Sanli, Toran MSc*†; Liu, Caiqiong MSc*; Rashid, Ayesha MSc*†; Hopmans, Sarah N. MSc‡; Tsiani, Evangelia PhD§; Schultz, Carrie BSc#; Farrell, Thomas PhD∥#; Singh, Gurmit PhD‡; Wright, James MD#†; Tsakiridis, Theodoros MD, PhD*†#
Radiotherapy is a widely used therapy in all stages of non-small cell lung cancer (NSCLC). However, NSCLC demonstrates intrinsic radioresistance that leads to failure of even high-dose thoracic radiation.1 Therefore, there is an urgent need for rational development of effective radiation sensitizers for NSCLC, which are able to inhibit molecular pathways mediating radiation resistance.
Ionizing radiation (IR) elicits signal transduction leading to cell survival, apoptosis, and cell cycle regulation.2 IR-induced DNA double-strand breaks are potentially lethal DNA damages leading to activation of phosphatidylinositol 3-kinase (PI3k)-like family protein kinases such as DNA-protein kinase and ataxia telangiectasia mutated (ATM).3 ATM mediates phosphorylation of p53 leading to stabilization of this tumor suppressor and cell cycle arrest at the G1-S or the G2-M check points through induction of the cip/kip family cyclin-dependent kinase inhibitor (CDKI) p21cip1.4 p27kip1, another cip/kip family CDKI, functions independently of the p53-p21cip1 pathway and inhibits cyclin E-cyclin-dependent kinase 2 complex and cycle progression through the G1-S checkpoint.4
Recently, we reported that the energy sensor AMP-activated kinase (AMPK), an established effector of the tumor suppressor LKB1, is activated by IR in a variety of epithelial cancer cells.5 IR activates AMPK in LKB1-independent but ATM-dependent manner leading to induction of p53 and p21cip1, cell cycle arrest at the G2-M checkpoint, and modulation of the sensitivity of cells to IR. IR is also shown to regulate mediators of the signaling pathway of epidermal growth factor receptor (EGFR),6,7 a well-established activator of cancer cell proliferation. IR activates the downstream effector pathways of EGFR such as the PI3k—Akt—mammalian target of rapamycin (mTOR) and the Raf—mitogen-activated protein kinase-kinase (Mek1)—mitogen-activated protein kinase p42/44 (also known as extracellular signal-regulated kinase [Erk1/2]) pathways. These are known to mediate cell survival and radiation resistance, gene expression, and protein synthesis.8,9
Small GTP-binding proteins of the Ras family such as Ras, Rac, and Rho (A/B) mediate signal transduction downstream of EGFR to activate the PI3k-Akt-mTOR and the Raf-Mek-Erk1/2 pathways. Ras mutations are frequent in lung cancer, and they occur in both the H- and K-Ras isoforms and were shown to induce radiation resistance in vitro.10,11 For that, extensive work is focused on targeting Ras family members with inhibitors of prenylation, a posttranslational modification required for membrane targeting and function of Ras.12
Members of the statin family of 3-hydroxy-3-methylgutaryl-CoA (HMG-CoA) reductase inhibitors are widely used anticholesterol agents that inhibit the conversion of HMG-CoA to mevalonate, a rate-limiting step of the mevalonate—cholesterol biosynthesis pathway.13,14 This pathway is also vital for the production of farnesyl and geranylgeranyl moieties required for the posttranslational modification and function of Ras and Rho, respectively.13 For this reason, statins have been studied extensively as antitumor agents.
During the past 20 years, a large amount of studies have demonstrated the antiproliferative and proapoptotic effects of statins both in vitro and in animal models of cancer. Growth inhibition, cell cycle arrest, and induction of apoptosis in cancer cells have been demonstrated convincingly.15 The interest in these drugs was enhanced by epidemiological studies indicating that patients on statins may have lower risk for development of colorectal carcinoma16 and lung cancer.17 The Veterans Affairs Health Care System study17 showed that use of statins for more than 6 months could offer a 55% risk reduction on the incidence of lung cancer, indicating that these agents may have significant chemoprevention action. Further, in prostate cancer, statin use is suggested to decrease the risk for advanced and metastatic cancer in epidemiological studies,18 to slow disease progression after radical prostatectomy,19 and, importantly, to reduce disease recurrence in patients treated with curative radiotherapy.20,21
Lovastatin is probably the most widely studied statin in cancer. It has been shown to possess anticancer properties in vitro and in vivo.14 The antiproliferative action of lovastatin has been demonstrated in lung cancer cells,22 but its role as a potential IR sensitizer or adjunct to radiation has not been examined in lung cancer models. In this study, we examined the effects of lovastatin on clonogenic survival of lung cancer cells treated with or without IR and explored the effects of this drug on cell cycle, apoptosis, and signaling pathways involved in IR resistance.
