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Chronic modulation of AMP-Kinase, Akt and mTOR pathways by ionizing radiation in human lung cancer xenografts

Chronic modulation of AMP-Kinase, Akt and mTOR pathways by ionizing radiation in human lung... Introduction: Earlier, we showed that in cancer cells, AMP-activated kinase (AMPK) participates in a signal cip1 transduction pathway involving ATM-AMPK-p53/p21 which is activated by ionizing radiation (IR) to mediate G2-M arrest and enhanced cytotoxicity. We also observed that AMPK modulates ATM expression and activity and the IR response of the Akt-mTOR pathway. Since the ATM, AMPK and Akt pathways are key targets of novel radio-sensitizing therapeutics, we examined the chronic modultion of expression and activity of those pathways by IR alone in xenograft models of lung cancer. Methods: Immuno-compromised mice were grafted with human lung A549 and H1299 cells, were treated with a single fraction of 0 or 10 Gy, and left to grow for 8 weeks. Extracted tumors were subjected to lysis and immunoblotting or fixation and immunohistochemical analysis. Results: IR inhibited significantly xenograft growth and was associated with increased expression of Ataxia Telengiectasia Mutated (ATM) and enhanced phosphorylation of two ATM targets, H2Ax and checkpoint kinase Chk2. Irradiated tumours showed increased total AMPK levels and phosphorylation of AMPK and its substrate Acetyl-CoA Carboxylase (ACC). IR led to enhanced expression and phosphorylation of p53 and cyclin dependent cip1 kip1 kinase inhibitors p21 and p27 . However, irradiated tumours had reduced phosphorylation of Akt, mTOR and it‘s target translation initiation inhibitor 4EBP1. Irradiated xenografts showed reduced microvessel density, reduced expression of CD31 but increased expression of hypoxia-induced factor 1A (HIF1a) compared to controls. Conclusion: IR inhibits epithelial cancer tumour growth and results in sustained expression and activation of cip1 kip1 ATM-Chk2, and AMPK-p53/p21 /p27 but partial inhibition of the Akt-mTOR signaling pathways. Future studies should examine causality between those events and explore whether further modulation of the AMPK and Akt- mTOR pathways by novel therapeutics can sensitize lung tumours to radiation. cip1 Keywords: Lung cancer, ATM, p53, 4-EBP1, p21 Introduction EGF receptor (EGFR), which modulates the activity In tumor cells ionizing radiation (IR) activates within of these molecules through a pathway involving phos- minutes the protein kinase B (Akt) and mammalian phatidylinositol 3-kinase (PI3k) and phosphoinositide- Target of Rapamycin (mTOR) pathway leading to radio- dependent kinase 1 (PDK1) [2]. Akt kinase acts as a resistance and tumor survival [1]. Akt and mTOR are main activator of mTOR, up regulation of which is established effectors of tyrosine kinase receptors such as known to occur by at least two different steps: i) phos- phorylation and inhibition of Tuberous Sclerosis Com- plex 2 (TSC2), that inactivates GTPase activity of the * Correspondence: theos.tsakiridis@jcc.hhsc.ca Translational Radiation Biology Laboratory, McMaster University, Hamilton, GTP-binding protein Rheb leading to mTOR activation Ontario, Canada 2 [3] and ii) stimulation of mTOR activity through phos- Juravinski Cancer Center, McMaster University, Hamilton, Ontario, Canada phorylation of PRAS40, a member of mTORC1, one of Full list of author information is available at the end of the article © 2012 Storozhuk et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Storozhuk et al. Radiation Oncology 2012, 7:71 Page 2 of 11 http://www.ro-journal.com/content/7/1/71 the two functional mTOR complexes, which also flank with 1x10 A549 or H1299 human lung adenocar- includes mLST8/Gbl and the scaffold protein Raptor [4]. cinoma cells. Once tumours reached 100 mm , animals To date, extensive published work demonstrated the im- were equally divided into non-irradiated (control: 0 Gy) pact of mTOR on cell growth, cancer cell proliferation or ionizing radiation (IR: 10 Gy) treated groups (n = 6 and resistance to cytotoxic agents [5] mTORC1 regu- per group). Tumour volume was measured every 3 days lates multiple growth and gene expression pathways with calliper according to the formula: V = Lenght*Width*- and specifically stimulates mRNA translation through Height*0.5236. Eight weeks after treatment, tumours were phosphorylation and activation of the ribosomal p70S6- extractedand snap-frozeninliquidnitrogenfor lysis, total s6k kinase (p70 ) and phosphorylation-induced inhibition protein extraction and immunoblotting or were formalin of the translation initiation inhibitor eIF4E binding pro- fixed and paraffin embedded for immunohistochemistry tein 1 (4EBP1) [5]. (IHC) analysis. Tumour lysates were prepared from frozen Recently, we showed that IR activates acutely the en- tumours that were sectioned, mechanically homogenized ergy sensor and tumor suppressor AMP-activated kinase in RIPA (Radio-Immunoprecipitation Assay) buffer and (AMPK) pathway, an evolutionally-preserved kinase that manually processed with Dounce homogenizer for total mediates a metabolic checkpoint on cell cycle when cells protein extraction. are under stress [6]. AMPK is an effector of Liver Kinase B 1 (LKB1), a tumour suppressor mutated in Peutz- Animal irradiation Jeghers syndrome, which is associated with benign and After appropriate dosimetry, conformal IR treatment malignant epithelial tumors [7]. AMPK is a heterotri- (10 Gy) was delivered to xenografts with a clinical radio- meric enzyme of α, β and γ subunits that senses low therapy unit while animals were anaesthetized and housed in energy levels through AMP binding on the γ subunit aPlexiglas tube equipped with High-Efficiency-Particulate- and is regulated by phosphorylation of the α subunit on Air-(HEPA) filters. Thr172 [8]. AMPK inhibits anabolic processes and pro- tein synthesis by inhibition of mTORC1 through differ- Immunoblotting ent mechanisms including, i) Ser1387 phosphorylation Immunoblotting was performed as described previously and activation of TSC2, leading to enhanced Rheb [6]. Antibodies for total AMPKα, P-AMPKα (Thr172), GTPase activity and mTOR inhibition and ii) by Raptor P-ACC(Ser79), ATM, γH2AX (Ser139), P-Chk2 (Thr68), phosphorylation [9]. In addition, AMPK mediates cell kip waf/cip P-p53 (Ser15), p27 , p21 , mTOR, P-mTOR cycle checkpoints through induction of p53 and the (Ser2448), Akt, P-Akt (S473), P-Akt (Thr308), P-4EBP1 cip1 cyclin-dependent kinase inhibitors (CDKI) p21 and (Thr37/46), CD31 and HIF1α were purchased from Cell kip1 p27 leading to cell cycle arrest [6,10]. Signalling Technology (Mississauga, Ontario, Canada). We have suggested that, apart from its metabolic Antibodies against p53 and β-actin were supplied by action, AMPK is activated by IR and may be a mediator Millipore (Etobicoke, Ontario, Canada). of DNA damage signals. We implicated AMPK in the mediation of IR-induced signal transduction through Immunohistochemistry an Ataxia Telengiectasia mutated (ATM)-AMPK-p53- cip1 Four μm thick tumour sections were mounted onto p21 pathway to facilitate G2/M cell cycle arrest and slides, deparaffinised, followed by antigen retrieval, mediate radiosensitization [6]. However, the effects of IR blocking with goat serum and incubated with primary on AMPK subunit expression and chronic regulation of antibody against P-AMPKα (Thr172) (1:200), anti-CD31 its activity have not been examined in human tumours. (1/500) dilution overnight and processed as described Furthermore, the levels of expression and activation of earlier [11]. the Akt and mTOR pathways have not been analyzed extensively in irradiated tumours long after treatment. Here, we analyzed in two different human non-small cell Statistical analysis lung cancer xenograft models the effects of a single frac- Quantitation and normalization of immunoblotting tion of IR on the long term expression and activation of results was pursued for all xenograft lysates and anti- the AMPK and the Akt-mTOR pathways, as well as their bodies (12 per tumour type and 6 per condition, Control upstream regulator ATM. vs irradiated). All density values of each immublotting band were first normalized against a value that for each Methods and materials blot was defined by the average density of the 6 control Animal treatments (untreated) lysates in each tumor type. Mean and SE Balb/c immune-compromised nude mice were obtained values were determined after this normalization. from Charles River (Mississauga, Ontario, Canada). At Paired t-test was performed to analyze the results from five weeks of age, animals were injected into the right immunoblotting experiments using SPSS software (SPSS, Storozhuk et al. Radiation Oncology 2012, 7:71 Page 3 of 11 http://www.ro-journal.com/content/7/1/71 Chicago, IL). Results are presented as Mean ± SEM. Stat- [25]. For that we examined here whether those effects istical significance was determined at p< 0.05 (*). of IR could be sustained in xenografts long after IR de- livery. The levels of total AMPKα, P-AMPK and P- Results Acetyl CoA Carboxylase (P-ACC), a substrate of AMPK Effects of IR on lung cancer xenograft growth indicating AMPK kinase activity, were examined in con- Within 15 days after IR treatment, xenografts began to trol and irradiated A549 and H1299 tumours. Basal show differences in growth kinetics that became statisti- levels of total AMPK α subunit increased in irradiated cally significant by day 25 (Figure 1). At the end of the xenografts along with activation of the enzyme marked 8 week period irradiated tumours were on average by phosphorylation on Thr172 residue (Figure 3A-C). 