Targeting PI3 Kinase/AKT/mTOR Signaling in Cancer

Transcription

1 Critical Reviews in Oncogenesis, 17(1), (2012) Targeting PI3 Kinase/AKT/mTOR Signaling in Cancer Karen E. Sheppard, 1,4 Kathryn M. Kinross, 1 Benjamin Solomon, 1,2,5 Richard B. Pearson, 1,4,5,7* & Wayne A. Phillips 1,3,5,6,7 1 Division of Cancer Research, 2 Division of Cancer Medicine, and 3 Division of Cancer Surgery, Peter MacCallum Cancer Centre, East Melbourne, Victoria, Australia; 4 Department of Biochemistry and Molecular Biology, 5 Sir Peter MacCallum Department of Oncology, and the 6 Department of Surgery (SVH), University of Melbourne, Parkville, Australia; 7 Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia *Address all correspondence to: Richard B. Pearson, Protein Chemistry Laboratory, Peter MacCallum Cancer Centre, Locked Bag 1, A Beckett Street, Melbourne, Victoria 8006, Australia; Tel.: (61) ; Fax: (61) ; rick.pearson@ petermac.org. ABSTRACT: The phosphatidylinositol 3 kinase (PI3K) pathway is one of the major pathways modulating cell growth, proliferation, metabolism, survival, and angiogenesis. Hyperactivation of this pathway is one of the most frequent occurrences in human cancer and is thus an obvious target for treatment of this disease. Currently there are 26 novel compounds targeting the PI3K pathway being assessed in more than 150 cancer-related clinical trials. Although this pathway is involved in many vital biologic functions, data emanating from these clinical trials indicate that these drugs are well tolerated. This review outlines the interaction of the PI3K pathway with other signaling cascades, highlights mechanisms involved in hyperactivation, discusses current therapeutics in cancer-related clinical trials that target this pathway, and, based on preclinical data, discusses possible leads on patient selection and combinational therapy, including targeting multiple components of the associated signaling network. KEY WORDS: phosphatidylinositol 3 kinase, anticancer drugs, targeted therapeutics, kinase s, combinational therapy, clinical trials ABBREVIATIONS ATP: Adenosine triphosphate; DNA-PK: DNA-dependent protein kinase; ERK: extracellular signal regulated kinase; GTP: guanosine triphosphate; HR: hazard ratio; MAPK: mitogen-activated protein kinase; mtor: mammalian target of rapamycin; mtorc1: mammalian target of rapamycin complex 1; mtorc2: mammalian target of rapamycin complex 2; PDK1: 3-phosphoinositide-dependent kinase 1;; PFS: progression-free survival; PI3K: phosphatidylinositol 3 kinase; PTEN: phosphatase and tensin homolog; RAPTOR: regulatory associated protein of mtor; RTKs: receptor tyrosine kinases; S6K1: p70 ribosomal protein S6 kinase; Ser: serine; Thr: threonine; TSC2: tuberous sclerosis complex 2 I. THE PI3K SIGNALING PATHWAY Phosphatidylinositol 3 kinases (PI3Ks) are a family of lipid kinases that share the primary biochemical function to phosphorylate the 3-hydroxyl group of inositol-containing phospholipids. There are 3 classes of PI3Ks grouped according to substrate preference and structure. Class I PI3Ks are further divided into classes IA and IB. Class IA PI3Ks are activated by receptor tyrosine kinases (RTKs) and G protein coupled receptors, whereas class IB PI3Ks are activated by G protein coupled receptors only. 1 Of the PI3Ks, class IA PI3Ks are the most clearly implicated in cancer. 2 5 These kinases are heterodimeric, comprising 1 of the 5 regulatory subunits (p85, p85, p55, p55, or p50 ) and 1 of the 3 catalytic subunits (p110, p110, or p110 ). Of these subunits, gain-of-function mutations occur most commonly in the catalytic subunit p110 and to a lesser extent in the regulatory subunit p85. 1 Generation of phosphatidylinositol (3,4,5)P 3 (PIP3) within the cell by class IA PI3Ks initiates a signaling cascade activating 3-phosphoinositide-dependent kinase 1 (PDK1), which in turn phosphorylates and activates AKT. Phosphatase and tensin homolog (PTEN) dephosphorylates PIP3 and thus decreases signaling via this pathway (Fig. 1). AKT activation is responsible for many of the biologic processes associated with increased PI3K signaling; however, there is increasing evidence that /12/$ by Begell House, Inc. 69

2 70 Sheppard et al. FIGURE 1. Phosphatidylinositol 3 kinase (PI3K)/AKT signaling cascade. Stimulation of receptor tyrosine kinases (RTKs) activate PI3K, initiating a signaling cascade via AKT and mammalian target of rapamycin complex 1 (mtorc1), regulating angiogenesis, cell survival, metabolism, growth, and proliferation. 4EBP1, eukaryotic translation initiation factor 4E binding protein; DNA-PK, DNA-dependent protein kinase; ERK, extracellular signal regulated kinase; MEK, ERK kinase; mtorc2, mammalian target of rapamycin complex 2; PHLPP, PH domain and leucine-rich repeat protein phosphatase; PIP2, phosphatidylinositol (4,5)P2; PIP3, phosphatidylinositol (3,4,5)P 3 ; PTEN, phosphatase and tensin homolog; S6K1, p70 ribosomal protein S6 kinase; TSC, tuberous sclerosis complex. both PI3K and PTEN can regulate other tumorigenic signaling pathways independently of AKT. PTEN is known to have nuclear functions that promote cell death through targeting a variety of transcription factors and regulating chromosome stability. 6 8 A recent study reported that the regulatory subunit of PI3K, p85, also could serve as a protein substrate of PTEN phosphatase, which represents an alternative mechanism for negative regulation of PI3K by PTEN. 9 In addition to AKT, the SGK family of kinases is activated by PI3K; they target many of the same substrates as AKT, can regulate cell proliferation and survival, and have been implicated in tumorigenesis There is also evidence for PI3K activation of the Ras proto-oncogene 13,14 and the small guanosine triphosphate (GTP) binding proteins CDC42 and RAC1, which can function as oncogenes when overexpressed. 15 Activation of PDK1 by PI3K can lead to increased activity of protein kinase C zeta, a kinase involved in a wide range of Critical Reviews in Oncogenesis

