Radiosensitization of metformin in pancreatic cancer cells via abrogating the G2 checkpoint and inhibiting DNA damage repair

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Original Articles

Radiosensitization of metformin in pancreatic cancer cells via abrogating the G2 checkpoint and inhibiting DNA damage repair Zheng Wang a,b, Song-Tao Lai a,b, Ning-Yi Ma a,b, Yun Deng c, Yong Liu c, Dong-Ping Wei c, Q1 Jian-Dong Zhao a,b, Guo-Liang Jiang a,b,d,* a

Department of Radiation Oncology, Fudan University Shanghai Cancer Center, 270 Dongan Road, Shanghai 200032, China Department of Oncology, Shanghai Medical College, Fudan University, 270 Dongan Road, Shanghai 200032, China c Cancer Research Institute, Fudan University Shanghai Cancer Center, 270 Dongan Road, Shanghai 200032, China d Department of Radiation Oncology, Shanghai Proton and Heavy Ion Center, 4365 Kangxin Road, Shanghai 201321, China b

A R T I C L E

I N F O

Article history: Received 11 April 2015 Received in revised form 14 July 2015 Accepted 18 August 2015 Keywords: Metformin Radiosensitizer Pancreatic cancer Wee1 Rad51 mTOR

A B S T R A C T

Recent evidences have demonstrated the potential of metformin as a novel agent for cancer prevention and treatment. Here, we investigated its ability of radiosensitization and the underlying mechanisms in human pancreatic cancer cells. In this study, we found that metformin at 5 mM concentration enhanced the radiosensitivity of MIA PaCa-2 and PANC-1 cells, with sensitization enhancement ratios of 1.39 and 1.27, respectively. Mechanistically, metformin caused abrogation of the G2 checkpoint and increase of mitotic catastrophe, associated with suppression of Wee1 kinase and in turn CDK1 Tyr15 phosphorylation. Furthermore, metformin inhibited both expression and irradiation-induced foci formation of Rad51, a key player in homologous recombination repair, ultimately leading to persistent DNA damage, as reflected by γ-H2AX and 53BP1 signaling. Finally, metformin-mediated AMPK/mTOR/ p70S6K was identified as a possible upstream pathway controlling translational regulation of Wee1 and Rad51. Our data suggest that metformin radiosensitizes pancreatic cancer cells in vitro via abrogation of the G2 checkpoint and inhibition of DNA damage repair. However, the in-vivo study is needed to further confirm the findings from the in-vitro study. © 2015 Published by Elsevier Ireland Ltd.

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Introduction Pancreatic cancer remains among the deadliest human malignancies with an overall 5-year survival rate for all patients of less than 5% [1]. This dismal outcome is mainly attributed to delayed diagnosis with the absence of specific early symptoms, and more crucially, its inherent propensity of resistance to most conventional therapies including radiotherapy [2], which is currently one of the important treatment modalities for pancreatic cancer, serving as either post-operative adjuvant treatment for resectable/borderline resectable disease or definitive chemoradiotherapy for locally advanced unresectable disease [3,4]. Therefore, it is imperative to identify novel agents, as effective sensitizers, to overcome this radioresistance, which might establish better treatment strategies and further improve therapeutic outcomes. Metformin is one of the commonly used oral anti-hyperglycemic drugs for the treatment of type 2 diabetes mellitus (T2DM) [5]. Recent epidemiological evidences have indicated that use of metformin can decrease both incidence and mortality of multiple

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* Corresponding author. Tel.: +86 21 64175590; fax: +86 21 64439052. E-mail address: [email protected] (G.-L. Jiang).