67 ± 3.4 % (A549) and 70 ± 4.2 % (H1299) smaller than P-ACC levels were also significantly higher in tumours their control (non-irradiated) counterparts. collected from irradiated xenografts compared to con- trol (Figure 3 A and B). Figure 3B shows the quantita- Effects of IR on the ATM expression and activity tion results of immunoblotting experiments of 6 We examined the effects of IR on the total protein levels xenografts per group. To examine whether increased and the activity of ATM. Eight weeks after IR treatment levels of P-AMPK (Thr172) signals are indeed attribu- A549 xenografts exhibited significantly increased levels ted to cancer cells, rather than to the surrounding of total ATM protein (Figure 2a). To evaluate the activity tumor microenvironment, we have performed immuno- of ATM we assessed the phosphorylation levels of two histochemistry analysis of xenografts using anti-P- established targets of this kinase, histone H2AX and the AMPK (Thr172) antibody (Figure 3C). In those experi- checkpoint kinase Chk2. In both A549 and H1299 xeno- ments we also observed significant increases in P- grafts we detected increased levels of phosphorylated AMPK in irradiated tumour cells compared to controls H2AX (γH2AX) in the irradiated tumours compared to that distributed both cytoplasm and nuclei of tumor untreated control tumours that were significantly higher cells of A549 origin but mainly in cytoplasm of H1299 in H1299 xenografts (Figure 2A-B). Similarly, irradiated tumour cells. A549 and H1299 xenografts showed increased Chk2 phosphorylation (P-Chk2). That was statistically signifi- cant in H1299 but not in A549 xenografts when all Regulation of steady state levels of p53 and CDKIs by IR tumours were analyzed (Figure 2). To examine the effects of IR treatment on cell cycle check- point regulators, lysates of control and IR-treated xenografts kip1 cip1 Chronic regulation of expression and activity of AMPK were probed with anti-p53, P-p53 (Ser15), p27 and p21 by IR antibodies. Figure 4A-C shows that a single fraction of IR kip1 cip1 In recent studies with tissue cultures of A549 cells, we induces a sustained significant increase, of p27 and p21 observed that within 24-48 h IR stimulates expression levels in irradiated A549 and H1299 tumours. We analyzed of AMPK subunits at both the mRNA and protein level total and phosphorylated (P-) p53 levels specifically in A549 700 CON A549 H1299 CON IR IR 500 900 0 0 0 5 10 15 20 25 30 35 40 45 50 55 60 Treatment Course (days) Treatment Course (days) Figure 1 Ionizing radiation (IR) suppresses A549 and H1299 lung cancer tumour growth in vivo. Twelve four week old male balb/c nude male mice were grafted with A549 or H1299 cells and left to 100 mm³ and were treated with a single fraction of 0 Gy (Control) or 10 Gy R. Tumour volume was monitored every 3 days for a period of 8 weeks. Representative graph of average tumour volume are shown. *p< 0.05 compared with control tumour volume. Average Tum our Volum e (m m ) Average Tumour Volume (mm ) Storozhuk et al. Radiation Oncology 2012, 7:71 Page 4 of 11 http://www.ro-journal.com/content/7/1/71 A549 H1299 ATM ATM H2AX(Ser139) H2AX(Ser139) P-Chk2 P-Chk2 -actin -actin Figure 2 Ionizing radiation (IR) induces sustained stimulation of the DNA damage response pathway.(A) Tumour tissue extracted from Control and IR–treated animals were subjected to immunoblotting analysis using ATM, P-Chk2 and γH2AX (Ser139) antibodies. Anti-actin was used as a loading control. Representative immunoblots from 6 independent experiments are shown. (B) Immunoblot densitometric values are shown as percent change in protein expression relative to the control group (*p< 0.05). tumours only as H1299 tumours lack p53 expression. Interest- (Figure 5). However, we observed that IR caused a sus- ingly, we detected highly significant increase in total and phos- tained reduction in the levels of P-AktS473 in both A549 phorylated (Ser15: 5.5-fold increase) p53 levels in irradiated and H1299 xenografts that reached significance in A549 tumours. but not in H1299 tumours. A trend for reduced P- AktT308 levels was also detected in irradiated tumours IR mediates a long term suppression of the Akt-mTOR of both types but that was not statistically significant pathway in either of them (30.0 + 6.4% and 55.0 + 10.9% vs We did not detect significant differences in the total 15.0 ± 4.3% and 42.0 ± 2.3% decrease for T308 and Akt levels between control and irradiated tumours S473 phosphorylation in A549 and H1299, respectively) Storozhuk et al. Radiation Oncology 2012, 7:71 Page 5 of 11 http://www.ro-journal.com/content/7/1/71 A549 H1299 A549 Control IR AMPK P AMPK (Thr172) H1299 Control IR PACC actin P-AMPK (Thr172) Figure 3 Ionizing Radiation (IR) upregulates AMPK expression and activity in A549 and H1299 lung xenografts in-vivo. (A) Control and IR-treated tumours were subjected to immunoblotting analysis using AMPKα, P-AMPK (Thr172), and P-ACC antibodies. Anti-actin was used as a loading control. A representative immunoblot of 6 independent experiments is shown. (B) Immunoblot densitometric values are shown as percent change in protein expression relative to the control group p (*p< 0.05; **p< 0.001). (C) A549 and H1299 tumours were fixed and immunohistochemistry analysis was performed using a specific P-AMPK antibody. (Figure 5B, D). Consistently, both IR-treated tumour xenografts showed significantly reduced levels CD31 and types showed reduced P-mTOR (Ser2448) levels without increased levels of HIF1α in comparison to untreated a significant change in total-mTOR levels. Irradiated tumours (Figure 6A, B). We performed immunohisto- xenografts of the two lung cancer types showed reduced chemistry experiments with the antibody against CD31 levels of phosphorylation of 4EBP1 (P-4EBP1) indicating to verify whether indeed the reduced expression of CD31 reduced mTOR activity (reduction by 81.0 ± 4.75% and levels corresponded to a reduced density of microvessels 47.0 ± 3.20% in A549 and H1299 xenografts, respectively) in irradiated tumours. All six tumours per group were (Figure 5A-B). analyzed. Figure 6C shows representative images from these experiments illustrating a significantly reduced Levels of microvasculature and hypoxia markers in density of microvessels in the irradiated A549 tumours. irradiated xenografts Since hypoxia is known to modulate tumour IR Discussion responses and ATM activity, we examined the levels of The Akt-mTOR pathway is an established mediator of the endothelial protein CD31, as a marker of microvas- radio-resistance and novel biological inhibitors of the culature density, and those of HIF1α, as marker of hyp- two kinases are shown to sensitize tumour cells to IR oxia, in control and irradiated xenografts from both lung [12,13]. On the other hand, AMPK is an emerging cancer A549 and H1299 xenografts. Figure 6A and B metabolic and genomic stress sensor that is also a illustrates representative immunoblots and quantitation promising target of novel cancer therapeutics such as of results from all xenografts. Both types of irradiated the anti-diabetic agent metformin. Metformin inhibits Control IR Storozhuk et al. Radiation Oncology 2012, 7:71 Page 6 of 11 http://www.ro-journal.com/content/7/1/71 A549 H1299 p53 P-p53 (Ser15) kip1 p27 kip1 p27 cip1 cip1 p21 p21 -actin -actin Figure 4 Ionizing radiation (IR) activates cell-cycle regulatory proteins in lung cancer tumour xenographts. (A) Tumour tissue kip waf/cip extracted from Control and IR–treated animals were subjected to immunoblotting analysis using p53, P-p53 (Ser15), p27 , and p21 antibodies. Anti-actin immunoblotting was used as a loading control. A representative immunoblot from 6 independent experiments is shown. (B) Immunoblot densitometric values are shown as percent change in protein expression relative to the control group (*p< 0.05; **p< 0.001). cancer cell proliferation and we have shown that it has mTOR and AMPK signaling pathways in tumours in order radio-sensitizing properties in lung cancer in-vitro [6] to understand better tumour radiation biology and as- These notions suggest a need to understand in depth the sist in a rational development of new effective radio- effects of IR on the expression and activity of the Akt- sensitizers. Here we analyzed the effects of a single IR Control Control IR Storozhuk et al. Radiation Oncology 2012, 7:71 Page 7 of 11 http://www.ro-journal.com/content/7/1/71 A549 H1299 Akt P-Akt(S473) P-Akt(Thr308) mTOR P-mTOR(Ser2448) P-4EBP1 -actin Figure 5 Ionizing Radiation (IR) inhibits Akt-mTOR pro-survival pathway in A549 and H1299 lung carcinoma xenografts.