Roswell Park Memorial Institute (RPMI) media, fetal bovine serum, trypsin, and antibiotic were purchased from Invitrogen (Burlington, ON). Antibodies against phospho-EGFR, phospho-Akt, phospho-Erk, p53, phospho-AMPK α-subunit, p21cip1, p27kip1, cleaved caspase 3, actin, and horseradish peroxidase-conjugated anti-rabbit secondary antibody were purchased from Cell Signal Technology (Mississauga, ON, Canada). Polyvinylidene difluoride membrane was purchased from Pall Corporation (Port Washington, NY). Lovastatin, mevalonate, and Hoechst 33258 were purchased from Sigma (Toronto, ON). A549 cells were from the American Type Culture Collection (Manassa, VA).
Cell Culture and Treatments
A549 cells were grown in Roswell Park Memorial Institute (RPMI) media containing 5 mM glucose, 10% (vol/vol) fetal bovine serum, and 1% (vol/vol) antibiotic-antimycotic at 37°C as described previously.23 Cells were treated with the indicated concentrations of lovastatin 24 hours before radiation.
A549 cells were subjected to clonogenic assays as described earlier.5 Briefly, 500 or 1000 cells were seeded into individual wells of a 6-well plate in triplicate and maintained at the indicated doses of lovastatin before radiation (2–8 Gy). After 7 days, cells were fixed and stained with methylene blue, and viable colonies (>50 cells) were counted. Results are expressed as cell survival fraction compared with untreated control. To evaluate radiation sensitization by lovastatin, data were fitted to the linear quadratic equation using Graphpad Prism version 5 software (La Jolla, CA) as described previously.24
Approximately 2500 cells were seeded into a 96-well plate and treated with the indicated concentrations of lovastatin before being exposed to 0, 2, or 8 Gy IR. Ninety-six hours later, the cells were washed with phosphate-buffered saline, distilled H2O was added to each well, and the plates were stored at −80°C until completely frozen. The plates were then thawed and stained with Hoechst working solution (20 μg/mL Hoechst 33258 in a Tris-Borate-EDTA buffer), and fluorescence was determined using the Cyto-Fluor Plate Reader (Applied Biosystems, Toronto, ON, Canada).
Twenty micrograms of protein was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane as described earlier.23 The primary antibody was detected with horseradish peroxidase-conjugated anti-rabbit secondary antibody and enhanced chemiluminescent detection reagent.
Cell Cycle Analysis
The propidium iodine method was used as described previously.5 Cells were treated with lovastatin (10 μM) before treatment with 0 or 8 Gy of IR and incubated for the indicated times and were then subjected to flow cytometric cell cycle analysis using a FACScan flow cytometer (Beckton Dickinson, Mississauga, Canada).
Cells grown on glass coverslips for 24 hours were treated with lovastatin (10 μM) for the indicated times. Then the cells were stained with Hoechst 33258, and images were obtained as described previously.5 Quantitation of apoptotic cells (showing nuclear fragmentation) was performed by counting the average proportion of apoptotic cells in four high-power fields on each slide (100 cells counted in each quadrant of each slide). Values were normalized to the untreated control.
Statistical analyses was performed with unpaired T-test, using SPSS version 16.0 software (Somers, NY) and are presented as mean ± SEM of at least three separate experiments.
Lovastatin Inhibits NSCLC Cell Survival and Enhances the Cytotoxicity of IR
We initiated our studies with clonogenic survival assays. Lovastatin alone caused a dose-dependent inhibition of clonogenic survival in A549 cells (Figure 1A). The drug began inhibiting clonogenic survival at a dose of 5 μM (10% reduction in survival), inhibited the majority of clonogenic survival at 25 μM (95% reduction in survival), and completely abolished survival at 50 μM (Figure 1A).