cancers [6], suggesting its potential as a novel agent for cancer prevention and treatment. Considering the bidirectional and complicated relationship between diabetes and pancreatic cancer, its role in chemoprevention and chemotherapy of pancreas cancer becomes an emerging focus of research. A latest meta-analysis from our group has reported that metformin is associated with reduced risk of pancreatic cancer in patients with T2DM [7]. However, as to whether metformin administration has any survival benefits for pancreatic cancer patients associated with diabetes, the only two retrospective cohort studies to date have yielded inconsistent conclusions [8,9]. Even so, some preclinical studies have been carried out and revealed its antitumor activities for pancreatic cancer in vitro and in vivo models. It was found that metformin effectively inhibited the abilities of proliferation, colony formation, migration and invasion in pancreatic cancer cell lines [10,11], also the growth of MIA PaCa-2 and PANC-1 pancreatic cancer xenografts [12]. In this context, several experimental investigations have been concentrated on the potential role of metformin as a sensitizer to irradiation (IR) for different malignancies [13–16]. The involved mechanisms mainly include enhancement of IR-induced apoptosis, inhibition of pro-survival pathway and DNA damage repair, as well as preferential eradication of cancer stem cells. However, so far, data for pancreas cancer is limited. Although a recently

http://dx.doi.org/10.1016/j.canlet.2015.08.015 0304-3835/© 2015 Published by Elsevier Ireland Ltd.

Please cite this article in press as: Zheng Wang, et al., Radiosensitization of metformin in pancreatic cancer cells via abrogating the G2 checkpoint and inhibiting DNA damage repair, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.08.015

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published in vitro study showed that metformin radiosensitized pancreatic cancer cells at micromolar concentration, which may be mediated through AMPK and correlated with increased H2AX foci 1 h post-IR [17], we report here our results of testing metformin at millimolar level for its ability to radiosensitize pancreatic cancer cell lines. Of note, our study provides more exact and intriguing mechanisms of the action, for the first time to our knowledge, the most important of which is that metformin clearly inhibits the expression of Wee1 kinase, a key regulator of G2 checkpoint, leading to premature mitotic entry and so-called synthetic lethality after exogenous DNA damage by IR in p53-deficient pancreatic cancer cells.

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Cell culture, IR and metformin solution

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Human pancreatic cancer cell lines MIA PaCa-2 and PANC-1 were purchased from the American Type Culture Collection (ATCC). Cells were maintained in DMEM medium (Gibco) supplemented with 10% Fetal Bovine Serum (Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco), at 37 °C in a humidified atmosphere with 5% carbon dioxide. Cells in a monolayer were X-ray irradiated at room temperature (RT) using Small Animal Radiation Research Platform (Gulmay Medical) operated at 220 kV and a dose rate of 3.845 Gy/min. Metformin was purchased from Sigma and freshly dissolved in DMEM medium before each use.

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Cell viability assay

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The cells were plated in a 96-well plate and the next day they were treated with increasing concentrations (2.5–160 mM) of metformin. The cells were stained after 24 h exposure to metformin using the Cell Counting Kit-8 (Dojindo) following the manufacturer’s protocol, and the absorbance was measured at 450 nm using a 96well plate reader.

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Colony formation assay

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Cells were treated with or without 5 mM metformin for 24 h before 0–6 Gy IR. Immediately after IR, cells were harvested, diluted and re-plated into 60 mm dishes in triplicate per treatment. After incubation in drug free medium for 10–12 days, cells were fixed and stained with 0.5% crystal violet in absolute methanol, and colonies containing more than 50 cells were counted under a dissection microscope. The surviving fractions were normalized for the cytotoxicity induced by metformin alone. Cell survival curves were fitted using the least squares regression by the linear quadratic model. Sensitization enhancement ratio (SER) was calculated as the dose needed to produce 10% of cell survival for IR alone divided by the dose for IR plus metformin.