3 PI3 Kinase/AKT/mTOR Signaling in Cancer 71 physiologic processes, including mitogenesis, protein synthesis, cell survival, and transcriptional regulation, all of which, when dysregulated, would contribute to the tumorigenic phenotype. 16 The AKT kinase family is comprised of 3 highly homologous isoforms: AKT1, AKT2, and AKT3, each of which has both distinct and overlapping functions. 17 Activation of the AKT kinases leads to phosphorylation of many factors that impact cell metabolism, angiogenesis, protein translation, cell proliferation, and survival. 18 The regulation of AKT is a dynamic process that depends on the balance between activation via phosphorylation by upstream kinases and inactivation via dephosphorylation by protein phosphatases. PDK1 phosphorylates AKT at Thr 308, which increases AKT activity, but maximal activity also requires phosphorylation of Ser473, although recent data suggests that phosphorylation of AKT at Thr308 is enough to promote tumorigenesis. 19 Several candidates are responsible for phosphorylating Ser473, including mammalian target of rapamycin (mtor) complex 2 (mtorc2) in response to both growth factors and mitogens 23 and DNA-dependant protein kinase (DNA-PK) 24 in response to DNA damage. 25,26 AKT signaling is decreased by protein phosphatase 2A and the PH domain and leucine-rich repeat protein phosphatases 1 and 2, which dephosphorylate Thr308 and serine 473, respectively. 27,28 Specificity of the downstream targets phosphorylated by AKT is, in part, a function of the activating upstream kinases 26,29 and the presence of the different AKT isoforms. 30,31 One of the major targets of AKT is mtor complex1 (mtorc1), which is a complex consisting of the serine/threonine kinase mtor, the regulatory associated protein of mtor (RAPTOR), prolinerich AKT substrate 40 (PRAS40), and mlst8 32 ; this complex acts as a master regulator of protein synthesis by integrating the effects of growth factors and the availability of nutrients and energy. 33 AKT activates mtorc1 via phosphorylation of tuberous sclerosis complex 2 (TSC2) 34 and PRAS40, 35 2 negative regulators of mtorc1. Activation of mtorc1 increases translation initiation, ribosome biogenesis, and cell cycle progression via the phosphorylation of p70 ribosomal protein S6 kinase (S6K1) and eukaryotic initiation factor 4E binding protein Phosphorylation of 4E binding protein 1 (4EBP1) inhibits its ability to bind eif4e, thus allowing this protein to assemble the cap-binding complex and initiate translation of proteins involved in cell growth and division, such as c-myc and cyclin D. Phosphorylation of S6K1 enhances its kinase activity, increasing ribosome biogenesis 36 and protein synthesis via acting on many substrates including 40S ribosomal protein S6, eif4b, 37 and SKAR. 38 In addition to AKT regulation of ribosome biogenesis via mtorc1, an mtorc1-independent AKT signaling pathway to this endpoint also exists. 39 Inhibition of mtorc1 occurs via feedback loops (these will be discussed later in greater detail) and in response to both glucose deprivation and hypoxia, which trigger adenosine monophosphate activated protein kinase to phosphorylate both RAPTOR 40 and TSC2, enhancing their y activity. 41 Amino acid deprivation also inhibits mtorc1 via Rag small guanosine triphosphatase modulation of Rheb-GTP s ability to activate mtorc1. 42 In addition to being a major component of the PI3K pathway, AKT impacts many cellular processes and has a myriad of putative substrate targets. 18,43 AKT not only promotes cell cycle progression via mtorc1 but also via inhibition of cyclin-dependent kinase s including p21cip1/waf1 44 and p27kip Inhibition of glycogen synthase kinase 3 by AKT-mediated phosphorylation leads to stabilization of cyclin D and cyclin E and the transcription factors c-jun and c-myc, all of which play a central role in the G1 to S phase cell-cycle transition Relevant to the tumorigenic phenotype is AKT s ability to promote cell survival and angiogenesis. AKT-induced cell survival occurs via several mechanisms, including blocking the function of proapoptotic proteins and processes through decreasing the function and expression of Bcl-2 homology domain 3 only proteins both directly and indirectly via FOXO and MDM2. 43 In addition, AKT increases the transcription of prosurvival genes through its activation of CREB and I B kinase, leading to increased nuclear factor- B activity, or inhibition of apoptosis signal-regulated kinase 1, leading to reduced JNK/p38 apoptotic signaling Volume 17, Number

4 72 Sheppard et al. AKT-induced angiogenesis is mediated via activation of endothelial nitric oxide synthase and subsequent release of nitric oxide and by increasing the production of the hypoxia inducible factor (HIF) transcription factors (HIF1 and HIF2 ), which induces the expression and subsequent secretion of vascular endothelial growth factor and other angiogenic factors II. CROSS-TALK AND FEEDBACK LOOPS PI3K signaling dynamics are influenced by positive and negative feedback loops and interaction with other signaling pathways (Fig. 2). S6K has been implicated as mediating negative feedback at several levels. S6K1 phosphorylation of insulin receptor substrates 1 and 2 62 destabilizes the protein and thus prevents growth factor signaling to PI3K. In addition, S6K phosphorylates mtorc2, reducing AKT activity and thus mtorc1 signaling. 63,64 In contrast, S6K1 has been shown to phosphorylate its direct upstream regulator mtor (on Thr2446 and Ser2448). 65 Considering that this region of the mtor proteins acts as a repressor domain, 66 it is likely this phosphorylation promotes mtorc1 activity. More recently, both mtorc1 and AKT have been implicated in negative feedback regulation of RTK signaling and expression. mtorc1 FIGURE 2. Cross-talk and feedback loops of the phosphatidylinositol 3 kinase (PI3K) pathway. The many cellular processes mediated by PI3K signaling are tightly regulated by a complex network of positive and negative feedback loops and cross-talk between other signaling nodes., positive physiologic regulation;, negative physiological regulation;, positive feedback;, negative feedback;, positive cross-talk between pathways;, negative cross-talk between pathways;, gene transcription; ERK, extracellular signal regulated kinase; GRB10, growth factor receptor bound protein 10; HB-EGF, heparin-bound epidermal growth factor; IRS, insulin receptor substrate; MEK, ; mtorc, mammalian target of rapamycin complex; RTK, receptor tyrosine kinase; S6K1, p70 ribosomal protein S6 kinase; TSC, tuberous sclerosis complex. Critical Reviews in Oncogenesis