Lovastatin sensitized A549 cells to IR (Figure 1B). Clonogenic assay values were fitted into a linear quadratic model. Both 5 and 10 μM of the drug showed significant radiosensitization of A549 cells to 2 to 8 Gy of IR. Almost complete inhibition of clonogenic survival was achieved with 10 μM lovastatin in combination with 8 Gy IR. In addition, we evaluated proliferation through DNA synthesis analysis using the Hoescht DNA staining method. Five micromolar of lovastatin inhibited basal cell proliferation (by 33%; compared with 0 Gy control) without affecting significantly the proliferation levels after IR (Figure 1C). However, at 10 μM, the drug inhibited dramatically cell proliferation in both control cells and those radiated with 2 or 8 Gy (by 63% and 90%, respectively).
Mevalonate Prevents Lovastatin-Induced Inhibition of Clonogenic Survival
We used mevalonate to examine the specificity of lovastatin for the mevalonate-cholesterol synthesis pathway (Figure 1D). Two gray of IR decreased clonogenic survival by 44% compared with control. Lovastatin alone (15 μM) inhibited survival by 75% and by 92% when combined with 2 Gy of IR. In these experiments, the higher concentration of 15 μM lovastatin was used to examine whether mevalonate is capable of reversing the effects of even high lovastatin doses. Mevalonate (100 μM) inhibited the lovastatin-induced decrease in cell survival in both nonradiated and radiated cells, suggesting that lovastatin mediates its cytotoxic action solely through inhibition of the mevalonate synthesis pathway.
Lovastatin Inhibits EGF-Stimulated Activation of EGFR and Akt
To analyze the mechanism of action of lovastatin, we examined first its effects on EGF-induced EGFR and downstream effector phosphorylation. EGF induced phosphorylation of EGFR and the Akt and Erk1/2 kinases (Figure 2). However, lovastatin inhibited EGFR and Akt phosphorylation, in a dose-dependent fashion, without affecting phosphorylation of Erk1/2 (Figure 2).
Modulation of IR-Stimulated Activation of Akt and AMPK
Control and lovastatin-treated cells were subjected to increasing doses of IR and were analyzed by immunoblotting. IR induced a consistent Akt phosphorylation, even with the lower dose of 2 Gy, but Erk phosphorylation was seen only after 4 to 6 Gy (Figure 3A–C). Interestingly, lovastatin abolished the IR activation of Akt but did not affect significantly Erk1/2 phosphorylation by IR (Figure 3A–C). The inhibition of IR-induced Akt phosphorylation by lovastatin was completely reversed by addition of mevalonate (Figure 3D–E), consistent with clonogenic survival results (Figure 1D). IR also caused a dose-dependent phosphorylation of AMPK that was accompanied by activation of this kinase shown by the enhanced phosphorylation of its established substrate acetyl CoA carboxylase, as observed earlier5 (Figure 4A–C). Interestingly, lovastatin enhanced significantly both basal and radiation-induced AMPK phosphorylation and activity.
Modulation of Cell Cycle Regulators and the Cell Cycle by Lovastatin
The Akt and AMPK pathways regulate cell cycle through modulation of p53 and CDKIs p27kip1 and p21cip1.25 Therefore, we examined whether lovastatin modulates the levels of these cell cycle inhibitors in control and IR-treated cells. We observed a significant increase in the expression of p53, p27kip1, and p21cip1 in response to IR (Figure 5A). However, lovastatin caused an early inhibition of the IR-induced expression of p53, p27kip1, and p21cip1, within 24 hours, and for that, we examined the levels of these three cell cycle regulators up to 96 hours after initiation of treatments. IR maintained enhanced p53, p27kip1, and p21cip1 levels up to 96 hours later (Figure 5A), but lovastatin inhibited this IR-induced expression, which was almost completely eliminated at 96 hours.
Effects on Cell Cycle Phase Distribution
Lovastatin alone did not affect significantly the distribution of cells in the phases of the cell cycle in the first 24 hours (Figure 5B). However, lovastatin caused a progressive significant shift of cells into G0/G1 phase, after 24 hours, compared with control, and eventually a marked induction of apoptosis by 72 to 96 hours (24 hours: 2%; 96 hours: 89%). IR alone caused a significant arrest of cells in G2/M phase at 48 hours (control: 0%; IR: 31%). However, lovastatin attenuated IR-induced G2/M arrest and shifted cells into G0/G1 phase and apoptosis (IR G2-M: 31%; Lovastatin + IR G2-M: 12%; Figure 5C).
