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May 16, 2015 - abnormalities and amplification (Leonard et al., 2013; Nam et al.,. 2010). ...... Andriatsilavo, M., Gervais, L., Fons, C., Bardin, A.J., 2013.
Mechanisms of Ageing and Development 149 (2015) 8–18

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Metformin inhibits age-related centrosome amplification in Drosophila midgut stem cells through AKT/TOR pathway Hyun-Jin Na a,1 , Joung-Sun Park a,1 , Jung-Hoon Pyo a , Ho-Jun Jeon a , Young-Shin Kim a , Robert Arking b , Mi-Ae Yoo a,∗ a b

Department of Molecular Biology, Pusan National University, Busan 609-735, South Korea 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 16 May 2015 Keywords: Drosophila intestinal stem cells (ISCs) Metformin Centrosome amplification AKT/TOR pathway DNA damage

a b s t r a c t We delineated the mechanism regulating the inhibition of centrosome amplification by metformin in Drosophila intestinal stem cells (ISCs). Age-related changes in tissue-resident stem cells may be closely associated with tissue aging and age-related diseases, such as cancer. Centrosome amplification is a hallmark of cancers. Our recent work showed that Drosophila ISCs are an excellent model for stem cell studies evaluating age-related increase in centrosome amplification. Here, we showed that metformin, a recognized anti-cancer drug, inhibits age- and oxidative stress-induced centrosome amplification in ISCs. Furthermore, we revealed that this effect is mediated via down-regulation of AKT/target of rapamycin (TOR) activity, suggesting that metformin prevents centrosome amplification by inhibiting the TOR signaling pathway. Additionally, AKT/TOR signaling hyperactivation and metformin treatment indicated a strong correlation between DNA damage accumulation and centrosome amplification in ISCs, suggesting that DNA damage might mediate centrosome amplification. Our study reveals the beneficial and protective effects of metformin on centrosome amplification via AKT/TOR signaling modulation. We identified a new target for the inhibition of age- and oxidative stress-induced centrosome amplification. We propose that the Drosophila ISCs may be an excellent model system for in vivo studies evaluating the effects of anti-cancer drugs on tissue-resident stem cell aging. © 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. Introduction Metformin, a biguanide drug, is clinically approved- and welltolerated for the treatment of type 2 diabetes, and is of interest for cancer prevention and therapy (Aljada and Mousa, 2011; Baur et al., 2010; Landman et al., 2009). The direct molecular target of metformin is mitochondrial respiratory complex 1 (Owen et al.,

Abbreviations: 4E-BP, eukaryotic translation initiation factor 4E-binding protein; 8-oxo-dG, 8-oxo-2 -deoxyguanosine; AMPK, 5 AMP-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 Natural Science, Pusan National University, Busan 609-735, South Korea. Tel.: +82 51 510 2278; fax: +82 51 513 9258. E-mail address: [email protected] (M.-A. Yoo). 1 These authors contributed equally to this article.

2000). Inhibition of this protein complex decreases ATP production, which increases the AMP/ATP ratio and leads to subsequent activation of AMP-activated protein kinase (AMPK), an inhibitor of mammalian target of rapamycin (mTOR) (Owen et al., 2000). Many studies support for the anti-tumor effects of metformin (Algire et al., 2010; Berstein, 2012; Kato et al., 2012; Landman et al., 2009; Saito et al., 2013). Metformin has been shown to inhibit human cancer cells proliferation, and it can be used to prevent and treat a variety of cancers (Alimova et al., 2009; Ashinuma et al., 2012; Cantrell et al., 2010). Metformin blocks the production of endogenous reactive oxygen species (ROS) (Halicka et al., 2011), and it inhibits pro-inflammatory responses and nuclear factor kappa B in human vascular wall cells (Isoda et al., 2006). However, the molecular mechanisms underlying the anti-tumor effects of metformin remain unclear. The centrosome is the major microtubule-organizing center and plays an important role in key cellular processes, including cell division, cell migration, and cell polarity (Bettencourt-Dias and Glover, 2007). Centrosome aberrations, such as centrosome amplification, lead to genomic instability (Rao et al., 2009). Centrosome amplification, which occurs when there is more than two centrosomes during mitosis, is a common feature of human cancers

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/).