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Flow cytometry

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Cells were exposed to 5 mM metformin for 24 h or not, and subjected to 6 Gy of IR or not. For cell cycle analysis, at 8 h and 24 h after IR, cells were trypsinized, washed in PBS, fixed in 70% ice-cold ethanol and stored at −20 °C overnight. Samples were then re-suspended in PBS, and stained with 50 μg/ml propidium iodide (PI) solution containing 0.2% Triton X-100 and 100 μg/ml DNase-free RNase A for analysis. For apoptosis analysis, at 16 h, 24 h and 48 h after IR, cells were harvested and stained using Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences) according to the manufacturer’s instructions. The cells showing positive surface annexin V staining without cell-permeable PI were identified as apoptotic cells. For γ-H2AX analysis, at 1 h and 24 h after IR, cells were harvested and stained using FlowCellect Histone H2A.X Phosphorylation Assay Kit (Millipore) following the manufacturer’s protocol. For quantification of γ-H2AX positive cell rate, a gate was arbitrarily set on control group to define a region of positive staining for γ-H2AX of around 5%, and this gate was then overlaid on the treated groups. Samples were analyzed on a Cytomics FC500 flow cytometer (Beckman Coulter) and quantified with CXP software.

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Immunofluorescence

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Cells cultured in 24-well plates with coated glass coverslips were pretreated with 5 mM metformin for 24 h or not, and then irradiated to 6 Gy or not. After an additional incubation for indicated times, cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 5 min and blocked with 5% bovine serum albumin (BSA) in PBS for 1 h at RT. For γ-H2AX, 53BP1 or Rad51 foci visualization, samples were then stained overnight at 4 °C with primary antibody against either γ-H2AX (Epitomics), 53BP1 or Rad51 (Abcam), followed by incubation with Alexa Fluor 488 or Alexa Fluor 568 goat anti-rabbit IgG (Invitrogen) for 45 min at RT; For mitotic catastrophe analysis, samples were stained overnight at 4 °C with Alexa Fluor 488-conjugated α-tubulin rabbit monoclonal antibody (Cell Signaling Technology). Subsequently, coverslips were mounted on glass slides and cell nuclei,

Materials and methods

meanwhile, were counterstained using DAPI Fluoromount-G (SouthernBiotech). Cells were visualized and images were captured using a TCS SP5 confocal fluorescence microscope (Leica). A total of 200 randomly selected cells were analyzed for each treatment group. The presence of two or more distinct nuclear lobes within a single cell was scored as positive for mitotic catastrophe.

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Western blot analysis

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Cells were exposed to 5 mM metformin for 24 h or not, and subsequently irradiated at a dose of 6 Gy or not. At 2 h, 8 h and 24 h after IR, cells were lysed with cold lysis buffer (Thermo) containing protease and phosphatase inhibitors (Roche). Equal amounts of total protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a nitrocellulose filter membrane. The membrane was blocked with 5% BSA for 2 h at RT and then incubated with primary antibodies overnight at 4 °C. Next day, the membrane was incubated for 1 h at RT with horseradish peroxidase-conjugated secondary antibodies. Protein bands were detected and visualized using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo). The following antibodies were used: CDK1 (Abcam), Wee1, phospho-CDK1 (Tyr15), Cyclin B1, Rad51, γ-H2AX (Ser139), AMPK, phospho-AMPK (Thr172), mTOR, phospho-mTOR (Ser2448), p70S6k, phospho-p70S6K (Thr389), β-Tubulin and GAPDH (Cell Signaling Technology).

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Quantitative real-time reverse transcription PCR

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Cells were treated with 5 mM metformin for 24 h or not. Total RNA was isolated using RNAprep Pure Cell Kit (Tiangen) and reverse transcribed into cDNA using PrimeScript RT reagent Kit (Takara). Real-time quantitative PCR was carried out in triplicate using SYBR Green I Master (Roche) on a LightCycler 480 Real-Time PCR System (Roche). The PCR primers were 5′-CTCTGTTGATGAGCAGAACGC-3′ and 5′TGATCATCTTCTGCCCACGC-3′ for Wee1; 5′-GCAGATGCAGCTTGAAGCAA-3′ and 5′CCACACTGCTCTAACCGTGA-3′ for Rad51; and 5′-TTGTTACAGGAAGTCCCTTGCC-3′ and 5′-ATGCTATCACCTCCCCTGTGTG-3′ for β-actin. Gene expression was normalized to β-actin expression. Analysis of relative gene expression was performed using the 2-ΔΔCt method [18].