Apoptosis Events Induced by Lovastatin
Finally, we examined the effects of lovastatin and IR on molecular and morphological markers of apoptosis. Cleaved caspase 3 levels, an established marker of apoptosis,26 was analyzed by immunoblotting. Lovastatin alone caused a significant increase in cleaved caspase 3 levels and further potentiated IR-induced expression of this protein at 12 hours (Figure 7A). IR induction of cleaved caspase 3 dissipated after 12 hours, but lovastatin enhanced cleaved caspase 3 levels for up to 48 hours later and decreased thereafter. We analyzed apoptotic events also with morphological analysis of cells treated with lovastatin for 0 to 72 hours. Consistent with induction of cleaved caspase 3 (Figure 6A) and the cell cycle results (Figure 5), lovastatin caused a time-dependent nuclear fragmentation and induction of apoptotic bodies (Figure 6B, C).
Lovastatin was shown to sensitize human cervix cancer cells to IR.27 Recently, another statin, simvastatin, was shown to inhibit small cell lung cancer growth in vitro and in vivo,28 and Bellini et al.29 showed that simvastatin inhibits the proliferation of A549 lung cancer cells. However, the potential benefit of combining statins with therapeutic doses of IR has not been examined in lung cancer models. To our knowledge, this is the first study to demonstrate that lovastatin acts as a radiation sensitizer in NSCLC cells.
Lovastatin Regulation of Clonogenic Survival in Control and Radiated Cells
We observed that lovastatin sensitized A549 lung cancer cells to therapeutic doses of IR of 2 to 8 Gy (Figure 1). This was mediated specifically through inhibition of the mevalonate pathway, as exogenous mevalonate completely reversed the decrease in lung cancer cell survival observed by lovastatin (Figure 1D). Fritz et al.27 examined the sensitivity of a number of cancer cell lines to lovastatin, but only a few of them showed sensitivity to lovastatin at high doses. HeLa cells required 20 to 50 μM of lovastatin to demonstrate radiosensitization.27 In this study, lung adenocarcinoma A549 cells showed higher sensitivity to the drug (at 5 and 10 μM), indicating that survival pathways in those cells may be more dependent on protein prenylation events.
Interestingly, in recent experiments investigating the effects of lovastatin in SK-MES lung cancer cells, a cell line of squamous cell carcinoma origin, we have observed an even greater sensitivity to the drug. These results are shown in Figure s1 (Supplemental Digital Content) and indicate a 20 to 50 times greater sensitivity of SK-MES cells to lovastatin compared with adenocarcinoma A549 cells. We are currently investigating in depth the molecular etiology of this higher sensitivity of SK-MES cells and its implications. However, overall, our results demonstrate that lung cancer cells show significant radiosensitization in response to lovastatin that should be explored further in preclinical in vivo and in clinical studies.
As other statins, beyond lovastatin, have shown antiproliferative effects in cancer cells,15 one wonders whether lung cancer cell radiosensitization is a phenomenon unique to lovastatin. For that, we began to explore the effects of other statins in A549 cells. In preliminary studies, we observed that simvastatin is also able to inhibit proliferation of A549 cells (as shown earlier29) and to sensitize lung cancer cells to IR. This indicates that radiosensitization is likely a common effect for this class of agents (Figure s2, Supplemental Digital Content).
Effects on EGFR and Effector Kinases
Adenocarcinoma A549 cells have a genetic profile that offers a survival advantage including a K-Ras (Gly12-Ser) mutation.30 K-Ras activates the PI3k-Akt pathway31 and that is required for NSCLC tumorigenesis in K-Ras mutant mice.32 Because it inhibits posttranslational modification of Ras GTP-binding proteins, lovastatin is expected to abrogate oncogenic K-Ras and EGFR signaling. In this study, we observed that lovastatin selectively abrogated EGF-stimulated phosphorylation of EGFR and Akt but not Erk1/2. This discrepancy was observed also by Mantha et al.33 in SCC9 head and neck tumor cells and suggests that (1) persistent EGFR phosphorylation may not be required for Erk1/2 activation and (2) activation of Erk1/2 alone is not adequate to confer radiation resistance. Our observations suggest that in lung cancer cells, lovastatin is able to inhibit selectively the key prosurvival pathway of Akt. This alone could account for the antiproliferative and proapoptotic effects of the drug.