5 PI3 Kinase/AKT/mTOR Signaling in Cancer 73 phosphorylation of growth factor receptor bound protein 10 stabilizes this protein, leading to feedback inhibition of both the PI3K and Ras/extracellular signal regulated kinase (ERK) pathways, 67,68 whereas AKT prevents nuclear localization of FOXO, thus inhibiting the FOXO-dependent increase in RTK expression. 69 Glycogen synthase kinase 3, a downstream target of AKT, recently has been identified as also being upstream of AKT, increasing phosphorylation; thus, it is a novel feed forward. 70 The PI3K pathway also interacts with multiple signaling pathways; the best characterized is the Ras/ERK pathway. Ras-GTP can bind directly and allosterically activate PI3K, whereas both ERKand p90 ribosomal S6 kinase mediated phosphorylation of TSC2 and ERK-mediated phosphorylation of RAPTOR promote mtorc1 activity; the latter leads to Ras-induced, PI3K-independent mtorc1 activation. Conversely, activated PI3K can either increase or decrease Ras-GTP activity. 13,14,77 Through the autocrine production of growth factors, such as heparin-binding epidermal growth factor, the ERK pathway also can potently induce both Ras and PI3K signaling. A recent kinome small interfering RNA phosphoproteomic screen identified a significant inverse correlation between activation of AKT and ERK, suggesting these pathways are tightly balanced and that there is extensive y crosstalk. 70 Inhibition of the ERK pathway by the PI3K pathway can occur via AKT inhibition of c-raf and through S6K signaling via an undefined mechanism The PI3K pathway also interacts with many other signaling pathways. AKT signaling can activate the nuclear factor- B transcription factor, promote prosurvival pathways, 52,53 and inhibit the stress-activated mitogen-activated protein kinases (MAPKs) JNK and p38, likely via inhibition of apoptosis signal-regulated kinase 1, which is an upstream activating kinase within the JNK and p38 pathways. 54 p38 MAPK signaling also can inhibit PI3K signaling via induction of PTEN expression. 82 The feedback loops within the PI3K pathway and the cross-talk with various signaling pathways will impact on the effectiveness of PI3K-targeted therapy and are likely to be specific to tumor type and underlying mutation status; thus, one treatment regime may be effective for one tumor type but not for all tumors. The extensive interactions within the PI3K pathway, and between this and other signaling pathways, also predicts that inhibition of the PI3K pathway will require targeting several of its components and that combinational therapy targeting other pathways will be necessary for more effective treatment. III. DYSREGULATION OF THE PI3K PATHWAY IN CANCER The PI3K pathway is activated more frequently by genomic aberrations than any other signaling pathway across many cancer types. Multiple pathway components are targeted by germ line or somatic mutation, amplification, rearrangement, methylation, overexpression, and aberrant splicing. 43,83 88 There are many excellent reviews highlighting the changes that lead to increased PI3K pathway signaling, 1,89 93 so this will only briefly be discussed here. PI3K is activated downstream of numerous RTKs. Under physiologic conditions, only a small proportion of PI3K is activated in response to RTK stimulation; thus increased signaling through these receptors can lead to substantial increases in PI3K activity. In cancer, gene amplifications, activating mutations in RTKs and increased expression of ligands for these receptors are common. In addition, a cancer cell often will have multiple RTKs activating PI3K, making targeting these receptors a challenge. 91 The 2 most common aberrations leading to the activation of the PI3K pathway are loss of PTEN function and a gain in class IA PI3K activity via amplification and mutations in the catalytic subunit p110 (which is coded for by the PIK3CA gene). Activating PIK3CA mutations occur in 2 hotspots: the helical domain (E545K and E542K) and the kinase domain (H1047R). Loss of heterozygosity, inactivating mutations, and epigenetic silencing account for PTEN dysfunction and lead to increased PIP3 and thus signaling. Activating mutations in the PIK3CB gene, which encodes the p110 catalytic subunit, have not been found, but gene amplification has been reported at Volume 17, Number

6 74 Sheppard et al. low frequency in both breast and ovarian cancers. 94 Mutations in the PIK3R1 gene, encoding the p85 regulatory subunit, are found infrequently and are thought to disrupt the y contact of p85 with p110, leading to constitutive PI3K activity. 95 Activating mutations in PDK1 are rare but amplification has been reported in breast cancer. 94 Of the 3 AKT isoforms, a rare activating mutation has been identified in AKT1 96 and AKT3, 97 and gene amplification of AKT1 and AKT2 occurs but is less common for AKT3. 93 The ubiquitous nature of PI3K pathway activation in cancer indicates that PI3K, AKT, and other components of this pathway are attractive targets for cancer therapy, and multiple PI3K pathway s are now under active clinical development. IV. PI3K PATHWAY INHIBITORS IN CLINICAL TRIALS Preclinical studies have highlighted that within the PI3K pathway there is a multitude of feedback loops as well as cross-talk with oncogenes in other signaling pathways, and these interactions are likely to be tumor specific and impact therapeutic intervention (Fig. 3). Thus, targeting several components within the PI3K pathway, combined treatment with s that target other signaling pathways or oncogenes, or both are likely to provide the way forward for the use of PI3K s in the treatment of cancer. To date there are 3 main points within the PI3K pathway that are actively being pursued as therapeutic targets: the catalytic subunits of the class 1 PI3Ks, AKT, and mtor. Inhibitors targeting each of these kinases, as well as dual PI3K/mTOR s, are currently being assessed in more than 150 clinical trials ( A. Targeting mtor Initial studies have focused on inhibition of mtorc1 with the natural product rapamycin (sirolimus). Rapamycin binds to its intracellular receptor FK506 binding protein 12, enabling interaction with and allosteric inhibition of mtorc1 and thus suppression of mtor-mediated phosphorylation of its downstream substrates, S6K1 and 4EBP1, 102,103 though not all the functions of mtorc1 are targeted by rapamycin. 104 Analogs of rapamycin (sometimes referred to as rapalogs), such as the intravenous agents temsirolimus (CCI-779, ) and ridaforolimus (AP23573) and the oral agent everolimus (RAD001, Afinitor, Novartis Pharmaceuticals Corp., East Hanover, NJ) also have been developed as anticancer drugs. These agents inhibit mtorc1 through the same mechanism as rapamycin but have better pharmacologic properties for clinical use in cancer. In preclinical models, both rapamycin and rapalogs inhibit cell proliferation and tumorigenesis. 105,106 Although, in general, mtor s are associated with limited single-agent activity in common solid tumors, the US Food and Drug Administration has provided approval for these agents in advanced renal cell carcinoma, pancreatic neuroendocrine tumors, and subependymal giant-cell astrocytomas associated with tuberous sclerosis, and promising activity has been reported for everolimus in combination with endocrine therapy in breast cancer. 107 In 2 phase III clinical trials conducted with patients with advanced renal cell carcinoma, both everolimus and temsirolimus significantly improved progression-free survival (PFS). In the first study, everolimus treatment resulted in an objective response rate of only 1.8% but significantly improved PFS compared with placebo (4.0 vs. 1.9 months; hazard ratio [HR], 0.33; P <.001). 108,109 In the second study, temsirolimus improved both PFS (3.8 vs. 1.9 months; P <.001) and overall survival (10.9 vs. 7.3 months; HR, 0.73; P <.008) compared with interferon alone, with an objective response rate of 8.6%. There was no additional benefit with the combination of temsirolimus and interferon. 110 These data led to the US Food and Drug Administration approval of both everolimus and temsirolimus for use in advanced renal cell carcinoma. In a placebo-controlled study of pancreatic neuroendocrine tumors, everolimus treatment was associated with an objective response rate of 5% and significantly improved PFS (11 months vs. 4.6 months; HR, 0.35; P <.001). 111 In patients with Critical Reviews in Oncogenesis