(A) Lysates from Control and IR treated tumours were subjected to immunoblot analysis using Akt, P-Akt (S473), P-Akt (Thr308), mTOR, P-mTOR (Ser2448) and P-4EBP1 antibodies. A representative immunoblot from 6 independent experiments is shown. (B) The immunoblot densitometric values are shown as percent change in protein expression relative to the control group (*p< 0.05; **p< 0.001). fraction of therapeutic IR (10 Gy) on the steady state levels K-Ras (G12S) oncogenic mutant and truncated LKB1-null of expression and activity of AMPK and Akt pathway but wild-type p53 vs H1299: p53-null, wild-type K-Ras and members. Tumours were extracted and analyzed 8 weeks LKB1) were used to examine whether detected chronic re- after radiation as this is a typical protocol in pre-clin- sponse of the AMPK-p53/CDKIs and Akt-mTOR pathways ical radio-sensitizer studies. Two different NSCLC to IR apply in lung cancer types with diverse oncogenic tumour models with distinct molecular defects (A549: genotypes. IR Control Storozhuk et al. Radiation Oncology 2012, 7:71 Page 8 of 11 http://www.ro-journal.com/content/7/1/71 A A549 H1299 A549 CD31 CD31 HIF1 HIF1 Control -actin actin IR Anti-CD31 IHC H1299 A549 Figure 6 Ionizing Radiation (IR) downregulates CD31 and enhances HIF1α levels in human lung cancer tumours. (A) Lysates from control and irradiated tumours were subjected to immunoblot analysis using CD31 and HIF1α antibodies. A representative immunoblot from 6 independent experiments is shown. (B) Average normalized densitometric values are shown as percent change in protein expression relative to the control group (*p< 0.05; **p< 0.001). (C) A549 tumours were fixed and immunohistochemistry analysis was performed using a specific anti-CD31 antibody. Treatment of human lung xenografts with a single The detection of a sustained enhancement of AMPKα fraction of IR (10 Gy) caused an expected significant protein levels and activity in tumours long after IR is a inhibition of tumour growth kinetics (Figure 1). Since novel finding in this study (Figure 3). Irradiated tumours our earlier studies suggested that AMPK is an effector had significantly higher levels of total and phosphory- of ATM [6] and other work pointed to direct modula- lated AMPK as well as P-ACC suggesting maintained tion of Akt activity by ATM [14] we explored the effect enhanced expression and activity of the enzyme. Since of IR on ATM expression and activity. Interestingly, we and others have shown that AMPK is a transducer we observed increased total ATM levels and increased of ATM signals [6,16] sustained activation of AMPK phosphorylation of two ATM targets, histone H2AX would be an expected finding in the presence of ATM and Chk2 (Figure 2). Both events are well described activation. However, our results also showed increased acute effects of IR. Enhanced levels of H2AX have AMPKα protein levels, suggesting that IR drives AMPKα also been described in human tumours 24 h after a gene expression. In recent studies with lung (A549) and clinical dose of radiotherapy of 2 Gy [15]. However, breast cancer cells (MCF7 and MB-231), we observed our results suggest a sustained increased activity of that within 24 and 48 hour IR enhances not only the ATM-γH2AX DNA damage response pathways long activity of AMPK but also the levels of mRNA and pro- after exposure to IR treatment which can be respon- tein of AMPKα, β and γ subunits [17] indicating that IR sible for the increased activity of the AMPK pathway regulates AMPK gene expression at both the transcrip- discussed below. tional and the translational level. Those results suggested Control IR Storozhuk et al. Radiation Oncology 2012, 7:71 Page 9 of 11 http://www.ro-journal.com/content/7/1/71 that IR stimulates significantly AMPK gene expression 4EBP1 phosphorylation that was more significant in within 24 – 48 h that is maintained long after the geno- A549 tumours (Figure 5). We have obtained similar toxic insult is delivered. The specific mechanism and results in PC3 prostate cancer xenografts (see Additional transcription factors involved in these events remain file 1: Figure S1) indicating that these are likely universal to be elucidated but studies suggest involvement of the responses of human epithelial tumours to IR that are in- p53-dependent stress-responsive genes Sestrin 1 and 2 dependent of K-Ras mutation status and LKB1 or p53 [18]. The regulation of AMPK gene expression and function. One could contribute the suppressed mTOR activity in response to IR is likely a universal pheno- activity in xenografts on the enhanced AMPK activity. menon in epithelial tumour cells. Similar to observations However, the mechanism of reduced phosphorylation in lung cancer xenografts, we have observed sustained of Akt remains unclear and needs to be elucidated by enhancement of total and phosphorylated AMPK α sub- future studies. Nevertheless, the concept of Akt inhib- unit levels in xenografts of PC3 prostate cancer cells ition in tumours by agents that activate the AMPK path- also, a cell line that lacks expression p53 (see Additional way has been described in earlier studies by our group file 1: Figure S1). Therefore, overall our results suggest and others [22,23]. It is possible that in irradiated that IR triggers acute and chronic expression of AMPK tumours conditions develop, long after delivery of IR, genes as well as activation of this enzyme that is likely that attenuate signal transduction between ATM and universal in epithelial cancer cells and is independent of Akt leading to suppression of Akt and mTOR activity p53. Currently, we analyze the exact role of sestrin genes despite enhanced ATM activation. In irradiated tumours in these processes. the combined effects of sustained increased expression cip1 kip1 Importantly, we observed that irradiated tumours of AMPK-p53-p21 /p27 pathway, that is shown to maintain significantly increased levels of total and phos- lead to inhibition of cell cycling, and inhibition of Akt- cip1 phorylated p53 and of CDK inhibitors p21 and mTOR-4EBP1 pathway, known to lead to gene tran- kip1 p27 (Figure 4). We also detected in irradiated scription and translation, may be capable of mediating tumours highly increased level of p53-Ser15 phosphoryl- an effective anti-proliferative action in those tumours, ation a post-translational modification believed to con- which may be adequate to mediate the cytotoxic action tribute to a greater stability of this protein [14]. These of IR [13]. Future studies should examine causality in results support the notion that IR activates the p53/ the relationship between these events. CDKI signaling pathways in tumours in a sustained fash- Our observation of sustained ATM activity in irra- ion probably through increased expression, phosphoryl- diated tumours is a significant finding of the present ation and stabilization of p53 and increased levels of study. Since ATM is suggested to be a common regula- kip1 cip1 cip1 cip1 kip1 CDKIs p27 and p21 (Figure 4). The p53-p21 tor of the activity of the AMPK-p53/p21 /p27 and pathway is an established target for ATM [19] and Akt-mTOR-4EBP1 pathways [6,14], future work should AMPK [6,8] both of which were suggested to phosphor- address the mechanism of this sustained activation of ylate p53. Earlier, we showed that induction of p53 and ATM in irradiated tissues. It