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Fig. 1. Inhibitory effect of metformin on age-related centrosome amplification in midgut ISCs. (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 Catn1 mutant flies (c–c” and g–g”) without (a–c”) or with (e–g”) 5 mM metformin (met) 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 Catn1 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 centrosomes (>2) per mitotic ISC in 14-day-old wild type, 45-day-old wild type, 14-day-old Catn1 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 cells) 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 centrosomes per gut in 14-day-old wild type, 45-day-old wild type, 14-day-old Catn1 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). Centrosome amplification is involved in the initial stages of tumorigenesis (Leonard et al., 2013; Nakajima et al., 2004; Nam et al., 2010). Centrosome amplification is caused by DNA damage (Nigg, 2002; Xu et al., 1999). Interestingly, DNA damage incurred during G2

phase leads to greater centrosome amplification than G1 phase damage (Inanc et al., 2010). Furthermore, centrosome amplification can switch asymmetric division of stem cells to symmetric division, leading to polyploid cells and hyperplasia in Drosophila (Basto et al., 2008). Therefore, centrosome amplification in adult

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Fig. 2. Inhibitory effect of metformin on centrosome amplification in ISCs of midgut with ISC/EB-specific activation of AKT/TOR signaling pathway. (A) Guts from 9-day-old esgts > GFP (a and i), esgts > GFP + InR (b and j), esgts > GFP + PTENRNAi (c and k), esgts > GFP+AKT (d and l), esgts > GFP + Rheb (e and m), esgts > GFP + Raptor (f and n), esgts > GFP + d4E-BPRNAi (g and o), and esgts > GFP + S6KSTDE (h and p) flies without (a–h) or with (i–p) 5 mM metformin (met) 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 esgts > GFP,

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stem cells, particularly those in high turnover tissues, could affect tissue homeostasis and regeneration. The AKT/TOR pathway is a central signaling pathway regulating lifespan (Haigis and Yankner, 2010). The TOR pathway controls cell proliferation, survival, and metabolism (Kapuria et al., 2012; Laplante and Sabatini, 2012). Hyperactivation of AKT/TOR signaling is observed in many human cancers (Alliouachene et al., 2008). Furthermore, TOR signaling-related factors, including insulin receptor (InR), ras homolog enriched in brain (Rheb), phosphatase and tensin homolog (PTEN), regulatory-associated protein of mTOR (Raptor), ribosomal protein S6 kinase (S6K), and the eukaryotic translation initiation factor 4E-binding protein (4E-BP), are also implicated in tumorigenesis (Alliouachene et al., 2008). In particular, the loss of PTEN is associated with centrosome abnormalities (Leonard et al., 2013; Levine et al., 1998; Nam et al., 2010). Constitutive activation of AKT has also been shown to cause centrosome abnormalities and amplification (Leonard et al., 2013; Nam et al., 2010). However, the effect of metformin on centrosome abnormalities and the modulation of molecular pathways have not been reported. Adult stem cells maintain tissue homeostasis and repair during adulthood through their ability to self-renew and produce differentiated cells (Amcheslavsky et al., 2011). Age-related changes in stem cells are associated with tissue aging and age-related diseases (Liu and Rando, 2011). Therefore, understanding age-related changes in stem cells may provide mechanistic insights into tissue homeostasis and stem cell-derived diseases, including cancer. Directed modulation of stem cell aging could result in the prevention of age-related diseases and thus promote healthy aging. The Drosophila midgut is an excellent model system to study adult stem cells (Andriatsilavo et al., 2013; Jiang and Edgar, 2011). The intestine is a sensitive organ and is exposed to various environmental stressors and damage during replication. The Drosophila intestinal stem cells (ISCs) undergo self-renewal and produce two major types of cells, enterocytes (ECs) and enteroendocrine cells (EEs), from enteroblasts (EBs) (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006, 2007). ISCs are only mitotic cells (Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006, 2007), and several conserved signaling pathways regulate proliferation of ISCs, such as the epidermal growth factor receptor (EGFR), Wnt, Janus kinase (JAK)/signal transducer and activator of transcription (STAT), c-Jun N-terminal kinase (JNK), Hippo, PVR/PVF2 and InR/TOR pathways (Beebe et al., 2009; Biteau et al., 2008, 2010; Choi et al., 2008; Jiang and Edgar, 2009; Jiang et al., 2011, 2009; O’Brien et al., 2011; Ren et al., 2010; Xu et al., 2011). Previous studies have reported age-associated increases in ISC hyperproliferation and mis-differentiation, leading to intestinal hyperplasia (Biteau et al., 2008; Choi et al., 2008; Park et al., 2009).