7 PI3 Kinase/AKT/mTOR Signaling in Cancer 75 FIGURE 3. Pharmacological inhibition of the phosphatidylinositol 3 kinase (PI3K) pathway results in loss of negative feedback loops. Inhibition of mammalian target of rapamycin (mtor) complex (mtorc) 1, mtor kinase, PI3K, and AKT each have been shown to perturb the normal negative feedback loops that control signaling activity through this pathway in both preclinical studies and in clinical samples. mtorc1 allosteric s (A) lead to loss of p70 ribosomal protein S6 kinase (S6K1 mediated inhibition of insulin receptor substrate (IRS), resulting in enhanced insulin receptor signaling to PI3K/AKT and the mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) pathways. In addition to its effects on mtorc1, mtor kinase s (B) also inhibit phosphorylation at Ser473 on AKT; however, in some models, increased Thr308 phosphorylation of AKT is observed, which enables increased AKT activity downstream of receptor tyrosine kinases (RTKs), IRS, or both. RTK expression can be upregulated after mtor inhibition by a currently undefined mechanism. Inhibition of PI3K (C) or AKT (D) activity leads to loss of repression of FOXO proteins, which are then able to activate transcriptionally a number of target genes, including RTKs. This increase in RTKs can then activate the MAPK/ERK pathway and, in the case of AKT s, also potentially can promote AKT-independent PI3K effects. Bolded or grey lines indicate increased or decreased protein activity, respectively, after therapy. Red and green arrows indicate decreased and increased phosphorylation of AKT, respectively, after therapy. 4EBP1, eukaryotic translation initiation factor 4E binding protein. Volume 17, Number

8 76 Sheppard et al. tuberous sclerosis (a multisystem genetic disease associated with germline TSC1/tuberin or TSC2/ hamartin mutations), everolimus treatment reduced seizure frequency and induced major tumor responses in more than 75% of patients with subependymal giant-cell astrocytomas. 112 Impressive activity also has been reported for the combination of mtor inhibition and hormonal therapy in breast cancer. A recent phase III study (BOLERO-2) comparing the addition of everolimus or placebo to exemestane in women with estrogen receptor positive breast cancer that had progressed with letrozole or anastrozole showed that the combination more than doubled PFS compared with exemestane treatment (10.6 months vs. 4.1 months; HR 0.36; P <.00001), with a significantly greater response rate (9.5% vs 0.4%; P <.001). 107 Although most cancers initially respond to therapeutic intervention, the development of acquired resistance, through a range of distinct mechanisms, is a major clinical problem. 113 Resistance to rapamycin 114 is caused in part by feedback loops that activate the Ras/ERK 101 and PI3K pathways. 62 Rapamycin induces AKT Ser473 phosphorylation and AKT activity, 100,115,116 thus attenuating its therapeutic effects. To overcome this problem, adenosine triphosphate (ATP) competitive s of mtor kinase that inhibit both mtorc1 and mtorc2 now have been developed, with the hope that such s will have greater antitumor activity than rapalogs because inhibition of mtorc2 will prevent feedback induction of AKT. Torin1, PP242, and PP30 are potent and selective ATP-competitive s of mtor, inhibiting both mtorc1 and mtorc2. In cell lines, these s are more effective than rapamycin at inhibiting cap-dependent translation and proliferation. 117,118 The enhanced activity of these mtor kinase s seems to be through more complete inhibition of mtorc1 activity 118 rather than inhibition of mtorc2. Currently there are 3 ATP-competitive mtor s (OSI-027, AZD8055, and INK128) in phase I clinical trials of patients with advanced solid tumors and lymphoma (Table 1). All compounds have been reported as being well tolerated and, out of 31 patients treated with OSI-027, 8 have achieved stable disease (>12 weeks). The tumor types in which stable disease has been achieved are 3 colorectal and one each of melanoma, neuroendocrine, endometrial, renal, and cervical. 119 Recent preclinical studies suggest that resistance to these mtor s is likely to occur via the establishment of a new steady state in which there is accumulation of activated AKT phosphorylated on Thr308, but not Ser Phosphorylation of AKT at Thr308 was shown to be sufficient for full reactivation of AKT signaling; however, this was reliant on RTK activation. Combined inhibition of mtor kinase and the induced RTKs fully abolished AKT signaling and resulted in substantial cell death and tumor regression in vivo. These findings suggest that, similar to rapalog resistance, resistance to the mtor kinase s may be mediated by increased AKT signaling, although the feedback mechanisms inducing AKT activation via the 2 s is likely to be different (Fig. 3). Thus, combining RTK s with mtor s probably will be necessary to overcome resistance to mtor kinase s. B. Targeting PI3K At present, there are 12 PI3K-targeted compounds in clinical trials (Table 1), of which 5 are dual PI3K/ mtor s. The dual s simultaneously target the catalytic domains (ATP binding sites) of mtor and the p110 subunits of class 1 PI3Ks and subsequently block mtorc1, mtorc2, and PI3K activity. 120 Targeting PI3K in addition to mtor potentially will overcome the increased AKT activation associated with the mtorc1 and mtorc2 s and thus this mechanism of resistance. Cell line and animal model studies featuring both normal and dysregulated PI3K signaling, including loss of PTEN function or gain of function PI3K mutations, clearly have demonstrated that the dual PI3K/ mtor s are more potent than rapamycin 121 and exhibit strong antiproliferative activity and, in some cases, tumor vascular reduction In mouse models, these compounds generally induce tumor stasis, 123,124,126 but they have been shown to cause tumor regression in a mouse model of lung cancer. 128 However, resistance to these s Critical Reviews in Oncogenesis

