AKT signaling pathway and cancer: an updated

Annals of Medicine, 2014; Early Online: 1–12 © 2014 Informa UK, Ltd. ISSN 0785-3890 print/ISSN 1365-2060 online DOI: 10.3109/07853890.2014.912836

REVIEW ARTICLE

PI3K/AKT signaling pathway and cancer: an updated review Miriam Martini, Maria Chiara De Santis, Laura Braccini, Federico Gulluni & Emilio Hirsch

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Molecular Biotechnology Center, Department of Molecular Biotechnology and Health Sciences, University of Turin, Italy

Despite development of novel agents targeting oncogenic pathways, matching targeted therapies to the genetic status of individual tumors is proving to be a daunting task for clinicians. To improve the clinical efficacy and to reduce the toxic side effects of treatments, a deep characterization of genetic alterations in different tumors is required. The mutational profile often evidences a gain of function or hyperactivity of phosphoinositide 3-kinases (PI3Ks) in tumors. These enzymes are activated downstream tyrosine kinase receptors (RTKs) and/or G proteins coupled receptors (GPCRs) and, via AKT, are able to induce mammalian target of rapamycin (mTOR) stimulation. Here, we elucidate the impact of class I (p110a, b, g, and d) catalytic subunit mutations on AKTmediated cellular processes that control crucial mechanisms in tumor development. Moreover, the interrelation of PI3K signaling with mTOR, ERK, and RAS pathways will be discussed, exploiting the potential benefits of PI3K signaling inhibitors in clinical use. Key words: Cancer, mTOR, PI3K, PI3K inhibitors, Ras, signaling

Key messages • The PI3K pathway is one of the most frequently activated signal transduction pathways in human cancer. • Alterations of the PI3K pathway may be responsible for drug sensitivity or resistance to specific therapeutic agents.

eukaryotic organisms. Vps34 was first discovered in yeast, and it is implicated in integrating cellular responses to changing nutritional status. In this review, we will analyze in details the physiological functions of different PI3Ks isoforms, their involvement in cancer development, and their therapeutic implications.

The phosphoinositide 3-kinases (PI3Ks) and their functions

Introduction

Class I PI3Ks

The phosphoinositide 3-kinases (PI3Ks) are a large family of lipid enzymes able to phosphorylate the 3’-OH group of phosphatidylinositols (PtdIns) on the plasma membrane. They were first discovered more than 25 years ago in association with the transforming ability of viral oncoproteins (1,2). Over the following years, studies established a key role for PI3Ks signaling in different cellular processes such as metabolism, inflammation, cell survival, motility, and cancer progression (3). This plethora of functions is exerted by distinct PI3K enzymes that are highly related to each other and that can be distinguished by substrate specificity, structure, and mechanism of activation/regulation. Three classes of PI3Ks (class I, class II, and class III) have been identified in mammals. Class I PI3Ks are further divided in class IA (p110α, p110β, and p110δ) and class IB (p110γ), and this is the best-characterized class implicated in cancer disease. Class II PI3K includes three different enzymes (PI3K-C2α, PI3K-C2β, and PI3K-C2γ), and it remains the most enigmatic among all PI3Ks, despite recent studies providing novel clues about its role in signal transduction. Finally, there is only one known class III PI3-kinase member named vacuolar protein sorting 34 (Vps34, also known as PI3K-C3), and it is also the only PI3-kinase expressed in all

Class I PI3Ks are heterodimeric enzymes which consist of a regulatory subunit and a catalytic subunit (p110) (1). Mammalian genome encodes four different p110 isoforms (α, β, γ, and δ) and several regulatory subunits. For p110α, p110β, and p110δ, the most common regulatory subunit is named p85, while p110γ associates with the p101 and p84/p87 regulatory subunits (1). Physiologically, class I PI3K occurs as obligatory dimers in cells (4) and can transduce signals received from activated tyrosine kinase receptors (RTKs), G protein-coupled receptors (GPCRs), and from activated RAS (5). Upon stimulation, the regulatory subunit may interact with the intracellular portion of the activated receptor. This interaction allows the activation of the catalytic subunit which, in turn, may associate with the lipid membranes to phosphorylate the PtdIns(4,5)P2 to PtdIns(3,4,5)P3 (5). The final product of this reaction, the PtdIns(3,4,5)P3, functions as an important second messenger in the cell and is the principal mediator of the class I PI3Ks activity (Figure 1). Together with PtdIns(3,4)P2, PtdIns(3,4,5)P3 constitutes a docking site for proteins that contain a pleckstrin homology (PH) domain, controlling their localization and activation. The PI3K signaling cascade is mainly mediated by the activation of AGC kinases such as the

Correspondence: Emilio Hirsch, Molecular Biotechnology Center, Dipartimento di Biotecnologie Molecolari e Scienze per la Salute, Via Nizza 52, Turin 10126, Italy. E-mail: [email protected] (Received 14 January 2014; accepted 31 March 2014)


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Figure 1. Overview of PI3K/AKT/mTOR signaling pathway. Class I PI3Ks are activated by growth factors through GPCR or RTK receptors. The PI3K activation results in the conversion of in PtdIns(4,5)P2 to PtdIns(3,4,5)P3, a process that is reversed by phosphatase PTEN. PtdIns(3,4,5)P3 constitutes a docking site for PH-containing proteins (PDK1 and AKT) recruitment and activation. Subsequently, AKT removes the inhibition on the mTOR/Raptor complex (also known as mTORC1), thus leading to mTORC1 activation. Other intracellular pathways also converge on the mTORC1 complex. One of these is constituted by the Ras-dependent LKB1 pathway, altered in Peutz–Jeghers syndrome. Upon LKB1 activation by Ras, this kinase is able to phosphorylate AMPK (AMP-activated protein kinase), that in turn activates mTORC1 inhibitor TSC2. Another mTORC1-converging pathway is mediated by nutrientregulated Vps34, which acts positively on mTOR/S6K1, thus integrating glucose and amino-acid inputs on the mTOR pathway. PtdIns(3,4,5)P3 constitutes a docking site also for other kinases such as TEC kinases and small GTPases (P-Rex1/Rac) involved in cell adhesion/migration, actin reorganization, and apoptosis.

phosphoinositide-3-kinase-protein kinase B/AKT (PKB/AKT), p70S6K, and serum- and glucocorticoid-induced protein kinase (SGK) (6,7). AKT is one of the major downstream effectors of PI3K, and it was originally identified as a crucial component of the insulin receptor intracellular signaling (6). Upon PI3K activation, AKT is translocated via its PH domain to the inner membrane, where it is phosphorylated by PDK1 on its activation loop (T308) (8). This AKT modification is sufficient to activate the mammalian target of rapamycin complex 1 (mTORC1) by the direct phosphorylation and activation of the proline-rich AKT substrate of 40 kDa (PRAS40) and the tuberous sclerosis protein 2 (TSC2). Activation of mTORC1 results in increased protein synthesis and cell survival by direct phosphorylation of its effectors, such as the ribosomal S6 kinase, 70 kDa, polypeptide (S6K1 and S6K2), and 4E-BPs (elF4E-binding proteins) to terminate binding to elF4E and relieve the block on translation (9,10). While mTORC1 conveys signals following PI3K-AKT activation, another mTOR complex, mTORC2, contributes to the full activation of AKT by phosphorylating its serine 473 (Figure 1). Complete AKT activation leads to additional substrate-specific phosphorylation events in both cytoplasm and nucleus, including inhibitory phosphorylation of the pro-apoptotic FOXO proteins. Besides AKT and PDK1, guanosine diphosphate (GDP)-GTP exchange factors for Rac and for ADP-ribosylation factors 6 (ARF6) have also been identified as key effectors in the PI3Kmediated cytoskeletal remodeling and membrane trafficking,

respectively (11). In particular, PtdIns(3,4,5)P3 is required to activate several Rac-specific GEFs, such as P-Rex and Vav, which in turn activate Rac to promote cell adhesion, migration, cell cycle progression, and transformation (12–14). Moreover, PI3K can trigger the activity of TEC tyrosine kinases, a family of nonreceptor protein-tyrosine kinases (PTKs) characterized by the presence of a PH domain (15). In order to be activated, TEC kinases have to be recruited to the cell membrane, via their PH domain, and need to be phosphorylated on tyrosine residues by members of the Src kinase family (16). Therefore, the TEC kinase family acts as signal integrators between phospholipid- and phosphotyrosine-mediated pathways. On the other hand, GAP inhibitors of Rho GTPases can also be regulated by PIP3 binding and PI3K-mediated signaling, thus controlling the balance between activating and inhibitory stimuli (17). PI3K-AKT signaling may be antagonized by the tumor suppressor phosphatase and tensin homolog (PTEN), that was identified as a frequently mutated gene in many types of tumors particularly endometrium, skin, brain, and prostate (18,19). Soon after its discovery, it was shown that PTEN has a strong phosphatase activity for the lipid-signaling second messenger PtdIns(3,4,5)P3 (Figure 1) (20). This kind of lipid phosphatase activity is the best characterized physiological function contributing to the tumor suppressor function of PTEN. It is not surprising that loss of PTEN activity has a great impact on different aspects of tumor development. PTEN is constitutively expressed in normal tissues,


