UAS-InR (#8248), UAS-AKT (#8191), UAS-Rheb (#9688), UAS-Raptor (#53726) and UAS-S6KSTDE (#6913) were acquired from Bloomington Drosophila Stock Center (Indiana University, Blooming-ton, IN, USA) UAS-PTENRNAi (#101475) and UAS-d4E-BPRNAi

(#100739) were obtained from the Vienna Drosophila RNAi Center (Vienna, Austria) Temperature-inducible Su(H)GBE-lacZ; esg-Gal4:tub-Gal80ts,UAS-GFP/CyO (esgts>GFP) was kindly provided by Benjamin Ohlstein (Ohlstein and Spradling, 2007) Catn1/+ flies were obtained from a cross of OR males and Catn1/TM3 females The esgts>GFP/+, esgts>GFP+InR, esgts>GFP+PTENRNAi, esgts>GFP+AKT, esgts>GFP+Rheb, esgts>GFP+Raptor,esgts>GFP+d4E-BPRNAi,andesgts>GFP+S6KSTDE

flieswere obtainedfrom a cross of esgts>GFP females and OR, UAS-InR,PTENRNAi,UAS-AKT,UAS-Rheb,UAS-Raptor,d4E-BPRNAi,and UAS-S6KSTDEmales

esg ts > GFP + InR, esg ts > GFP + PTEN RNAi , esg ts > GFP+AKT, esg ts > GFP + Rheb, esg ts > GFP + Raptor, esg ts > GFP + d4EBP RNAi , and esg ts > GFP + S6K STDE flies with (black bars) or without (gray bars) metformin feeding for 7 days Metformin treatment flies reduced mitotic ISCs in esg ts > GFP + InR (21–9.7), esg ts > GFP + PTEN RNAi (38–20), esg ts > GFP + AKT (35–15), esg ts > GFP + Rheb (23–6), esg ts > GFP + Raptor (16–8), esg ts > GFP + d4E-BP RNAi (18–7), and esg ts > GFP + S6K STDE flies (16–5) The number in parentheses indicate PH3 + cell per gut.

N is the number of observed guts and n is the number of observed PH3-positive cells (C) The frequency of supernumerary centrosome (>2) per mitotic ISC in guts The centrosome numbers were counted in mitotic ISCs from 9-day-old esg ts > GFP, esg ts > GFP + InR, esg ts > GFP + PTEN RNAi , esg ts > GFP + AKT, esg ts > GFP + Rheb, esg ts > GFP + Raptor, esg ts > GFP + d4EBP RNAi , and esg ts > GFP + S6K STDE flies with (black bars) or without (gray bars) metformin feeding for 7 days Metformin treatment flies reduced the number of mitotic ISCs with supernumerary centrosomes in esg ts > GFP + InR (19–3%), esg ts > GFP + PTEN RNAi (17–8%), esg ts > GFP + AKT (17–5%), esg ts > GFP + Rheb (16–7%), esg ts > GFP + Raptor (24–8%), esg ts > GFP + d4E-BP RNAi (16–4%), and esg ts > GFP + S6K STDE flies (22–2%) The number in parentheses indicate abnormal ␥-tubulin + cell (>2) per PH3 + cell (%) N is the number of observed guts and n is the number of observed ␥-tubulin-positive cells (D) The frequency of mitotic ISCs with supernumerary centrosomes per gut in 9-day-old esg ts > GFP, esg ts > GFP + InR, esg ts > GFP + PTEN RNAi , esg ts > GFP + AKT, esg ts > GFP + Rheb, esg ts > GFP + Raptor, esg ts > GFP + d4EBP RNAi , and esg ts > GFP + S6K STDE fly with (black bars) or without (gray bars) metformin feeding for 7 days Metformin treatment also reduced the number of mitotic ISCs with supernumerary centrosomes per gut in esg ts > GFP + InR (3.7–0.5), esg ts > GFP + PTEN RNAi (7–1.6), esg ts > GFP + AKT (6–0.9), esg ts > GFP + Rheb (3.5–0.7), esg ts > GFP + Raptor (4.5–0.9), esg ts > GFP + d4E-BP RNAi (3–0.6), and esg ts > GFP + S6K STDE

flies (3–0.3) The number in parentheses indicate abnormal ␥-tubulin + cell (>2) per gut N is the number of observed guts The error bar represents standard error p-values were calculated using Student’s t-test (E) Cryosectioned guts from 9-day-old esg ts > GFP + InR (b and j), esg ts > GFP + PTEN RNAi (c and k), esg ts > GFP + AKT (d and l), esg ts > GFP + Rheb (e and m), esg ts > GFP + Raptor (f and n), esg ts > GFP + d4EBP RNAi (g and o), and esg ts > GFP + S6K STDE (h and p) flies with or without 5 mM metformin feeding for 7 days were stained with rhodamine-phalloidin (red, actin, indicating visceral muscle), anti-GFP (green), and DAPI (blue) (a–h, without metformin; i–p, with 5 mM metformin) Original magnification is 400×.