9 PI3 Kinase/AKT/mTOR Signaling in Cancer 77 TABLE 1. PI3K Continued Pathway Inhibitors in Clinical Trials Targets Compound Other Targets (IC50) Company Mechanism Trial Reported Efficacy Responsive Tumor Type Tumor Type in Trials Combined With Therapy Type (Therapeutic) Clinical Trials (n) PI3K and mtor PF Pfizer Catalytic PF Pfizer Catalytic BEZ-235 DNA-PK Novartis Catalytic (20nM) 1184 SF-1126 DNA-PK, PIM1, PLK1, CK2, ATM Semaphore Catalytic GDC-0980 Genentech Catalytic XL765 Exelixis Catalytic GSK Glaxo Smith- Kline Catalytic PI3K BKM120 Novartis Catalytic Phase I Solid MEKi (PD ): endocrine (letrozole) Phase I Solid MEKi (PD ): cytotoxic (irinotecan) Phase I/II 2 PR: PR: Lung, 28% SD 133 breast Solid, breast, endometrial MEKi (MEK162): endocrine (letrozole): cytotoxic (paclitaxel): paclitaxel + HER2i (trastuzumab) Phase I 58% SD 131 Solid 1 Phase I/II 4 PR 135 PR: Mesothelioma, GIST Solid, non- Hodgkin s lymphoma, breast, RCC Phase I/II 20%SD 132 Solid, NSCLC, glioma, breast Phase I 2 PR 136 PR: Kidney, bladder Phase I/II 2 PR: 40% PR: Breast Solid, SD 140 prostate, colorectal, breast, glioma, endometrial, leukemia, ovarian, RCC, NSCLC Cytotoxic (fluoropyrimidine, paclitaxel, carboplatin, capecitabine): VEGF-Ai (bevacizumab): Paclitaxel and carboplatin ± bevacizumab or HER2i (trastuzumab): mtorc1i (everolimus): endocrine (fulvestrant) Cytotoxic (temozolomide): endocrine (letrozole): EGFRi (erlotinib) Solid MEKi (GSK ) 2 Cytotoxic (carboplatin + paclitaxel, irinotecan, capecitabine, docetaxel, pemetrexed): Endocrine (fulvestrant, letrozole): VEGFR-Ai (bevacizumab):meki (GSK , MEK162): paclitaxel +/- HER2i (trastuzumab) Volume 17, Number

10 78 Sheppard et al. TABLE 1. Continued Targets Compound Other Targets (IC50) Company Mechanism Trial Reported Efficacy Responsive Tumor Type Tumor Type in Trials Combined With Therapy Type (Therapeutic) Clinical Trials (n) PX-866 Oncothyreon Catalytic: inhibits p110 p110 and p110 XL147 Exelixis Catalytic GDC-0941 mtor (413nM), DNA-PK (335nM) 119 ZSTK474 mtor (377nM), DNA-PK (436nM) 119 Genentech Catalytic Zenyaku Kogyo Catalytic Cal101 Calistoga Catalytic: inhibits p110 Phase I/II 23% SD 145 Solid, glioblastoma, prostate, colorectal, squamous cell Phase I/II 1 PR: PR: Lung Solid, 20% SD 143 lymphoma, breast, NSCLC, endometrial, ovarian, glioma Phase I/II 5 PR 141,142 PR: Endocervical, breast, melanoma, ovarian, GIST Solid, non- Hodgkin lymphoma, breast, NSCLC Cytotoxic (docetaxel): EGFRi (cetuximab) Cytotoxic (paclitaxel + carboplatin): HER2i (trastuzumab) ± paclitaxel: endocrine (letrozole): EGFRi (erlotinib): MEKi (MSC B) MEKi (GDC-0973/ XL518): HER2i (trastuzumab): EGFRi ( erlotinib): cytotoxic (paclitaxel) + VEGFR-Ai (bevacizumab): paclitaxel + carboplatin ± bevacizumab: endocrine (fulvestrant) Phase I Solid 1 Phase I/II 62% PR 147 : PR: 33% PR 148 Non- Hodgkin lymphoma (62%), CLL (33%) Haematological CD20i (ofatumumab, rituximab): cytotoxic (bendamustine, fludarabine): rituximab + bendamustine Critical Reviews in Oncogenesis

11 PI3 Kinase/AKT/mTOR Signaling in Cancer 79 TABLE 1. Continued Targets Compound Other Targets (IC50) Company Mechanism Trial Reported Efficacy Responsive Tumor Type Tumor Type in Trials Combined With Therapy Type (Therapeutic) mtor OSI-027 OSI Pharmaceuticals Catalytic AZD-8055 Astra Zeneca Catalytic INK-128 Intellikine Catalytic Phase I 26% SD 119 SD: Colorectal, melanoma, neuroendocrine, endometrial, renal, cervical Solid, lymphoma Phase I/II Solid, glioma, hepatocellular Phase I Solid, breast, lymphoma, MM, WM Cytotoxic (paclitaxel) ± HER2i (trastuzumab) mtorc1 Sirolimus (Rapamycin) Pfizer Allosteric FDA approved (organ rejection) Reviewed in Targeted therapy, standard Alvarado 185 chemotherapy, and radiation therapy for many tumor types Temsirolimus (CCI-779) Everolimus (RAD001) Pfizer Allosteric Novartis Allosteric FDA approved FDA approved Reviewed in Targeted therapy, standard Alvarado 185 chemotherapy, and radiation therapy for many tumor types Reviewed in Targeted therapy, stan- Alvarado 185 dard chemotherapy, and radiation therapy for many tumor types Clinical Trials (n) >600* >600* >600* Volume 17, Number