PI3K/Akt signaling pathway and cancer 3 but its mRNA and protein levels are finely regulated. A number of factors including transforming growth factor β (TGFβ), insulinlike growth factor 2 (IGF-2), peroxisome proliferation-activated receptor γ (PPRγ), and the tumor suppressor p53 may transcriptionally control PTEN mRNA levels (21). Also different microRNAs, frequently upregulated in cancer, such as miR-21, miR-221, and miR-222, contribute to a post-transcriptional control of PTEN (21). At the protein level, the C-terminal tail of PTEN contains several phosphorylation sites (22). The phosphorylation leads to a closed state of PTEN and contributes to the protein stability, while its dephosphorylation opens the PTEN phosphatase domain, increasing its activity (22). However, the open state of PTEN is more susceptible to ubiquitin-mediated proteosome degradation. Moreover, it is known that other phosphatases, besides PTEN, are involved in the termination of signaling downstream PI3Ks by degradation of PtdIns, for instance Src-homology 2 (SH2)-containing inositol 5′-phosphatase (SHIP), which dephosphorylates PtdIns(3,4,5)P3 into PtdIns(3,4)P2, and inositol polyphosphate 4-phosphatase type II (INPP4B) which hydrolyzes the 4-position of PtdIns(3,4)P2. The family of SHIP phosphatases includes two members, SHIP-1 and SHIP-2. The SHIP-1 isoform is expressed in endothelial cells and in the hematopoietic lineage (23,24), while SHIP-2 is ubiquitously expressed. These enzymes are known to play a key role in the negative regulation of AKT activity (25,26). On the other hand, INPP4 has been originally characterized as a Mg2⫹-independent enzyme that catalyzes the hydrolysis of the 4-position phosphate of PtdIns(3,4)P2 (27). Two INPP4 phosphatases have been described, INPP4A (also known as type I) and INPP4B (type II), showing different expression patterns, tissue distribution, and subcellular localization. Similarly to PTEN, INPP4A and INPP4B possess one and two C2 domains, respectively, that allow the interaction with the plasma membrane via electrostatic and phosphoinositide interactions (28,29). As both PtdIns(3,4)P2 and PtdIns(3,4,5)P3, produced by PI3Ks, are necessary for the full activation of AKT, INPP4A and INPP4B have the potential negatively to regulate AKT activation following insulin and IGF-1 (28,30). Different studies show that INPP4A is mainly involved in endocytosis, and its function is critical for neuronal function. In contrast, the unique report about the role of INPP4B in growth factor signaling showed that INPP4B acts as suppressor of the insulin-mediated PI3K signaling (31). Knocking down of INPP4B in cells displays an increment in the magnitude and duration of AKT activation following insulin stimulation (31). Integrating a constitutive activation of the PI3K-AKT pro-survival pathway and the loss of phosphatase activity, tumors acquire a peculiar way to evade apoptosis and promote cell growth and proliferation.

Class II PI3Ks Class II PI3Ks are monomers of high molecular mass that differ from class I and class III for their long N- and C-terminal domains (32). In mammals, three different class II members have been identified: the ubiquitously expressed PI3K-C2α and PI3KC2β, and the liver-specific PI3K-C2γ (32). Contrarily to class I, class II PI3Ks are predominantly involved in regulating vesicular trafficking and recognize as substrate PtdIns and PtdIns(4)P producing PtdIns(3)P and PtdIns(3,4)P2, respectively. Several stimuli such as hormones, growth factors, chemokines, and cytokines may activate class II PI3Ks through different membrane receptors including RTKs (EGFR and PDGFR) and GPCRs (32). Among class II PI3Ks, the best studied is PI3K-C2α. At the cellular level, PI3K-C2α has been demonstrated to regulate full translocation of the glucose transporter GLUT4 to the plasma membrane of muscle cells, a key event for the regulation of glucose homeosta-

sis (33). In addition, a recent report established PI3K-C2α as an essential spatio-temporal regulator of clathrin-mediated endocytosis through the localized production of PtdIns(3,4)P2 at the plasma membrane (34). At a physiological level, a study in mice first reported an essential role for PI3K-C2α in angiogenesis and vascular barrier function (35). Global or endothelial cell-specific deficiency of PI3K-C2α resulted in embryonic lethality caused by defects in sprouting angiogenesis and vascular maturation (35). In a xenograft model, a role of PI3K-C2α in tumor growth was also reported. Pik3c2a endothelial-restricted knock-out mice had reduced tumor volumes/weights compared to controls, suggesting that the in vivo pro-angiogenetic function of PI3K-C2α is required for tumor growth and maintenance (35). Although less well characterized than class I PI3Ks at the functional level, C2β has recently been implicated as being important in cell migration in several epithelial lines and in the differentiation of HL-60 hematopoietic cells by retinoic acid (36,37). The null mouse reported for its coding gene Pik3c2b is viable and fertile (38). Its kinase activity has been associated to cell migration since its downregulation inhibits the LPA-dependent migration of ovarian and cervical cancer cell lines. PI3K-C2β overexpression was also associated to inhibition of apoptosis induced by downregulation of intersectin in a neuroblastoma cell line, suggesting that PI3K-C2β may be involved in survival of neuronal cells (39). Recently, it was also reported that the intersectin 1 (ITSN1) scaffold stimulates RAS activation on endocytic vesicles without activating classic RAS effectors but involving PI3K-C2β. A direct association between PI3K-C2β and nucleotide-free RAS was shown, suggesting a novel role for nucleotide-free RAS in cell signaling in which PI3K-C2β stabilizes nucleotide-free RAS and that the interaction of RAS and PI3K-C2β mutually inhibits each another (40). At present, there is no evidence regarding a specific intracellular function for PI3K-C2γ, the third member of class II PI3K, which remains the least characterized PI3K enzyme.

Class III PI3K Vacuolar protein sorting 34 (Vps34) represents the unique member of class III, and it is mainly involved in generation of PtdIns(3) P, a central phospholipid for membrane trafficking processes (41). The first known function of Vps34 was indeed the regulation of vesicular trafficking in the endo-lysosomal system (42,43). Its production of PtdIns(3)P recruits effector proteins containing PtdIns(3)P binding domains that control membrane docking and fusion during the formation of internal vesicles required for recycling and autophagy pathways (41). More recently, Vps34 has also been implicated in other intracellular processes such as nutrient sensing in the mTOR pathway in mammalian cells and MAPK (mitogen-activated protein kinase) signaling in yeast (41,44,45).

Genetic alterations in PI3K pathway The PI3K pathway is a theater with several actors: during good performances all characters play roles that have been assigned to them, but sometimes one or more actors start to wear the wrong disguise so that the balance of the show and the story-line are lost. Only in some cases, an external intervention can save the show. The systematic characterization of somatic mutations in cancer genomes is essential for understanding the disease, and the discovery of the genes mutated in human cancer is useful for developing targeted therapeutics for clinical intervention. Thanks to their ability to control the survival/proliferative AKT-mediated pathway, biochemical and genetic evidences strongly connect class I PI3K activation with cancer (46). Pioneer


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studies revealed a tight link between PI3K activity and different tyrosine kinase oncoproteins which are activated by the polyoma virus middle T protein, and in turn phosphorylate several tyrosine residues of middle T, causing the recruitment and activation of the p85–p110α dimer. The same cell-transforming activity of tyrosine kinase oncoproteins seems to be related to their ability to associate with class I PI3Ks, especially p110α. A direct oncogenic potential was properly referred to p110α, whose gene PIK3CA is frequently mutated in several tumors (Figure 2). These mutations in PIK3CA usually result in a constitutively active state of p110α which is no more dependent on upstream stimuli by growth factors, thus promoting uncontrolled pro-survival/proliferative signaling in cells. Several studies underlined the clinical relevancy of the oncoprotein p110α as a fundamental drug target in different kind of tumors. While it is well established that p110α is the most frequently deregulated member of the PI3K family in cancer, so far little is known about genetic alterations in class II and III PI3Ks (3), whereas genetic modifications of PI3K pathway components, such as AKT and PTEN (47), are well characterized (Table I). Therefore it is becoming increasingly clear that deregulation of multiple signaling pathways, rather than individual genes, is required to promote tumorigenesis and the maintenance of the transformed phenotype (48).

p85 In addition to oncogenic mutations in PIK3CA, recent cancer genome analysis has identified recurrent somatic mutations in the PIK3R1 gene, which encodes for p85α, the regulatory subunit of class I PI3K (49–52). In particular, PIK3R1 mutations have been reported in several types of cancer, including endometrioid

endometrial cancers (EEC), non-endometrioid endometrial cancers (NEEC), glioblastomas (GBM), and breast, ovarian, and colon tumors (51,53). The mutations preferentially cluster in the inter-SH2 (iSH2) domain, involving residues that interact with the C2 domain of the catalytic subunit p110α (50). These activating mutations disrupt the iSH2–C2 interaction, releasing the inhibitory effect on p110α (54,55), while N564 and D560 mutations mimic the effects of the substitutions N345 in the C2 domain of p110α, promoting its activity (50). PIK3R1 mutations promote AKT phosphorylation on Ser473, even if novel somatic mutations of PIK3R1 and PIK3R2 genes demonstrate gain-of-function activity resulting in destabilized PTEN protein (52).

p110 In cancer, genetic lesions occurring in p110α are mostly missense mutations, whereas no truncating mutations have been observed. Mutations affecting the PIK3CA gene were identified in GBMs, breast, colorectal, gastric, acute leukemia, hepatocellular, and lung cancers (56–58). The mutations cluster into two major ‘hot spots’, exon 9 of the helical domain (33%, aa E542K, E545K) and exon 20 of the kinase domain (47%, aa H1047R), and are known to induce oncogenic transformation by constitutive phosphorylation of AKT, S6K, and 4E-BP1 (56). The mechanisms through which helical and kinase domain mutations trigger gainof-function are quite different (59). In fact, exon 9 mutations are independent from p85-binding, but they require the interaction with RAS-GTP, while the exon 20 mutation is highly dependent on p85 interaction also in the absence of RAS-GTP. Somatic mutations in the PIK3CA gene exhibit also a distinctive pattern in gender and tissue specificity. For instance, in colorectal cancer (CRC), PIK3CA mutations occur at higher frequencies in

Figure 2. Mechanism of action of pharmacological inhibitors in cancer. Schematic representation of PI3K/AKT/mTOR signaling evidencing activating mutations (signed by orange stars) and loss of function mutations (signed by red interdiction symbols), that are often associated with cancer promotion. Since the literature is conflicting about the effects of PI3Kγ mutations on tumors, in this scheme the mutations affecting this isoform are represented by a question mark (in blue). Furthermore, inhibitors against PI3K, mTOR, or MEK/ERK describe the rationale of combinatorial therapies in cancer treatment.