Lovastatin Regulation of IR-Activated Signals
Effects on Akt
Similar to EGF-induced signals, lovastatin attenuated IR-activation of Akt, in a mevalonate-dependent fashion but did not affect IR-activation of Erk1/2 (Figure 3A–C). Similarly, Mistafa and Stenius34 found that statins primarily target the Akt pathway to sensitize pancreatic cancer cells to chemotherapeutic drugs, without effecting Erk. Studies in K-Ras mutant cells, including A549, have shown that in these cells, activation of the EGFR-PI3k-Akt pathway confers radioresistance35 and that inhibition of this axis by EGFR inhibitors sensitizes cells to IR.36 Further, Akt is an established mediator of radiation resistance in many cancer cells.37 The effect of lovastatin to inhibit IR activation of Akt illustrates a key property of this drug that luckily mediates its radiosensitization action.
Effects on AMPK
A549 cells also carry a point mutation of the LKB1 gene (codon 37 [Q-Ter]) that generates a truncated LKB1 product.38 Therefore, these cells lack LKB1-regulated AMPK activation, an event that is shown to lead to aberrant activation of the Akt—mTOR pathway activating protein synthesis and survival.39 In this study, in agreement with earlier studies with statins,40 we observed that lovastatin alone activated AMPK. However, we observed that it also potentiated its activation by IR (Figure 4C). Recently, we observed that IR activates AMPK in LKB1 null A549 cells.5 Our observations in the same cells here suggest that lovastatin also activates AMPK in an LKB1-independent fashion. These observations are significant because AMPK is (1) shown to dephosphorylate and inhibit Akt through increased protein phosphatase 2A activity41 and (2) to inhibit the mTOR pathway by directly phosphorylating either its upstream regulator tuberous sclerosis 2 or its binding partner Raptor.39
Importantly, AMPK activation also mimics statin action because this kinase is known to inhibit HMG-CoA reductase.42 Therefore, AMPK activation by stimuli such as IR can work synergistically with lovastatin to augment the effects of inhibition of the mevalonate pathway. Taking these notions together with the discussion earlier, lovastatin seems to be a highly attractive agent with dual potential to enhance the activity of AMPK and inhibit the Akt pathway through a number of potential molecular steps.
Modulation of Cell Cycle
Cell Cycle Regulators
IR regulates cell cycle through the induction of p53 and CDKIs, p21cip1 and p27kip1, expression to mediate mainly an arrest at the G2-M checkpoint.43 We hypothesized that lovastatin's antiproliferative effects may involve arrest of the cell cycle through enhanced expression of p53 and CDKIs. Although we did observe a potent induction of p53, p21cip1, and p27kip1 expression by IR alone, lovastatin inhibited IR induction of p53 and CDKI expression (Figure 5A). This may be due to either (1) effects of the drug on global gene transcription and translation or (2) a dependence of p53 and CDKI expression on specific events inhibited by lovastatin. Statins were shown to inhibit mTOR-dependent phosphorylation or deactivation of the translational repressor eukaryotic initiation factor 4E-binding protein, leading to suppression of initiation of cap-dependent mRNA translation.44 It should be stressed that we did not detect in our study any significant effects on the levels of any other proteins, including signaling molecules or actin, suggesting that a global effect on gene expression is unlikely. Conversely, Akt activity, which is inhibited by lovastatin, is required for the DNA damage–induced stabilization of p53,45 and this mechanism may be active in lovastatin-treated cells. A decrease in p21cip1 levels with statin treatment was observed by other investigators in A549 cells.22 Consistent with observations in HeLa cells,27 our work suggests that the mechanism of radiosensitization of A549 cells is independent of p53 and the CDKIs p21cip1 and p27kip1.
