The Role of the Akt/Mtor Pathway in Tobacco Carcinogen– Induced Lung Tumorigenesis

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Published OnlineFirst December 22, 2009; DOI: 10.1158/1078-0432.CCR-09-0234 Published Online First on December 22, 2009 as 10.1158/1078-0432.CCR-09-0234

Molecular Pathways Clinical Cancer Research The Role of the Akt/mTOR Pathway in Tobacco Carcinogen– Induced Lung Tumorigenesis

Regan M. Memmott and Phillip A. Dennis

Abstract Lung cancer is the leading cause of cancer-related death in the United States, and 85 to 90% of lung cancer cases are associated with tobacco use. Tobacco components promote lung tumorigenesis through genotoxic effects, as well as through biochemical modulation of signaling pathways such as the Akt/mammalian target of rapamycin (mTOR) pathway that regulates cell proliferation and survival. This review will describe cell surface receptors and other upstream components required for tobacco carcinogen–induced activation of Akt and mTOR. Preclinical studies show that inhibitors of the Akt/mTOR pathway inhibit tumor formation in mouse models of carcinogen-induced lung tumorigenesis. Some of these inhibitors will be highlighted, and their clinical potential for the treatment and prevention of lung cancer will be discussed. Clin Cancer Res; 16(1); 4–10. ©2010 AACR.

Background of these pathways is prevalent in lung cancer. Although tobacco components have pleiotropic effects on signaling The complex biological effects of tobacco smoke could pathways in cells, arguably the most important mediator of be attributed to the large number of chemical agents it con- tobacco carcinogen–induced lung tumorigenesis is the Akt/ tains. At least 60 carcinogens are present in tobacco smoke mTOR pathway. that can be categorized into three groups: polycyclic aromatic hydrocarbons, nitrosamines, and aromatic amines. Tobacco Components Stimulate Multiple Additionally, nicotine, a precursor of many tobacco carcinogens, also promotes lung tumorigenesis (reviewed in Upstream Activators of the Akt Pathway ref. 1). Metabolic activation of tobacco components, such as the nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)- Activation of the Akt pathway by tobacco components 1-butanone (NNK), generates metabolites that bind DNA increases lung epithelial cell proliferation and survival, and promote adduct formation. The persistence of DNA and inhibits apoptosis in response to DNA damage. To- adducts results in somatic mutations in oncogenes and tu- bacco components activate the Akt pathway by modulat- mor suppressor genes such as K-Ras and TP53, which are ing the expression or activity of upstream regulators of Akt critical to the formation of lung cancer. Although the gen- (Fig. 1). Immediately upstream of Akt is the lipid kinase otoxic effects of tobacco carcinogens are well established, PI3K. PI3K phosphorylates phosphoinositides at the D3 tobacco components can also promote lung tumorigenesis position, generating the biologically active lipids phos- through modulation of signal transduction pathways that phatidylinositol-3,4-bisphosphate [PI(3, 4)P2] and phos- regulate cell proliferation, transformation, and survival. phatidylinositol-3,4,5-triphosphate [PI(3,4,5,)P3]. For example, nicotine and NNK are potent agonists of nic- Generation of these lipids, and subsequent binding to otinic acetylcholine receptors (nAChR), which are ex- the pleckstrin homology domain of Akt, is critical to the pressed in lung epithelial cells and can activate multiple activation of Akt. Activating mutations or amplification PIK3CA signaling pathways such as Akt/mammalian target of rapa- of , the gene that encodes the catalytic subunit of α mycin (mTOR), protein kinase C (PKC; ref. 2), mitogen- PI3K, p110 , occur in a subset of smoking-related lung activated protein/extracellular signal-regulated kinase cancers (6). The tumor suppressor phosphatase and tensin kinase (MEK/ERK; ref. 3, 4), and janus-activated kinase/sig- homolog (PTEN) is a lipid phosphatase that opposes ∼ nal transducer and activator of transcription (JAK/STAT) this activity of PI3K. Loss of PTEN occurs in 70% of pathways (5). Overexpression or activation of components non-small cell lung cancer (NSCLC) through inactivating mutations, or more frequently, through promoter methylation (7, 8). Studies done using an inhibitor of PI3K, Authors' Affiliation: Medical Oncology Branch, Center for Cancer LY294002, showed that nicotine and NNK-induced acti- Research, National Cancer Institute, Bethesda, Maryland vation of Akt in lung epithelial cells is dependent on Corresponding Author: Phillip A. Dennis, National Cancer Institute/Navy Medical Oncology, Room 5101, Building 8, 8901 Wisconsin Avenue, PI3K (9, 10). Moreover, pretreatment of lung epithelial cells Bethesda, MD 20889. Phone: 301-496-0929; Fax 301-496-0047; E-mail: with LY294002 decreases tobacco component–induced [email protected]. proliferation and increases apoptosis. doi: 10.1158/1078-0432.CCR-09-0234 PI3K itself is activated by tobacco components through ©2010 American Association for Cancer Research. multiple, upstream mechanisms. PI3K is composed of a