is possible that ATM acti- cip1 p21 in response to IR is dependent on AMPK and vation is the result of sustained, IR-induced DNA dam- that AMPK activity is required for the mediation of IR- age or genomic instability that remains in tumours induced G2-M checkpoint and IR cytotoxicity [6]. long after irradiation. Other mechanisms of ATM acti- AMPK may indeed mediate the inhibitory effects of IR vation have been described, including hypoxia. Since IR on xenograft growth through regulation of p53 and is known to damage tumour vascular supply one could CDKIs. Similar to our earlier observation on the acute hypothesize that the sustained ATM activity of irra- cip1 response of p21 to IR in A549 and H1299 cell cul- diated tumours may be the result of hypoxia develop- tures [6], the induction of this CDKI in irradiated xeno- ing in these tissues rather than sustained DNA grafts does not appear to depend on p53 as it was damage. Conceivably, the reduced vascular supply and observed in p53-null H1299 xenografts also (Figure 4 A). CD31 expression we observed in irradiated xenografts IR is known to mediate a rapid activation of Akt [20] here would be responsible for local tumour hypoxia and recent studies showed that ATM can function as an and the enhanced expression of HIF1α we observed activating Akt kinase that phosphorylates rapidly Akt- (Figure 6). Interestingly, Cam et al. [24] showed that in S473 [21]. Despite that, and the detection of increased hypoxic conditions ATM mediates phosphorylation of ATM activity in radiated xenografts (Figure 2), we HIF1α leading to activation of this molecule and inhib- observed significantly reduced levels of Akt-S473 phos- ition of mTORC1. phorylation in both types of lung cancer xenografts and a trend for reduced AktT308 phosphorylation. Consist- Conclusions ently, mTOR phosphorylation was partially reduced and This study explored in tumours the long-term regulation so was the activity of this key enzyme indicated by lower by IR of two key tumour suppression or growth pathways Storozhuk et al. Radiation Oncology 2012, 7:71 Page 10 of 11 http://www.ro-journal.com/content/7/1/71 that are targets of promising therapeutics. Despite estab- Concession Street, Hamilton, Ontario, Canada, L8V 5C2. Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, lished acute activation of both the AMPK and Akt- Canada. Department of Medicine, McMaster University, Hamilton, Ontario, mTOR pathways by IR, irradiated tumours showed a sus- Canada. Department of Medical Physics and Applied Radiation Science, tained expression and activation of the AMPK-p53/ McMaster University, Hamilton, Ontario, Canada. cip1 kip1 p21 /p27 but inhibition of the activity of the Akt- Received: 11 January 2012 Accepted: 08 April 2012 mTOR-4EBP1 pathway. This was associated with increased Published: 18 May 2012 expression and sustained activity of the upstream regula- tor of the two pathways ATM that may be associated References with the development of hypoxia in irradiated tumours 1. Bussink J, van der Kogel AJ, Kaanders JH: Activation of the PI3-K/AKT pathway and implications for radioresistance mechanisms in head and or with potential genomic instability. These molecular neck cancer. Lancet Oncol 2008, 9:288–296. responses of irradiated tumours do not appear to be 2. 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Whelan T, Wright JR: Association of phosphorylated epidermal growth factor receptor with survival in patients with locally advanced non-small Competing interests cell lung cancer treated with radiotherapy. J Thorac Oncol 2008, The authors declare no competing interests. 3:716–722. 12. Chen H, Ma Z, Vanderwaal RP, Feng Z, Gonzalez-Suarez I, Wang S, Zhang J, Roti Roti JL, Gonzalo S: The mTOR inhibitor rapamycin suppresses DNA Authors’ contributions double-strand break repair. Radiat Res 2011, 175:214–224. YS pursued some of the animal handling and treatments, carried out the majority of the analysis of the xenograft tissue and helped draft the 13. Konstantinidou G, Bey EA, Rabellino A, Schuster K, Maira MS, Gazdar AF, manuscript. TS aided in the animal handling and treatment studies and Amici A, Boothman DA, Scaglioni PP: Dual phosphoinositide 3-kinase/ helped draft the manuscript. SNH pursued most of the animal grafting, care mammalian target of rapamycin blockade is an effective radiosensitizing and radiation treatment handling. CS helped deliver animal radiation. TF strategy for the treatment of non-small cell lung cancer harboring K-RAS performed the radiation dosimetry study and supervised animal radiation. J- mutations. Cancer Res 2009, 69:7644–7652. CC helped optimize the immunohistochemistry protocols and reviewed 14. Lavin MF, Gueven N: The complexity of p53 stabilization and activation. slides. GS, JW and GRS provided scientific support. TT conceived the study, Cell Death Differ 2006, 13:941–950. directed the study design, supervised all experimental work and prepared 15. Olive PL, Banath JP: Kinetics of H2AX phosphorylation after exposure to the manuscript. All authors have read and approved the final manuscript. cisplatin. Cytometry B Clin Cytom 2009, 76:79–90. 16. Fu X, Wan S, Lyu YL, Liu LF, Qi H: Etoposide induces ATM-dependent Acknowledgements mitochondrial biogenesis through AMPK activation. PLoS ONE 2008, This work was supported by grants from the RAZCER program of the 3:e2009. Canadian Association of Radiation Oncologists and the Prostate Cancer 17. Sanli T, Storozhuk Y, Linher-Melville K, Bristow RG, Laderout K, Viollet B, Canada Foundation to T.T. and the Canadian Institutes of Health Research to Wright J, Singh G, Tsakiridis T: Ionizing radiation regulates the expression G.S.. We greatly appreciate the help of Dr. Eric Seidlitz on animal handling of AMP-activated protein kinase (AMPK) in epithelial cancer cells: methods. We thank Dr. Robert Bristow, Princess Margaret Hospital, Toronto, modulation of cellular signals regulating cell cycle and survival. Radiother ON, for scientific advice. Oncol 2012, 102:459–465. 18. Budanov AV, Karin M: p53 target genes sestrin1 and sestrin2 connect Author details genotoxic stress and mTOR signaling. Cell 2008, 134:451–460. Translational Radiation Biology Laboratory, McMaster University, Hamilton, 19. Smith J, Tho LM, Xu N, Gillespie DA: The ATM-Chk2 and ATR-Chk1 Ontario, Canada. Juravinski Cancer Center, McMaster University, Hamilton, pathways in DNA damage signaling and cancer. Adv Cancer Res 2010, Ontario, Canada. Department of Oncology, McMaster University, 699 108:73–112. Storozhuk et al. Radiation Oncology 2012, 7:71 Page 11 of 11 http://www.ro-journal.com/content/7/1/71 20. Valerie K, Yacoub A, Hagan MP, Curiel DT, Fisher PB, Grant S, Dent P: Radiation-induced cell signaling: inside-out and outside-in. Mol Cancer Ther 2007, 6:789–801. 21. Li Y, Yang DQ: The ATM inhibitor KU-55933 suppresses cell proliferation and induces apoptosis by blocking Akt in cancer cells with overactivated Akt. Mol Cancer Ther 2010, 9:113–125. 22. King TD, Song L, Jope RS: AMP-activated protein kinase (AMPK) activating agents cause dephosphorylation of Akt and glycogen synthase kinase-3. Biochem Pharmacol 2006, 71:1637–1647. 23. Sanli T, Liu C, Rashid A, Hopmans S, Tsiani E, Schultz C, Farrell T, Singh G, Wright J, Tsakiridis T: Lovastatin sensitizes lung cancer cells to ionizing radiation. Modulation of growth and tumour suppressor signalling pathways and induction of apoptosis. J Thorac Oncol. 2011, 63:439-50 24. Cam H, Easton JB, High A, Houghton PJ: mTORC1 signaling under hypoxic conditions is controlled by ATM-dependent phosphorylation of HIF- 1alpha. Mol Cell 2010, 40:509–520. 25. Sanli T, Storozhuk Y, Linher-Melville K, Bristow RG, Laderout K, Viollet B, Wright J, Singh G, Tsakiridis T: Ionizing radiation regulates the expression of AMP-activated protein kinase (AMPK) in epithelial cancer cells: modulation of cellular signals regulating cell cycle and survival. Radiother Oncol. 2012, 102(3):459-65. doi:10.1186/1748-717X-7-71 Cite this article as: Storozhuk et al.: Chronic modulation of AMP-Kinase, Akt and mTOR pathways by ionizing radiation in human lung cancer xenografts. Radiation Oncology 2012 7:71. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Radiation Oncology Springer Journals

Chronic modulation of AMP-Kinase, Akt and mTOR pathways by ionizing radiation in human lung cancer xenografts

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Springer Journals
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Copyright © 2012 by Storozhuk et al.; licensee BioMed Central Ltd.