Lovastatin was shown to inhibit cell cycle progression at G0/G1 phase and promote apoptosis in thyroid cancer,46 breast cancer,47 glioblastoma,48 cervical cancer cells,27 and squamous cell carcinomas.49 In this study, we observed that lovastatin treatment shifted cells into G0/G1 phase with a markedly increased proportion of cells moving into apoptosis after 48 hours of treatment (Figure 5B). Prolonged treatment with lovastatin (96 hours) induced marked induction of apoptosis in nonradiated cells and caused a reversal of the G2-M checkpoint arrest induced by IR and a G0/G1 and apoptotic distribution (Figure 5B, C). It is possible that inhibition of the IR-induced G2-M arrest by lovastatin induced radiosensitization through prevention of DNA repair and induction of genomic instability.
Induction of Apoptosis
Consistent with the cell cycle analysis results, we observed that lovastatin alone induced cleaved caspase 3, a significant contributor to protein degradation. Although IR caused a reversible induction of this marker that was not detectable after 24 hours, lovastatin enhanced and prolonged the IR-induced cleaved caspase 3 formation for up to 72 hours (Figure 6A). Furthermore, morphological analysis verified a progressive formation of apoptotic bodies with continued incubation with lovastatin (Figure 6B, C). Overall, our results are consistent with other studies,22,50 suggesting apoptosis as major mechanism of the cytotoxic action of lovastatin and suggests that this is also a predominant mode of action of the drug when combined with radiation in lung cancer cells.
Potential for Clinical Development in Lung Cancer in Combination with Radiotherapy
A number of clinical studies explored the potential of lovastatin to achieve tumoricidal doses in human patients. Typical doses of lovastatin aiming to control cholesterol levels in humans are approximately 1 mg/kg/d and are shown to yield serum concentrations in the range of 0.15 to 0.3 μM.51 Early phase dose-escalation studies have explored a number of regiments, and in a study of 7 consecutive days treatment, in 4-week cycles, doses up to 25 mg/kg/d were tolerated without severe myopathy.51 Ubiquinone is used to address myopathy. Under these conditions, maximum tolerated doses were not reached, and systemic drug concentrations reached 0.1 to 3.92 μM.51 In a study with end-stage head and neck and cervix cancers patients,52 a regiment of 7.5 mg/kg/d for 21 consecutive days in 4-week cycles was defined as maximum tolerated doses in patients with good renal function. Although no objective responses were seen in this study, where lovastatin was used as a single agent, the authors still reported a 23% rate of stable disease at 3 months,52 which is indeed encouraging in patients with end-stage disease.
The aforementioned studies suggest that it is possible to achieve safely tumoricidal and radiosensitizing doses of lovastatin in cancer patients. Our work indicates that some lung cancer tumors may exhibit sensitivity to lovastatin even in the high nanomolar range (Figure s1, Supplemental Digital Content and discussion earlier) making it even more plausible that lovastatin will sensitize tumors to IR in human patients. Overall, these data indicate that this drug deserves further investigation with in vivo preclinical and clinical studies. Although, other statins may also be able to radiosensitize lung tumor cells (Figure s2, Supplemental Digital Content and discussion above), lovastatin remains the best studied agent in this class, in both the preclinical and the clinical setting and, therefore, is the most favorable candidate for further development.
Figure 7 illustrates our model of the action of lovastatin in lung cancer cells. Our work suggests that lovastatin is a promising agent with significant antitumor properties as a single agent and a radiation sensitizer. Lovastatin seems to function mainly through induction of apoptosis. This effect may be mediated by a unique simultaneous inhibition of the prosurvival Akt and activation of the tumor suppressor AMPK pathways. This work presents compelling evidence that support further investigation of lovastatin as a radiation sensitizer in vivo. Work in animal models of lung cancer will expedite the development of this drug to the clinical setting in early phase studies in combination with radiotherapy.
Supported by funds from the Juravinski Cancer Center Foundation. We appreciate the support by Dr. William K. Evans (CEO, Juravinski Cancer Center), in the pursuit of this study.