PI3K/Akt signaling pathway and cancer 5 Table I. Summary of genetic alterations in PI3Ks pathway in human cancer. Isoform p110α p85 (PIK3R1) p110β (PIK3CB) p110γ p110δ PTEN INPP4B SHIP-1 (INPP5D) AKT1 AKT2 AKT3

Genetic mutation mutation amplification mutation mutation amplification amplification mutation amplification mutation deletion deletion mutation mutation amplification amplification mutation

E542K, E545K, H1047R E633K

E1021K G129E

E17K, Q79K

Cancer type

Ref.

GBM, breast, CRC, gastric, AML, hepatocellular, and lung ovarian, cervical, head and neck, squamous cell lung, gastric breast, colon, ovarian, GBM, EEC, NEEC breast colon CML, invasive breast, PDAC marginal zone lymphoma AML prostate, GBM, melanoma, endometrial urothelial breast, ovarian AML breast, CRC, ovarian, bladder gastric ovarian, breast, CRC, pancreatic sporadic melanomas

(132) (64,65) (50) (77) (68) (71) (76) (74) (47) (78) (47) (47) (78) (84) (133) (91)


AML ⫽ acute myeloid leukemia; CML ⫽ chronic myeloid leukemia; CRC ⫽ colorectal cancer; EEC ⫽ endometrioid endometrial cancers; GBM ⫽ glioblastoma multiforme; NEEC ⫽ non-endometrioid endometrial cancers; PDAC ⫽ pancreatic ductal adenocarcinoma.

women, whereas male breast cancers display an overall frequency similar to female breast tumors (60). Interestingly, a meta-analysis of PIK3CA mutations occurring in breast, colorectal, gastric, and endometrial cancers reported that the ratio between mutation prevalence in exons 9 and 20 can be considered as a signature of cancer type (61). In particular, exon 9 is significantly more affected than exon 20 in CRC, while the opposite pattern was described for endometrial cancer, suggesting that different mutations could exert specific effects on the downstream oncogenic signaling. Furthermore, the coexistence of helical and kinase domain mutations causes a synergistic gain-of-function of p110α activity, enhancing its tumorigenic effects in vitro (59). This synergism has also been described in CRC patients, in which concomitant PIK3CA mutations in exons 9 and 20, but not PIK3CA mutation in either exon 9 or 20 alone, significantly reduce overall survival (62). In addition, amplification of the chromosomal regions containing PIK3CA has been identified in several human cancers, including ovarian, cervical, head and neck, and gastric cancers (63,64), whereas this is not a common mechanism of activation in CRC (56). Several preclinical studies have already described a role for the PIK3CB gene, which encodes for the p110β catalytic subunit, in breast cancer initiation (65–67). Although mutations in p110β are rarely found in tumors, a recent study described, for the first time, the functional consequence of PIK3CB tumor-associated mutation (68). This missense substitution (E633K) is located in the helical domain of p110β, and it increases the basal activity of the protein, enhancing cell proliferation and transformation. Similarly to p110α H1047R mutation, E633K shows an enhanced membrane targeting and, consequently, a decreased dependency on Ras activation. Beside the well-documented tumorigenic potential of p110α, overexpression of the γ isoform is also able to induce oncogenic transformation in cell culture (69). In human cancer, elevated expression of p110γ has been reported in chronic myeloid leukemia (70) and in invasive breast carcinoma (71). Recently, Edling and colleagues reported that p110γ expression is increased in pancreatic ductal adenocarcinoma (PDAC) tissue compared with normal ducts and that its downregulation, through siRNA, reduces cell proliferation, highlighting a critical role for p110γ in pancreatic cancer progression (72). Consistent with its specific expression in immune and hematopoietic cells, p110δ is mainly involved in hematological cancers. High levels of p110δ expression has been reported in blast cells derived from acute myeloblastic leukemia (AML) patients, and point mutations have been described in a panel

of diffuse large B-cell lymphomas (DLBCL) (73,74). Moreover, Angulo and colleagues described a somatic mutation affecting the p110δ catalytic subunit (E1021K) that is associated with recurrent infections and progressive airway damage (75). In addition, a recent high-throughput mutational analysis identified novel somatic mutations affecting p110γ (N66K, D161E, R178L, S348I, K364N, T503M, R542W, E602V, and E740K) and p110δ (V397A) in different types of tumors, including breast, lung, ovarian, and prostate cancer (76). Since none of these mutations affects homologous residues on p110α, it will be important to assess the effects on kinase activity and the oncogenic potential of p110γ and p110δ mutants. The presence of PIK3CD somatic mutations, together with its tissue-specific expression, makes p110δ a potential therapeutic target in specific inflammatory conditions in cancer.

PTEN and other phosphatases PI3Ks pathway is negatively regulated by a group of lipid phosphatases of which PTEN is the main representative. Moreover, among all phosphatases, PTEN is the most clearly involved in oncogenesis. In particular, PTEN is frequently mutated in various tumors including those of prostate, GBM, melanoma, and endometrial carcinoma (47). Deletions of 10q, including PTEN region, are found in 24%– 58% of invasive urothelial carcinomas, whereas mutations on the retained allele of PTEN are not detected frequently (77). In patients with Cowden’s syndrome, the G129E mutation has been identified in the catalytic domain. This mutation alters the lipidphosphatase activity but does not affect PTEN’s ability to dephosphorylate protein targets, indicating that loss of lipid-phosphatase activity is sufficient to cause the clinical cancer phenotype (78). Instead, PTEN-inactivating mutations have been found in approximately 30% of primary GBMs (79). Moreover, germline mutations in PTEN cause autosomal dominant hamartoma tumor syndromes, and PTEN protein levels correlate with disease severity, suggesting that PTEN is functionally haploinsufficient (80). Apart from PTEN, other enzymes that regulate the production and degradation of PIP3 signals are involved in cancer. For example, SHIP-1, encoded by INPP5D, is a hematopoietic-specific 5-phosphatase, mainly expressed in B cells, T cells, macrophages, and mast cells. Mutations in INPP5D have been found in patients with acute myeloid leukemia. The reduction in SHIP-1 levels is correlated with chronic myeloid leukemia (CML), and its activation by small molecules has an inhibitory effect in hematopoietic

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and myeloma cells: this suggests an inhibitory role of SHIP-1 in the PI3Ks pathway. On the contrary, a different role of SHIP-2 (encoded by INPPL1) has been proposed, as its expression is increased in a panel of breast cancer cell lines (47). Mutations in INPP5D have been identified in patients with acute myeloid leukemia. The 4-phosphatase INPP4B is considered a tumor suppressor (31), and it is frequently deleted in breast cancers: loss of heterozygosity at the INPP4B locus has been found in most basal-like breast cancers, as well as in a substantial proportion of ovarian cancers, and this correlates with lower overall patient survival (47).


AKT The central role of AKT in the PI3Ks pathway makes it one of the most activated downstream effectors in the oncogenic landscape. The AKT kinase family includes three members, AKT1, AKT2, and AKT3, that are structurally homologous but exhibit distinct features (81). While AKT1 and AKT2 are ubiquitously expressed, AKT3 expression is more confined in terms of tissue distribution (82). It is becoming increasingly clear that AKT isoforms carry specific genetic aberrations in different tumor types, including colorectal, breast, lung, and pancreatic cancers. AKT1 amplification has been detected in gastric carcinomas where it correlates with resistance to cisplatinum treatment (83). On the other hand, somatic mutations occurring on the AKT1 gene have been described in breast, colorectal, ovarian, lung, and bladder cancers (77). In particular, AKT1 E17K is an activating mutation that results in the constitutive localization of the protein at the plasma membrane and promotes hyper-phosphorylation at serine-473 and threonine-308 in a growth factor-independent way (84). This mutation is also sufficient to transform cells in culture and to induce leukemia in mouse models. In addition, the AKT1 E17K variant is mutually exclusive with PIK3CA E454K and H1047R alleles in breast cancer (ductal and lobular histotypes), suggesting that AKT mutations may occur in a tissue-specific fashion (85). Moreover, a novel AKT1 mutation (Q79K) has been recently identified in melanoma patients, and it is critically involved in the acquired resistance to BRAF inhibitors (86). Amplification of AKT2 has been frequently detected in several types of tumors, including ovarian, breast, colorectal, and pancreatic tumors, where it positively correlates with aggressiveness and poor prognosis (87–89). Finally, a selective activation of the AKT3 isoform in combination with loss of PTEN has been described in 43% to 60% of sporadic melanomas, showing an increase of active AKT3 in the advanced stages of the disease (90). Therefore, the genetic alterations occurring in the three AKT isoforms underline their distinct functional role in cancer development and progression.