12 80 Sheppard et al. TABLE 1. Continued Targets Compound Other Targets (IC50) Company Mechanism Trial Reported Efficacy Responsive Tumor Type Tumor Type in Trials AKTi Perifosine (KRX-0401) Ridaforolimus Merck & Ariad Allosteric Triciribine (VQD-002) Transduction pathways, including JNK Keryx PIP3- binding VioQuest PIP3-binding Phase I/ II/III Phase I/ II/III Reviewed in Solid, Alvarado 185 breast, prostate, NSCLC, glioma, sarcoma, endometrial, hematologic, ovarian, head and neck, colon, PR 161,162 PR: Sarcoma, WM Phase I PR 158 PR: Squamous cell carcinoma Solid, NSCLC, breast, ovarian, endometrial, prostate, head and neck, pancreatic, WM, glioma, colon; sarcoma, liver, kidney, lung, hematologic Cancer, hematologic GSK Glaxo SmithKline Catalytic Phase I 1 PR and PR: Anal Solid, SD 175 ovarian, lymphoma Combined With Therapy Type (Therapeutic) Clinical Trials (n) HER2i (trastuzumab): VGFR-Ai (bevacizumab): IGFRi (dalotuzumab): endocrine (bicalutamide): Notchi (MK-0752): AKTi ( MK-2206): HDACi (vorinostat): cytotoxic (doxorubicin, paclitaxel, carboplatin) 17 Cytotoxic (gemcitabine, paclitaxel, capecitabine, docetaxel): docetaxel ± prednisolone: TKi (imatinib, sunitinib, sorafenib): steroid (dexamethasone) ± proteasome (bortezomib): dexamethasone + lenalidomide: CHK1i (UCN-01): TORC1i (temsirolimus) 27 2 MEKi (GSK ) 3 Critical Reviews in Oncogenesis

13 PI3 Kinase/AKT/mTOR Signaling in Cancer 81 TABLE 1. Continued Targets Compound Other Targets (IC50) Company Mechanism Trial Reported Efficacy Responsive Tumor Type Tumor Type in Trials Combined With Therapy Type (Therapeutic) Clinical Trials (n) GSK Glaxo SmithKline Catalytic GDC0068 Genentech Catalytic Phase I/II Haematologic Proteasome (bortezomib) + steroid (dexamethasone) Phase I Solid Cytotoxic (docetaxel): regime (LOLFOX6) 3 2 MK-2206 Merck Allosteric RX-0201 Rexahn AKT1 antisense Phase I/II 3 PR: 32% PR: Pancreatic, SD 172,173 melanoma, neuroendocrine Solid, NSCLC, breast, ovarian, prostate, neuroendocrine, pancreatic, WM, glioma, colon, gastric, oesophageal, GIST; sarcoma, kidney; melanoma, hematologic Cytotoxic therapy (carboplatin + paclitaxel, docetaxel): HER2i (trastuzumab): paclitaxel + trastuzumab: endocrine (anastrozol, letrozole, exemestane, fulvestrant, goserelin, bicalutamide): EGFRi (erlotinib): IGFRi (dalotuzumab): HER2i (lapatinib): cytotoxic (bendamustine) + CD20i (rituximab): MEKi (AZD6244): mtorc1i (ridaforolimus, everolimus) Phase I/II Pancreatic Cytotoxic (gemcitabine) 34 1 * More than 600 trials including all 3 mtorc1 s: sirolimus, temsirolimus, and everolimus. ATM, ataxia telangiectasia mutated protein kinase; ATP, adenosine triphosphate; CD20i, binds and inhibits CD20 protein; CHK1i, checkpoint kinase 1 ; CK2, casein kinase 2; CLL, chronic lymphocytic leukemia; DNA-PK, DNA-dependent protein kinase; EGFRi, epidermal growth factor receptor ; ERK, extracellular signal regulated kinase; FDA, US Food and Drug Administration; GIST, gastrointestinal stromal tumor; HDACi, histone deacetylase ; HER2i, HER2 receptor ; IGFRi, insulin-like growth factor ; MEKi, MEK1/2 ; MM, multiple myeloma; mtor, mammalian target of rapamycin; mtorc1, mammalian target of rapamycin complex 1; NSCLC, non small-cell lung cancer; PI3K, phosphatidylinositol 3 kinase; PIM1, protooncogene serin/threonine protein kinase 1; PIP3, phosphatidylinositol (3,4,5)P3; PLK1, polo-like kinase 1; PR, partial response; RCC, renal cell carcinoma; SD, stable disease; WM, Waldenström macroglobulinemia; VEGFR-Ai, vascular endothelial growth factor receptor A. Volume 17, Number

14 82 Sheppard et al. has been reported in preclinical studies. A recurring theme is that activation of the Ras/ERK pathway decreases the efficacy of these compounds These observations are not surprising given the extensive cross-talk between the PI3K and Ras/ERK pathways; the latter can promote signaling along the PI3K pathway at several points (discussed earlier), and ERK signaling plays a major role in cell growth and proliferation. SF-1126, XL765, BEZ235, PF , PF , GDC-0980, and GSK are all dual PI3K/mTOR s that are currently in clinical trials. SF-1126 is a prodrug of LY that is linked to an integrin antagonist and designed to target tumor vasculature. In a phase I study of SF-1126, this compound was tolerated well and, although no objective responses were observed, disease stabilization was seen in 19 out of 33 patients (58%) across multiple solid tumors. 131 One patient, whose disease had progressed while taking the rapalog temsirolimus, showed prolonged stable disease (>1 year) while taking SF-1126, suggesting that, similar to the preclinical models in which the dual s were more effective than rapalogs, these s may also be more effective in patients. XL765 so far has been reported as being well tolerated and of 79 patients treated, 18 had stable disease (>16 weeks). 132 In biopsies taken after treatment, there was evidence of PI3K pathway inhibition (by 60 80%) and, notably, a concurrent decrease in phospho- ERK (by 50 80%). 132 In clinical trials, BEZ235 has induced a partial response in 2 patients one with lung cancer and the other with breast cancer and induced stable disease in 28% of patients. 133 It is well tolerated, but because of poor pharmacodynamics, additional clinical trials using alternative formulations are now in progress. 134 GDC-0980 has demonstrated single-agent antitumor activity in 3 patients with mesothelioma and one gastrointestinal stromal tumor 135 and currently is under further evaluation in a range of solid tumors. Partial responses have been reported in patients treated with GSK : one with renal cell carcinoma (>11 months) and one with bladder cancer. This compound also has entered into a clinical trial in combination with a ERK kinase (MEK). So far there are no published reports on the two Pfizer dual PI3K/ mtor s, PF and PF , that are currently in Phase 1 clinical trials as single agents and in combination with a MEK ( The PI3K selective s can be divided into isoform-specific s or pan-pi3k s, which target all class I PI3Ks. Preclinical studies suggest that, compared with the dual PI3K/mTOR s, they are not as effective at inhibiting cell proliferation 137,138 and, similar to the dual PI3K/ mtor s, these compounds tend to induce tumor stasis in vivo rather than tumor regression. 139 The clinical data presented so far on these s as single agents are more promising than might be expected from preclinical results, with some patients showing partial tumor regression. Several of these compounds are now in clinical trials in combination with various agents including an AKT (GSK ), cytotoxic agents (carboplatin, paclitaxel), specific MEK s, RTK s (both specific, e.g., the HER2 monoclonal antibody trastuzumab and the pan-rtk XL647), endocrine therapy (letrozole, fulvestrant), antiangiogenics (bevacizumab), and DNA synthesis s (capecitabine). BKM120, GDC-0941, XL-147, and GSK are all pan-pi3k s that target class I PI3K enzymes with no mtor y activity. In a phase I clinical trial, treatment with BKM120 resulted in partial tumor responses in 2 breast cancer patients. Interestingly, the tumor of one patient had a KRAS mutation, a predictor of resistance based on preclinical studies. Forty percent of patients (18 of 45 evaluable) had stable disease (>16 weeks), including 8 patients who had tumors with an activated PI3K pathway. A trend toward better activity was observed in the higher-dose cohorts. 140 On the basis of these promising data, BKM120 is under evaluation in 19 clinical trials in combination with various agents (Table 1). GDC-0941 has been assessed in 2 phase 1 clinical trials. Of 49 patients, 2 have exhibited partial responses: one with endocervical cancer (with a PIK3CA mutation) and one with estrogen receptor positive HER2 breast cancer. Additional signs of antitumor activity included Critical Reviews in Oncogenesis