1. Schuurbiers OC, Kaanders JH, van der Heijden HF, et al. The PI3-K/AKT-pathway and radiation resistance mechanisms in non-small cell lung cancer. J Thorac Oncol
2. Bernhard EJ, Maity A, Muschel RJ, et al. Effects of ionizing radiation on cell cycle progression. A review. Radiat Environ Biophys
3. Hartlerode AJ, Scully R. Mechanisms of double-strand break repair in somatic mammalian cells. Biochem J
4. Vidal A, Koff A. Cell-cycle inhibitors: three families united by a common cause. Gene
5. Sanli T, Rashid A, Liu C, et al. Ionizing radiation activates AMP-activated kinase (AMPK): a target for radiosensitization of human cancer cells. Int J Radiat Oncol Biol Phys
6. Park CM, Park MJ, Kwak HJ, et al. Ionizing radiation enhances matrix metalloproteinase-2 secretion and invasion of glioma cells through Src/epidermal growth factor receptor-mediated p38/Akt and phosphatidylinositol 3-kinase/Akt signaling pathways. Cancer Res
7. Zimmermann M, Zouhair A, Azria D, et al. The epidermal growth factor receptor (EGFR) in head and neck cancer: its role and treatment implications. Radiat Oncol
8. Nakamura JL, Karlsson A, Arvold ND, et al. PKB/Akt mediates radiosensitization by the signaling inhibitor LY294002 in human malignant gliomas. J Neurooncol
9. Le Tourneau C, Siu LL. Molecular-targeted therapies in the treatment of squamous cell carcinomas of the head and neck. Curr Opin Oncol
10. Fritz G, Kaina B. Rho GTPases: promising cellular targets for novel anticancer drugs. Curr Cancer Drug Targets
11. Bernhard EJ, Stanbridge EJ, Gupta S, et al. Direct evidence for the contribution of activated N-ras and K-ras oncogenes to increased intrinsic radiation resistance in human tumor cell lines. Cancer Res
12. Lerner EC, Zhang TT, Knowles DB, et al. Inhibition of the prenylation of K-Ras, but not H- or N-Ras, is highly resistant to CAAX peptidomimetics and requires both a farnesyltransferase and a geranylgeranyltransferase I inhibitor in human tumor cell lines. Oncogene
13. Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature
14. Chan KK, Oza AM, Siu LL. The statins as anticancer agents. Clin Cancer Res
15. Sassano A, Platanias LC. Statins in tumor suppression. Cancer Lett
16. Poynter JN, Gruber SB, Higgins PD, et al. Statins and the risk of colorectal cancer. N Engl J Med
17. Khurana V, Bejjanki HR, Caldito G, et al. Statins reduce the risk of lung cancer in humans: a large case-control study of US veterans. Chest
18. Platz EA, Leitzmann MF, Visvanathan K, et al. Statin drugs and risk of advanced prostate cancer. J Natl Cancer Inst
19. Hamilton RJ, Banez LL, Aronson WJ, et al. Statin medication use and the risk of biochemical recurrence after radical prostatectomy: results from the Shared Equal Access Regional Cancer Hospital (SEARCH) Database. Cancer
20. Kollmeier MA, Katz MS, Mak K, et al. Improved biochemical outcomes with statin use in patients with high-risk localized prostate cancer treated with radiotherapy. Int J Radiat Oncol Biol Phys
21. Gutt R, Tonlaar N, Kunnavakkam R, et al. Statin use and risk of prostate cancer recurrence in men treated with radiation therapy. J Clin Oncol
22. Maksimova E, Yie TA, Rom WN. In vitro mechanisms of lovastatin on lung cancer cell lines as a potential chemopreventive agent. Lung
23. Tsakiridis T, Vranic M, Klip A. Disassembly of the actin network inhibits insulin-dependent stimulation of glucose transport and prevents recruitment of glucose transporters to the plasma membrane. J Biol Chem
24. Liu SK, Coackley C, Krause M, et al. A novel poly(ADP-ribose) polymerase inhibitor, ABT-888, radiosensitizes malignant human cell lines under hypoxia. Radiother Oncol
25. Motoshima H, Goldstein BJ, Igata M, et al. AMPK and cell proliferation—AMPK as a therapeutic target for atherosclerosis and cancer. J Physiol
26. Janicke RU, Sprengart ML, Wati MR, et al. Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis. J Biol Chem
27. Fritz G, Brachetti C, Kaina B. Lovastatin causes sensitization of HeLa cells to ionizing radiation-induced apoptosis by the abrogation of G2 blockage. Int J Radiat Biol