PI3K pathway status as genetic determinants of therapeutic response in clinical trials In the previous section, we discussed alterations occurring in the PI3K pathway in human cancers, including mutations and/ or amplifications of PI3K catalytic and regulatory subunits, the PI3K effector AKT, and deletions or epigenetic silencing of negative regulators of PI3K signaling, such as PTEN and INPP4B (31,91–93). Since PI3K/AKT/mTOR axis has been classified among the most frequently activated pathways in cancer, members of the cascade represent an attractive target for cancer therapeutics (94). A number of molecules targeting members of the PI3K axis have been developed and evaluated in preclinical studies as well as in clinical trials (Table II). These targeted drugs include isoform-specific

(p110α, p110β, p110γ, p110δ) or pan-class I PI3K inhibitors, dual PI3K/mTOR inhibitors, mTOR inhibitors, and AKT inhibitors, which are all currently in clinical development. Rapalogs (rapamycin analogs) were the first PI3K pathway inhibitors to be tested in clinical trials for the treatment of cancer. Although mTOR inhibitors were initially developed as immune suppressants for patients undergoing transplantation, subsequent studies demonstrated that by suppressing cell proliferation and angiogenesis, these agents have efficacy in various cancer types, including renal cell carcinoma (RCC). The first drug in this class to gain US Food and Drug Administration (FDA) approval for first-line treatment of metastatic RCC (mRCC) was temsirolimus. The survival advantage of temsirolimus was demonstrated in a phase III study comparing the drug alone to interferon-alfa or to a combination of the two drugs (95–97). Phase III trials of temsirolimus in the second-line setting are underway or completed but have not yet been reported. In the oncology setting, everolimus has been approved for the treatment of advanced breast cancer, pancreatic neuroendocrine tumor (pNET), and non-malignant kidney and brain tumors (98). However, the genetic determinants of rapalog inhibition in RCC may be very different from other tumors, since RCCs rarely present mutations in the PI3K pathway members. The sensitivity to rapalogs may be due to the fact that RCCs mainly rely on angiogenesis and hypoxia, two cellular processes regulated by mTORC1 (99,100). Several preclinical and clinical studies suggested that PIK3CA mutations may be predictive for response to selected targeted therapies in several tumor types, including CRC and head and neck squamous cell carcinoma (HNSCC) (101,102). On the other hand, no statistically significant interaction between PIK3CA mutations in exon 9/20 and response to irinotecan-based adjuvant chemotherapy has been reported in a cohort of 627 stage III colon carcinomas (103). Similar results were observed in 572 chemotherapy-refractory metastatic CRC (mCRC) randomly treated with cetuximab or best supportive care (BSC), in which PIK3CA mutation or PTEN loss were not predictors of response to targeted therapy (104). Nevertheless, it has been shown that PIK3CA mutations are associated with poor survival in mCRC patients treated with anti-EGFR monoclonal antibodies (MoAbs) (105). In addition, meta-analyses performed on 576 mCRC reported that PIK3CA exon 20 mutations may be associated with resistance to MoAbs in KRAS wild-type tumors (106). Taken together, these data suggest that the predictive role of PIK3CA mutations is still controversial in single-agent clinical trials; however, they seem to be promising biomarkers in combinatorial therapies. A recent phase I trial of liposomal doxorubicin, bevacizumab, and temsirolimus reported a high percentage of responders (PR, prolonged Stable disease) in gynecologic and breast patients carrying PI3K pathway aberrations (PIK3CA mutations and PTEN loss) (107). A recent study by Janku and colleagues reported that the presence of PIK3CA mutations is associated with increased sensitivity to regimens that include PI3K/mammalian target of rapamycin (mTOR)/AKT inhibitors in a variety of tumors (108). Patients with breast, ovary, and endometrial tumors were sequenced for the presence of PIK3CA mutations and treated in combination with different rapalogs or the PI3K inhibitor PX866 in a prospective phase I clinical trial (108). Patients with PIK3CA mutations treated with PI3K/AKT/mTOR inhibitors demonstrated a partial response (30%) in contrast to patients whose tumors were PIK3CA wild-type. In addition, it is emerging that also the type of PIK3CA mutation can be relevant for the response to targeted treatments. In patients with advanced cancers, PIK3CA exon 20 mutation H1047R may predict response to PI3K/AKT/mTOR inhibitors compared with other PIK3CA

mTOR mTOR mTOR mTOR Akt Akt Akt Akt

AZD8055 INK128 OSI-027 AZD5363 GDC-0068 GSK690693 MK-2206

Class I PI3K Class I PI3K

BAY 80-6946 XL-147

RAD-001 (everolimus)

Class I PI3K

PX866

PI3K and mTOR PI3K and mTOR PI3K and mTOR PI3K and mTOR mTOR

Class I PI3K

BKM120

BEZ235 GDC-0980 NVP-BEZ235 PF-04691502 CCI-779 (temsirolimus)

p110α Class I PI3K

BYL719 GDC-0941

breast, ovarian Non-Hodgkin’s lymphoma, solid cancers, breast advanced breast advanced solid mRCC, solid, refractory diffuse large B-cell lymphoma, breast, mRCC advanced breast, pNET, non-malignant kidney and brain, advanced melanoma, CRC, SCLC solid advanced solid, breast solid, lymphoma advanced breast solid lymphoma solid, breast

breast, CRC, MM, NSCLC, pancreatic, prostate, ovarian advanced solid lymphoma, glioblastoma, NCLC, solid, breast

AML, CLL, Hodgkin’s and non-Hodgkin’s lymphoma, MCL solid, CRC breast, melanomas, MM, non-Hodgkin’s lymphoma, NSCLC, ovarian breast, CRC, endometrial, GIST, GBM, leukemia, melanoma, NSCLC, pancreatic, prostate

p110δ

CAL-101 (idelalisib)

Cancer type solid advanced solid hematological malignancies

p110α p110β p110γ and p110δ

GDC-0032 GSK2636771 IPI-145

Isoform selectivity Clinical trial status

I-II, advanced solid cancers I, lymphoma, glioblastoma, non-small-cell lung cancer, solid tumors; I-II, breast I-II, breast I, Non-Hodgkin’s lymphoma, solid cancers, breast I-II, advanced breast cancer I, advanced solid cancers I, solid tumors; II, refractory diffuse large B-cell lymphoma and breast; III, mRCC I, advanced breast cancer; II, advanced melanoma, CRC, SCLC I, solid tumors I, advanced solid tumors; I-II, breast I, solid tumor and lymphoma I-II, advanced breast I, solid cancer I, lymphoma I, solid cancer and breast

I-II, breast, CRC, endometrial, GIST, GBM, leukemia, melanoma, NSCLC, pancreatic, renal cell, HNSCC, TCC I-II, CRC, GBM, NSCLC, HNSCC

I-II, solid tumors I-IIa, advanced solid cancers with PTEN deficiency I-IIa, advanced hematological malignancies, III CLL, SLL I-II-III, AML, CLL, Hodgkin’s and non-Hodgkin’s lymphoma, MCL, MM I-II, advanced solid tumor, CRC I-II, breast, non-Hodgkin’s lymphoma, NSCLC

Reference

(146) www.clinicaltrials.gov www.clinicaltrials.gov (147) www.clinicaltrials.gov www.clinicaltrials.gov (148)

(99,130,143–145)

www.clinicaltrials.gov www.clinicaltrials.gov www.clinicaltrials.gov (141) (96–98,108,142)

www.clinicaltrials.gov (140)

www.clinicaltrials.gov

(139)

www.clinicaltrials.gov www.clinicaltrials.gov

(137,138)

(134) www.clinicaltrials.gov (135,136)

AML ⫽ acute myeloid leukemia; CLL ⫽ chronic lymphocytic leukemia; CRC ⫽ colorectal cancer; GBM ⫽ glioblastoma multiforme; GIST ⫽ gastrointestinal stromal tumors; MCL ⫽ mantle cell lymphoma; MM ⫽ multiple myeloma; mRCC ⫽ metastatic renal cell carcinoma; NSCLC ⫽ non-small-cell lung carcinoma; pNET ⫽ pancreatic neuroendocrine tumor; HNSCC ⫽ head and neck squamous cell carcinoma; SCLC ⫽ small-cell lung cancer; SLL ⫽ small lymphocytic lymphoma.

AKT

mTOR

Dual PI3K/mTOR

Pan-class I PI3K

Isoform specific

Compound

Table II. PI3K inhibitors tested in preclinical and clinical models.


PI3K/Akt signaling pathway and cancer 7

8

M. Martini et al.

mutations or PIK3CA wild-type (109). However, further studies are needed to define clearly the relationship between drug resistance/sensitivity and specific mutants.