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Medicine & Public Health; Oncology; Radiotherapy
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1748-717X
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10.1186/1748-717X-7-71
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22607554
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Abstract

Introduction: Earlier, we showed that in cancer cells, AMP-activated kinase (AMPK) participates in a signal cip1 transduction pathway involving ATM-AMPK-p53/p21 which is activated by ionizing radiation (IR) to mediate G2-M arrest and enhanced cytotoxicity. We also observed that AMPK modulates ATM expression and activity and the IR response of the Akt-mTOR pathway. Since the ATM, AMPK and Akt pathways are key targets of novel radio-sensitizing therapeutics, we examined the chronic modultion of expression and activity of those pathways by IR alone in xenograft models of lung cancer. Methods: Immuno-compromised mice were grafted with human lung A549 and H1299 cells, were treated with a single fraction of 0 or 10 Gy, and left to grow for 8 weeks. Extracted tumors were subjected to lysis and immunoblotting or fixation and immunohistochemical analysis. Results: IR inhibited significantly xenograft growth and was associated with increased expression of Ataxia Telengiectasia Mutated (ATM) and enhanced phosphorylation of two ATM targets, H2Ax and checkpoint kinase Chk2. Irradiated tumours showed increased total AMPK levels and phosphorylation of AMPK and its substrate Acetyl-CoA Carboxylase (ACC). IR led to enhanced expression and phosphorylation of p53 and cyclin dependent cip1 kip1 kinase inhibitors p21 and p27 . However, irradiated tumours had reduced phosphorylation of Akt, mTOR and it‘s target translation initiation inhibitor 4EBP1. Irradiated xenografts showed reduced microvessel density, reduced expression of CD31 but increased expression of hypoxia-induced factor 1A (HIF1a) compared to controls. Conclusion: IR inhibits epithelial cancer tumour growth and results in sustained expression and activation of cip1 kip1 ATM-Chk2, and AMPK-p53/p21 /p27 but partial inhibition of the Akt-mTOR signaling pathways. Future studies should examine causality between those events and explore whether further modulation of the AMPK and Akt- mTOR pathways by novel therapeutics can sensitize lung tumours to radiation. cip1 Keywords: Lung cancer, ATM, p53, 4-EBP1, p21 Introduction EGF receptor (EGFR), which modulates the activity In tumor cells ionizing radiation (IR) activates within of these molecules through a pathway involving phos- minutes the protein kinase B (Akt) and mammalian phatidylinositol 3-kinase (PI3k) and phosphoinositide- Target of Rapamycin (mTOR) pathway leading to radio- dependent kinase 1 (PDK1) [2]. Akt kinase acts as a resistance and tumor survival [1]. Akt and mTOR are main activator of mTOR, up regulation of which is established effectors of tyrosine kinase receptors such as known to occur by at least two different steps: i) phos- phorylation and inhibition of Tuberous Sclerosis Com- plex 2 (TSC2), that inactivates GTPase activity of the * Correspondence: theos.tsakiridis@jcc.hhsc.ca Translational Radiation Biology Laboratory, McMaster University, Hamilton, GTP-binding protein Rheb leading to mTOR activation Ontario, Canada 2 [3] and ii) stimulation of mTOR activity through phos- Juravinski Cancer Center, McMaster University, Hamilton, Ontario, Canada phorylation of PRAS40, a member of mTORC1, one of Full list of author information is available at the end of the article © 2012 Storozhuk et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Storozhuk et al. Radiation Oncology 2012, 7:71 Page 2 of 11 http://www.ro-journal.com/content/7/1/71 the two functional mTOR complexes, which also flank with 1x10 A549 or H1299 human lung adenocar- includes mLST8/Gbl and the scaffold protein Raptor [4]. cinoma cells. Once tumours reached 100 mm , animals To date, extensive published work demonstrated the im- were equally divided into non-irradiated (control: 0 Gy) pact of mTOR on cell growth, cancer cell proliferation or ionizing radiation (IR: 10 Gy) treated groups (n = 6 and resistance to cytotoxic agents [5] mTORC1 regu- per group). Tumour volume was measured every 3 days lates multiple growth and gene expression pathways with calliper according to the formula: V = Lenght*Width*- and specifically stimulates mRNA translation through Height*0.5236. Eight weeks after treatment, tumours were phosphorylation and activation of the ribosomal p70S6- extractedand snap-frozeninliquidnitrogenfor lysis, total s6k kinase (p70 ) and phosphorylation-induced inhibition protein extraction and immunoblotting or were formalin of the translation initiation inhibitor eIF4E binding pro- fixed and paraffin embedded for immunohistochemistry tein 1 (4EBP1) [5]. (IHC) analysis. Tumour lysates were prepared from frozen Recently, we showed that IR activates acutely the en- tumours that were sectioned, mechanically homogenized ergy sensor and tumor suppressor AMP-activated kinase in RIPA (Radio-Immunoprecipitation Assay) buffer and (AMPK) pathway, an evolutionally-preserved kinase that manually processed with Dounce homogenizer for total mediates a metabolic checkpoint on cell cycle when cells protein extraction. are under stress [6]. AMPK is an effector of Liver Kinase B 1 (LKB1), a tumour suppressor mutated in Peutz- Animal irradiation Jeghers syndrome, which is associated with benign and After appropriate dosimetry, conformal IR treatment malignant epithelial tumors [7]. AMPK is a heterotri- (10 Gy) was delivered to xenografts with a clinical radio- meric enzyme of α, β and γ subunits that senses low therapy unit while animals were anaesthetized and housed in energy levels through AMP binding on the γ subunit aPlexiglas tube equipped with High-Efficiency-Particulate- and is regulated by phosphorylation of the α subunit on Air-(HEPA) filters. Thr172 [8]. AMPK inhibits anabolic processes and pro- tein synthesis by inhibition of mTORC1 through differ- Immunoblotting ent mechanisms including, i) Ser1387 phosphorylation Immunoblotting was performed as described previously and activation of TSC2, leading to enhanced Rheb [6]. Antibodies for total AMPKα, P-AMPKα (Thr172), GTPase activity and mTOR inhibition and ii) by Raptor P-ACC(Ser79), ATM, γH2AX (Ser139), P-Chk2 (Thr68), phosphorylation [9]. In addition, AMPK mediates cell kip waf/cip P-p53 (Ser15), p27 , p21 , mTOR, P-mTOR cycle checkpoints through induction of p53 and the (Ser2448), Akt, P-Akt (S473), P-Akt (Thr308), P-4EBP1 cip1 cyclin-dependent kinase inhibitors (CDKI) p21 and (Thr37/46), CD31 and HIF1α were purchased from Cell kip1 p27 leading to cell cycle arrest [6,10]. Signalling Technology (Mississauga, Ontario, Canada). We have suggested that, apart from its metabolic Antibodies against p53 and β-actin were supplied by action, AMPK is activated by IR and may be a mediator Millipore (Etobicoke, Ontario, Canada). of DNA damage signals. We implicated AMPK in the mediation of IR-induced signal transduction through Immunohistochemistry an Ataxia Telengiectasia mutated (ATM)-AMPK-p53- cip1 Four μm thick tumour sections were mounted onto p21 pathway to facilitate G2/M cell cycle arrest and slides, deparaffinised, followed by antigen retrieval, mediate radiosensitization [6]. However, the effects of IR blocking with goat serum and incubated with primary on AMPK subunit expression and chronic regulation of antibody against P-AMPKα (Thr172) (1:200), anti-CD31 its activity have not been examined in human tumours. (1/500) dilution overnight and processed as described Furthermore, the levels of expression and activation of earlier [11]. the Akt and mTOR pathways have not been analyzed extensively in irradiated tumours long after treatment. Here, we analyzed in two different human non-small cell Statistical analysis lung cancer xenograft models the effects of a single frac- Quantitation and normalization of immunoblotting tion of IR on the long term expression and activation of results was pursued for all xenograft lysates and anti- the AMPK and the Akt-mTOR pathways, as well as their bodies (12 per tumour type and 6 per condition, Control upstream regulator ATM. vs irradiated). All density values of each immublotting band were