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Recently, we reported that foci of the DNA double strand break marker Drosophila ortholog of ␥H2AX (␥H2AvD) and the oxidative DNA damage marker 8-oxo-dG, accumulates 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 et al., 2014). Based on these data, 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 recently reported that metformin inhibits age-related accumulation of DNA damage in Drosophila ISCs and hyperplasia (Na et al., 2013). In the present study, we investigated the effects of metformin on centrosome amplification in Drosophila ISCs and the mechanism of its anti-cancer effect. Our results showed that metformin prevents age- and oxidative stress-induced centrosome amplification in Drosophila midgut ISCs, which is correlated with the inhibition of DNA damage accumulation via down-regulation of the AKT/TOR signaling pathway. 2. Materials and methods 2.1. Fly stocks Fly stocks were maintained at 25 ◦ C on standard food under a ∼12 h/12 h 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 overpopulation, 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, Bloomington, 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 + AKT, esgts > GFP + Rheb, esgts > GFP + PTENRNAi , ts ts RNAi esg > GFP + Raptor, esg > GFP + d4E-BP , and esgts > GFP + S6KSTDE flies were obtained from a cross of esgts > GFP females and OR, UASInR, UAS-PTENRNAi , UAS-AKT, UAS-Rheb, UAS-Raptor, UAS-d4E-BPRNAi , and UAS-S6KSTDE males. 2.2. Fly genotypes Fig. 1A–D: +; +; +/+. +; +; Catn1 /+.

esgts > GFP + InR, esgts > GFP + PTENRNAi , esgts > GFP+AKT, esgts > GFP + Rheb, esgts > GFP + Raptor, esgts > GFP + d4E-BPRNAi , and esgts > GFP + S6KSTDE flies with (black bars) or without (gray bars) metformin feeding for 7 days. Metformin treatment flies reduced mitotic ISCs in esgts > GFP + InR (21 to 9.7), esgts > GFP + PTENRNAi (38 to 20), esgts > GFP + AKT (35 to 15), esgts > GFP + Rheb (23 to 6), esgts > GFP + Raptor (16 to 8), esgts > GFP + d4E-BPRNAi (18 to 7), and esgts > GFP + S6KSTDE flies (16 to 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 centrosomes (>2) per mitotic ISC in guts. The centrosome numbers were counted in mitotic ISCs from 9-day-old esgts > GFP, esgts > GFP + InR, esgts > GFP + PTENRNAi , esgts > GFP + AKT, esgts > GFP + Rheb, esgts > GFP + Raptor, esgts > GFP + d4E-BPRNAi , and esgts > GFP + S6KSTDE 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 esgts > GFP + InR (19% to 3%), esgts > GFP + PTENRNAi (17% to 8%), esgts > GFP + AKT (17% to 5%), esgts > GFP + Rheb (16% to 7%), esgts > GFP + Raptor (24% to 8%), esgts > GFP + d4E-BPRNAi (16% to 4%), and esgts > GFP + S6KSTDE flies (22% to 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 esgts > GFP, esgts > GFP + InR, esgts > GFP + PTENRNAi , esgts > GFP + AKT, esgts > GFP + Rheb, esgts > GFP + Raptor, esgts > GFP + d4E-BPRNAi , and esgts > GFP + S6KSTDE 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 esgts > GFP + InR (3.7 to 0.5), esgts > GFP + PTENRNAi (7 to 1.6), esgts > GFP + AKT (6 to 0.9), esgts > GFP + Rheb (3.5 to 0.7), esgts > GFP + Raptor (4.5 to 0.9), esgts > GFP + d4E-BPRNAi (3 to 0.6), and esgts > GFP + S6KSTDE flies (3 to 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 esgts > GFP + InR (b and j), esgts > GFP + PTENRNAi (c and k), esgts > GFP + AKT (d and l), esgts > GFP + Rheb (e and m), esgts > GFP + Raptor (f and n), esgts > GFP + d4E-BPRNAi (g and o), and esgts > GFP + S6KSTDE (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×.

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Fig. 3. Inhibitory effect of metformin on DNA damage accumulation by activation of TOR signal pathway in midgut ISCs/EBs. (A) Guts from 9-day-old esgts > GFP (a–a’ and i–i’), esgts > GFP + InR (b–b’ and j–j’), esgts > GFP + PTENRNAi (c–c’ and k–k’), esgts > GFP + AKT (d–d’ and l–l’), esgts > GFP + Rheb (e–e’ and m–m’), esgts > GFP + Raptor (f–f’ and n–n’), esgts > GFP + d4E-BPRNAi (g–g’ and o–o’), and esgts > GFP + S6KSTDE (h–h’ and p–p’) flies without (a–h’) or with (i–p’) 5 mM metformin (met) 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 esgts > GFP (a–a’ and i–i’), esgts > GFP + InR (b–b’ and j–j’), esgts > GFP + PTENRNAi (c–c’ and k–k’), esgts > GFP + AKT (d–d’ and l–l’), esgts > GFP + Rheb (e–e’ and m–m’), esgts > GFP + Raptor (f–f’ and n–n’), esgts > GFP + d4E-BPRNAi (g–g’ and o–o’), and esgts > GFP + S6KSTDE (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’,

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Fig. 2A–E, Fig. 3A–D, and Supplementary Fig. 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. 4A and B: +; +; +/+. +; +; Catn1 /+. Fig. 4C and D: Su(H)GBE-lacZ; esg-Gal4:tub-Gal80ts , UAS-GFP/+; +/+.