28. Khanzada UK, Pardo OE, Meier C, et al. Potent inhibition of small-cell lung cancer cell growth by simvastatin reveals selective functions of Ras isoforms in growth factor signalling. Oncogene
29. Bellini MJ, Polo MP, de Alaniz MJ, et al. Effect of simvastatin on the uptake and metabolic conversion of palmitic, dihomo-gamma-linoleic and alpha-linolenic acids in A549 cells. Prostaglandins Leukot Essent Fatty Acids
30. Valenzuela DM, Groffen J. Four human carcinoma cell lines with novel mutations in position 12 of c-K-ras oncogene. Nucleic Acids Res
31. Shaw RJ, Cantley LC. Ras, PI(3)K and mTOR signalling controls tumour cell growth. Nature
32. Gupta S, Ramjaun AR, Haiko P, et al. Binding of ras to phosphoinositide 3-kinase p110alpha is required for ras-driven tumorigenesis in mice. Cell
33. Mantha AJ, Hanson JE, Goss G, et al. Targeting the mevalonate pathway inhibits the function of the epidermal growth factor receptor. Clin Cancer Res
34. Mistafa O, Stenius U. Statins inhibit Akt/PKB signaling via P2X7 receptor in pancreatic cancer cells. Biochem Pharmacol
35. Toulany M, Dittmann K, Kruger M, et al. Radioresistance of K-Ras mutated human tumor cells is mediated through EGFR-dependent activation of PI3K-AKT pathway. Radiother Oncol
36. Toulany M, Kasten-Pisula U, Brammer I, et al. Blockage of epidermal growth factor receptor-phosphatidylinositol 3-kinase-AKT signaling increases radiosensitivity of K-RAS mutated human tumor cells in vitro by affecting DNA repair. Clin Cancer Res
37. Gupta AK, McKenna WG, Weber CN, et al. Local recurrence in head and neck cancer: relationship to radiation resistance and signal transduction. Clin Cancer Res
38. Sanchez-Cespedes M, Parrella P, Esteller M, et al. Inactivation of LKB1/STK11 is a common event in adenocarcinomas of the lung. Cancer Res
39. Steinberg GR, Kemp BE. AMPK in health and disease. Physiol Rev
40. Sun W, Lee TS, Zhu M, et al. Statins activate AMP-activated protein kinase in vitro and in vivo
41. Kim KY, Baek A, Hwang JE, et al. Adiponectin-activated AMPK stimulates dephosphorylation of AKT through protein phosphatase 2A activation. Cancer Res
42. Shackelford DB, Shaw RJ. The LKB1-AMPK pathway: metabolism and growth control in tumour suppression. Nat Rev Cancer
43. Niculescu AB III, Chen X, Smeets M, et al. Effects of p21(Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions: pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication. Mol Cell Biol
44. Woodard J, Sassano A, Hay N, et al. Statin-dependent suppression of the Akt/mammalian target of rapamycin signaling cascade and programmed cell death 4 up-regulation in renal cell carcinoma. Clin Cancer Res
45. Boehme KA, Kulikov R, Blattner C. p53 stabilization in response to DNA damage requires Akt/PKB and DNA-PK. Proc Natl Acad Sci USA
46. Laezza C, Fiorentino L, Pisanti S, et al. Lovastatin induces apoptosis of k-ras-transformed thyroid cells via inhibition of ras farnesylation and by modulating redox state. J Mol Med
47. Keyomarsi K, Sandoval L, Band V, et al. Synchronization of tumor and normal cells from G1 to multiple cell cycles by lovastatin. Cancer Res
48. Bouterfa HL, Sattelmeyer V, Czub S, et al. Inhibition of Ras farnesylation by lovastatin leads to downregulation of proliferation and migration in primary cultured human glioblastoma cells. Anticancer Res
49. Dimitroulakos J, Ye LY, Benzaquen M, et al. Differential sensitivity of various pediatric cancers and squamous cell carcinomas to lovastatin-induced apoptosis: therapeutic implications. Clin Cancer Res
50. Wu J, Wong WW, Khosravi F, et al. Blocking the Raf/MEK/ERK pathway sensitizes acute myelogenous leukemia cells to lovastatin-induced apoptosis. Cancer Res
51. Sleijfer S, van der Gaast A, Planting AS, et al. The potential of statins as part of anti-cancer treatment. Eur J Cancer
52. Knox JJ, Siu LL, Chen E, et al. A phase I trial of prolonged administration of lovastatin in patients with recurrent or metastatic squamous cell carcinoma of the head and neck or of the cervix. Eur J Cancer