15 PI3 Kinase/AKT/mTOR Signaling in Cancer 83 CA-125 responses in 3 patients with ovarian cancer, one of whom had a known high PIK3CA gene copy number, was on-study for 15 months, and had a 23% decrease in the target lesion. 141 In a second study, 42 patients were enrolled and signs of clinical activity included a partial response in 3 patients: one with a melanoma (V600E BRAF mutant), one a with small-bowel gastrointestinal stromal tumor, and one with ovarian cancer (PTEN negative). 142 In clinical trials, XL-147 has demonstrated a partial response in a lung cancer patient and 9 patients had stable disease ( 24 weeks). 143 PX-866 is more limited in target inhibition, inhibiting p110, p110, and p110 with single-digit nanomolar IC50 values 144 ; only at much higher concentrations does it inhibit p110ß. Phase I clinical trial results showed that treatment inhibited S6 ribosomal protein and mtor phosphorylation, and stable disease was achieved in 23% of patients. 145 CAL-101 is a p110 -selective. The p110 isoform is restricted to cells of hematopoietic origin, thus making it potentially useful for the treatment of hematologic malignancies. 146 In phase I studies, single-agent CAL-101 showed high response rates of 62% in patients with non-hodgkin lymphoma and 33% in patients with chronic lymphocytic leukemia. 147,148 Overall, both the PI3K s and the dual PI3K/mTOR s are generally well tolerated, and the pharmacodynamic markers indicate that all the agents currently in clinical trials are effectively hitting the target and down regulating the PI3K pathway. In one study, phospho-erk also was shown to decrease in tumor biopsies, highlighting the crosstalk between these 2 pathways. 132 In many preclinical models, these drugs primarily promote tumor cell stasis or delay tumor growth without induction of apoptosis, 124,126,149,150 and this may explain why, in clinical trials where there was an effect of these therapeutics, they most often induced stable disease rather than tumor regression. Many of the PI3K s have off-target effects, 151 and these potentially could cause unwanted side effects or, conversely, promote antitumor activity. A common additional target of these s is DNA-PK, a kinase that is a crucial component of the nonhomologous end joining pathway that repairs double-strand DNA breaks and, when inhibited, renders cells acutely sensitive to ionizing radiation. 152 Thus it may be possible to combine PI3K s with this off-target effect with radiation therapy. In addition, DNA-PK can phosphorylate and activate AKT, again strengthening the argument that off-target effects of some of these agents may improve antitumor activity. C. Targeting AKT AKT is a crucial component in the PI3K pathway that not only signals to the mtorc1 complex to regulate protein translation and proliferation but also directly targets many proteins that impact cell metabolism, proliferation, and survival. 18,43 Cell line and animal model studies have demonstrated that AKT s are effective at inhibiting cell proliferation and xenograft tumor growth A large number of AKT s have been developed, which can be grouped into ATP mimetics and noncatalytic-site AKT s, including lipid-based phosphatidylinositol analogs and allosteric s. Most of the AKT s are nonselective, targeting all 3 AKT isoforms. An exception is RX-0201, which is a 20-mer antisense oligonucleotide with sequence homology to AKT-1 messenger RNA and is designed to specifically inhibit AKT-1 expression. In 2 human cell xenograft models, RX-0201 significantly inhibited tumor growth, 156 and it is currently in a phase I study in combination with gemcitabine. The 2 most clinically advanced AKT s are perifosine and triciribine. Both of these agents target the PH domain of all AKT isoforms, preventing AKT membrane translocation and thus activation. 153,157 Triciribine has been used in phase I and phase II studies. In 2 metastatic squamous cell carcinoma patients, triciribine at high doses induced a complete response (>19 months) and a partial response (>5 months) 158 but in many other cancers has little effect. 159,160 Because of its significant side effect profile, including hepatotoxicity, hyperglycemia, and hypocalcemia, its clinical use has been limited. Perifosine is currently in clinical trials as a single agent or in combination with various drugs to treat multiple types of cancers. Single-agent activity with perifosine has been reported in patients with Volume 17, Number