Intact adult guts were dissected, fixed at room temperature for 1 h in 4% para-formaldehyde (Sigma–Aldrich), washed with PBST [0.1% Triton X-100 in phosphate-buffered saline (PBS)], and incubated overnight with primary antibody at 4 ◦ C after 1 h of incubation at 25 ◦ C. The samples were then incubated for 2 h with secondary antibodies at 25 ◦ C, washed in PBST, mounted with Vectashield and analyzed using a Zeiss Axioskop 2plus microscope (Carl Zeiss Inc.).

2.3. Temperature-controlled expression

2.8. Antisera

For transgene expression at specific developmental stages, the Gal80ts technique was used (McGuire et al., 2004). The flies were set up and maintained at 22 ◦ C until adulthood. After maintaining the flies at 29 ◦ C for 7 days, the midguts were dissected and analyzed. Genotype-by-environment (GxE) interactions have been known (Lachance et al., 2013; Lewontin, 2000). Although the GxE interactions could not to be controlled, to address a possibility of phenotypic effects due to differences in genetic background, flies were assayed without the change in temperature (at 22 ◦ C) as a partial control. In the experiments at 22 ◦ C, we observed no phenotypic effect due to differences in genetic background (Supplementary Fig. S1).

Antibodies were diluted as follows: rabbit anti-phospho-histone H3 (PH3, mitotic ISC marker), 1:1000 (Millpore Corporation, Billerica, 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 Immunochemicals Inc., Gilbertsville, PA, USA); mouse anti-␥-tubulin, 1:500 (Sigma–Aldrich); mouse anti-8-oxo-dG, 1:100, (Trevigen, Gaithersburg, MD, USA); mouse anti-Delta, 1:100, mouse anti-␤gal, 1:100 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA); and rabbit anti-phospho-4E-BP1 (T37/46), 1:100 (Cell Signaling Technologies, Beverly, MA, USA). For the detection of visceral muscle, we used rhodamine-phalloidin 1:300, (Molecular Probes). Secondary antibodies included Cy3-conjugated goat antimouse, FITC-conjugated goat anti-rabbit, FITC-conjugated goat anti-mouse, FITC-conjugated goat anti-rat, Cy3-conjugated goat anti-rabbit and goat anti-rabbit Alexa Fluor® 647 (Jackson Immuno Research, West Grove, PA, USA); used at a 1:500 dilution.

2.4. Metformin feeding assay Seven-day-old wild type flies, 38-day-old wild type flies, and 7-day-old Catn1 mutant flies were fed 5 mM metformin (Sigma–Aldrich, St. Louis, MO, USA) mixed in standard food for 7 days at 25 ◦ C. Two-day-old esgts > GFP flies were fed 5 mM metformin mixed in standard food for 7 days at 29 ◦ C. Flies were transferred to new metformin-containing food vials every 2–3 days. 2.5. Paraquat feeding assay Seven-day-old wild type flies pre-treated with 5 mM metformin for 6 days were fed 10 mM paraquat (PQ, methyl viologen, Sigma–Aldrich) in standard media for 18–20 h at 25 ◦ C. The midgut guts of the flies were analyzed by immunostaining. 2.6. Cryosection The midguts were dissected, fixed in 4% para-formaldehyde for 1 h at room temperature and infiltrated with 20% sucrose overnight at 4 ◦ C. After flash-freezing in Tissue-Tek optimal cutting temperature (OCT) medium (SAKURA, Tokyo, Japan), sections (10 ␮m) were cut on a Cryostat (Leica CM1850, Leica Microsystems, GmbH, Wetzlar, Germany) at −20 ◦ C. Sections were then blocked in 5% BSA for 1 h and incubated overnight with primary antibody. After treatment with secondary antibody conjugated to fluorescent dye, samples were mounted with Vectashield (Vector Laboratories,

Burlingame, CA, USA) on microscope slide (Thermo Fisher Scientific Inc., Waltham, MA, USA) and analyzed using a Zeiss Axioskop 2plus microscope (Carl Zeiss Inc., Gottingen, Germany). 2.7. Immunochemistry

2.9. Quantitative analysis To quantitatively analyze PH3-positive cells, the number of PH3positive cells in the whole gut was counted. To quantitatively analyze centrosome amplification, the number of ␥-tubulin stained spots per PH3-positive cell in the whole midguts was determined. To quantitatively analyze Delta-positive cells, Su(H)-positive cells and p4E-BP-positive cells, the numbers of cells were counted in the microscopic fields at a magnification of 400× of the posterior midgut. Quantified data are expressed as the mean ± SE. Significant differences were identified using the Student’s t test. Sigma Plot 10.0 (Systat Software Inc., San Jose, CA, USA) was used for analysis of standard error. 2.10. Quantification of H2AvD and 8-oxo-dG fluorescence The ␥H2AvD and 8-oxo-dG images were captured at the same exposure time in each experiment using a microscope with the Axio Vision Rel 4.8 program (Carl Zeiss Inc.). We used Adobe Photoshop CS5.1 extended software (Adobe System Incorporated, San Jose, CA, USA) to measure the fluorescence level of ␥H2AvD and 8-oxo-dG foci. The fluorescence level was measured within the nucleus based