57

58

59

60

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

106

107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128

129

130

131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152

ARTICLE IN PRESS

G Model

MAD 10767 1–11

H.-J Na et al / Mechanisms of Ageing and Development xxx (2015) xxx–xxx 5

Fig 3. Inhibitory effect of metformin on DNA damage accumulation by activation of TOR signal pathway in midgut ISCs/EBs (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(A) Guts from 9-day-old esg ts > GFP (a–a’ and i–i’), esg ts > GFP + InR (b–b’ and j–j’), esg ts > GFP + PTEN RNAi (c–c’ and k–k’), esg ts > GFP + AKT (d–d’ and l–l’), esg ts > GFP + Rheb (e–e’ and m–m’), esg ts > GFP + Raptor (f–f’ and n–n’), esg ts > GFP + d4EBP RNAi (g–g’ and o–o’), and esg ts > GFP + S6K STDE (h–h’ and p–p’) flies without (a–h’) or with (i–p’) 5 mM metformin feeding for 7 days were stained with anti-␥-H2AvD (red), anti-GFP (green), and DAPI (blue) a’, b’, c’, d’, e’, f’, g’, h’, i’, j’, k’, l’, m’, n’, o’ and p’ indicates enlarged ␥H2AvD staining images Arrowheads indicate ␥H2AvD-positive and GFP-positive cells Original magnification is 400× (B) Guts from 9-day-old esg ts > GFP (a–a’ and i–i’), esg ts > GFP + InR (b–b’ and j–j’), esg ts > GFP + PTEN RNAi (c–c’ and k–k’), esg ts > GFP + AKT (d–d’ and l–l’), esg ts > GFP + Rheb (e–e’ and m–m’), esg ts > GFP + Raptor (f–f’ and n–n’), esg ts > GFP + d4EBP RNAi (g–g’ and o–o’), and esg ts > GFP + S6K STDE (h–h’ and p–p’) flies 5 mM metformin feeding for 7 days were stained with anti-8-oxo-dG (red), anti-Arm (green), and DAPI (blue) a’, b’, c’,

2.2 Flygenotypes

Fig.1A–D:+;+;+/+.+;+;Catn1/+

Fig.2A–E,Fig.3A–D,andSupplementaryFig.S1:

Su(H)GBE-lacZ;esg-Gal4:tub-Gal80ts,UAS-GFP/+;+/+

Su(H)GBE-lacZ;esg-Gal4:tub-Gal80ts,UAS-GFP/UAS-InR;+/+

Su(H)GBE-lacZ; esg-Gal4:tub-Gal80ts,UAS-GFP/UAS-PTENRNAi;

+/+

Su(H)GBE-lacZ;esg-Gal4:tub-Gal80ts,UAS-GFP/UAS-Akt1;+/+

Su(H)GBE-lacZ;esg-Gal4:tub-Gal80ts,UAS-GFP/+;UAS-Rheb/+

Su(H)GBE-lacZ;esg-Gal4:tub-Gal80ts,UAS-GFP/UAS-Raptor;+/+

Su(H)GBE-lacZ; esg-Gal4:tub-Gal80ts,UAS-GFP/ UAS-d4E-BPRNAi;

+/+

Su(H)GBE-lacZ; esg-Gal4:tub-Gal80ts,UAS-GFP/+;

UAS-S6k.STDE/+

Fig.4AandB:+;+;+/+.+;+;Catn1/+

Fig.4 andD:Su(H)GBE-lacZ;esg-Gal4:tub-Gal80ts,UAS-GFP/+;

+/+

2.3 Temperature-controlledexpression

Fortransgeneexpressionatspecificdevelopmentalstages,the

Gal80tstechniquewasused(McGuireetal.,2004).Theflieswere

setupand maintainedat22◦Cuntiladulthood.After

maintain-ingthefliesat29◦Cfor7days,themidgutsweredissectedand

analyzed.Genotype-by-environment(GxE)interactionshavebeen

known(Lachanceetal.,2013;Lewontin,2000).AlthoughtheG×E

interactionscouldnottobecontrolled,toaddressapossibilityof

phenotypiceffectsduetodifferencesingeneticbackground,flies

wereassayedwithoutthechangeintemperature(at22◦C)asa

par-tialcontrol.Intheexperimentsat22◦C,weobservednophenotypic

effectduetodifferencesingeneticbackground(SupplementaryFig

S1)

2.4 Metforminfeedingassay

Seven-day-old wild type flies, 38-day-old wild type flies,

and 7-day-old Catn1 mutant flies were fed 5mM metformin

(Sigma–Aldrich,St.Louis,MO,USA;workingconcentration)mixed

instandardfoodfor7daysat25◦C.Two-day-oldesgts>GFPflies

werefed5mMmetforminmixedinstandard foodfor7daysat

29◦C Fliesweretransferredtonewmetformin-containingfood

vialsevery2–3days

2.5 Paraquatfeedingassay

Seven-day-old wild type flies pre-treated with 5mM

met-forminfor6dayswerefed10mMparaquat(methylviologen,PQ,

Sigma–Aldrich)instandardmediafor18–20hat25◦C.Themidgut

gutsoftheflieswereanalyzedbyimmunostaining

2.6 Cryosection

Themidguts were dissected, fixedin 4% para-formaldehyde

for 1h at room temperature and infiltrated with 20% sucrose

overnightat4◦C.Afterflash-freezinginTissue-Tekoptimal

cut-tingtemperature(OCT)medium(SAKURA,Tokyo,Japan),sections

(10␮M)werecutonaCryostat(LeicaCM1850,LeicaMicrosystems,

GmbH,Wetzlar,Germany)at−20◦C.Sectionswerethenblocked

in5%BSAfor1handincubatedovernightwithprimaryantibody Aftertreatmentwithsecondaryantibodyconjugatedtofluorescent dye,samplesweremountedwithVectashield(VectorLaboratories, Burlingame,CA,USA)onmicroscopeslide(ThermoFisherScientific Inc.,Waltham,MA,USA)andanalyzedusingaZeissAxioskop2plus microscope(CarlZeissInc.,Gottingen,Germany)

2.7 Immunochemistry Intactadult guts weredissected, fixedat roomtemperature for 1h in 4%para-formaldehyde (Sigma–Aldrich), washed with PBST[0.1%TritonX-100inphosphate-bufferedsaline(PBS)],and incubatedovernightwithprimaryantibodyat4◦Cafter1hof incu-bationat 25◦C The sampleswere then incubated for 2h with secondaryantibodiesat25◦C,washedinPBST,mountedwith Vec-tashieldand analyzedusing aZeiss Axioskop 2plusmicroscope (CarlZeissInc.)

