CANCER TREATMENT REVIEWS (2004) 30, 193–204
`
`www.elsevierhealth.com/journals/ctrv
`
`LABORATORY–CLINIC INTERFACE
`
`PI3K/Akt signalling pathway and cancerq
`
`Juan Angel Fresno Vara, Enrique Casado, Javier de Castro,
`Paloma Cejas, Cristobal Belda-Iniesta, Manuel Gonzalez-Baron*
`
`Catedra de Oncologıa y Medicina Paliativa, Servicio de Oncologıa Medica,
`Hospital Universitario La Paz, Paseo de la Castellana 261, Madrid 28046, Spain
`
`KEYWORDS
`PI3K;
`Akt;
`Signalling;
`Apoptosis;
`Proliferation;
`Chemotherapy;
`Resistance;
`New cancer drugs
`
`Phosphatidylinositol-3 kinases, PI3Ks, constitute a lipid kinase family
`Summary
`characterized by their ability to phosphorylate inositol ring 30-OH group in inositol
`phospholipids to generate the second messenger phosphatidylinositol-3,4,5-tris-
`phosphate (PI-3,4,5-P3). RPTK activation results in PI(3,4,5)P3 and PI(3,4)P2
`production by PI3K at the inner side of the plasma membrane. Akt interacts with
`these phospholipids, causing its translocation to the inner membrane, where it is
`phosphorylated and activated by PDK1 and PDK2. Activated Akt modulates the
`function of numerous substrates involved in the regulation of cell survival, cell cycle
`progression and cellular growth. In recent years, it has been shown that PI3K/Akt
`signalling pathway components are frequently altered in human cancers. Cancer
`treatment by chemotherapy and c-irradiation kills target cells primarily by the
`induction of apoptosis. However, the development of resistance to therapy is an
`important clinical problem. Failure to activate the apoptotic programme represents
`an important mode of drug resistance in tumor cells. Survival signals induced by
`several receptors are mediated mainly by PI3K/Akt, hence this pathway may
`decisively contribute to the resistant phenotype. Many of the signalling pathways
`involved in cellular transformation have been elucidated and efforts are underway
`to develop treatment strategies that target these specific signalling molecules or
`their downstream effectors. The PI3K/Akt pathway is involved in many of the
`mechanisms targeted by these new drugs, thus a better understanding of this
`crossroad can help to fully exploit the potential benefits of these new agents.
`
`c 2003 Elsevier Ltd. All rights reserved.
`
`Introduction
`
`Carcinogenesis process is the result of a disturbed
`balance between cell division and growth on one
`hand, and programmed cell death (i.e., apoptosis),
`on the other. In the context of this delicate bal-
`
`ance, proteins and signalling pathways regulating
`cell growth, differentiation and development un-
`dergo oncogenic changes far more often than do
`other molecule groups. In recent years, it has been
`shown that PI3K/Akt signalling pathways, involved
`in the above and other processes, are frequently
`disturbed in many human cancers. This pathway
`plays a major role not only in tumor development
`but also in the tumor’s potential response to cancer
`treatment. Many of the new “targeted agents” have
`been specifically designed to act on PI3K/Akt-re-
`lated targets. Therefore, a better understanding of
`
`q We apologize for not citing original work of many colleagues
`because of space constraints.
`* Corresponding author. Tel./Fax: +34-917-277-118.
`E-mail address: mgonzalezb.hulp@salud.madrid.org
`Gonzalez-Baron).
`
`(M.
`
`doi:10.1016/j.ctrv.2003.07.007
`
`0305-7372/$ - see front matter c 2003 Elsevier Ltd. All rights reserved.
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`J.A.F. Vara et al.
`
`PI3K/Akt signalling pathway may improve the on-
`cologist’s prognosis ability and accuracy of predic-
`tion as to response to treatment. In the present
`review, we discuss PI3K/Akt pathway components
`and functions as well as its alteration in cancer and
`relationship with the different therapeutic ap-
`proaches. Moreover, we describe the current status
`of the different pharmacological compounds that
`target this important pathway.