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Small interfering RNA transfection

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Wee1 and mTOR siRNA kits were purchased from RiboBio (Guangzhou, China). PANC-1 cells were seeded in 6-well plates and transfected at 60% confluence with siRNA duplexes or negative control siRNA by lipofectamine 2000 (Invitrogen) according to manufacturer’s protocol. After 24 h transfection, cells were harvested and lysed for western blotting analysis described above.

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Statistical analysis

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Data were presented as the mean ± standard error from three independent experiments unless indicated otherwise. Statistical analyses were performed by Student’s t-test or one-way ANOVA using SPSS 20.0 and GraphPad Prism 5.0 software. All tests were two-sided and p values less than 0.05 were considered statistically significant.

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Results Metformin sensitizes pancreatic cancer cells to IR To identify the optimal concentration of metformin for colony formation assay, its cytotoxicity was first measured using CCK-8 assay. Metformin of 2.5–160 mM for 24 h inhibited cell viability, and the IC50 values for MIA PaCa-2 and PANC-1 were 43.03 and 46.65 mM, respectively (Fig. 1A). 5 mM metformin, which is approximately equal to IC10 value for both cell lines, was used to further investigate its radiosensitization effect. The colony formation assay showed that 5 mM metformin for 24 h prior to IR significantly enhanced the radiosensitivity of both MIA PaCa-2 and PANC-1 cells. The SER, at survival fraction level of 0.1, were 1.39 and 1.27 for MIA PaCa-2 and PANC-1, respectively (Fig. 1B). Metformin does not increase IR-induced apoptosis To explore the mechanisms underlying the radiosensitization effect of metformin in vitro we began by detecting whether apoptosis was enhanced by metformin in irradiated cells. At 16 h, 24 h and 48 h after 6 Gy of IR, apoptosis rate was raised to some extent in both cell lines, whereas cells pretreated with 5 mM metformin did not further increase the level of apoptosis induced by IR

Please cite this article in press as: Zheng Wang, et al., Radiosensitization of metformin in pancreatic cancer cells via abrogating the G2 checkpoint and inhibiting DNA damage repair, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.08.015

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metformin for 24 h caused cell cycle arrest in G0/G1 phase, while significantly decreased the proportion of S phase cells, the most radioresistant phase, in both MIA PaCa-2 and PANC-2 cells (Fig. 2A), which contributes at least in part to its radiosensitization. For MIA PaCa-2 cells, the mean percentage of S phase cells in control and metformin group were 54.32% ± 3.44% and 38.60% ± 1.29%, respectively (p = 0.0001), and for PANC-1 cells, 51.77% ± 2.16% and 33.33% ± 4.62% (p < 0.0001). Furthermore, the cell cycle regulatory effect of metformin combined with IR was explored. As expected, exposing cells to 6 Gy IR dramatically activated G2 checkpoint and resulted in G2/M phase arrest. Although exposure of metformin alone did not interfere the percentage of G2/M phase cells, pretreatment with it for 24 h significantly attenuated IR-induced G2/M phase accumulation, which were observed 8 h after IR in MIA PaCa-2 cells (p = 0.0025) and 24 h in PANC-1 cells (p = 0.0009), respectively (Fig. 2B). Given that lack of functional G1/S checkpoint causes G2/M arrest to be the major cell cycle response after IR, G1/S checkpoint status of two pancreatic cancer cell lines was then tested. At 8 h and 24 h after 6 Gy of IR, no increase of G0/G1 phase cells, but a significant decrease of that, was observed in both MIA PaCa-2 (8 h, p = 0.0057; 24 h, p = 0.03) and PANC-1 cells (8 h, p = 0.0019; 24 h, p = 0.0021) compared with the control group (Fig. 2C), which probably resulted from activation of G2/M arrest with reduced cells exiting from mitosis, suggesting G1/S checkpoint deficiency of these two cell lines. Metformin attenuates phosphorylated CDC2 via Wee-1 inhibition, and Rad51 expression As we found that metformin did abrogate IR-induced G2/M arrest by cell cycle analysis, the expression of G2 checkpoint-related proteins, Wee1, phospho-CDK1 and cyclin B1, were next studied by Western blot study. Our results showed that pretreatment of metformin significantly decreased the expression of Wee1 and in turn pCDK1 at various time points in both irradiated MIA PaCa-2 and PANC-1 cells (Fig. 3A). Furthermore, down-regulation of Cyclin B1 by metformin was found in PANC-1, but not in MIA PaCa-2 cells (Fig. 3A). Because Wee1 and CDK1 have recently been implicated in regulation of homologous recombination (HR) [20], we further examined whether the expression of Rad51, a key mediator of HR pathway, were influenced by metformin. As hypothesized, we found that metformin caused a persistent decrease in Rad51 expression in both cell lines (Fig. 3A). In addition to a reduction in total protein level, we also observed that Rad51 foci formation in response to IR was greatly inhibited by metformin in PANC-1 cells using immunofluorescence method (Fig. 3B). 1 2 3 4 5 6 7