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Fig. 1. Upstream activators and downstream mediators of the Akt pathway in tobacco carcinogen–induced lung tumorigenesis. Tobacco components stimulate cell surface receptors, such as members of the erbB family of receptor tyrosine kinases (RTK) and nicotinic acetycholine receptors (nAChR), which activate PI3K. PI3K can also be activated through direct interaction with K-Ras, which is commonly mutated in smoking-related lung cancers. The lipid kinase PI3K is required for tobacco component–induced activation of Akt. PI3K phosphorylates the phosphoinositides PI(4)P and PI(4, 5)P2 at their D3 position, generating PI(3, 4)P2 and PI(3,4,5)P3, respectively. The tumor suppressor PTEN opposes this activity of PI3K. PI(3, 4)P2 and PI(3,4,5)P3 bind to the pleckstrin homology domain of Akt, promoting its translocation to the cell membrane and subsequent activation. Akt activation in response to tobacco components increases cell survival by direct phosphorylation and inactivation of the pro-apoptotic proteins Bad and Bax. Additionally, Akt increases cell survival by indirectly inducing the anti-apoptotic protein survivin and the transcription factor NFκB. Tobacco component–induced activation of Akt can also increase cell growth and proliferation through activation of the mTOR pathway. Akt activates mTORC1 by at least two mechanisms. Akt can activate mTORC1 indirectly by phosphorylation and inactivation of TSC2, which suppresses the activity of the Rheb GTPase, an activator of mTORC1. Akt also directly activates mTORC1 through phosphorylation of PRAS40, a component of mTORC1. Drugs that inhibit the Akt/mTOR pathway could be effective in the treatment or prevention of tobacco carcinogen–induced lung tumorigenesis. The best-characterized inhibitors of this pathway (shown in bold) are rapamycin and its analogs, myoinositol, I3C, and deguelin. Rapamycin inhibits mTOR by binding to the immunophilin FKBP12. This complex, in turn, inhibits the activity of mTORC1. Conversely, myoinositol, I3C, and deguelin inhibit this pathway upstream of Akt, but whether PI3K is the direct target of these drugs is unclear (this is indicated by the dotted lines). catalytic subunit (p110) and a regulatory subunit (p85) human lung adenocarcinomas (13). Also, exposure of that contains Src-homology 2 (SH2) domains. Interaction A/J mice to the tobacco-specific carcinogen NNK induces of these domains with phosphotyrosine residues located K-Ras mutations, which promote lung tumorigenesis in on growth factor tyrosine kinase receptors, such as erbB this model (14). Immunohistochemical analysis of the family members, activates PI3K. In vitro studies showed lung adenomas and adenocarcinomas that develop in that NNK-induced transformation of bronchial epithelial these mice showed that Akt was activated in these lesions, cells increases the expression of epidermal growth factor which correlated with tumor progression (10). These receptor (EGFR; ref. 11). Additionally, overexpression of studies show the importance of erbB family members EGFR and erb-B3 occur in the bronchial epithelium of and K-Ras in mediating tobacco carcinogen–induced smokers (12). PI3K activation can also occur by direct inter- activation of the PI3K/Akt pathway. action of p110 with activated Ras. Activating mutations in Another mechanism by which tobacco components can K-Ras occur in approximately 25% of smoking-associated activate the PI3K/Akt pathway is via stimulation of