2.8 Antisera Antibodiesweredilutedasfollows:rabbitanti-phospho-histone H3(PH3,mitoticISCmarker),1:1000(MillporeCorporation, Bil-lerica, MA, USA); mouse anti-GFP, 1:1000 (Molecular Probes, Eugene, OR, USA); rat anti-GFP, 1:1000 (Nacalai Tesque Inc., Kyoto,Japan); rabbit anti-␥H2AvD, 1:1000 (Rockland Immuno-chemicals Inc., Gilbertsville, PA, USA); mouse anti-␥-tubulin, 1:500 (Sigma–Aldrich); mouse anti-8-oxo-dG, 1:100, (Trevigen, Gaithersburg,MD,USA);mouseanti-Delta,1:100,mouseanti- ␤-gal, 1:100 (DevelopmentalStudies Hybridoma Bank, Iowa City,

IA,USA); and rabbit anti-phospho-4E-BP1 (T37/46),1:100 (Cell SignalingTechnologies, Beverly,MA,USA) For thedetection of visceralmuscle,weusedrhodamine-phalloidin1:300,(Molecular Probes).SecondaryantibodiesincludedCy3-conjugatedgoat anti-mouse, FITC-conjugated goat anti-rabbit, FITC-conjugated goat anti-mouse, FITC-conjugated goat anti-rat,Cy3-conjugated goat anti-rabbitandgoatanti-rabbitAlexaFluor®647(JacksonImmuno Research,WestGrove,PA,USA);usedata1:500dilution

2.9 Quantitativeanalysis

ToquantitativelyanalyzePH3-positivecells,thenumberof PH3-positive cells in thewhole gut was counted To quantitatively analyzecentrosomeamplification,thenumberof␥-tubulinstained spotsperPH3-positivecellinthewholemidgutswasdetermined

ToquantitativelyanalyzeDelta-positivecells,Su(H)-positivecells andp4E-BP-positivecells,thenumbersofcellswerecountedin themicroscopicfieldsatamagnificationof400×oftheposterior midgut.Quantifieddataareexpressedasthemean±SE.Significant differenceswereidentifiedusingtheStudent’sttest.SigmaPlot 10.0(SystatSoftwareInc.,SanJose,CA,USA)wasusedforanalysis

ofstandarderror

2.10 QuantificationofH2AvDand8-oxo-dGfluorescence The␥H2AvDand8-oxo-dGimageswerecapturedatthesame exposuretimeineachexperimentusingamicroscopewiththeAxio VisionRel4.8program(CarlZeissInc.).WeusedAdobePhotoshop

d’, e’, f’, g’, h’, i’, j’, k’, l’, m’, n’, o’ and p’ indicates enlarged 8-oxo-dG staining images Original magnification is 400× (C) Graph showing the average fluorescence intensity of

␥H2AvD in GFP-positive cells in 9-day-old esg ts > GFP, esg ts > GFP + InR, esg ts > GFP + PTEN RNAi , esg ts > GFP + AKT, esg ts > GFP + Rheb, esg ts > GFP + Raptor, esg ts > GFP + d4EBP RNAi , and esg ts > GFP + S6K STDE flies without or with 5 mM metformin feeding for 7 days N is the number of observed guts and n is the number of observed cells The number of observed cells were 8–20 per gut (D) Graph showing average fluorescence intensity of 8-oxo-dG staining in small cells from 9-day-old esg ts > GFP, esg ts > GFP + InR, esg ts > GFP + PTEN RNAi , esg ts > GFP + AKT, esg ts > GFP + Rheb, esg ts > GFP + Raptor, esg ts > GFP + d4EBP RNAi , and esg ts > GFP + S6K STDE flies without or with 5 mM metformin feeding for 7 days N is the number

of observed guts and n is the number of observed cells The number of observed cells were 7–21 per gut The error bar represents standard error p-values were calculated using Student’s t-test.

153

154

155

156

157

158

159

160

161

162

163

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

198

199

200

201

202 203 204 205 206 207 208

209

210 211 212 213 214 215 216 217

218

219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235

236

237 238 239 240 241 242 243 244 245 246 247

248

249 250 251

ARTICLE IN PRESS

G Model

MAD 10767 1–11

H.-J Na et al / Mechanisms of Ageing and Development xxx (2015) xxx–xxx 7

Fig 4.Inhibitory effect of metformin on age-related TOR activity in age- and oxidative stress-related midguts (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

(A) Guts from 14-day-old wild type flies (a–a” and d–d”), 45-day-old wild type (b–b” and e–e”) and 14-day-old Cat n1 mutant flies (c–c” and f–f”) with-out (a–c”) or with (d–f”) 5 mM metformin feeding for 7 days were stained with anti-p4E-BP (green), anti-Delta (red) and DAPI (blue) a’, b’, c’, d’, e’ and f’ indicates enlarged Delta staining images a”, b”, c”, d”, e” and f” indicates enlarged p4E-BP staining images Arrowheads indicate p4E-BP-positive