Fig. 1. Metformin sensitizes pancreatic cancer cells to IR. (A) MIA PaCa-2 and PANC-1 cells were treated with various concentrations of metformin, as indicated, for 24 h. Effect of metformin on cellular viability was then measured by CCK-8 assay. (B) Cells were treated with 5 mM metformin and IR as illustrated and then assayed for clonogenic survival. Cell survival curves were plotted after normalizing for the cytotoxicity induced by metformin alone. Each data point shown represents the mean survival fraction with SE of three independent experiments.

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(Supplementary Fig. S1), indicating that apoptotic cell death may not be involved in the mechanism of radiosensitization by metformin. Metformin alters cell cycle distribution and abrogates IR-induced G2/M phase arrest Since the cell cycle phase is one of important determinants for radiosensitivity [19], we next tested whether metformin could change cell cycle distribution leading to the increased radiosensitivity. Compared to the control group, treatment with 5 mM

Metformin inhibits the repair of IR-induced DNA damage in PANC-1 cells Given that G2-abrogation can theoretically shorten the time for DNA repair and inhibition of Rad51 expression also affects HR repair, we examined whether metformin could affect γ-H2AX, an indirect marker of DNA double-strand breaks (DSBs), in PANC-1 cells by both western blot and flow cytometric analysis. Using western blot, we found that metformin caused persistent γ-H2AX expression, at 2 h and 24 h after IR, in irradiated cells compared to cells treated with IR alone (Fig. 4A). Flow cytometric analysis indicated that higher mean fluorescence intensity (MFI) and more γ-H2AX-positive cells were consistently observed at 24 h after IR in the combination group than that in IR alone, whereas no difference was found at 1 h after IR, when cells probably just began to repair DNA damage (Fig. 4B and C). To confirm the inter-group difference of residual DNA damage, we further detected nuclear foci formation of γ-H2AX and another

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Fig. 2. The effects of metformin on cell cycle distribution. (A) The cell cycle distribution of MIA PaCa-2 and PANC-1 cells treated with 5 mM metformin for 24 h, or not, was determined by flow cytometry. Data shown are mean ± SD from three independent experiments. *, statistical significance versus control is indicated (p < 0.01). (B) Cells were exposed to 5 mM metformin, or not 24 h before 6 Gy IR, and collected at indicated time points for analysis of cell-cycle phase distribution. The mean percentage ± SD of G2/M phase cells from three independent experiments are presented. *, statistical significance versus IR is indicated (p < 0.01). (C) The changes of cell proportion in G0/G1 phase after 6 Gy IR were assessed. Shown are mean value of three independent experiments with SD. *, statistical significance versus control is indicated (p < 0.05).