first normalized against a value that for each Methods and materials blot was defined by the average density of the 6 control Animal treatments (untreated) lysates in each tumor type. Mean and SE Balb/c immune-compromised nude mice were obtained values were determined after this normalization. from Charles River (Mississauga, Ontario, Canada). At Paired t-test was performed to analyze the results from five weeks of age, animals were injected into the right immunoblotting experiments using SPSS software (SPSS, Storozhuk et al. Radiation Oncology 2012, 7:71 Page 3 of 11 http://www.ro-journal.com/content/7/1/71 Chicago, IL). Results are presented as Mean ± SEM. Stat- [25]. For that we examined here whether those effects istical significance was determined at p< 0.05 (*). of IR could be sustained in xenografts long after IR de- livery. The levels of total AMPKα, P-AMPK and P- Results Acetyl CoA Carboxylase (P-ACC), a substrate of AMPK Effects of IR on lung cancer xenograft growth indicating AMPK kinase activity, were examined in con- Within 15 days after IR treatment, xenografts began to trol and irradiated A549 and H1299 tumours. Basal show differences in growth kinetics that became statisti- levels of total AMPK α subunit increased in irradiated cally significant by day 25 (Figure 1). At the end of the xenografts along with activation of the enzyme marked 8 week period irradiated tumours were on average by phosphorylation on Thr172 residue (Figure 3A-C). 67 ± 3.4 % (A549) and 70 ± 4.2 % (H1299) smaller than P-ACC levels were also significantly higher in tumours their control (non-irradiated) counterparts. collected from irradiated xenografts compared to con- trol (Figure 3 A and B). Figure 3B shows the quantita- Effects of IR on the ATM expression and activity tion results of immunoblotting experiments of 6 We examined the effects of IR on the total protein levels xenografts per group. To examine whether increased and the activity of ATM. Eight weeks after IR treatment levels of P-AMPK (Thr172) signals are indeed attribu- A549 xenografts exhibited significantly increased levels ted to cancer cells, rather than to the surrounding of total ATM protein (Figure 2a). To evaluate the activity tumor microenvironment, we have performed immuno- of ATM we assessed the phosphorylation levels of two histochemistry analysis of xenografts using anti-P- established targets of this kinase, histone H2AX and the AMPK (Thr172) antibody (Figure 3C). In those experi- checkpoint kinase Chk2. In both A549 and H1299 xeno- ments we also observed significant increases in P- grafts we detected increased levels of phosphorylated AMPK in irradiated tumour cells compared to controls H2AX (γH2AX) in the irradiated tumours compared to that distributed both cytoplasm and nuclei of tumor untreated control tumours that were significantly higher cells of A549 origin but mainly in cytoplasm of H1299 in H1299 xenografts (Figure 2A-B). Similarly, irradiated tumour cells. A549 and H1299 xenografts showed increased Chk2 phosphorylation (P-Chk2). That was statistically signifi- cant in H1299 but not in A549 xenografts when all Regulation of steady state levels of p53 and CDKIs by IR tumours were analyzed (Figure 2). To examine the effects of IR treatment on cell cycle check- point regulators, lysates of control and IR-treated xenografts kip1 cip1 Chronic regulation of expression and activity of AMPK were probed with anti-p53, P-p53 (Ser15), p27 and p21 by IR antibodies. Figure 4A-C shows that a single fraction of IR kip1 cip1 In recent studies with tissue cultures of A549 cells, we induces a sustained significant increase, of p27 and p21 observed that within 24-48 h IR stimulates expression levels in irradiated A549 and H1299 tumours. We analyzed of AMPK subunits at both the mRNA and protein level total and phosphorylated (P-) p53 levels specifically in A549 700 CON A549 H1299 CON IR IR 500 900 0 0 0 5 10 15 20 25 30 35 40 45 50 55 60 Treatment Course (days) Treatment Course (days) Figure 1 Ionizing radiation (IR) suppresses A549 and H1299 lung cancer tumour growth in vivo. Twelve four week old male balb/c nude male mice were grafted with A549 or H1299 cells and left to 100 mm³ and were treated with a single fraction of 0 Gy (Control) or 10 Gy R. Tumour volume was monitored every 3 days for a period of 8 weeks. Representative graph of average tumour volume are shown. *p< 0.05 compared with control tumour volume. Average Tum our Volum e (m m ) Average Tumour Volume (mm ) Storozhuk et al. Radiation Oncology 2012, 7:71 Page 4 of 11 http://www.ro-journal.com/content/7/1/71 A549 H1299 ATM ATM H2AX(Ser139) H2AX(Ser139) P-Chk2 P-Chk2 -actin -actin Figure 2 Ionizing radiation (IR) induces sustained stimulation of the DNA damage response pathway.(A) Tumour tissue extracted from Control and IR–treated animals were subjected to immunoblotting analysis using ATM, P-Chk2 and γH2AX (Ser139) antibodies. Anti-actin was used as a loading control. Representative immunoblots from 6 independent experiments are shown. (B) Immunoblot densitometric values are shown as percent change in protein expression relative to the control group (*p< 0.05). tumours only as H1299 tumours lack p53 expression. Interest- (Figure 5). However, we observed that IR caused a sus- ingly, we detected highly significant increase in total and phos- tained reduction in the levels of P-AktS473 in both A549 phorylated (Ser15: 5.5-fold increase) p53 levels in irradiated and H1299 xenografts that reached significance in A549 tumours. but not in H1299 tumours. A trend for reduced P- AktT308 levels was also detected in irradiated tumours IR mediates a long term suppression of the Akt-mTOR of both types but that was not statistically significant pathway in either of them (30.0 + 6.4% and 55.0 + 10.9% vs We did not detect significant differences in the total 15.0 ± 4.3% and 42.0 ± 2.3% decrease for T308 and Akt levels between control and irradiated tumours S473 phosphorylation in A549 and H1299, respectively) Storozhuk et al. Radiation Oncology 2012, 7:71 Page 5 of 11 http://www.ro-journal.com/content/7/1/71 A549 H1299 A549 Control IR AMPK P AMPK (Thr172) H1299 Control IR PACC actin P-AMPK (Thr172) Figure 3 Ionizing Radiation (IR) upregulates AMPK expression and activity in A549 and H1299 lung xenografts in-vivo. (A) Control and IR-treated tumours were subjected to immunoblotting analysis using AMPKα, P-AMPK (Thr172), and P-ACC antibodies. Anti-actin was used as a loading control. A representative immunoblot of 6 independent experiments is shown. (B) Immunoblot densitometric values are shown as percent change in protein expression relative to the control group p (*p< 0.05; **p< 0.001). (C) A549 and H1299 tumours were fixed and immunohistochemistry analysis was performed using a specific P-AMPK antibody. (Figure 5B, D). Consistently, both IR-treated tumour xenografts showed significantly reduced levels CD31 and types showed reduced P-mTOR (Ser2448) levels without increased levels of HIF1α in comparison to untreated a significant change in total-mTOR levels. Irradiated tumours (Figure 6A, B). We performed immunohisto- xenografts of the two lung cancer types showed reduced chemistry experiments with the antibody against CD31 levels of phosphorylation of 4EBP1 (P-4EBP1) indicating to verify whether indeed the reduced expression of CD31 reduced mTOR activity (reduction by 81.0 ± 4.75% and levels corresponded to a reduced density of microvessels 47.0 ± 3.20% in A549 and H1299 xenografts, respectively) in irradiated tumours. All six tumours per group were (Figure 5A-B). analyzed. Figure 6C shows representative images from these experiments illustrating a significantly reduced Levels of microvasculature and hypoxia markers in density of microvessels in the irradiated A549 tumours. irradiated xenografts Since hypoxia is known to modulate tumour IR Discussion responses and ATM activity, we examined the levels of The Akt-mTOR pathway is an established mediator of the endothelial protein CD31, as a marker of microvas- radio-resistance and novel biological inhibitors of the culature density, and those of HIF1α, as marker of hyp- two kinases are shown to sensitize tumour cells to IR oxia, in control and irradiated xenografts from both lung [12,13]. On the other hand, AMPK is an emerging cancer A549 and H1299 