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 esgts > GFP, esgts > GFP + InR, esgts > GFP + PTENRNAi , esgts > GFP + AKT, esgts > GFP + Rheb, esgts > GFP + Raptor, esgts > GFP + d4E-BPRNAi , and esgts > GFP + S6KSTDE 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 esgts > GFP, esgts > GFP + InR, esgts > GFP + PTENRNAi , esgts > GFP + AKT, esgts > GFP + Rheb, esgts > GFP + Raptor, esgts > GFP + d4E-BPRNAi , and esgts > GFP + S6KSTDE 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.

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Fig. 4. Inhibitory effect of metformin on age-related TOR activity in age- and oxidative stress-related midguts. (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 Catn1 mutant flies (c–c” and f–f”) without (a–c”) or with (d–f”) 5 mM metformin (met) 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

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on the boundaries defined using quick selection tool of Photoshop CS5.1 from the DAPI channel. The mean fluorescence was analyzed after exclusion of the mean of the background region (from two spot excluding the nucleus portion in the posterior midgut). The fluorescence mean of background region was not significantly different with secondary only control. Significant differences were identified using the Student’s t test. Sigma Plot 10.0 was used for analysis of standard error.

3. Results 3.1. Inhibitory effect of metformin on age-related centrosome amplification in midgut ISCs We investigated the effect of metformin on age- and oxidative stress-related centrosome amplification in ISCs. Age- and oxidative stress-related centrosome amplification with supernumerary centrosomes (>2) has been reported in ISCs (Park et al., 2014). In the present study, we confirmed that centrosome amplification increased in midgut ISCs after aging and oxidative stress exposure. We stained cells with anti-␥-tubulin, a centrosome marker, and anti-PH3, a marker of mitotic cells (ISCs), and counted the number of cells displaying centrosome amplification. Supernumerary centrosomes were observed in 7% of mitotic ISCs in 45-day-old wild type flies and in 9.4% of mitotic ISCs in 14-day-old Catn1 mutant flies, a model of intrinsic oxidative stress (Choi et al., 2008; Griswold et al., 1993; Park et al., 2012), as compared to 1.2% in 14-day-old wild type flies (Fig. 1A(a–c”) and C). The number of mitotic ISCs with supernumerary centrosomes per gut was 1.63 in 45-day-old wild type flies and 3.1 in 14-day-old Catn1 mutant flies, as compared to 0.16 in 14-day-old wild type flies (Fig. 1D). Interestingly, metformin treatment reduced PH3-positive cells and the number of ISCs with supernumerary centrosomes by 3.6% in 45-day-old wild type and 3.4% in 14-day-old Catn1 mutant flies (Fig. 1A(e–g”), B and C). The number of mitotic ISCs with supernumerary centrosomes per gut was reduced by 0.56 in 45-day-old wild type flies and by 0.83 in 14-day-old Catn1 mutant flies, whereas there was no change in 14-day-old wild type flies (Fig. 1D). To confirm that metformin inhibits oxidative stress-induced centrosome amplification in the midgut, we applied extrinsic oxidative stress. Seven-day-old wild type flies with or without 5 mM metformin treatment for 6 days were treated with 10 mM PQ for 20 h. Both mitotic ISCs and mitotic ISCs with supernumerary centrosomes increased in the PQ-treated wild type flies, as compared to control flies (1.2% to 12%; Fig. 1A(d–d”), B and C). Furthermore, the number of mitotic ISCs with supernumerary centrosomes per gut increased in PQ-treated flies (0.16 to 2.6; Fig. 1D). However, in metformin pre-treated wild type flies, the number of PH3-positive cell and mitotic ISCs with supernumerary centrosomes decreased after PQ treatment (12% to 4%; Fig. 1A(h–h”), B and C). Furthermore, the number of mitotic ISCs with supernumerary centrosomes per gut was reduced after PQ treatment, as compared to PQ-treated flies without metformin pretreatment (2.6 to 0.5; Fig. 1D).