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well-known sensor protein of DNA damage, 53BP1, using immunofluorescence staining. As a result, metformin significantly increased the number of persistent γ-H2AX and 53BP1 foci at 24 h after IR in irradiated PANC-1 cells (Fig. 4D).

seems to be the major contributor to radiosensitization effect of metformin in the present study. Metformin decrease Wee1 and Rad51 protein synthesis via AMPK/ mTOR/p70S6K pathway

Metformin enhances mitotic catastrophe after IR in PANC-1 cells Based on the findings of abrogation of IR-induced G2/M arrest and premature entry of cells into mitosis with lots of unrepaired DNA damage by metformin, we went on to determine whether it could further increase mitotic catastrophe, the most frequent type of cell death after IR in solid tumors, in PANC-1 cells. Compared to IR alone, combination treatment of metformin and IR dramatically increased the percentage of cells positive for mitotic catastrophe, while metformin alone had no effect on it (Fig. 5A and B), which

As metformin has previously been shown to exert anti-tumor effects through activating AMPK and inhibiting mTOR [21], which is known to regulate cap-dependent mRNA translation [22], we hypothesized that metformin might inhibit Wee1 and Rad51 protein synthesis via mTOR signaling pathway. At first, we found that metformin alone or in combination with IR significantly did increase p-AMPK levels, and decrease p-mTOR and its downstream target p-p70S6K levels in PANC-1 cells (Fig. 6A). And then, the protein levels of both Wee1 and Rad51, but not their mRNA levels, were

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Fig. 3. The effects of metformin on G2 checkpoint- and DNA damage repair-related proteins. MIA-PaCa-2 and PANC-1 cells were exposed to 5 mM metformin for 24 h or not, and subsequently irradiated at a dose of 6 Gy or not. At various time points after IR, samples were lysed or fixed for western blot or immunofluorescence analysis, respectively. (A) Representative immunoblots probed with indicated antibodies are shown. (B) Representative immunofluorescence images (cell nuclei: blue; Rad51 foci: green) and the mean number ± SE of Rad51 foci per nucleus from three independent experiments. *, statistical significance versus IR is indicated (p < 0.05).

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shown to decrease by metformin treatment (Fig. 6B and C). To further test our hypothesis, we inhibited mTOR expression using siRNA transfection in PANC-1 cells, which resulted in down-regulation of both Wee1 and Rad51 protein (Fig. 6D). In addition, Wee1 knockdown by siRNA also induced a moderate decrease of Rad51 protein expression (Fig. 6E).

Discussion In this work, we explored the possibility to use metformin, a classical oral anti-diabetic drug, as a new radiosensitizing agent in pancreatic cancer cells. We found that pretreatment with 5 mM metformin sensitized both MIA PaCa-2 and PANC-1 cells to IR to

Please cite this article in press as: Zheng Wang, et al., Radiosensitization of metformin in pancreatic cancer cells via abrogating the G2 checkpoint and inhibiting DNA damage repair, Cancer Letters (2015), doi: 10.1016/j.canlet.2015.08.015

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Fig. 4. Metformin treatment leads to persistent IR-induced DNA damage. PANC-1 cells were harvested or fixed at different time points after 6 Gy IR with or without 24 h pretreatment of 5 mM metformin. (A) Representative immunoblots of γ-H2AX expression are shown. (B) The mean fluorescence intensity (MFI) ± SD (n = 3) of γ-H2AX staining by flow cytometry. *, statistical significance versus IR is indicated (p < 0.05). (C) The mean percentage ± SD (n = 3) of γ-H2AX-positive cells defined by the gate as described in Methods using flow cytometry analysis. *, statistical significance versus IR is indicated (p < 0.05). (D) Representative immunofluorescence micrographs (cell nuclei: blue; γ-H2AX foci: green; 53BP1 foci: red) and the mean number ± SE of γ-H2AX and 53BP1 foci per nucleus from three independent experiments. *, statistical significance versus IR is indicated (p < 0.05).

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Fig. 5. Metformin increases mitotic catastrophe in irradiated cells. PANC-1 cells were untreated or treated with 5 mM metformin for 24 h and exposed to 6 Gy IR. Metformin was washed out and, 72 h after IR, cells were stained for α-Tubulin (green) and DAPI (blue). (A) Representative immunofluorescence images for evaluating cells undergoing mitotic catastrophe. White arrows: cells experiencing normal mitosis; Yellow arrows: cells with normal nucleus; Red arrows: cells undergoing mitotic catastrophe. (B) The mean percentage ± SE (n = 3) of cells defined as positive for mitotic catastrophe. *, statistical significance versus IR is indicated (p < 0.05).