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nAChR. These receptors are prototypic ligand-gated ion 27). Another important mechanism by which Akt pro- channels that consist of either homo-pentamers derived motes tobacco carcinogen–induced lung tumorigenesis is from α7toα10 subunits or hetero-pentamers derived through activation of the mTOR pathway. from a combination of α1toα6 and β2toβ4 subunits. Similar to Akt, mTOR regulates cellular processes critical nAChR are important mediators of fast synaptic transmis- to tumorigenesis such as cell growth, proliferation, and me- sion in neurons, but they are also expressed in many non- tabolism, and many cancers are characterized by aberrant neuronal cell types such as immune cells, keratinocytes, activation of mTOR, including lung cancer (reviewed in and epithelial and endothelial cells (reviewed in ref. 15). ref. 28). mTOR functions in two distinct complexes in cells, Reverse transcriptase (RT)-PCR and microarray analyses mTORC1 and mTORC2. mTORC1 increases protein syn- showed that lung epithelial cells vary in their expression thesis and cell growth through activation of S6K1 (p70 ri- of nAChR (9, 16). Epithelial cells of the small airways se- bosomal protein S6 kinase) and inactivation of 4E-BP1 lectively express α2 and α4 subunits, whereas large airway (eIF4E binding protein 1). Although the role of mTORC2 epithelial cells express α3 and α5 subunits. Both cell types in regulating cellular processes is not well understood, express α7toα10, β2, and β4 subunits. Quantitative PCR mTORC2 directly phosphorylates members of the AGC and microarray analysis showed that the α4 and β4 sub- family of kinases, such as PKCα and Akt itself, which could units are preferentially expressed by NSCLC cells com- promote tumorigenesis (29–31). Akt activates mTORC1 by pared with normal lung epithelium (17). Additionally, at least two mechanisms. Akt activates mTORC1 indirectly analysis of tumors from patients with NSCLC showed dif- by phosphorylating and inhibiting the tumor suppressor ferential expression of nAChR between smokers and non- TSC2 (32, 33). Because TSC2 suppresses the activity of smokers, with higher expression of the α6β3 receptor in the Ras-related GTPase Rheb, a selective activator of the tumors from nonsmokers. Genome-wide association mTORC1, inhibition of TSC2 by Akt results in activation studies have suggested that individual nAChR confer an of mTORC1 (34). Additionally, Akt-mediated phosphory- increased risk for tobacco-related lung cancer. Specifically, lation of PRAS40, a component of mTORC1, attenuates its the gene locus 15q24 was associated with increased lung inhibitory effect on mTORC1 (35, 36). cancer risk and nicotine dependence (18–20). This locus Preclinical studies have shown that mTOR promotes to- contains genes that encode for the α3, α5, and β4 subunits bacco component–induced lung tumorigenesis. In vitro of nAChR. Collectively, these studies support the role of studies done in lung epithelial or NSCLC cells showed that nAChR in mediating tobacco-induced lung carcinogenesis. nicotine and NNK activate mTORC1, as assessed by in- Subunit composition of nAChR determines agonist- creased phosphorylation of S6K and 4E-BP1 (9, 21). Nico- specific responsiveness. For example, nicotine and NNK tine can also activate mTORC1 by increasing the expression are potent agonists of hetero-pentameric and α7 homo- of the matrix glycoprotein fibronectin (37). In these stud- pentameric nAChR, respectively. Studies done using nAChR ies, the ability of nicotine or NNK to activate mTORC1 was isoform-specific antagonists showed that inhibitors of α3- dependent on the Akt/mTOR pathway because treatment and α4-containing nAChR decreased nicotine-induced acti- of NSCLC cells with Akt siRNA or the mTOR inhibitor vation of Akt, whereas inhibitors of α7-containing nAChR rapamycin inhibited fibronectin-induced cell proliferation blocked NNK-induced activation of Akt (9). These receptors (37). A preferential role for mTOR in tobacco carcinogen– are also required for tobacco component–induced Akt acti- induced lung tumorigenesis was also suggested by analysis vation in NSCLC cells, which promotes resistance to che- of NNK-induced lung lesions in A/J mice. Phenotypic pro- motherapy and radiation (21). Although the ability of gression of NNK-induced lung lesions correlated with nAChR to activate Akt is dependent on PI3K, the mecha- increased activation of Akt and mTOR, but not other sub- nism by which these receptors activate PI3K is still unclear. strates of Akt such as GSK-3β (10). Clinical data also validate the importance of the Akt/ mTOR pathway in tobacco carcinogen–induced lung tumor- mTOR Is a Critical Mediator of Tobacco igenesis. In hyperplastic and dysplastic lung lesions from Carcinogen–Induced, Akt-Driven Lung heavy smokers, Akt and mTOR are progressively activated Tumorigenesis (38–41). Additionally, activation of Akt is associated with poor prognosis in patients with early-stage NSCLC (42). Akt can promote tobacco carcinogen–induced lung tu- These studies suggest that activation of mTOR downstream morigenesis by regulation of multiple signaling pathways of the PI3K/Akt pathway might be an important and early (Fig. 1). For example, Akt increases lung epithelial cell sur- event in tobacco carcinogen–induced lung tumorigenesis. vival in response to NNK and nicotine by phosphorylation and inactivation of the pro-apoptotic proteins Bad and Clinical-Translational Advances Bax (16, 22–24), as well as through induction of the anti-apoptotic protein survivin (16, 25). Additionally, Inhibitors of the Akt/mTOR pathway for the treatment or Akt activates the transcription factor nuclear factor kappa prevention of tobacco carcinogen–induced lung tumorigen- B(NFκB), which increases NSCLC cell survival in vitro esis. Because there is strong rationale to target the Akt/ (21), and promotes tumor growth and angiogenesis via mTOR pathway in the treatment or prevention of tobacco vascular endothelial growth factor (VEGF) in vivo (26, carcinogen–induced lung tumorigenesis, inhibitors of this