USA)tomeasurethefluorescencelevelof␥H2AvDand8-oxo-dG

foci.Thefluorescencelevelwasmeasuredwithinthenucleusbased

ontheboundariesdefinedusingquickselectiontoolofPhotoshop

CS5.1fromtheDAPIchannel.Themeanfluorescencewasanalyzed

afterexclusionofthemeanofthebackgroundregion(fromtwospot

excludingthenucleusportionintheposteriormidgut).The

fluo-rescencemeanofbackgroundregionwasnotsignificantlydifferent

withsecondaryonlycontrol.Significantdifferenceswereidentified

usingtheStudent’sttest.SigmaPlot10.0wasusedforanalysisof

standarderror

3 Results

3.1 Inhibitoryeffectofmetforminonage-relatedcentrosome

amplificationinmidgutISCs

Weinvestigatedtheeffectofmetforminonage-andoxidative

stress-relatedcentrosomeamplificationinISCs.Age-and

oxida-tivestress-relatedcentrosomeamplificationwithsupernumerary

centrosomes(>2)hasbeenreportedinISCs(Parketal.,2014).In

thepresentstudy, weconfirmedthat centrosome amplification

increasedinmidgutISCsafteragingandoxidativestressexposure

Westainedcellswithanti-␥-tubulin,acentrosomemarker,and

anti-PH3,amarkerofmitoticcells(ISCs),andcountedthenumber

ofcellsdisplayingcentrosomeamplification.Supernumerary

cen-trosomeswereobservedin7%ofmitoticISCsin45-day-oldwild

typefliesandin9.4%ofmitoticISCsin14-day-oldCatn1mutant

flies,amodelofintrinsicoxidativestress(Choietal.,2008;Griswold

etal.,1993;Parketal.,2012),ascomparedto1.2%in14-day-old

wildtypeflies(Fig.1A(a–c”)andC).ThenumberofmitoticISCs

withsupernumerarycentrosomepergutwas1.63in45-day-old

wildtypefliesand3.1in14-day-oldCatn1mutantflies,as

com-paredto0.16in14-day-oldwildtypeflies(Fig.1D).Interestingly,

metformintreatmentreducedPH3-positivecellsandthenumberof

ISCswithsupernumerarycentrosomesby3.6%in45-day-oldwild

typeand3.4%in14-day-oldCatn1mutantflies(Fig.1A(e–g”),Band

C).ThenumberofmitoticISCswithsupernumerarycentrosomes

pergutwasreducedby0.56in45-day-oldwildtypefliesandby

0.83in14-day-oldCatn1mutantflies,whereastherewasnochange

in14-day-oldwildtypeflies(Fig.1D)

Toconfirm that metformin inhibitsoxidative stress-induced

centrosome amplification in the midgut, we applied extrinsic

oxidative stress.Seven-day-old wildtype flieswithor without

5mMmetformintreatmentfor6daysweretreatedwith10mM

PQfor20h.BothmitoticISCsandmitoticISCswithsupernumerary

centrosomesincreasedinthePQ-treatedwildtypeflies,as

com-paredtocontrolflies(1.2–12%;Fig.1A(d–d”),BandC).Furthermore,

thenumberofmitoticISCswithsupernumerarycentrosomesper

gutincreasedinPQ-treatedflies(0.16–2.6;Fig.1D).However,in

metforminpre-treatedwildtypeflies,thenumberofPH3-positive

cellandmitoticISCswithsupernumerarycentrosomesdecreased

afterPQtreatment(1–4%;Fig.1A(h–h”),BandC).Furthermore,the

numberofmitoticISCswithsupernumerarycentrosomespergut

wasreducedafterPQtreatment,ascomparedtoPQ-treatedflies

withoutmetforminpretreatment(2.6–0.5;Fig.1D)

These results indicated that metformin partially suppresses age- and oxidative stress-induced centrosome amplification in DrosophilamidgutISCs

3.2 Inhibitoryeffectofmetforminoncentrosomeamplificationin ISCsofmidgutwithISC/EB-specificactivationofAKT/TOR signalingpathway

To determine if the inhibitory effect of metformin on age-relatedcentrosomeamplificationisassociatedwiththeAKT/TOR pathway, we overexpressed componentsof the AKT/TOR path-way in ISCs and EBs usingheat-inducible esg-Gal4;tub-Gal80ts system (McGuire et al., 2004; Ohlstein and Spradling, 2006, 2007).ForISC-andEB-specificexpression,2-day-oldesgts>GFP, esgts>GFP+InR, esgts>GFP+PTENRNAi, esgts>GFP+AKT, esgts>GFP+Rheb, esgts>GFP+Raptor, esgts>GFP+d4E-BPRNAi, andesgts>GFP+S6KSTDEflieswerecultured at29◦Cfor 7days

In all cases, GFP-positivecells increased in thegut of the flies

in which theAKT/TOR pathwaywasactivated underesgts>GFP

as compared tothe control flies(Fig.2A(a–h)) The number of PH3-positivecellsincreasedin thegutofthefliesinwhichthe AKT/TOR pathway wasactivatedunder esgts>GFP as compared

to thecontrol flies(Fig 2B) The number of mitotic ISCs with supernumerarycentrosomesincreasedin thegutofthefliesin which theAKT/TOR pathwaywasactivatedunder esgts>GFPas comparedtothecontrol(Fig.2C).ThenumberofmitoticISCswith abnormalcentrosomes pergut increasedinthe gutof theflies

in which theAKT/TOR pathwaywasactivated underesgts>GFP

ascomparedtothecontrol(Fig.2D).Wealsoobservedintestinal hyperplasia with a multilayered epithelium in cross-sectioned guts of the fliesin which the AKT/TORpathway was activated underesgts>GFP(Fig.2Ea-h)