Small inhibitors and complexity of PI3K network In the fight against cancer, the development of new pharmacological compounds targeting well-known molecular actors and the search of new supporting actors that play a part in the drama are ongoing challenges. Among the participants, p110α is a leading player, and it recently appears that its role in tumor promotion is, at least in part, due to the fundamental interaction with the RAS protein (110,111). Mutations in the p110α RAS-binding domain (RBD) are, in fact, reported to be of fundamental importance in both tumor initiation (i.e. KRAS-induced lung cancer and HRAS skin cancer formation) and in tumor progression. Thus RBD mutations are demonstrated to lead to tumor regression or long-term stasis in KRAS-driven lung adenocarcinoma. The binding with HRas has been shown to induce a conformational change activating p110γ protein (112), thus it would be supposed that the direct interaction of p110α with RAS might determine an allosteric modification altering the oncogenic potential of p110α. These findings open the way to the development of putative small molecules, able to inhibit p110α/RAS interaction, as a valid alternative to conventional p110α blocker treatment. The future design of compounds able to disrupt the binding of a small GTPase to the RBD of a PI3K could be also extended to the p110β isoform. Indeed, it has been recently reported that p110β interacts with Rho family GTPases Rac1 and Cdc42 and that RBD point mutations affecting these interactions also abrogate the proliferative potential of p110β (113). Until PI3K’s RBD-specific and less toxic anti-cancer agents are successfully achieved, cancer treatment is still focused on PI3K blockade. The ‘undruggability’ of the KRAS oncogene has in fact led researchers to design therapy against KRAS downstream targets. It is well known that KRAS controls not only the PI3K-AKT axis but also different pathways involved in cell growth, survival, and proliferation, such as BRAF-MEK-ERK. Even if pan-PI3K class I agents such as BMK120 are in clinical trials to treat patients with solid tumors, evidence from cancer treatment with different pharmacological agents, that synergize each other or with additive effects, nevertheless supports the view that combinatorial therapy represents the path forward. Many clinical studies are ongoing to verify the efficacy of mTOR and PI3K concomitant pharmacological inhibition. An example of this strategy is the combined use of everolimus and the dual PI3K/mTOR inhibitor NVP-BEZ235 on solid tumors. Even if everolimus alone has been demonstrated useful in some cancer cell lines, resistance has been evidenced in other tumoral cells. Several mechanisms are conferring resistance to everolimus and other mTOR inhibitors in cancer cells. One is based on the feedback loop regulation between mTORC1 and PI3K, due to a downregulation of RTK and IRS1 activity mediated by mTORC1 (114,115). Thus mTOR blocking with everolimus is reported determine PI3K upregulation. However, it has been reported that tumoral cells carrying activating mutation on p110α (H1047R and E545K) are sensitive to everolimus when these mutations on PI3K are not associated with KRAS mutations (102). Thus it appears that in patients with KRAS mutation, despite treatment with everolimus, a proliferative advantage is furnished by mTOR-independent protein synthesis promotion by p90S6K (also known as RSK1) activation. The poor efficacy in proliferation inhibition of rapalog compounds is also explained considering that mTORC1 is able negatively to modulate ERK1/2 through the S6K1/PI3K/RAS-dependent pathway (116). In

addition, in cancer therapy it should also be taken into account that many KRAS-mutated tumors have alterations in other genes that may co-operate with tumor progression, which illustrates the complexity of the network that controls tumor development. An example of this is furnished by tumors with concomitant KRAS mutation and LKB1 loss that are resistant to treatment with the antimitotic drug docetaxel plus MEK1/2 inhibitor AZD6244 in combinatory therapy. It appears that the insensitivity to AZD6244 therapy is due to PI3K/AKT pathway activation, following tumor suppressor LKB1 ablation. In this context, it is interestingly that triple combination of dasatinib (BMS-354825; Bristol Myers Squibb, Princeton, New Jersey, USA), an agent able to induce SRC and TGF-β downregulation, plus AZD6244 (MEK inhibitor) and NVP-BEZ235 determine tumor regression in KRAS/LKB1 lung cancer (117). Interrelation between mTOR, PI3K, and LKB1 pathways is also confirmed by a recent observation that mice with concomitant deletion of both Lkb1 and Pten genes and mTOR dysregulation develop ovarian cancer with 100% penetrance (118). The relief of the synergic effect of these tumor suppressors on mTOR can be contrasted by using dual PI3K/mTOR inhibitor NVP-BEZ235 that leads to tumor regression (118). In this study it is also reported, surprisingly, that single treatment with mTOR inhibitor everolimus determines an antitumoral effect comparable to that of NVP-BEZ235, suggesting that in some cancer types the LKB1 and PI3K pathways are converging on mTOR modulation. Another factor that increases the complexity of molecular networks controlling tumor promotion is represented by the effect of the LKB1 protein on cell metabolism. It is, in fact, demonstrated that LKB1 loss determines an increase in lactate production through anaerobic glycolysis, known also as ‘Warburg effect’ in ErbB2-mediated breast cancer (119). In this context, it is to be considered that different PI3K isoforms, such as PI3Kα and PI3Kβ, are key regulators of glucose metabolism. Thus LKB1 and PI3K-regulated metabolic reactions can co-operate to furnish an additional selective advantage to cancer cells. A better understanding of complex molecular mechanisms that confer sensitivity or resistance to pharmacological compounds is the base of future cancer treatment. The classification of patients in relation to alterations in PI3K, mTOR, RAS, ERK, and other key molecules in cell survival, apoptosis, and proliferation control will permit personalized therapies to be defined.

Discussion and conclusion Alteration of the PI3K/AKT/mTOR pathway is strongly implicated in cancer pathogenesis, and targeting the effectors of this pathway is a promising therapeutic approach. The growing interest in developing PI3K inhibitors clearly indicates the central role of PI3Ks in cancer treatment. In light of currently available data, the correlation of PI3K status with the clinical outcome of targeted therapies is challenging for a number of reasons. Preclinical studies have not yet conclusively demonstrated that tumor cells harboring PI3K mutations show increased sensitivity to PI3K/ mTOR and pan-PI3K inhibitors (102,120–122). The lack of a strong correlation between PI3K mutations and response to therapy could also be related to a partial characterization of genetic lesions occurring in the tumor. In fact, tumors scored as ‘negative’ for PI3K pathway aberrations may be positive for alterations in other related cancer genes that were not analyzed in the cohort. For instance, activation of ERK signaling is associated with limited efficacy of PI3K/mTOR inhibitors in several studies (102,123,124). Brachmann et al. reported that resistance to NVPBEZ235 could be due to increased ERK pathway activity, caused by concomitant KRAS mutations or PTEN loss, in breast cancer


PI3K/Akt signaling pathway and cancer 9 cells (120). In addition, alterations of upstream activators of PI3K pathway, IGF1R, or its downstream effectors, such as AKT1 and the RSK family, can promote resistance to rapalogs (NVP-BEZ235) or pan-PI3K (BKM120, GDC-0941) treatment in breast cancer preclinical models (125). Interestingly, resistance can be overcome with the use of NVP-MEK162 and dihydropteridinone, specific inhibitors of MEK- and pan-RSK, respectively. In this regard, several inhibitors of the PI3K pathway are now currently used in advanced phases of clinical trials in combined therapies. A protocol based on MEK and PI3K pathway inhibitors demonstrated some efficacy in early clinical trials, even if toxic side effects caused by MEK inhibitors represent one of the major limits to the efficacy of the treatment (126,127). In addition, a combination of PI3K inhibitors and liposomal doxorubicin showed a higher response rate in PIK3CA-mutated patients (108), suggesting that PI3K mutations may enhance sensitivity to doxorubicin, and explaining the observed effect after chemotherapy treatment (128). On the other hand, a randomized phase II breast cancer study reported no differential benefits in wild-type and mutant PI3K tumors after treatment with everolimus and aromatase inhibitor, excluding a possible PI3K status-related phenotype (129). Taken together, these data suggest that several confounding elements make it difficult to determine whether there is a robust relationship between PI3K mutations and sensitivity to mTOR and/ or pan-PI3K inhibitors. Therefore, a comprehensive analysis of genetic alterations occurring in the tumor will be useful to define the complex genomic network in which PI3K mutations can exert a predictive role in response to targeted treatments (130, 131). Finally, besides targeting the class I PI3Ks, another promising approach will be to design inhibitors targeting the class II PI3Ks. The role of PI3K-C2α in development and angiogenesis suggests that this PI3K isoform might play a critical role in cancer onset and progression. Although further analyses need to be performed, this possibility opens up new horizons in cancer treatment.

Acknowledgements We thank Dr Sharmila Fagoonee for careful and critical reading critically of the manuscript. This work was supported by a grant from Compagnia di San Paolo and Associazione Italiana Ricerca Cancro (AIRC). Declaration of interest: E.H. is co-founder of Kither Biotech, a company involved in the development of PI3K inhibitors. The other authors declare no conflict of interest.

References 1. Kaplan DR, Whitman M, Schaffhausen B, et al. Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity. Cell. 1987;50: 1021–9. 2. Whitman M, Kaplan DR, Schaffhausen B, et al. Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation. Nature. 1985;315:239–42. 3. Vanhaesebroeck B, Guillermet-Guibert J, Graupera M, Bilanges B. The emerging mechanisms of isoform-specific PI3K signalling. Nat Rev Mol Cell Biol. 2010;11:329–41. 4. Geering B, Cutillas PR, Nock G, et al. Class IA phosphoinositide 3-kinases are obligate p85-p110 heterodimers. Proc Natl Acad Sci U S A. 2007;104:7809–14. 5. Zhao L, Vogt PK. Class I PI3K in oncogenic cellular transformation. Oncogene. 2008;27:5486–96. 6. Burgering BM, Coffer PJ. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature. 1995;376:599–602. 7. Pearce LR, Komander D, Alessi DR. The nuts and bolts of AGC protein kinases. Nat Rev Mol Cell Biol. 2010;11:9–22.