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pathway have been evaluated in mouse models of lung lar macrophage function and modulation of chemokine cancer and in clinical trials. The inhibitors of the Akt/ signaling contributed to inhibition of lung-tumorigenesis mTOR pathway that are best characterized are rapamycin by CCI-779. Collectively, these studies show that mTOR and its analogs, myoinositol, indole-3-carbinol (I3C), inhibitors are effective in the treatment or prevention of and deguelin (Fig. 1). tobacco carcinogen–induced lung tumorigenesis by inhi- mTOR inhibitors. The most clinically developed inhibi- biting the mTOR pathway in tumor cells, as well as by tors of the Akt/mTOR pathway are the mTOR inhibitors affecting the tumor microenvironment. rapamycin (sirolimus) and its analogs, CCI-779 (temsiro- Because mTOR inhibitors could be effective in the treat- limus) and RAD001 (everolimus). Rapamycin is a macro- ment of tobacco carcinogen–induced lung tumorigenesis, lide antibiotic that is U.S. Food and Drug Administration rapamycin and its analogs are currently being evaluated in (FDA) approved as an immunosuppressant for the preven- clinical trials for the treatment of NSCLC. However, the tion of renal allograft rejection. Rapamycin inhibits the therapeutic response in these trials has been modest (re- activity of mTORC1 by binding to the immunophilin viewed in ref. 51), and prolonged administration of these FKBP12. This complex, in turn, binds mTORC1 and inhi- drugs could be limited because of toxicities in patients. bits its phosphorylation of substrates, such as S6K and Preclinical and clinical studies also suggest that the efficacy 4E-BP1. Although originally reported to be insensitive to of mTOR inhibitors as anticancer agents could be limited rapamycin, long-term treatment of mammalian cells with because of feedback activation of Akt (52). Improved clin- rapamycin also inhibits mTORC2 (43). Preclinical studies ical response might be achieved by combining mTOR in- have shown that rapamycin and rapamycin analogs are hibitors with inhibitors of upstream components of the effective in the treatment or prevention of tobacco-induced Akt/mTOR pathway that could negate feedback activation. lung tumorigenesis in multiple mouse models. mTOR inhibitors might also be more effective in the set- Exposure of A/J mice to the tobacco-specific carcinogen ting of lung cancer chemoprevention. NNK causes K-Ras-mediated lung tumorigenesis (44), Myoinositol. Myoinositol is an isomer of glucose that is which is characterized by inflammation of the lung and structurally similar to the head group of phosphatidylino- increased activation of the mTOR pathway in tumors. Ad- sitol, the endogenous substrate of PI3K in cells. Studies ministration of rapamycin to A/J mice 26 weeks after ex- done using compounds related to myoinositol showed posure to NNK led to a 50% decrease in tumor volume, that this class of compounds inhibits the PI3K/Akt path- which correlated with decreased proliferation and inhibi- way, which is required for their anticancer activity (53, tion of the mTOR pathway in tumors (45). Additionally, 54). Additionally, treatment of human