Compared to the non-treated group, metformin treatment reducedmitoticISCsandthenumberofmitoticISCswith super-numerarycentrosomesinthegutofthefliesinwhichtheAKT/TOR pathway was activated under esg>GFP (Fig 2A(i–p) and B–C) Metformin treatment alsoreduced the number of mitotic ISCs withsupernumerarycentrosomespergutin thegutoftheflies

in which the AKT/TOR pathway was activated under esg>GFP (Fig.2D).Furthermore,metformintreatmentreduced the multi-layeredepithelium in theflies in which theAKT/TOR pathway was activated under esgts>GFP as compared to untreated flies (Fig.2E(i–p)).Incontrast,metformintreatmentdidnotaffectthe intestinalmorphologyof9-day-oldesgts>GFPflies(Fig.2E(a)and (i))

Theseresultsindicatethatcentrosome amplificationandISC hyperproliferationincreased in ISCs byAKT/TOR pathway acti-vation Furthermore, metforminreduced TORpathway-induced increasesinISCproliferation,mitoticISCswithsupernumerary cen-trosomes,andmultilayeredepithelium

3.3 InhibitoryeffectofmetforminonDNAdamageaccumulation

byactivationofTORsignalpathwayinmidgutISCs/EBs Centrosomeamplificationisassociatedwithcellcyclearrest, particularlyduringG2-Mphase,duetoDNAdamage(Nigg,2002;

and Delta-positive cells (B) Graph showing the ratio of p4E-BP-positive cells to Delta-positive cells in 14-day-old wild type flies, 45-day-old wild type and 14-day-old Cat n1

mutant flies without (gray bars) or with (black bars) metformin (met) feeding The error bar is standard error p-values were calculated using Student’s t-test N is the number

of observed guts n is the number of observed Delta-positive cells The number of observed cells were 10–45 per gut (C) Guts of 10-day-old Su(H)GBE-lacZ; esg ts flies (a–a” and c–c”) and 40-day-old Su(H)GBE-lacZ; esg ts flies (b–b” and d–d”) without (a–b”) or with (c–d”) 5 mM metformin feeding were stained with anti-p4E-BP (green), anti-␤-gal (red) and DAPI (blue) a’, b’, c’ and d’ indicates enlarged ␤-gal staining images a”, b”, c” and d” indicates enlarged p4E-BP staining images Arrowhead indicate p4E-BP-positive and Su(H)-positive cells Original magnification is 400× (D) Graph showing the ratio of p4E-BP-positive cells to Su(H)-positive cells The numbers of p4E-BP-positive cells to Su(H)-positive cells were counted in 10-day-old Su(H)GBE-lacZ; esg ts flies and 40-day-old Su(H)GBE-lacZ; esg ts flies without (gray bars) or with (black bars) metformin (met) feeding N is the number of observed guts n is the number of observed Su(H)-positive cells The number of observed cells were 7–111 per gut The error bar is standard error p-values were calculated using Student’s t-test.

252

253

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

301

302

303

304

305 306 307

308 309 310

311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352

353 354

355 356

ARTICLE IN PRESS

G Model

MAD 10767 1–11

H.-J Na et al / Mechanisms of Ageing and Development xxx (2015) xxx–xxx 9

Xuetal.,1999).ThecorrelationbetweenDNAdamageand

centro-someamplificationwasassessed,consideringtheinhibitoryeffects

ofmetformin.Metforminreducedtheaccumulationof␥H2AvD,as

amarkerofDNAdamage,inthemidgutsofaging,oxidative

stress-induced,andAKToverexpressingflies(Naetal.,2013).Basedonthe

aboveobservation,weassessedwhetheractivationoftheAKT/TOR

signalpathwayinducedDNAdamage,andascertainedtheeffect

ofmetforminontheenhancementofDNAdamagecausedbyTOR

signalingpathwayactivationinmidgutISCs/EBs.The␥H2AvD

flu-orescencelevelofesg-positivecellsincreasedinthegutoftheflies

inwhichtheAKT/TORpathwaywasactivatedunderesgts>GFPas

comparedtothecontrolflies(Fig.3A(a–h’)andgraybarsin3C).The

8-oxo-dGfluorescencelevelalsoincreasedupto2–4.5foldinthe

gutofthefliesinwhichtheAKT/TORpathwaywasactivatedunder

esgts>GFPascomparedtocontrolflies(Fig.3B(a–h’)andgraybars

in3D)

However, metformin reduced ␥H2AvD fluorescence in

esg-positive cells by 20–60% in the gut of the flies in which the

AKT/TORpathwaywasactivatedunderesgts>GFPascomparedto

non-treatedflies(Fig.3A(i–p’)andblackbarsin3C).Inaddition,

comparedtotheuntreatedflies,metforminalsoreduced8-oxo-dG

fluorescenceby15–37%inthegutofthefliesinwhichtheAKT/TOR

pathwaywasactivatedunderesgts>GFP(Fig.3B(i–p’)andblack

barsin3D)