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These results indicated that metformin partially suppresses age- and oxidative stress-induced centrosome amplification in Drosophila midgut ISCs. 3.2. Inhibitory effect of metformin on centrosome amplification in ISCs of midgut with ISC/EB-specific activation of AKT/TOR signaling pathway To determine if the inhibitory effect of metformin on age-related centrosome amplification is associated with the AKT/TOR pathway, we overexpressed components of the AKT/TOR pathway in ISCs and EBs using heat-inducible esg-Gal4; tubGal80ts system (McGuire et al., 2004; Ohlstein and Spradling, 2006, 2007). For ISC- and EB-specific expression, 2-day-old esgts > GFP, esgts > GFP + InR, esgts > GFP + PTENRNAi , esgts > GFP + AKT, esgts > GFP + Rheb, esgts > GFP + Raptor, esgts > GFP + d4E-BPRNAi , and esgts > GFP + S6KSTDE flies were cultured at 29 ◦ C for 7 days. In all cases, GFP-positive cells increased in the gut of the flies in which the AKT/TOR pathway was activated under esgts > GFP as compared to the control flies (Fig. 2A(a–h)). The number of PH3-positive cells increased in the gut of the flies in which the AKT/TOR pathway was activated under esgts > GFP as compared to the control flies (Fig. 2B). The number of mitotic ISCs with supernumerary centrosomes increased in the gut of the flies in which the AKT/TOR pathway was activated under esgts > GFP as compared to the control (Fig. 2C). The number of mitotic ISCs with abnormal centrosomes per gut increased in the gut of the flies in which the AKT/TOR pathway was activated under esgts > GFP as compared to the control (Fig. 2D). We also observed intestinal hyperplasia with a multilayered epithelium in cross-sectioned guts of the flies in which the AKT/TOR pathway was activated under esgts > GFP (Fig. 2E(a-h)). Compared to the non-treated group, metformin treatment reduced mitotic ISCs and the number of mitotic ISCs with supernumerary centrosomes in the gut of the flies in which the AKT/TOR pathway was activated under esgts > GFP (Fig. 2A(i–p) and B–C). Metformin treatment also reduced the number of mitotic ISCs with supernumerary centrosomes per gut in the gut of the flies in which the AKT/TOR pathway was activated under esgts > GFP (Fig. 2D). Furthermore, metformin treatment reduced the multilayered epithelium in the flies in which the AKT/TOR pathway was activated under esgts > GFP as compared to untreated flies (Fig. 2E(i–p)). In contrast, metformin treatment did not affect the intestinal morphology of 9-day-old esgts > GFP flies (Fig. 2E(a) and (i)). These results indicate that centrosome amplification and ISC hyperproliferation increased in ISCs by AKT/TOR pathway activation. Furthermore, metformin reduced TOR pathway-induced increases in ISC proliferation, mitotic ISCs with supernumerary centrosomes, and multilayered epithelium. 3.3. Inhibitory effect of metformin on DNA damage accumulation by activation of TOR signal pathway in midgut ISCs/EBs Centrosome amplification is associated with cell cycle arrest, particularly during G2-M phase, due to DNA damage (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 Catn1 mutant flies without (gray bars) or with (black bars) metformin 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; esgts flies (a–a” and c–c”) and 40-day-old Su(H)GBE-lacZ; esgts 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; esgts flies and 40-day-old Su(H)GBE-lacZ; esgts flies without (gray bars) or with (black bars) metformin 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.