bronchial epitheli- treatment of A/J mice with rapamycin following exposure al cells (HBEC) with myoinositol inhibits basal levels of to NNK, but prior to the development of tumors, reduced Akt activity as well as nicotine-induced activation of Akt, lung tumorigenesis by 90%. Rapamycin similarly pre- which correlates with inhibition of cell proliferation (41). vented lung tumorigenesis in A/J mice exposed to another Interestingly, myoinositol does not inhibit Akt nor inhibit tobacco carcinogen, benzo(a)pyrene (BaP; ref. 46). the proliferation of NSCLC cells in vitro, suggesting that Rapamycin could also inhibit tobacco carcinogen– myoinositol might be more effective in preventing the de- induced lung tumorigenesis by affecting the tumor velopment of lung cancer. Preclinical studies done using microenvironment. For example, administration of NNK A/J mice exposed to a mixture of tobacco carcinogens, significantly increases the number of lung-associated NNK, and BaP showed that myoinositol significantly pre- Foxp3+ regulatory T cells (Treg) prior to the development vented lung tumorigenesis when administered concurrent- of tumors in A/J mice (47). Treg are a subset of CD4+ ly or following exposure to these carcinogens (55, 56). T cells that express the transcription factor Foxp3 and sup- Assessment of P-Akt (S473) in lung tissues from these press autoreactive T cells, thus preventing autoimmunity mice showed only modest inhibition by myoinositol. (48, 49). Preclinical studies suggest that Treg may play However, this analysis was done on whole lung lysates, an important role in limiting the development of an effec- which would not distinguish between the effects of myo- tive immune response against cancer (reviewed in ref. 50). inositol on Akt activity in tumor cells and normal lung ep- Interestingly, rapamycin rapidly reverses the induction of ithelium. Additionally, phosphorylation of Akt at S473 lung-associated Treg by NNK, and maintains depletion of alone may not be an accurate readout for the activity of Foxp3+ Treg throughout treatment. These results suggest Akt (57). Taken together, these studies suggest that myo- that rapamycin inhibits NNK-induced lung tumorigenesis inositol could prevent tobacco carcinogen–induced lung by inhibition of mTOR in tumor cells and by decreasing tumorigenesis. lung- and tumor-associated Treg. A phase I clinical prevention trial with myoinositol in Similar results were obtained in a genetically engineered heavy smokers showed that it was well tolerated even at mouse model that genocopies and phenocopies tobacco doses as high as 18 g/d, and on the basis of historical com- carcinogen–induced lung tumorigenesis. Studies done us- parisons, the investigators suggested that treatment with ing mice bearing a mutant K-Ras allele, K-RASLA1,also myoinositol caused regression of preexisting bronchial showed that CCI-779 decreased tumor growth, in part, dysplastic lesions (58). To elucidate possible mechanisms by modulating components of the immune system (40). by which myoinositol might prevent lung tumorigenesis Specifically, these studies showed that inhibition of alveo- in smokers, immunohistochemical analysis was done on