TheseresultsshowthatmetformincanreducetheDNA

dam-ageaccumulationthatresultsfromenhancedTORsignalinginthe

midgut

3.4 Inhibitoryeffectofmetforminonage-relatedTORactivityin

age-andoxidativestress-relatedmidguts

Werecentlyreportedthatmetformininhibitsage-andoxidative

stress-inducedincreaseofAKTactivityintheDrosophilamidgut

ISCs/EBs(Naetal.,2013).Toconfirmtheeffectofmetforminon

TORsignalinginmidgutISCs/EBs,p4E-BP,aTORactivitymarker

inadultmidgut,wasevaluatedbyimmunostaining.Thep4E-BP

expressionincreasedin89%ofDelta-positivecells(ISCs)in

45-old-daywildtypeand85%ofDelta-positivecellsin14-day-oldCatn1

mutantflies, andin 84%of Su(H)-positivecells (EBs)in

40-old-daySu(H)GBE-lacZflies,ascomparedtocontrolflies(Fig.4A(a–c”),

B, C(a–b”) and D) Metformin reduced the age-related increase

ofp4E-BPby16%ofDelta-positivecellsin45-old-daywildtype

and23%ofDelta-positivecellsin14-day-oldCatn1 mutantflies,

aswellasin37%ofSu(H)-positivecells(progenitorcells)in

40-old-daySu(H)GBE-lacZflies, ascompared tonon-treatedgroups

(Fig.4A(d–f”),B,C(c–d”)andD)

Thisresultindicatesthat theage-or oxidativestress-related

activationoftheTORpathwayinISCs/EBscanbeinhibitedby

met-formin

4 Discussion

Werecentlyreportedthatcentrosomeamplificationincreases

inDrosophilaISCswithage(Parketal.,2014).Inthepresentstudy,

wereportthattheinhibitoryeffectofmetforminoncentrosome

amplificationinDrosophilaISCsastheunderlyingmechanismofits

anti-cancereffect

The major findings of the current study are: (1) metformin

inhibits age-related centrosome amplification in midgut ISCs,

which is a common feature of many cancer cells (2)

Met-formininhibitsAKT/TORsignal-inducedcentrosomeamplification

inmidgutISCs,and(3)metformininhibitsAKT/TORsignal-induced

DNAdamageaccumulationinmidgutISCs

Weshowedthatmetforminreducedcentrosomeamplification

inmidgutISCs fromfliesundergoingagingand intrinsic(Catn1

mutant)andextrinsic(PQ)oxidativestresses.TheCatn1 heterozy-gousmutantfliesasamodelofintrinsicoxidativestressisbased

onpreviousreportsshowingagenedosage-dependenteffecton catalase activity(Griswold et al.,1993), higher ROS generation (Choiet al., 2008) and increased 8-oxo-dGand ␥H2AvD levels

inthemidguts (Park etal.,2012).Metforminhasemerged asa promisinganti-cancerdrug(Algireetal.,2012;EmamiRiedmaier

et al., 2013) However, the molecular mechanisms underlying itsanti-cancereffectsremainunclear.Centrosomeamplification leads to genomic instability, a hallmark of cancer cells and is involved in the initial stages of tumorigenesis (Leonard et al., 2013;Nakajimaetal.,2004;Nametal.,2010).Metforminactivates AMPKinadultDrosophila(Slacketal.,2012).Metforminhasbeen reportedtosuppressthephosphorylationofinsulin-likegrowth factorreceptor1(IGF1R)/InR,AKT,mTORandribosomalprotein (rpS6)(AljadaandMousa,2011;Owenetal.,2000).Upregulation

ofAKT/TORsignalingisassociatedwithage-relateddiseases,such

ascancer(Fengetal.,2005;Foster,2009;YangandMing,2012)

Werecentlyreportedthatmetformininhibitsage-andoxidative stress-associatedincreaseofAKTactivityintheDrosophilamidgut ISCsand progenitorcells(Naetal.,2013).In thepresent study,

weshowedthat p4E-BPexpression,a TORactivitymarker,was increasedinthemidgutofagedandoxidativestress-treatedflies, andthatmetforminreducedthiseffect

Wefurthershowedthatcentrosomeamplificationisincreased

by hyperactivation of the TOR signal-pathway in midgut ISCs, andthatmetforminreducesAKT/TORsignal-inducedcentrosome amplification.Inmammals,constitutiveactivationofAKThasbeen showntocausecentrosomeabnormalitiesandamplification(Nam

etal.,2010), andlossofPTEN,aninhibitoroftheAKTpathway,

isassociatedwithcentrosomeabnormalities(Leonardetal.,2013; Levineetal.,1998;Nametal.,2010).Therefore,ourdatasuggest thattheinhibitoryeffectofmetforminonage-relatedcentrosome amplificationinmidgutISCsismediatedbydown-regulationofthe AKT/TORpathwayinthemidgut.Basedontheseobservations,we proposethatmetforminexertsitsanti-cancereffectby suppress-ingcentrosome amplificationthrough aninhibition ofAKT/TOR signaling

Metformin has been also reported in mammalian cultured cells to inhibit TORkinase in Rag GTpases-dependent manner whichisTSC1/2-independentTORactivationpathway(Kalender

etal.,2010).RagGTpases-mediatedTORactivationisconservedin DrosophilaandRagproteinsregulatestarvationresponses(Efeyan

etal.,2012).Therefore,weshouldnoteapossibilitythatmetformin inhibitscentrosomeamplificationinRagGTpases-dependent man-ner.Metforminhasabeneficialeffectinpathogenicmoldinfection (Shirazietal.,2014).Metforminmodulatesmetabolismof micro-biotainC.elegans(Cabreiroetal.,2013).Ithasbeenknownthat microbiotaacceleratedysplasiainthegut(Brodericketal.,2014; Buchonetal.,2009).Whethertheinhibitoryeffectisdependent

onalteredmicrobiotacausedbymetforminisnotaddressedinthe presentstudy.Therefore,wecannotexcludeapossibilitythatthe inhibitoryeffectofmetforminoncentrosomeamplificationmaybe associatedwithalteredmicrobiotabyadministrationofmetformin Metforminhasbeenshowntohavephysiologicaleffectssimilarto thoseofdietaryrestriction(Shirazietal.,2014).Weobservedthat

inagreementwiththepreviousreport(Slacketal.,2012), concen-trationof5mMmetforminusedinourexperimentsdidnotdisrupt intestinalphysiologyinanalysesofflyexcreta(SupplementaryFig S2).However,inthepresentstudy,wecannotcompletelyexclude thepossibledietaryrestrictioneffectofmetformin.Whetherornot dietaryrestrictioncaninhibitscentrosomeamplificationwouldbe

animportantquestionthatwarrantsfurtherexploration

HowdoesmetforminactuallysuppresstheAKT/TORpathway? Metforminis knowntoactivate AMPK,whichis aninhibitorof mTOR(Owenetal.,2000).However,inapreviousstudy,weshowed