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varying degrees. Furthermore, our mechanistic studies suggested three ways through which metformin may differentially regulate cellular radiation sensitivity. First, metformin treatment led to a significant increase in G0/ G1-phase cell population and a corresponding decrease in S-phase for both pancreatic cancer cell lines. The cell cycle phase plays an important role in the radiosensitivity of cancer cells, and cells in S phase are considered to be most resistant to IR [19]. Several previous studies have showed that metformin caused a dose- and timedependent G1 arrest, which was mostly at the expense of the S-phase population, in various types of cancer cell lines. And mechanistically, coincident decreases in the G1 checkpoint protein levels of CDKs (2, 4 and 6) and cyclins (D1 and E) were also confirmed [23–25]. Therefore, these outcomes together with our observations demonstrate that metformin may result in enhanced radiosensitivity, at least in part, through G1 arrest and subsequently down-regulating the proportion of radioresistant S-phase cells. Second, metformin treatment abrogated the G2 checkpoint through inhibition of Wee1 kinase in the presence of IR, which is thought to be the most important mechanism of its radiosensitizing activity in this study. Cancer cells usually lack a functional G1 checkpoint, due to for instance a p53 mutation, and rely on an intact G2 checkpoint, allowing extended time for DNA repair prior to mitosis, for survival in response to DNA-damaging agents [26]. Therefore, forced abrogation of the G2 checkpoint, releasing cells with unrepaired DNA lesions into premature mitosis and inducing subsequent cell death, represents an attractive strategy for tumorspecific radiosensitization [27]. Recently, overexpression of Wee1 kinase, a key regulator in the prevention of G2/M transition by directly inactivating tyrosine (Y15) phosphorylation of CDK1 in complex with Cyclin B, has been observed in several types of cancer [28–31], and high expression of that was showed to be correlated with poor disease-free survival in malignant melanoma [32]. In this context, targeting Wee1, particularly in combination with radiation therapy, has been extensively investigated [33–36]. Preclinical studies with cancer cell lines and animal models have demonstrated that inhibition of Wee1 by either siRNA or small molecular compounds, such as PD0166285 and MK1775, does show an expected attenuation of the IR-induced G2 block and increase in radiosensitization [29,30,37,38]. Although previous reports have de-

scribed the possible mechanisms of metformin-mediated radiosensitization, here we offer a novel finding that millimolar concentration of metformin can significantly suppress the expressions of Wee1 and p-CDK1 in both pancreatic cancer cell lines. It has also been found that after IR, cells accumulate in a predominant G2/M arrest, the abrogation of which by metformin effectively enhances IR-induced mitotic catastrophe and in turn clonogenic death. Despite having no relevant data of normal cell lines in our study, metformininduced radiosensitization might be tumor-specific because both MIA PaCa-2 and PANC-1 cells harbor p53 mutations and their G1 checkpoints are deficient, which has been confirmed in the present as well as previous studies [39,40]. Additionally, it is worth noting that metformin also induces down-regulation of Cyclin B1 in PANC-1 rather than MIA PaCa-2 cells, which, we believe, may in part explain their differential radiosensitization [41]. Third, our data show that metformin treatment significantly reduces not only overall Rad51 protein levels, but also IR-induced nuclear Rad51 foci formation, ultimately leading to persistent DNA damage in pancreatic cancer cells. The Rad51 protein plays a central role in DNA HR repair and its expression increases at S and G2 phases of cell cycle, in which predominant activity of HR was observed concomitantly [42]. Tumor cells often have an increased reliance on HR for IR-induced DNA DSBs repair, due to their aberrant G1 checkpoint where non-homologous end joining (NHEJ) mainly occurs [43]. Thus, inhibition of HR repair by targeting Rad51 seems to be another tumor-selective radiosensitizing mechanism. Mounting evidence from several studies supports this hypothesis. Down-regulation of the expression of Rad51 by ribozyme, antisense oligonucleotide or siRNA approach has been shown to sensitize various cancer cells to IR [44–46]. Two small molecule anti-cancer drugs, imatinib (a c-Abl tyrosine kinase inhibitor) and PCI-24781 (a histone deacetylase inhibitor), have also been found to enhance the radiosensitivity of cancer cells by suppressing Rad51 expression and subsequently impairing HR repair [47,48]. Furthermore, Gasparini P et al. recently discovered that overexpression of miR-155 decreased the efficiency of HR repair and increased sensitivity to IR through directly targeting Rad51 in breast cancer in vitro and in vivo [49]. In the present study, our mechanism investigations show that, in addition to Wee1, Rad51 is another important target of metformin, which together with delayed dispersal of IR-induced γ-H2AX and 53BP1 foci in the presence of metformin, could also be used to