xenografts. Figure 6A and B metabolic and genomic stress sensor that is also a illustrates representative immunoblots and quantitation promising target of novel cancer therapeutics such as of results from all xenografts. Both types of irradiated the anti-diabetic agent metformin. Metformin inhibits Control IR Storozhuk et al. Radiation Oncology 2012, 7:71 Page 6 of 11 http://www.ro-journal.com/content/7/1/71 A549 H1299 p53 P-p53 (Ser15) kip1 p27 kip1 p27 cip1 cip1 p21 p21 -actin -actin Figure 4 Ionizing radiation (IR) activates cell-cycle regulatory proteins in lung cancer tumour xenographts. (A) Tumour tissue kip waf/cip extracted from Control and IR–treated animals were subjected to immunoblotting analysis using p53, P-p53 (Ser15), p27 , and p21 antibodies. Anti-actin immunoblotting was used as a loading control. A representative immunoblot from 6 independent experiments is shown. (B) Immunoblot densitometric values are shown as percent change in protein expression relative to the control group (*p< 0.05; **p< 0.001). cancer cell proliferation and we have shown that it has mTOR and AMPK signaling pathways in tumours in order radio-sensitizing properties in lung cancer in-vitro [6] to understand better tumour radiation biology and as- These notions suggest a need to understand in depth the sist in a rational development of new effective radio- effects of IR on the expression and activity of the Akt- sensitizers. Here we analyzed the effects of a single IR Control Control IR Storozhuk et al. Radiation Oncology 2012, 7:71 Page 7 of 11 http://www.ro-journal.com/content/7/1/71 A549 H1299 Akt P-Akt(S473) P-Akt(Thr308) mTOR P-mTOR(Ser2448) P-4EBP1 -actin Figure 5 Ionizing Radiation (IR) inhibits Akt-mTOR pro-survival pathway in A549 and H1299 lung carcinoma xenografts.(A) Lysates from Control and IR treated tumours were subjected to immunoblot analysis using Akt, P-Akt (S473), P-Akt (Thr308), mTOR, P-mTOR (Ser2448) and P-4EBP1 antibodies. A representative immunoblot from 6 independent experiments is shown. (B) The immunoblot densitometric values are shown as percent change in protein expression relative to the control group (*p< 0.05; **p< 0.001). fraction of therapeutic IR (10 Gy) on the steady state levels K-Ras (G12S) oncogenic mutant and truncated LKB1-null of expression and activity of AMPK and Akt pathway but wild-type p53 vs H1299: p53-null, wild-type K-Ras and members. Tumours were extracted and analyzed 8 weeks LKB1) were used to examine whether detected chronic re- after radiation as this is a typical protocol in pre-clin- sponse of the AMPK-p53/CDKIs and Akt-mTOR pathways ical radio-sensitizer studies. Two different NSCLC to IR apply in lung cancer types with diverse oncogenic tumour models with distinct molecular defects (A549: genotypes. IR Control Storozhuk et al. Radiation Oncology 2012, 7:71 Page 8 of 11 http://www.ro-journal.com/content/7/1/71 A A549 H1299 A549 CD31 CD31 HIF1 HIF1 Control -actin actin IR Anti-CD31 IHC H1299 A549 Figure 6 Ionizing Radiation (IR) downregulates CD31 and enhances HIF1α levels in human lung cancer tumours. (A) Lysates from control and irradiated tumours were subjected to immunoblot analysis using CD31 and HIF1α antibodies. A representative immunoblot from 6 independent experiments is shown. (B) Average normalized densitometric values are shown as percent change in protein expression relative to the control group (*p< 0.05; **p< 0.001). (C) A549 tumours were fixed and immunohistochemistry analysis was performed using a specific anti-CD31 antibody. Treatment of human lung xenografts with a single The detection of a sustained enhancement of AMPKα fraction of IR (10 Gy) caused an expected significant protein levels and activity in tumours long after IR is a inhibition of tumour growth kinetics (Figure 1). Since novel finding in this study (Figure 3). Irradiated tumours our earlier studies suggested that AMPK is an effector had significantly higher levels of total and phosphory- of ATM [6] and other work pointed to direct modula- lated AMPK as well as P-ACC suggesting maintained tion of Akt activity by ATM [14] we explored the effect enhanced expression and activity of the enzyme. Since of IR on ATM expression and activity. Interestingly, we and others have shown that AMPK is a transducer we observed increased total ATM levels and increased of ATM signals [6,16] sustained activation of AMPK phosphorylation of two ATM targets, histone H2AX would be an expected finding in the presence of ATM and Chk2 (Figure 2). Both events are well described activation. However, our results also showed increased acute effects of IR. Enhanced levels of H2AX have AMPKα protein levels, suggesting that IR drives AMPKα also been described in human tumours 24 h after a gene expression. In recent studies with lung (A549) and clinical dose of radiotherapy of 2 Gy [15]. However, breast cancer cells (MCF7 and MB-231), we observed our results suggest a sustained increased activity of that within 24 and 48 hour IR enhances not only the ATM-γH2AX DNA damage response pathways long activity of AMPK but also the levels of mRNA and pro- after exposure to IR treatment which can be respon- tein of AMPKα, β and γ subunits [17] indicating that IR sible for the increased activity of the AMPK pathway regulates AMPK gene expression at both the transcrip- discussed below. tional and the translational level. Those results suggested Control IR Storozhuk et al. Radiation Oncology 2012, 7:71 Page 9 of 11 http://www.ro-journal.com/content/7/1/71 that IR stimulates significantly AMPK gene expression 4EBP1 phosphorylation that was more significant in within 24 – 48 h that is maintained long after the geno- A549 tumours (Figure 5). We have obtained similar toxic insult is delivered. The specific mechanism and results in PC3 prostate cancer xenografts (see Additional transcription factors involved in these events remain file 1: Figure S1) indicating that these are likely universal to be elucidated but studies suggest involvement of the responses of human epithelial tumours to IR that are in- p53-dependent stress-responsive genes Sestrin 1 and 2 dependent of K-Ras mutation status and LKB1 or p53 [18]. The regulation of AMPK gene expression and function. One could contribute the suppressed mTOR activity in response to IR is likely a universal pheno- activity in xenografts on the enhanced AMPK activity. menon in epithelial tumour cells. Similar to observations However, the mechanism of reduced phosphorylation in lung cancer xenografts, we have observed sustained of Akt remains unclear and needs to be elucidated by enhancement of total and phosphorylated AMPK α sub- future studies. Nevertheless, the concept of Akt inhib- unit levels in xenografts of PC3 prostate cancer cells ition in tumours by agents that activate the AMPK path- also, a cell line that lacks expression p53 (see Additional way has been described in earlier studies by our group file 1: Figure S1). Therefore, overall our results suggest and others [22,23]. It is possible that in irradiated that IR triggers acute and chronic expression of AMPK tumours conditions develop, long after delivery of IR, genes as well as activation of this enzyme that is likely that attenuate signal transduction between ATM and universal in epithelial cancer cells and is independent of Akt leading to suppression of Akt and mTOR activity p53. Currently, we analyze the exact role of sestrin genes despite enhanced ATM activation. In irradiated tumours in these processes. the combined effects of sustained increased expression cip1 kip1 Importantly, we observed that irradiated tumours of AMPK-p53-p21 /p27 pathway, that is shown to maintain significantly increased levels of total and phos- lead to inhibition of cell cycling, and inhibition of Akt- cip1 phorylated p53 and of CDK inhibitors p21 and mTOR-4EBP1 pathway, known to lead to gene tran- kip1 p27 (Figure 4). We also detected in irradiated scription and translation, may be capable of mediating tumours highly increased level of p53-Ser15 phosphoryl- an effective anti-proliferative action in those tumours, ation a post-translational modification believed to con- which may be adequate to mediate the cytotoxic action tribute to a greater stability of this protein [14]. These of IR [13]. Future studies should examine causality