357

358

359

360

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378

379

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

396

397

398

399

400

401

402

403

404

405

406

407

408

409

410

411

412

413

414

415

416

417

418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483

thatpAMPK islocalizedinenteroendocrinecells butnot inthe

ISCs/EBs(Naetal.,2013).Incontrast,pAKTactivityincreasesin

ISCs/EBswithageandoxidativestress,suggestingthatthe

suppres-siveeffectofmetforminontheAKT/TORpathwayisnotdirectly

associatedwithAMPKactivation.Metforminreducesintracellular

ROSlevels(Halickaetal.,2011).Itwasreportedthatmetformin

reducesthePQ-inducedROSproductioninanAMPK-independent

manner(Algireetal.,2012).TheAKT/TORpathwaypromotes

pro-teinsynthesisandcellgrowth,leadingtoROSproduction,thereby

increasingmitochondrialROSproduction(WellenandThompson,

2011).Therefore,metforminmaysuppresstheAKT/TORpathway

byreducingintracellularROSlevelsintheDrosophilamidgut

Furthermore, we showed that metformin inhibits AKT/TOR

signal-inducedDNA damage accumulationin midgutISCs

Sev-eralrecentstudieshavereportedarelationshipbetweenthelevel

of AKT/TOR signaling and that of DNA damage accumulation

PTEN-deficientcellshavebeenreportedtoshowincreasedDNA

damage(Mingand He,2012).TheAKThyperactivation leadsto

DNAdamageaccumulationinmidgutISCsvia␥H2AvD/8-oxo-dG

accumulation(Naetal.,2013).TherpS6mutantsarereportedto

showincreasedstainingof␥H2AXinmammals(Khalailehetal.,

2013).TheAKT/TORsignalingpathwayisanegativeregulatorof

autophagy.Itwasreportedthatalleliclossofbeclin1,theessential

autophagyregulator,increasesDNAdamageaccumulation(␥H2AX

foci)(Karantza-Wadsworthetal.,2007;Mathewetal.,2007)and

inducessupernumerarycentrosomes(Mathewetal.,2007), and

beclin1isproposedasabiomarkerofgoodhealth,basedonits

highlevelsinhealthycentenarians(Emanueleetal.,2014).Itwas

alsoreportedthatmicewithadeletionofAtg5andAtg7displayed

increased8-oxo-dGand␥H2AXfoci(Takamuraetal.,2011)

There-fore,AKT/TORsignalingcouldincreaseDNAdamageaccumulation

viainhibitingautophagy

Centrosomeamplificationcan beinducedby DNAdamaging

agents, includingionizing radiation(Inanc and Morrison,2011;

LevisandMarin,1963;Saladinoetal.,2009;Yihetal.,2006).We

alsoreportedcentrosomeamplificationinDrosophilaISCsby2Gy

irradiation(Pyoetal.,2014).Centrosomeamplificationis

associ-atedwithcellcyclearrest,especiallyduringG2-Mphase,dueto

DNAdamage(Nigg,2002;Xuetal.,1999).Althoughseveral

stud-ieshavereportedthatAKT/TORsignalingfactorsarelocalizedin

thecentrosome(Goshimaetal.,2007;Rossietal.,2007;Wakefield

etal.,2003),AKT/TORsignaling-inducedDNAdamage

accumula-tionmayalsobeamajorcauseofcentrosomeamplificationdueto

thealteredAKT/TORsignalpresentinsuchcases

In summary, the present data demonstrate that metformin

reducesage-,andoxidativestress-,andTORsignaling-induced

cen-trosome amplificationin Drosophila ISCs Our datasuggest that

metforminmaybeabeneficialanti-cancerdrugviaitsinhibition

ofDNAdamage-inducedcentrosomeamplificationby

hyperactiva-tionofAKT/TORsignaling.OurstudysuggeststhattheDrosophila

midgutisasuitablemodelsystemforinvivoevaluationof

anti-cancerdrugsinagingtissue-residentstemcells

Uncited references

Q5

CabreiroandGems(2013)andCognignietal.(2011)

Acknowledgements

WewouldliketothankBenjaminOhlsteinforhisgenerousgift

oftheflystrains.WewouldalsoliketothanktheDevelopmental

StudiesHybridomaBankfortheantibodiesandtheBloomington

StockCenterandViennaDrosophilaRNAiCenterfortheDrosophila

stocks.WethankProf.ByungP.Yu(UniversityofTexasHealth

Sci-enceCenteratSanAntonio,Texas)for invaluablecommentson

themanuscript This workwassupported bythe BasicScience Q6

References

Algire, C., Amrein, L., Zakikhani, M., Panasci, L., Pollak, M., 2010 Metformin blocks the stimulative effect of a high-energy diet on colon carcinoma growth in vivo and is associated with reduced expression of fatty acid synthase Endocr.

Related Cancer 17, 351–360.