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Xu et al., 1999). The correlation between DNA damage and centrosome amplification was assessed, considering the inhibitory effects of metformin. Metformin reduced the accumulation of ␥H2AvD, as a marker of DNA damage, in the midguts of aging, oxidative stressinduced, and AKT overexpressing flies (Na et al., 2013). Based on the above observation, we assessed whether activation of the AKT/TOR signal pathway induced DNA damage, and ascertained the effect of metformin on the enhancement of DNA damage caused by TOR signaling pathway activation in midgut ISCs/EBs. The ␥H2AvD fluorescence level of esg-positive cells increased in the gut of the flies in which the AKT/TOR pathway was activated under esgts > GFP as compared to the control flies (Fig. 3A(a–h’) and gray bars in 3C). The 8-oxo-dG fluorescence level also increased up to 2–4.5 fold in the gut of the flies in which the AKT/TOR pathway was activated under esgts > GFP as compared to control flies (Fig. 3B(a–h’) and gray bars in 3D). However, metformin reduced ␥H2AvD fluorescence in esgpositive cells by 20–60% in the gut of the flies in which the AKT/TOR pathway was activated under esgts > GFP as compared to non-treated flies (Fig. 3A(i–p’) and black bars in 3C). In addition, compared to the untreated flies, metformin also reduced 8-oxo-dG fluorescence by 15–37% in the gut of the flies in which the AKT/TOR pathway was activated under esgts > GFP (Fig. 3B(i–p’) and black bars in 3D). These results show that metformin can reduce the DNA damage accumulation that results from enhanced TOR signaling in the midgut. 3.4. Inhibitory effect of metformin on age-related TOR activity in age- and oxidative stress-related midguts We recently reported that metformin inhibits age- and oxidative stress-induced increases of AKT activity in the Drosophila midgut ISCs/EBs (Na et al., 2013). To confirm the effect of metformin on TOR signaling in midgut ISCs/EBs, p4E-BP, a TOR activity marker in adult midgut, was evaluated by immunostaining. The p4E-BP expression increased in 89% of Delta-positive cells (ISCs) in 45-dayold wild type and 85% of Delta-positive cells in 14-day-old Catn1 mutant flies, and in 84% of Su(H)-positive cells (EBs) in 40-dayold Su(H)GBE-lacZ flies, as compared to control flies (Fig. 4A(a–c”), B, C(a–b”) and D). Metformin reduced the age-related increase of p4E-BP by 16% of Delta-positive cells in 45-day-old wild type and 23% of Delta-positive cells in 14-day-old Catn1 mutant flies, as well as in 37% of Su(H)-positive cells (progenitor cells) in 40old-day Su(H)GBE-lacZ flies, as compared to non-treated groups (Fig. 4A(d–f”), B, C(c–d”) and D). This result indicates that the age- or oxidative stress-related activation of the TOR pathway in ISCs/EBs can be inhibited by metformin. 4. Discussion We recently reported that centrosome amplification increases in Drosophila ISCs with age (Park et al., 2014). In the present study, we report the inhibitory effect of metformin on centrosome amplification in Drosophila ISCs as the underlying mechanism of its anti-cancer effect. 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) Metformin inhibits AKT/TOR signal-induced centrosome amplification in midgut ISCs, and (3) metformin inhibits AKT/TOR signal-induced DNA damage accumulation in midgut ISCs. We showed that metformin reduced centrosome amplification in midgut ISCs from flies undergoing aging and intrinsic (Catn1

mutant) and extrinsic (PQ) oxidative stresses. The Catn1 heterozygous mutant flies as a model of intrinsic oxidative stress is based on previous reports showing a gene dosage-dependent effect on catalase activity (Griswold et al., 1993), higher ROS generation (Choi et al., 2008) and increased 8-oxo-dG and ␥H2AvD levels in the midguts (Park et al., 2012). Metformin has emerged as a promising anti-cancer drug (Algire et al., 2012; Emami Riedmaier et al., 2013). However, the molecular mechanisms underlying its anticancer effects remain unclear. Centrosome amplification leads to genomic instability, a hallmark of cancer cells and is involved in the initial stages of tumorigenesis (Leonard et al., 2013; Nakajima et al., 2004; Nam et al., 2010). Metformin activates AMPK in adult Drosophila (Slack et al., 2012). Metformin has been reported to suppress the phosphorylation of insulin-like growth factor receptor 1 (IGF1R)/InR, AKT, mTOR and ribosomal protein (rp S6) (Aljada and Mousa, 2011; Owen et al., 2000). Upregulation of AKT/TOR signaling is associated with age-related diseases, such as cancer (Feng et al., 2005; Foster, 2009; Yang and Ming, 2012). We recently reported that metformin inhibits age- and oxidative stressassociated increases of AKT activity in the Drosophila midgut ISCs and progenitor cells (Na et al., 2013). In the present study, we showed that p4E-BP expression, a TOR activity marker, was increased in the midgut of aged and oxidative stress-treated flies, and that metformin reduced this effect. We further showed that centrosome amplification is increased by hyperactivation of the TOR signal-pathway in midgut ISCs, and that metformin reduces AKT/TOR signal-induced centrosome amplification. In mammals, constitutive activation of AKT has been shown to cause centrosome abnormalities and amplification (Nam et al., 2010), and loss of PTEN, an inhibitor of the AKT pathway, is associated with centrosome abnormalities (Leonard et al., 2013; Levine et al., 1998; Nam et al., 2010). Therefore, our data suggest that the inhibitory effect of metformin on age-related centrosome amplification in midgut ISCs is mediated by down-regulation of the AKT/TOR pathway in the midgut. Based on these observations, we propose that metformin exerts its anti-cancer effect by suppressing centrosome amplification through an inhibition of AKT/TOR signaling. Metformin has been also reported in mammalian cultured cells to inhibit TOR kinase in Rag GTPases-dependent manner which is TSC1/2-independent TOR activation pathway (Kalender et al., 2010). Rag GTPases-mediated TOR activation is conserved in Drosophila and Rag proteins regulate starvation responses (Efeyan et al., 2012). Therefore, we should note a possibility that metformin inhibits centrosome amplification in Rag GTPases-dependent manner. Metformin has a beneficial effect in pathogenic mold infection (Shirazi et al., 2014). Metformin modulates metabolism of microbiota in C. elegans (Cabreiro et al., 2013). It has been known that microbiota accelerate dysplasia in the gut (Broderick et al., 2014; Buchon et al., 2009). Whether the inhibitory effect is dependent on altered microbiota caused by metformin is not addressed in the present study. Therefore, we cannot exclude a possibility that the inhibitory effect of metformin on centrosome amplification may be associated with altered microbiota by administration of metformin. Metformin has been shown to have physiological effects similar to those of dietary restriction (Shirazi et al., 2014). We observed that in agreement with the previous report (Slack et al., 2012), concentration of 5 mM metformin used in our experiments did not disrupt intestinal physiology in analyses of fly excreta (Supplementary Fig. S2). However, in the present study, we cannot completely exclude the possible dietary restriction effect of metformin. Whether or not dietary restriction can inhibit centrosome amplification would be an important question that warrants further exploration. How does metformin actually suppress the AKT/TOR pathway? Metformin is known to activate AMPK, which is an inhibitor of mTOR (Owen et al., 2000). However, in a previous study, we showed