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Fig. 6. Metformin inhibits Wee1 and Rad51 mRNA translation via inactivation of mTOR pathway. (A) PANC-1 cells were treated with 5 mM metformin for 24 h prior to 6 Gy IR and collected 2 h later for Western blot analysis of the indicated proteins. (B and C) PANC-1 cells were treated with or without 5 mM metformin for 24 h. The protein expression and relative mRNA level of Wee1 and Rad51 were detected using Western blot and qPCR analysis, respectively. (D and E) PANC-1 cells were transfected with siRNA duplexes specific to mTOR, Wee1 or negative oligo for 24 h. Whole cell extracts were collected for western blot analysis using mTOR, Wee1 and Rad51 antibodies.

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reasonably explain metformin-enhanced radiosensitivity. Coincidentally, a latest study published by Havelek R et al. explored the effects of co-targeting Wee1 kinase and Rad51 recombinase using specific small molecular inhibitors on radiation sensitivity in human leukemic T-cells. In conformity with our data, they found that the combination treatment resulted in a further enhancement of radiosensitivity, which was associated with the abrogation of G2 cell cycle arrest and impairment of DNA damage repair [50]. Finally, since enhanced radiosensitivity observed in metformintreated cells is considered resulting mainly from Wee1 and Rad51 inhibition, we attempt to dissect the possible upstream pathways that in turn regulate them. Previous studies as well as our data suggested that metformin could activate AMPK and thereby inactivate mTOR in pancreatic cancer cells. Given that two major pathways regulate mRNA translation, one of which is cap-dependent controlled by mTOR [22], suppression of mTOR-mediated p70S6K phosphorylation with unaffected Wee1 and Rad51 mRNA expression but decreased protein expression together implicated possible interference with mRNA translation rather than transcription in cells

exposed to metformin. More than that, mTOR was further confirmed as an upstream regulator of Wee1 and Rad51 protein by siRNA approach in our study. In conclusion, our results have demonstrated for the first time that metformin at millimolar concentration potently enhances radiosensitivity in pancreatic cancer cell lines. It may be caused by down-regulation of Wee1 and Rad51, leading to abrogation of the G2 checkpoint and impairment in the repair of DSBs. These effects were probably due to the simultaneous inhibition of upstream mTOR signaling pathway (Fig. 7). However, our data are based on in vitro studies and the biological effect of metformin in vivo may be even more complicated. Therefore, the combination of metformin with IR should be further validated using animal models for potential translation into clinical trials.

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Funding This study was supported by a grant from “985 project” (985IIIYPT06), Ministry of Education, China.

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Fig. 7. Schematic diagram illustrating the potential molecular mechanisms of metformin-mediated radiosensitization. Metformin activates AMPK and thereby inactivates mTOR and its downstream p70S6K, resulting in inhibition of Wee1 and Rad51 protein synthesis. On the one hand, decreased Wee1 causes impaired phosphorylation of CDK1 (Y15), leading to activation of CDK1. Active CDK1, together with Cyclin B, is the key regulator of the G2/M cell-cycle transition. On the other hand, decreased Rad51 impairs HR repair. In addition, inhibition of Wee1 by siRNA moderately down-regulates Rad51.

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