8. Wick MJ, Dong LQ, Riojas RA, et al. Mechanism of phosphorylation of protein kinase B/Akt by a constitutively active 3-phosphoinositidedependent protein kinase-1. J Biol Chem. 2000;275:40400–6. 9. Nave BT, Ouwens M, Withers DJ, et al. Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation. Biochem J. 1999;344(Pt 2):427–31. 10. Aoki M, Blazek E, Vogt PK. A role of the kinase mTOR in cellular transformation induced by the oncoproteins P3k and Akt. Proc Natl Acad Sci U S A. 2001;98:136–41. 11. Hawkins PT, Anderson KE, Davidson K, Stephens LR. Signalling through Class I PI3Ks in mammalian cells. Biochem Soc Trans. 2006;34:647–62. 12. Qiu RG, Chen J, Kirn D, et al. An essential role for Rac in Ras transformation. Nature. 1995;374:457–9. 13. Welch HC, Coadwell WJ, Ellson CD, et al. P-Rex1, a PtdIns(3,4,5)P3and Gbetagamma-regulated guanine-nucleotide exchange factor for Rac. Cell. 2002;108:809–21. 14. Han J, Luby-Phelps K, Das B, et al. Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav. Science. 1998;279:558–60. 15. Lindvall J, Islam TC. Interaction of Btk and Akt in B cell signaling. Biochem Biophys Res Commun. 2002;293:1319–26. 16. Rameh LE, Arvidsson A, Carraway KL 3rd, et al. A comparative analysis of the phosphoinositide binding specificity of pleckstrin homology domains. J Biol Chem. 1997;272:22059–66. 17. Di Paolo G, De Camilli P. Phosphoinositides in cell regulation and membrane dynamics. Nature. 2006;443:651–7. 18. Li J, Yen C, Liaw D, et al. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science. 1997;275:1943–7. 19. Ali IU, Schriml LM, Dean M. Mutational spectra of PTEN/MMAC1 gene: a tumor suppressor with lipid phosphatase activity. J Natl Cancer Inst. 1999;91:1922–32. 20. Maehama T, Dixon JE. The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem. 1998;273:13375–8. 21. Zhang JG, Wang JJ, Zhao F, et al. MicroRNA-21 (miR-21) represses tumor suppressor PTEN and promotes growth and invasion in nonsmall cell lung cancer (NSCLC). Clin Chim Acta. 2010;411:846–52. 22. Leslie NR, Downes CP. PTEN function: how normal cells control it and tumour cells lose it. Biochem J. 2004;382:1–11. 23. Damen JE, Liu L, Rosten P, et al. The 145-kDa protein induced to associate with Shc by multiple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5-triphosphate 5-phosphatase. Proc Natl Acad Sci U S A. 1996;93:1689–93. 24. Tu Z, Ninos JM, Ma Z, et al. Embryonic and hematopoietic stem cells express a novel SH2-containing inositol 5’-phosphatase isoform that partners with the Grb2 adapter protein. Blood. 2001;98:2028–38. 25. Liu Q, Sasaki T, Kozieradzki I, et al. SHIP is a negative regulator of growth factor receptor-mediated PKB/Akt activation and myeloid cell survival. Genes Dev. 1999;13:786–91. 26. Locke NR, Patterson SJ, Hamilton MJ, et al. SHIP regulates the reciprocal development of T regulatory and Th17 cells. J Immunol. 2009;183:975–83. 27. Norris FA, Majerus PW. Hydrolysis of phosphatidylinositol 3,4bisphosphate by inositol polyphosphate 4-phosphatase isolated by affinity elution chromatography. J Biol Chem. 1994;269:8716–20. 28. Murray D, Honig B. Electrostatic control of the membrane targeting of C2 domains. Mol Cell. 2002;9:145–54. 29. Shearn CT, Norris FA. Biochemical characterization of the type I inositol polyphosphate 4-phosphatase C2 domain. Biochem Biophys Res Commun. 2007;356:255–9. 30. Ma J, Sawai H, Matsuo Y, et al. IGF-1 mediates PTEN suppression and enhances cell invasion and proliferation via activation of the IGF-1/ PI3K/Akt signaling pathway in pancreatic cancer cells. J Surg Res. 2010;160:90–101. 31. Gewinner C, Wang ZC, Richardson A, et al. Evidence that inositol polyphosphate 4-phosphatase type II is a tumor suppressor that inhibits PI3K signaling. Cancer Cell. 2009;16:115–25. 32. Falasca M, Maffucci T. Regulation and cellular functions of class II phosphoinositide 3-kinases. Biochem J. 2012;443:587–601. 33. Falasca M, Hughes WE, Dominguez V, et al. The role of phosphoinositide 3-kinase C2alpha in insulin signaling. J Biol Chem. 2007; 282:28226–36. 34. Posor Y, Eichhorn-Gruenig M, Puchkov D, et al. Spatiotemporal control of endocytosis by phosphatidylinositol-3,4-bisphosphate. Nature. 2013;499:233–7.


10

M. Martini et al.

35. Yoshioka K, Yoshida K, Cui H, et al. Endothelial PI3K-C2alpha, a class II PI3K, has an essential role in angiogenesis and vascular barrier function. Nat Med. 2012;18:1560–9. 36. Maffucci T, Cooke FT, Foster FM, et al. Class II phosphoinositide 3-kinase defines a novel signaling pathway in cell migration. J Cell Biol. 2005;169:789–99. 37. Visnjic D, Crljen V, Curic J, et al. The activation of nuclear phosphoinositide 3-kinase C2beta in all-trans-retinoic acid-differentiated HL-60 cells. FEBS Lett. 2002;529:268–74. 38. Harada K, Truong AB, Cai T, Khavari PA. The class II phosphoinositide 3-kinase C2beta is not essential for epidermal differentiation. Mol Cell Biol. 2005;25:11122–30. 39. Das M, Scappini E, Martin NP, et al. Regulation of neuron survival through an intersectin-phosphoinositide 3’-kinase C2beta-AKT pathway. Mol Cell Biol. 2007;27:7906–17. 40. Wong KA, Russo A, Wang X, et al. A new dimension to Ras function: a novel role for nucleotide-free Ras in Class II phosphatidylinositol 3-kinase beta (PI3KC2beta) regulation. PLoS One. 2012;7:e45360. 41. Backer JM. The regulation and function of Class III PI3Ks: novel roles for Vps34. Biochem J. 2008;410:1–17. 42. Brown WJ, DeWald DB, Emr SD, et al. Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells. J Cell Biol. 1995;130:781–96. 43. Schu PV, Takegawa K, Fry MJ, et al. Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science. 1993;260:88–91. 44. Nobukuni T, Joaquin M, Roccio M, et al. Amino acids mediate mTOR/ raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase. Proc Natl Acad Sci U S A. 2005;102:14238–43. 45. Byfield MP, Murray JT, Backer JM. hVps34 is a nutrient-regulated lipid kinase required for activation of p70 S6 kinase. J Biol Chem. 2005 ;280:33076–82. 46. Yuan TL, Cantley LC. PI3K pathway alterations in cancer: variations on a theme. Oncogene. 2008;27:5497–510. 47. Bunney TD, Katan M. Phosphoinositide signalling in cancer: beyond PI3K and PTEN. Nat Rev Cancer. 2010;10:342–52. 48. Wood LD, Parsons DW, Jones S, et al. The genomic landscapes of human breast and colorectal cancers. Science. 2007;318:1108–13. 49. Shekar SC, Wu H, Fu Z, et al. Mechanism of constitutive phosphoinositide 3-kinase activation by oncogenic mutants of the p85 regulatory subunit. J Biol Chem. 2005;280:27850–5. 50. Jaiswal BS, Janakiraman V, Kljavin NM, et al. Somatic mutations in p85alpha promote tumorigenesis through class IA PI3K activation. Cancer Cell. 2009;16:463–74. 51. Parsons DW, Jones S, Zhang X, et al. An integrated genomic analysis of human glioblastoma multiforme. Science. 2008;321:1807–12. 52. Cheung LW, Hennessy BT, Li J, et al. High frequency of PIK3R1 and PIK3R2 mutations in endometrial cancer elucidates a novel mechanism for regulation of PTEN protein stability. Cancer Discov. 2011;1: 170–85. 53. Urick ME, Rudd ML, Godwin AK, et al. PIK3R1 (p85alpha) is somatically mutated at high frequency in primary endometrial cancer. Cancer Res. 2011;71:4061–7. 54. Sun M, Hillmann P, Hofmann BT, et al. Cancer-derived mutations in the regulatory subunit p85alpha of phosphoinositide 3-kinase function through the catalytic subunit p110alpha. Proc Natl Acad Sci U S A. 2010;107:15547–52. 55. Wu H, Shekar SC, Flinn RJ, et al. Regulation of Class IA PI 3-kinases: C2 domain-iSH2 domain contacts inhibit p85/p110alpha and are disrupted in oncogenic p85 mutants. Proc Natl Acad Sci U S A. 2009;106:20258–63. 56. Samuels Y, Wang Z, Bardelli A, et al. High frequency of mutations of the PIK3CA gene in human cancers. Science. 2004;304:554. 57. Levine DA, Bogomolniy F, Yee CJ, et al. Frequent mutation of the PIK3CA gene in ovarian and breast cancers. Clin Cancer Res. 2005;11:2875–8. 58. Lee JW, Soung YH, Kim SY, et al. PIK3CA gene is frequently mutated in breast carcinomas and hepatocellular carcinomas. Oncogene. 2005;24:1477–80. 59. Zhao L, Vogt PK. Helical domain and kinase domain mutations in p110alpha of phosphatidylinositol 3-kinase induce gain of function by different mechanisms. Proc Natl Acad Sci U S A. 2008;105:2652–7. 60. Benvenuti S, Frattini M, Arena S, et al. PIK3CA cancer mutations display gender and tissue specificity patterns. Hum Mutat. 2008;29:284–8. 61. Barbi S, Cataldo I, De Manzoni G, et al. The analysis of PIK3CA mutations in gastric carcinoma and metanalysis of literature suggest that exon-selectivity is a signature of cancer type. J Exp Clin Cancer Res. 2010;29:32.