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that pAMPK is localized in enteroendocrine cells but not in the ISCs/EBs (Na et al., 2013). In contrast, pAKT activity increases in ISCs/EBs with age and oxidative stress, suggesting that the suppressive effect of metformin on the AKT/TOR pathway is not directly associated with AMPK activation. Metformin reduces intracellular ROS levels (Halicka et al., 2011). It was reported that metformin reduces the PQ-induced ROS production in an AMPK-independent manner (Algire et al., 2012). The AKT/TOR pathway promotes protein synthesis and cell growth, leading to ROS production, thereby increasing mitochondrial ROS production (Wellen and Thompson, 2011). Therefore, metformin may suppress the AKT/TOR pathway by reducing intracellular ROS levels in the Drosophila midgut. Furthermore, we showed that metformin inhibits AKT/TOR signal-induced DNA damage accumulation in midgut ISCs. Several recent studies have reported a relationship between the level of AKT/TOR signaling and that of DNA damage accumulation. PTEN-deficient cells have been reported to show increased DNA damage (Ming and He, 2012). The AKT hyperactivation leads to DNA damage accumulation in midgut ISCs via ␥H2AvD/8-oxo-dG accumulation (Na et al., 2013). The rp S6 mutants are reported to show increased staining of ␥H2AX in mammals (Khalaileh et al., 2013). The AKT/TOR signaling pathway is a negative regulator of autophagy. It was reported that allelic loss of beclin 1, an essential autophagy regulator, increases DNA damage accumulation (␥H2AX foci) (Karantza-Wadsworth et al., 2007; Mathew et al., 2007) and induces supernumerary centrosomes (Mathew et al., 2007), and beclin 1 is proposed as a biomarker of good health, based on its high levels in healthy centenarians (Emanuele et al., 2014). It was also reported that mice with a deletion of Atg5 and Atg7 displayed increased 8-oxo-dG and ␥H2AX foci (Takamura et al., 2011). Therefore, AKT/TOR signaling could increase DNA damage accumulation via inhibiting autophagy. Centrosome amplification can be induced by DNA damaging agents, including ionizing radiation (Inanc and Morrison, 2011; Levis and Marin, 1963; Saladino et al., 2009; Yih et al., 2006). We also reported centrosome amplification in Drosophila ISCs by 2 Gy irradiation (Pyo et al., 2014). Centrosome amplification is associated with cell cycle arrest, especially during G2-M phase, due to DNA damage (Nigg, 2002; Xu et al., 1999). Although several studies have reported that AKT/TOR signaling factors are localized in the centrosome (Goshima et al., 2007; Rossi et al., 2007; Wakefield et al., 2003), AKT/TOR signaling-induced DNA damage accumulation may also be a major cause of centrosome amplification due to the altered AKT/TOR signal present in such cases. In summary, the present data demonstrate that metformin reduces age-, oxidative stress-, and TOR signaling-induced centrosome amplification in Drosophila ISCs. Our data suggest that metformin may be a beneficial anti-cancer drug via its inhibition of DNA damage-induced centrosome amplification by hyperactivation of AKT/TOR signaling. Our study suggests that the Drosophila midgut is a suitable model system for in vivo evaluation of anticancer drugs in aging tissue-resident stem cells.

Acknowledgements We would like to thank Benjamin Ohlstein for his generous gift of the fly strains. We would also like to thank the Developmental Studies Hybridoma Bank for the antibodies and the Bloomington Stock Center and Vienna Drosophila RNAi Center for the Drosophila stocks. We thank Prof. Byung P. Yu (University of Texas Health Science Center at San Antonio, Texas) for invaluable comments on the manuscript. This work was supported by the Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Education (NRF-2013R1A1A2012029) and the JSPS Core-to Core Program, B. Asia-Africa Science Platforms.

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