in results support the notion that IR activates the p53/ the relationship between these events. CDKI signaling pathways in tumours in a sustained fash- Our observation of sustained ATM activity in irra- ion probably through increased expression, phosphoryl- diated tumours is a significant finding of the present ation and stabilization of p53 and increased levels of study. Since ATM is suggested to be a common regula- kip1 cip1 cip1 cip1 kip1 CDKIs p27 and p21 (Figure 4). The p53-p21 tor of the activity of the AMPK-p53/p21 /p27 and pathway is an established target for ATM [19] and Akt-mTOR-4EBP1 pathways [6,14], future work should AMPK [6,8] both of which were suggested to phosphor- address the mechanism of this sustained activation of ylate p53. Earlier, we showed that induction of p53 and ATM in irradiated tissues. It is possible that ATM acti- cip1 p21 in response to IR is dependent on AMPK and vation is the result of sustained, IR-induced DNA dam- that AMPK activity is required for the mediation of IR- age or genomic instability that remains in tumours induced G2-M checkpoint and IR cytotoxicity [6]. long after irradiation. Other mechanisms of ATM acti- AMPK may indeed mediate the inhibitory effects of IR vation have been described, including hypoxia. Since IR on xenograft growth through regulation of p53 and is known to damage tumour vascular supply one could CDKIs. Similar to our earlier observation on the acute hypothesize that the sustained ATM activity of irra- cip1 response of p21 to IR in A549 and H1299 cell cul- diated tumours may be the result of hypoxia develop- tures [6], the induction of this CDKI in irradiated xeno- ing in these tissues rather than sustained DNA grafts does not appear to depend on p53 as it was damage. Conceivably, the reduced vascular supply and observed in p53-null H1299 xenografts also (Figure 4 A). CD31 expression we observed in irradiated xenografts IR is known to mediate a rapid activation of Akt [20] here would be responsible for local tumour hypoxia and recent studies showed that ATM can function as an and the enhanced expression of HIF1α we observed activating Akt kinase that phosphorylates rapidly Akt- (Figure 6). Interestingly, Cam et al. [24] showed that in S473 [21]. Despite that, and the detection of increased hypoxic conditions ATM mediates phosphorylation of ATM activity in radiated xenografts (Figure 2), we HIF1α leading to activation of this molecule and inhib- observed significantly reduced levels of Akt-S473 phos- ition of mTORC1. phorylation in both types of lung cancer xenografts and a trend for reduced AktT308 phosphorylation. Consist- Conclusions ently, mTOR phosphorylation was partially reduced and This study explored in tumours the long-term regulation so was the activity of this key enzyme indicated by lower by IR of two key tumour suppression or growth pathways Storozhuk et al. Radiation Oncology 2012, 7:71 Page 10 of 11 http://www.ro-journal.com/content/7/1/71 that are targets of promising therapeutics. Despite estab- Concession Street, Hamilton, Ontario, Canada, L8V 5C2. Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, lished acute activation of both the AMPK and Akt- Canada. Department of Medicine, McMaster University, Hamilton, Ontario, mTOR pathways by IR, irradiated tumours showed a sus- Canada. Department of Medical Physics and Applied Radiation Science, tained expression and activation of the AMPK-p53/ McMaster University, Hamilton, Ontario, Canada. cip1 kip1 p21 /p27 but inhibition of the activity of the Akt- Received: 11 January 2012 Accepted: 08 April 2012 mTOR-4EBP1 pathway. This was associated with increased Published: 18 May 2012 expression and sustained activity of the upstream regula- tor of the two pathways ATM that may be associated References with the development of hypoxia in irradiated tumours 1. Bussink J, van der Kogel AJ, Kaanders JH: Activation of the PI3-K/AKT pathway and implications for radioresistance mechanisms in head and or with potential genomic instability. These molecular neck cancer. Lancet Oncol 2008, 9:288–296. responses of irradiated tumours do not appear to be 2. 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Whelan T, Wright JR: Association of phosphorylated epidermal growth factor receptor with survival in patients with locally advanced non-small Competing interests cell lung cancer treated with radiotherapy. J Thorac Oncol 2008, The authors declare no competing interests. 3:716–722. 12. Chen H, Ma Z, Vanderwaal RP, Feng Z, Gonzalez-Suarez I, Wang S, Zhang J, Roti Roti JL, Gonzalo S: The mTOR inhibitor rapamycin suppresses DNA Authors’ contributions double-strand break repair. Radiat Res 2011, 175:214–224. YS pursued some of the animal handling and treatments, carried out the majority of the analysis of the xenograft tissue and helped draft the 13. Konstantinidou G, Bey EA, Rabellino A, Schuster K, Maira MS, Gazdar AF, manuscript. TS aided in the animal handling and treatment studies and Amici A, Boothman DA, Scaglioni PP: Dual phosphoinositide 3-kinase/ helped draft the manuscript. 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Fu X, Wan S, Lyu YL, Liu LF, Qi H: Etoposide induces ATM-dependent Acknowledgements mitochondrial biogenesis through AMPK activation. PLoS ONE 2008, This work was supported by grants from the RAZCER program of the 3:e2009. Canadian Association of Radiation Oncologists and the Prostate Cancer 17. Sanli T, Storozhuk Y, Linher-Melville K, Bristow RG, Laderout K, Viollet B, Canada Foundation to T.T. and the Canadian Institutes of Health Research to Wright J, Singh G, Tsakiridis T: Ionizing radiation regulates the expression G.S.. We greatly appreciate the help of Dr. Eric Seidlitz on animal handling of AMP-activated protein kinase (AMPK) in epithelial cancer cells: methods. We thank Dr. Robert Bristow, Princess Margaret Hospital, Toronto, modulation of cellular signals regulating cell cycle and survival. Radiother ON, for scientific advice. Oncol 2012, 102:459–465. 18. Budanov AV, Karin M: p53 target genes sestrin1 and sestrin2 connect Author details genotoxic stress and mTOR signaling. Cell 2008, 134:451–460. Translational Radiation Biology Laboratory, McMaster University, Hamilton, 19. Smith J, Tho LM, Xu N, Gillespie DA: The ATM-Chk2 and ATR-Chk1 Ontario, Canada. Juravinski Cancer Center, McMaster University, Hamilton, pathways in DNA damage signaling and cancer. Adv Cancer Res 2010, Ontario, Canada. Department of Oncology, McMaster University, 699 108:73–112. Storozhuk et al. Radiation Oncology 2012, 7:71 Page 11 of 11 http://www.ro-journal.com/content/7/1/71 20. Valerie K, Yacoub A, Hagan MP, Curiel DT, Fisher PB, Grant S, Dent P: Radiation-induced cell signaling: inside-out and outside-in. Mol Cancer Ther 2007, 6:789–801. 21. Li Y, Yang DQ: The ATM inhibitor KU-55933 suppresses cell proliferation and induces apoptosis by blocking Akt in cancer cells with overactivated Akt. Mol Cancer Ther 2010, 9:113–125. 22. King TD, Song L, Jope RS: AMP-activated protein kinase (AMPK) activating agents cause dephosphorylation of Akt and glycogen synthase kinase-3. Biochem Pharmacol 2006, 71:1637–1647. 23. Sanli T, Liu C, Rashid A, Hopmans S, Tsiani E, Schultz C, Farrell T, Singh G, Wright J, Tsakiridis T: Lovastatin sensitizes lung cancer cells to ionizing radiation. Modulation of growth and tumour suppressor signalling pathways and induction of apoptosis. J Thorac Oncol. 2011, 63:439-50 24. Cam H, Easton JB, High A, Houghton PJ: mTORC1 signaling under hypoxic conditions is controlled by ATM-dependent phosphorylation of HIF- 1alpha. Mol Cell 2010, 40:509–520. 25. Sanli T, Storozhuk Y, Linher-Melville K, Bristow RG, Laderout K, Viollet B, Wright J, Singh G, Tsakiridis T: Ionizing radiation regulates the expression of AMP-activated protein kinase (AMPK) in epithelial cancer cells: modulation of cellular signals regulating cell cycle and survival. Radiother Oncol. 2012, 102(3):459-65. doi:10.1186/1748-717X-7-71 Cite this article as: Storozhuk et al.: Chronic modulation of AMP-Kinase, Akt and mTOR pathways by ionizing radiation in human lung cancer xenografts. Radiation Oncology 2012 7:71. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit

Journal

Radiation Oncology – Springer Journals

Published: May 18, 2012

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