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206 paired premalignant lesions from these patients using to BaP or a mixture of NNK and BaP, respectively (64, an antibody specific for P-Akt (T308), a site important to 65). Immunohistochemical analysis showed that inhibi- the activation of Akt. Prior to treatment with myoinositol, tion of tumorigenesis correlated with decreased P-Akt active Akt was detected in 71% of hyperplastic and/or (S473) in bronchial epithelium and lung adenomas. Al- metaplastic lesions and 90% of dysplastic lesions, and cor- though these studies suggest that deguelin could be effec- related with phenotypic progression (41). Regression of tive in the prevention of tobacco carcinogen–induced lung dysplastic lesions by myoinositol correlated with de- tumorigenesis, the clinical potential of deguelin might be creased phosphorylation of Akt. In addition, myoinositol limited because of toxicities (66). Efforts are currently un- decreased ERK phosphorylation in these lesions, a compo- derway to synthesize derivatives of deguelin that are less nent of the MEK/ERK pathway that is also activated during toxic but retain its anticancer activity. tobacco component–induced lung tumorigenesis. These studies suggest that inhibition of signaling pathways by Conclusion myoinositol underlies its ability to prevent lung tumorigenesis in smokers. A phase II trial is currently being con- Modulation of signaling pathways by tobacco compo- ducted with myoinositol in current and former smokers to nents is an important aspect of tumor promotion. The further evaluate its ability to reverse bronchial dysplasia. Akt/mTOR pathway is an especially intriguing target for Indole-3-carbinol. I3C is a metabolite of glucobrassicins tobacco-related lung tumorigenesis, based on the pre- that are prevalent in cruciferous vegetables. Epidemiologi- clinical efficacy of pathway inhibitors and the results of cal studies indicate that consumption of cruciferous vege- early phase clinical trials. Drugs such as rapamycin, myo- tables inversely correlates with lung cancer risk (59). inositol, I3C, and deguelin that inhibit the pathway at Preclinical studies done using A/J mice exposed to NNK different steps have clinical potential. As the complexity and BaP showed that I3C inhibited lung tumorigenesis in of mTOR regulation increases, options for new drugs to a dose-dependent manner when it was administered either intervene in tobacco-related carcinogenesis will also in- during or 1 week following exposure to these carcinogens crease. For example, activation of the LKB1/AMPK path- (60). Immunohistochemical and immunoblotting analy- way inhibits mTOR in an Akt-independent manner. ses of lung tissues from these mice showed that I3C signif- Activators of AMPK such as the antidiabetic drug metfor- icantly decreased P-Akt (S473) in tumors, as well as min might therefore also inhibit tobacco carcinogen– phosphorylation of the pro-apoptotic protein Bad, a sub- induced lung tumorigenesis through inhibition of mTOR. strate of Akt. However, quantitative proteomic analyses Future studies will determine the best method to counteract showed that I3C modulated the expression of multiple the effects of tobacco components on signaling pathways. proteins altered by the carcinogens in this model (61), and the relative contribution of Akt inhibition to the anti- Disclosure of Potential Conflicts of Interest cancer activity of this compound is unclear. Also, the clinical utility of I3C may be limited because I3C can promote No potential conflicts of interest were disclosed. colon and hepatic carcinogenesis in other mouse models (reviewed in ref. 62). Deguelin. Deguelin is a naturally occurring rotenoid that Acknowledgments can be isolated from several plant species. Studies done using premalignant and malignant HBECs showed that The costs of publication of this article were defrayed in deguelin inhibits the PI3K/Akt pathway, which was re- part by the payment of page charges. This article must quired for its ability to inhibit cell proliferation and in- therefore be hereby marked advertisement in accordance duce apoptosis (63). In A/J mice, deguelin significantly with 18 U.S.C. Section 1734 solely to indicate this fact. decreased tumor multiplicity, volume, and overall tumor Received 9/9/09; accepted 9/10/09; published burden when administered before or following exposure OnlineFirst 12/22/09.

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10 Clin Cancer Res; 16(1) January 1, 2010 Clinical Cancer Research

The Role of the Akt/mTOR Pathway in Tobacco Carcinogen− Induced Lung Tumorigenesis

Regan M. Memmott and Phillip A. Dennis

Clin Cancer Res Published OnlineFirst December 22, 2009.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-09-0234

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