62. Liao X, Morikawa T, Lochhead P, et al. Prognostic role of PIK3CA mutation in colorectal cancer: cohort study and literature review. Clin Cancer Res. 2012;18:2257–68. 63. Shayesteh L, Lu Y, Kuo WL, et al. PIK3CA is implicated as an oncogene in ovarian cancer. Nat Genet. 1999;21:99–102. 64. Engelman JA, Luo J, Cantley LC. The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism. Nat Rev Genet. 2006;7:606–19. 65. Ciraolo E, Iezzi M, Marone R, et al. Phosphoinositide 3-kinase p110beta activity: key role in metabolism and mammary gland cancer but not development. Sci Signal. 2008;1:ra3. 66. Jia S, Liu Z, Zhang S, et al. Essential roles of PI(3)K-p110beta in cell growth, metabolism and tumorigenesis. Nature. 2008;454:776–9. 67. Benistant C, Chapuis H, Roche S. A specific function for phosphatidylinositol 3-kinase alpha (p85alpha-p110alpha) in cell survival and for phosphatidylinositol 3-kinase beta (p85alpha-p110beta) in de novo DNA synthesis of human colon carcinoma cells. Oncogene. 2000; 19:5083–90. 68. Dbouk HA, Khalil BD, Wu H, et al. Characterization of a tumor-associated activating mutation of the p110beta PI 3-kinase. PLoS One. 2013;8:e63833. 69. Kang S, Denley A, Vanhaesebroeck B, Vogt PK. Oncogenic transformation induced by the p110beta, -gamma, and -delta isoforms of class I phosphoinositide 3-kinase. Proc Natl Acad Sci U S A. 2006;103:1289–94. 70. Hickey FB, Cotter TG. BCR-ABL regulates phosphatidylinositol 3-kinase-p110gamma transcription and activation and is required for proliferation and drug resistance. J Biol Chem. 2006;281:2441–50. 71. Xie Y, Abel PW, Kirui JK, et al. Identification of upregulated phosphoinositide 3-kinase gamma as a target to suppress breast cancer cell migration and invasion. Biochem Pharmacol. 2013;85:1454–62. 72. Edling CE, Selvaggi F, Buus R , et al. Key role of phosphoinositide 3-kinase class IB in pancreatic cancer. Clin Cancer Res. 2010;16: 4928–37. 73. Sujobert P, Bardet V, Cornillet-Lefebvre P, et al. Essential role for the p110delta isoform in phosphoinositide 3-kinase activation and cell proliferation in acute myeloid leukemia. Blood. 2005;106:1063–6. 74. Zhang J, Grubor V, Love CL, et al. Genetic heterogeneity of diffuse large B-cell lymphoma. Proc Natl Acad Sci U S A. 2013;110:1398–403. 75. Angulo I, Vadas O, Garcon F, et al. Phosphoinositide 3-kinase delta gene mutation predisposes to respiratory infection and airway damage. Science. 2013;342:866–71. 76. Kan Z, Jaiswal BS, Stinson J, et al. Diverse somatic mutation patterns and pathway alterations in human cancers. Nature. 2010;466:869–73. 77. Knowles MA, Platt FM, Ross RL, Hurst CD. Phosphatidylinositol 3-kinase (PI3K) pathway activation in bladder cancer. Cancer Metastasis Rev. 2009;28:305–16. 78. Vivanco I, Sawyers CL. The phosphatidylinositol 3-Kinase AKT pathway in human cancer. Nat Rev Cancer. 2002;2:489–501. 79. Lino MM, Merlo A. PI3Kinase signaling in glioblastoma. J Neurooncol. 2011;103:417–27. 80. Carracedo A, Alimonti A, Pandolfi PP. PTEN level in tumor suppression: how much is too little? Cancer Res. 2011;71:629–33. 81. Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell. 2007;129:1261–74. 82. Agarwal E, Brattain MG, Chowdhury S. Cell survival and metastasis regulation by Akt signaling in colorectal cancer. Cell Signal. 2013;25:1711–19. 83. Liu LZ, Zhou XD, Qian G, et al. AKT1 amplification regulates cisplatin resistance in human lung cancer cells through the mammalian target of rapamycin/p70S6K1 pathway. Cancer Res. 2007;67:6325–32. 84. Carpten JD, Faber AL, Horn C, et al. A transforming mutation in the pleckstrin homology domain of AKT1 in cancer. Nature. 2007; 448:439–44. 85. Bleeker FE, Felicioni L, Buttitta F, et al. AKT1(E17K) in human solid tumours. Oncogene. 2008;27:5648–50. 86. Shi H, Hong A, Kong X, et al. A novel AKT1 mutant amplifies an adaptive melanoma response to BRAF inhibition. Cancer Discov. 2014;4:69–79. 87. Bellacosa A, de Feo D, Godwin AK, et al. Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas. Int J Cancer. 1995;64:280–5. 88. Parsons DW, Wang TL, Samuels Y, et al. Colorectal cancer: mutations in a signalling pathway. Nature. 2005;436:792. 89. Cheng JQ, Ruggeri B, Klein WM, et al. Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA. Proc Natl Acad Sci U S A. 1996; 93:3636–41.


PI3K/Akt signaling pathway and cancer 11 90. Stahl JM, Sharma A, Cheung M, et al. Deregulated Akt3 activity promotes development of malignant melanoma. Cancer Res. 2004; 64:7002–10. 91. Forbes SA, Bindal N, Bamford S, et al. COSMIC: mining complete cancer genomes in the Catalogue of Somatic Mutations in Cancer. Nucleic Acids Res. 2011;39:D945–50. 92. Fedele CG, Ooms LM, Ho M, et al. Inositol polyphosphate 4-phosphatase II regulates PI3K/Akt signaling and is lost in human basal-like breast cancers. Proc Natl Acad Sci U S A. 2010;107:22231–6. 93. Hollander MC, Blumenthal GM, Dennis PA. PTEN loss in the continuum of common cancers, rare syndromes and mouse models. Nat Rev Cancer. 2011;11:289–301. 94. Miled N, Yan Y, Hon WC, et al. Mechanism of two classes of cancer mutations in the phosphoinositide 3-kinase catalytic subunit. Science. 2007;317:239–42. 95. Zustovich F, Lombardi G, Nicoletto O, Pastorelli D. Second-line therapy for refractory renal-cell carcinoma. Crit Rev Oncol Hematol. 2012;83:112–22. 96. Hudes G, Carducci M, Tomczak P, et al. Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. N Engl J Med. 2007;356:2271–81. 97. Hudes GR, Berkenblit A, Feingold J, et al. Clinical trial experience with temsirolimus in patients with advanced renal cell carcinoma. Semin Oncol. 2009;36(Suppl 3):S26–36. 98. Lebwohl D, Anak O, Sahmoud T, et al. Development of everolimus, a novel oral mTOR inhibitor, across a spectrum of diseases. Ann N Y Acad Sci. 2013;1291:14–32. 99. Thomas GV, Tran C, Mellinghoff IK, et al. Hypoxia-inducible factor determines sensitivity to inhibitors of mTOR in kidney cancer. Nat Med. 2006;12:122–7. 100. Linehan WM, Srinivasan R, Schmidt LS. The genetic basis of kidney cancer: a metabolic disease. Nat Rev Urol. 2010;7:277–85. 101. Lui VW, Hedberg ML, Li H, et al. Frequent mutation of the PI3K pathway in head and neck cancer defines predictive biomarkers. Cancer Discov. 2013;3:761–9. 102. Di Nicolantonio F, Arena S, Tabernero J, et al. Deregulation of the PI3K and KRAS signaling pathways in human cancer cells determines their response to everolimus. J Clin Invest. 2010;120: 2858–66. 103. Ogino S, Liao X, Imamura Y, et al. Predictive and prognostic analysis of PIK3CA mutation in stage III colon cancer intergroup trial. J Natl Cancer Inst. 2013;105:1789–98. 104. Karapetis CS, Jonker D, Daneshmand M, et al. PIK3CA, BRAF, and PTEN status and benefit from cetuximab in the treatment of advanced colorectal cancer—results from NCIC CTG/AGITG CO.17. Clin Cancer Res. 2014;20:744–53. 105. Wu S, Gan Y, Wang X, et al. PIK3CA mutation is associated with poor survival among patients with metastatic colorectal cancer following anti-EGFR monoclonal antibody therapy: a meta-analysis. J Cancer Res Clin Oncol. 2013;139:891–900. 106. Mao C, Yang ZY, Hu XF, et al. PIK3CA exon 20 mutations as a potential biomarker for resistance to anti-EGFR monoclonal antibodies in KRAS wild-type metastatic colorectal cancer: a systematic review and meta-analysis. Ann Oncol. 2012;23:1518–25. 107. Moroney JW, Schlumbrecht MP, Helgason T, et al. A phase I trial of liposomal doxorubicin, bevacizumab, and temsirolimus in patients with advanced gynecologic and breast malignancies. Clin Cancer Res. 2011;17:6840–6. 108. Janku F, Wheler JJ, Westin SN, et al. PI3K/AKT/mTOR inhibitors in patients with breast and gynecologic malignancies harboring PIK3CA mutations. J Clin Oncol. 2012;30:777–82. 109. Janku F, Wheler JJ, Naing A, et al. PIK3CA mutation H1047R is associated with response to PI3K/AKT/mTOR signaling pathway inhibitors in early-phase clinical trials. Cancer Res. 2013;73:276–84. 110. Der CJ, Van Dyke T. Stopping ras in its tracks. Cell. 2007; 129:855–7. 111. Castellano E, Sheridan C, Thin MZ, et al. Requirement for interaction of PI3-kinase p110alpha with RAS in lung tumor maintenance. Cancer Cell. 2013;24:617–30. 112. Pacold ME, Suire S, Perisic O, et al. Crystal structure and functional analysis of Ras binding to its effector phosphoinositide 3-kinase gamma. Cell. 2000;103:931–43. 113. Fritsch R, de Krijger I, Fritsch K, et al. RAS and RHO families of GTPases directly regulate distinct phosphoinositide 3-kinase isoforms. Cell. 2013;153:1050–63. 114. O’Reilly KE, Rojo F, She QB, et al. mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt. Cancer Res. 2006;66:1500–8.