16 84 Sheppard et al. sarcoma and Waldenström macroglobulinemia, 161,162 but the response rates of common solid tumors to perifosine as a single agent have been poor and treatment has been associated with frequent adverse events MK2206, an allosteric AKT, has high y potency against both AKT1 and AKT2 with less activity toward AKT3. In preclinical models it was able to overcome completely the feedback activation of AKT from temsirolimus-induced mtor suppression, and the 2 s synergistically inhibited thyroid cancer cell growth. 168 In addition, enhanced antitumor effects of MK2206 also are apparent when used in combination with both targeted and cytotoxic agents MK2206 is the first allosteric AKT used in clinical studies and, as a single agent, it induced stable disease in 6 of 19 patients with advanced solid tumors. 172 In a separate trial, evidence for tumor shrinkage was observed in patients with pancreatic, melanoma, and neuroendocrine tumors. 173 Currently, multiple studies with various drug combinations, including both targeted and cytotoxic agents, are ongoing. In a phase I study in HER2+ treatment-refractory breast and gastroesophageal cancers, treatment with MK2206 and concurrent trastuzumab resulted in complete remission in a breast cancer patient and prolonged stable disease in 4 of 24 patients. 174 GSK is an ATP-competitive AKT kinase, which targets all 3 AKT isoforms. In a phase 1 clinical trial, tumor regression and stable disease have been observed in patients with an activated PI3K pathway. 175 When combined with a MEK, GSK also has shown clinical activity (3 of 13 patients): 2 patients with ovarian cancer and one with endometrial cancer had tumor regression after 8 weeks of treatment. 176 GDC-0068 is also an ATP-competitive, pan-akt that is highly selective over other kinases and is currently in phase I clinical trials. In summary, the initial studies with AKT s that target the PH domain and thus inhibit AKT translocation to the membrane have shown poor responses overall in clinical trials. There is promise with the allosteric and ATP-competitive, pan-akt kinase s, with some patients showing tumor regression; however, as with the PI3K s, most patients demonstrate stable disease. V. RESISTANCE AND SENSITIVITY TO PI3K PATHWAY TARGETED THERAPIES Mechanisms conferring resistance to PI3K pathway targeted therapies are likely to fall into several categories: (1) efflux by transmembrane proteins, drug metabolism, or extracellular sequestration by plasma proteins, resulting in low drug exposure; (2) activating mutations in or gene amplification of kinases downstream of the targeted therapy; (3) acquisition of gatekeeper mutations, which block binding of the ; (4) increased signaling through parallel pathways (these could be primary or acquired); and (5) loss of negative feedback loops and cross-talk with other pathways 177 (Fig. 3). In terms of this last point, preclinical studies have demonstrated that mtorc1 inhibition results in feedback activation of AKT 100,115 and the Ras/ERK pathway. 101 Upregulation of these 2 pathways also has been observed in patients treated with rapalogs. 101,114,178,179 The concordance between these preclinical and clinical studies validates the use of current cell and mouse models in predicting responses and resistance mechanisms to targeted therapy. Concomitant inhibition of RTK, PI3K, or AKT blocks the rapamycin-induced AKT activation and enhances inhibition of tumor cell growth. 100,115,180,181 Such combinational strategies are currently being implemented in phase I clinical trials. Preclinical models also predict that increased signaling through the MAPK/ERK pathway will confer resistance to PI3K pathway s On the basis of this data there are several clinical trials underway in which pan-pi3k and dual PI3K/ mtor s are being combined with MEK s (Table 1). Undoubtedly, preclinical data will reveal more resistance mechanisms and facilitate the rational development of effective combinational therapies for PI3K therapeutics. Identifying patients who are likely to respond to treatment is essential to produce greater effectiveness with less toxicity and will likely reduce the cost of cancer care. For kinase s, one of the best predictors of success so far has been the presence Critical Reviews in Oncogenesis

17 PI3 Kinase/AKT/mTOR Signaling in Cancer 85 of an activating mutation or other genomic alteration in the targeted kinase. For example, the use of vemurafenib (PLX4032) for melanoma patients with BRAF mutations 182 and gefitinib for lung cancer patients with mutations of EGFR. 183 This suggests that PI3K and mtor s should be effective in tumors featuring activating mutations in p110 or loss of PTEN, whereas AKT mutations would be expected to sensitize a tumor to AKT s. Preclinical data on the PI3K pathway s indicate that mutations in the pathway do predict sensitivity to these agents. 124,130,154 In contrast, the majority of patients with detected PTEN loss or PIK3CA mutations have not responded to monotherapy. There could be several reasons for this, one being that the current s do not lead to complete shutdown of the targeted kinase and the residual signaling is enough to sustain tumorigenesis. Alternatively, the presence of additional mutations in upstream RTKs or in parallel pathways such as Ras/ERK could lead to resistance to PI3K pathway inhibition. Despite extensive preclinical and clinical evaluation of PI3K pathway s, there are currently no reliable biomarkers that successfully select patients who are likely to respond. Understanding the complex interplay, both within the PI3K pathway and in the broader associated signaling network, will be crucial in designing effective combinational therapies for PI3K therapeutics. In addition, extensive biochemical and genomic tumor profiling likely will be necessary for appropriate patient selection and hence greater success in achieving patient responses. VI. CONCLUSIONS There are many PI3K pathway s currently in clinical trials both as monotherapy and in various combinations with other targeted therapies and chemotherapy. To date the therapeutics targeting the PI3K pathway are relatively well tolerated and, in some patients, induce stable disease but rarely induce tumor regression. At least in part, this seems to be because of incomplete inactivation of key components of the PI3K-dependent signaling network. Given the dysregulation of the PI3K pathway in many malignant phenotypes, targeting the pathway for the treatment of cancer is still warranted. To achieve better patient outcomes, several strategies could be employed, including optimization of drug administration to more effectively inhibit the pathway, identification of patients who are most likely to benefit, identification of drug combinations that increase activity or overcome mechanisms of resistance, and development of more effective s including both isoform or mutant-specific s. REFERENCES 1. Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet. 2006;7(8): Zhao JJ, Liu Z, Wang L, Shin E, Loda MF, Roberts TM. The oncogenic properties of mutant p110alpha and p110beta phosphatidylinositol 3-kinases in human mammary epithelial cells. Proc Natl Acad Sci U S A. 2005;102(51): Isakoff SJ, Engelman JA, Irie HY, Luo J, Brachmann SM, Pearline RV, Cantley LC, Brugge JS. Breast cancer-associated PIK3CA mutations are oncogenic in mammary epithelial cells. Cancer Res. 2005;65(23): Kang S, Bader AG, Vogt PK. Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenic. Proc Natl Acad Sci U S A. 2005;102(3): Bader AG, Kang S, Vogt PK. Cancer-specific mutations in PIK3CA are oncogenic in vivo. Proc Natl Acad Sci U S A. 2006;103(5): Puc J, Keniry M, Li HS, Pandita TK, Choudhury AD, Memeo L, Mansukhani M, Murty VV, Gaciong Z, Meek SE, Piwnica-Worms H, Hibshoosh H, Parsons R. Lack of PTEN sequesters CHK1 and initiates genetic instability. Cancer Cell. 2005;7(2): Shen WH, Balajee AS, Wang J, Wu H, Eng C, Pandolfi PP, Yin Y. Essential role for nuclear PTEN in maintaining chromosomal integrity. Cell. 2007;128(1): Gu T, Zhang Z, Wang J, Guo J, Shen WH, Yin Y. CREB is a novel nuclear target of PTEN phosphatase. Cancer Res. 2011;71(8): He J, de la Monte S, Wands JR. The p85beta regulatory subunit of PI3K serves as a substrate for PTEN protein phosphatase activity during insulin Volume 17, Number