Algire, C., Moiseeva, O., Deschenes-Simard, X., Amrein, L., Petruccelli, L., Birman, E., Viollet, B., Ferbeyre, G., Pollak, M.N., 2012 Metformin reduces endogenous reactive oxygen species and associated DNA damage Cancer Prev Res 5, 536–543.

Alimova, I.N., Liu, B., Fan, Z., Edgerton, S.M., Dillon, T., Lind, S.E., Thor, A.D., 2009.

Metformin inhibits breast cancer cell growth, colony formation and induces cell cycle arrest in vitro Cell Cycle 8, 909–915.

Aljada, A., Mousa, S.A., 2011 Metformin and neoplasia: implications and indications Pharmacol Ther 133, 108–115.

Alliouachene, S., Tuttle, R.L., Boumard, S., Lapointe, T., Berissi, S., Germain, S., Jaubert, F., Tosh, D., Birnbaum, M.J., Pende, M., 2008 Constitutively active Akt1 expression in mouse pancreas requires S6 kinase 1 for insulinoma formation J Clin Invest 118, 3629–3638.

Amcheslavsky, A., Ito, N., Jiang, J., Ip, Y.T., 2011 Tuberous sclerosis complex and Myc coordinate the growth and division of Drosophila intestinal stem cells J.

Cell Biol 193, 695–710.

Andriatsilavo, M., Gervais, L., Fons, C., Bardin, A.J., 2013 [The Drosophila midgut as

a model to study adult stem cells] Int J Med Sci 29, 75–81.

Ashinuma, H., Takiguchi, Y., Kitazono, S., Kitazono-Saitoh, M., Kitamura, A., Chiba, T., Tada, Y., Kurosu, K., Sakaida, E., Sekine, I., Tanabe, N., Iwama, A., Yokosuka, O., Tatsumi, K., 2012 Antiproliferative action of metformin in human lung cancer cell lines Oncol Rep 28, 8–14.

Basto, R., Brunk, K., Vinadogrova, T., Peel, N., Franz, A., Khodjakov, A., Raff, J.W.,

2008 Centrosome amplification can initiate tumorigenesis in flies Cell 133, 1032–1042.

Baur, D.M., Klotsche, J., Hamnvik, O.P., Sievers, C., Pieper, L., Wittchen, H.U., Stalla, G.K., Schmid, R.M., Kales, S.N., Mantzoros, C.S., 2010 Type 2 diabetes mellitus and medications for type 2 diabetes mellitus are associated with risk for and mortality from cancer in a German primary care cohort Metab Clin Exp 60, 1363–1371.

Beebe, K., Lee, W.C., Micchelli, C.A., 2009 JAK/STAT signaling coordinates stem cell proliferation and multilineage differentiation in the Drosophila intestinal stem cell lineage Dev Biol 338, 28–37.

Berstein, L.M., 2012 Metformin in obesity, cancer and aging: addressing controversies Aging 4, 320–329.

Bettencourt-Dias, M., Glover, D.M., 2007 Centrosome biogenesis and function:

centrosomics brings new understanding Nat Rev 8, 451–463.

Biteau, B., Hochmuth, C.E., Jasper, H., 2008 JNK activity in somatic stem cells causes loss of tissue homeostasis in the aging Drosophila gut Cell Stem Cell 3, 442–455.

Biteau, B., Karpac, J., Supoyo, S., Degennaro, M., Lehmann, R., Jasper, H., 2010.

Lifespan extension by preserving proliferative homeostasis in Drosophila PLoS Genet 6, e1001159.

Broderick, N.A., Buchon, N., Lemaitre, B., 2014 Microbiota-induced changes in drosophila melanogaster host gene expression and gut morphology mBio 5, e01117–01114.

Buchon, N., Broderick, N.A., Chakrabarti, S., Lemaitre, B., 2009 Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila Genes Dev 23, 2333–2344.

Cabreiro, F., Au, C., Leung, K.Y., Vergara-Irigaray, N., Cocheme, H.M., Noori, T., Weinkove, D., Schuster, E., Greene, N.D., Gems, D., 2013 Metformin retards aging in C elegans by altering microbial folate and methionine metabolism.

Cell 153, 228–239.

Cabreiro, F., Gems, D., 2013 Worms need microbes too: microbiota, health and aging in Caenorhabditis elegans EMBO Mol Med 5, 1300–1310.

Cantrell, L.A., Zhou, C., Mendivil, A., Malloy, K.M., Gehrig, P.A., Bae-Jump, V.L., 2010 Metformin is a potent inhibitor of endometrial cancer cell proliferation – implications for a novel treatment strategy Gynecol Oncol 116, 92–98.

Choi, N.H., Kim, J.G., Yang, D.J., Kim, Y.S., Yoo, M.A., 2008 Age-related changes in Drosophila midgut are associated with PVF2, a PDGF/VEGF-like growth factor Aging Cell 7, 318–334.

Cognigni, P., Bailey, A.P., Miguel-Aliaga, I., 2011 Enteric neurons and systemic signals couple nutritional and reproductive status with intestinal homeostasis Cell Metab 13, 92–104.

D’Assoro, A.B., Lingle, W.L., Salisbury, J.L., 2002 Centrosome amplification and the development of cancer Oncogene 21, 6146–6153.

484

485

486

487

488

489

490

491

492

493

494

495

496

497

498

499

500

501

502

503

504

505

506

507

508

509

510

511

512

513

514

515

516

517

518

519

520

521

522

523

524

525

526

527

528

529

530

531

532

533

534

535

536

537

538

539

540

541

542

543

544

545 546 547 548 549

550

551 552

553

554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623