115. Wan X, Harkavy B, Shen N, et al. Rapamycin induces feedback activation of Akt signaling through an IGF-1R-dependent mechanism. Oncogene. 2007;26:1932–40. 116. Carracedo A, Ma L, Teruya-Feldstein J, et al. Inhibition of mTORC1 leads to MAPK pathway activation through a PI3K-dependent feedback loop in human cancer. J Clin Invest. 2008;118:3065–74. 117. Carretero J, Shimamura T, Rikova K, et al. Integrative genomic and proteomic analyses identify targets for Lkb1-deficient metastatic lung tumors. Cancer Cell. 2010;17:547–59. 118. Tanwar PS, Mohapatra G, Chiang S, Engler DA, Zhang L, KanekoTarui T, et al. Loss of LKB1 and PTEN tumor suppressor genes in the ovarian surface epithelium induces papillary serous ovarian cancer. Carcinogenesis. 2014;35:546–53. 119. Dupuy F, Griss T, Blagih J, et al. LKB1 is a central regulator of tumor initiation and pro-growth metabolism in ErbB2-mediated breast cancer. Cancer Metab. 2013;1:18. 120. Brachmann SM, Hofmann I, Schnell C, et al. Specific apoptosis induction by the dual PI3K/mTor inhibitor NVP-BEZ235 in HER2 amplified and PIK3CA mutant breast cancer cells. Proc Natl Acad Sci U S A. 2009;106:22299–304. 121. O’Brien C, Wallin JJ, Sampath D, et al. Predictive biomarkers of sensitivity to the phosphatidylinositol 3’ kinase inhibitor GDC-0941 in breast cancer preclinical models. Clin Cancer Res. 2010;16: 3670–83. 122. Weigelt B, Warne PH, Downward J. PIK3CA mutation, but not PTEN loss of function, determines the sensitivity of breast cancer cells to mTOR inhibitory drugs. Oncogene. 2011;30:3222–33. 123. Garrido-Laguna I, Hong DS, Janku F, et al. KRASness and PIK3CAness in patients with advanced colorectal cancer: outcome after treatment with early-phase trials with targeted pathway inhibitors. PLoS One. 2012;7:e38033. 124. Dienstmann R, Serpico D, Rodon J, et al. Molecular profiling of patients with colorectal cancer and matched targeted therapy in phase I clinical trials. Mol Cancer Ther. 2012;11:2062–71. 125. Serra V, Eichhorn PJ, Garcia-Garcia C, et al. RSK3/4 mediate resistance to PI3K pathway inhibitors in breast cancer. J Clin Invest. 2013;123:2551–63. 126. Hoeflich KP, Merchant M, Orr C, et al. Intermittent administration of MEK inhibitor GDC-0973 plus PI3K inhibitor GDC-0941 triggers robust apoptosis and tumor growth inhibition. Cancer Res. 2012;72:210–19. 127. Kummar S, Chen HX, Wright J, et al. Utilizing targeted cancer therapeutic agents in combination: novel approaches and urgent requirements. Nat Rev Drug Discov. 2010;9:843–56. 128. Juric D, Baselga J. Tumor genetic testing for patient selection in phase I clinical trials: the case of PI3K inhibitors. J Clin Oncol. 2012;30:765–6. 129. Baselga J, Semiglazov V, van Dam P, et al. Phase II randomized study of neoadjuvant everolimus plus letrozole compared with placebo plus letrozole in patients with estrogen receptor-positive breast cancer. J Clin Oncol. 2009;27:2630–7. 130. Garnett MJ, Edelman EJ, Heidorn SJ, et al. Systematic identification of genomic markers of drug sensitivity in cancer cells. Nature. 2012;483:570–5. 131. Sartore-Bianchi A, Martini M, Molinari F, Veronese S, Nichelatti M, Artale S, et al. PIK3CA mutations in colorectal cancer are associated with clinical resistance to EGFR-targeted monoclonal antibodies. Cancer Res. 2009;69:1851–7. 132. Kang S, Bader AG, Vogt PK. Phosphatidylinositol 3-kinase mutations identified in human cancer are oncogenic. Proc Natl Acad Sci U S A. 2005;102:802–7. 133. Ndubaku CO, Heffron TP, Staben ST, et al. Discovery of 2-{3-[2-(1isopropyl-3-methyl-1H-1,2-4-triazol-5-yl)-5,6-dihydrobenzo[f ] imidazo[1, 2-d][1,4]oxazepin-9-yl]-1H-pyrazol-1-yl}-2-methylpropanamide (GDC-0032): a beta-sparing phosphoinositide 3-kinase inhibitor with high unbound exposure and robust in vivo antitumor activity. J Med Chem. 2013;56:4597–610. 134. IPI-145 shows promise in CLL patients. Cancer Discov. 2014;4:136. 135. Chang JE, Kahl BS. PI3-kinase inhibitors in chronic lymphocytic leukemia. Curr Hematol Malig Rep. 2014;9:33–43. 136. Gopal AK, Kahl BS, de Vos S, Wagner-Johnston ND, Schuster SJ, Jurczak WJ, et al. PI3Kdelta inhibition by idelalisib in patients with relapsed indolent lymphoma. N Engl J Med. 2014;370:1008–18. 137. Furman RR, Sharman JP, Coutre SE, Cheson BD, Pagel JM, Hillmen P, et al. Idelalisib and rituximab in relapsed chronic lymphocytic leukemia. N Engl J Med. 2014;370:997–1007. 138. Brachmann SM, Kleylein-Sohn J, Gaulis S, et al. Characterization of the mechanism of action of the pan class I PI3K inhibitor NVPBKM120 across a broad range of concentrations. Mol Cancer Ther. 2012;11:1747–57.

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M. Martini et al.


139. Shapiro GI, Rodon J, Bedell C, et al. Phase I safety, pharmacokinetic, and pharmacodynamic study of SAR245408 (XL147), an oral pan-class I PI3K inhibitor, in patients with advanced solid tumors. Clin Cancer Res. 2014;20:233–45. 140. Yuan J, Mehta PP, Yin MJ, et al. PF-04691502, a potent and selective oral inhibitor of PI3K and mTOR kinases with antitumor activity. Mol Cancer Ther. 2011;10:2189–99. 141. Chan S, Scheulen ME, Johnston S, et al. Phase II study of temsirolimus (CCI-779), a novel inhibitor of mTOR, in heavily pretreated patients with locally advanced or metastatic breast cancer. J Clin Oncol. 2005;23:5314–22. 142. Tabernero J, Rojo F, Calvo E, et al. Dose- and schedule-dependent inhibition of the mammalian target of rapamycin pathway with everolimus: a phase I tumor pharmacodynamic study in patients with advanced solid tumors. J Clin Oncol. 2008;26:1603–10. 143. Motzer RJ, Escudier B, Oudard S, et al. Efficacy of everolimus in advanced renal cell carcinoma: a double-blind, randomised, placebocontrolled phase III trial. Lancet. 2008;372:449–56.

144. Morrow PK, Wulf GM, Ensor J, et al. Phase I/II study of trastuzumab in combination with everolimus (RAD001) in patients with HER2-overexpressing metastatic breast cancer who progressed on trastuzumab-based therapy. J Clin Oncol. 2011;29: 3126–32. 145. Maira SM, Stauffer F, Brueggen J, et al. Identification and characterization of NVP-BEZ235, a new orally available dual phosphatidylinositol 3-kinase/mammalian target of rapamycin inhibitor with potent in vivo antitumor activity. Mol Cancer Ther. 2008;7: 1851–63. 146. Davies BR, Greenwood H, Dudley P, et al. Preclinical pharmacology of AZD5363, an inhibitor of AKT: pharmacodynamics, antitumor activity, and correlation of monotherapy activity with genetic background. Mol Cancer Ther. 2012;11:873–87. 147. Hudis C, Swanton C, Janjigian YY, et al. A phase 1 study evaluating the combination of an allosteric AKT inhibitor (MK-2206) and trastuzumab in patients with HER2-positive solid tumors. Breast Cancer Res. 2013;15:R110.