`
`PI3K/Akt pathway components and
`functions
`
`PI3K
`
`Phosphatidylinositol-3 kinases, PI3Ks, constitute a
`lipid kinase family characterized by their ability to
`phosphorylate inositol ring 30-OH group in inositol
`phospholipids.1 Class-I PI3Ks are heterodimers
`composed of a catalytic subunit (p110) and an
`adaptor/regulatory subunit (p85). This class is
`further divided into the subclass IA, which is acti-
`vated by receptors with protein tyrosine kinase
`activity (Receptor Protein Tyrosine Kinase, RPTK),
`and the subclass IB, which is activated by receptors
`
`coupled with G proteins. The substrate for class I
`PI3Ks is phosphatidylinositol-4,5-bisphosphate (PI-
`4,5-P2) to generate the second messenger phos-
`phatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P3).
`Three isoforms of catalytic subunit p110, named a,
`b and c, and seven adapting protein generated by
`alternative splicing of three genes (p85a, p85b and
`p55c) are known for PI3K class IA.
`
`Activation mechanism
`
`RPTK activation results in the association of PI3K with
`the receptor through one or two SH2 domains in the
`adaptor unit to phosphotyrosine consensus motifs.
`This leads to allosteric activation of the catalytic
`subunit.2 PI3K activation leads to PI-3,4,5-P3 pro-
`duction in a few seconds (Fig. 1). Polyphosphoinosi-
`tide effects on cells are mediated through the
`specific binding to at least two distinct protein-lipid
`binding domains, namely, the FYVE and pleckstrin-
`homology (PH) domains (PH).3 Proteins containing
`the latter domain represent critical mediators for
`PI3K Class IA-induced signalling. PH domains are
`found in many proteins, including protein serine/
`treonine kinase 30-phosphoinositide-dependent klin-
`ase1 (PDK1) and Akt/PKB, both central for the
`transforming effects of deregulated PI3K activity.
`
`Figure 1 Activation of growth factor receptor protein tyrosine kinases results in autophosphorylation on tyrosine
`residues. PI3K is recruited to the membrane by directly binding to phosphotyrosine consensus residues of growth factor
`receptors or adaptors through one or both SH2 domains in the adaptor subunit. This leads to allosteric activation of the
`catalytic subunit. Activation results in production of the second messenger phosphatidylinositol-3,4,5-trisphosphate
`(PIP3). The lipid product of PI3K, PIP3, recruits a subset of signalling proteins with pleckstrin homology (PH) domains to
`the membrane, including PDK1 and Akt. PTEN, is a PI-3,4,5-P3 phosphatase, which negatively regulates the PI3K/Akt
`pathway. Once activated, Akt mediates the activation and inhibition of several targets, resulting in cellular survival,
`growth and proliferation through various mechanisms.
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`Akt signalling and cancer
`
`PKB/Akt
`
`PKB/Akt was characterized following the isolation
`of two genes termed akt1 and akt2, identified as
`the human homologues for the viral oncogene
`v-akt, which is known to be responsible for a type
`of leukemia in mice.4 Later, two unrelated studies
`revealed that v-akt and its human homologues en-
`coded a protein kinase bearing some resemblance
`to kinase protein C (PKC) and protein kinase A
`(PKA);5–7 therefore, it was called PKB. To date,
`three members of this PKB family have been iso-
`lated, named PKBa (Akt1), PKBb (Akt2), and PKBc
`(Akt3).8 Although they are product of different
`genes, they are closely related to each other, with
`up to 80% of amino acid homology. The three genes
`are expressed differentially, with a broader ex-
`pression for PKBa/Akt1 and PKBb/Akt2 and a more
`restricted expression for PKBc/Akt3. Each isoform
`presents a plekstrin homology (PH) domain of ap-
`proximately 100 amino acids in the N-terminal re-
`gion. There is then the kinase domain, which is very
`similar to those of PKA and PKC.9;10 In this region, a
`treonine residue (T308 in Akt1) is found, whose
`phosphorylation is required for Akt activation.
`Following the kinase domain is a hydrophobic
`C-terminal tail containing a second regulatory
`phosphorylation site (S473 in Akt1). T308 and S473
`phosphorylation occurs in response to growth fac-
`tors and other extracellular stimuli, and is essential
`to maximal Akt activation.11
`
`Activation mechanism
`
`RPTK activation results in PI(3,4,5)P3 and PI(3,4)P2
`production by PI3K at the inner side of the plasma
`membrane. Akt interacts with these phospholipids,
`causing its translocation to the inner membrane,
`where PDK1 is located (Fig. 1). The interaction of
`Akt PH domain with 30-hosphoinositides is thought
`to provoke conformational changes in Akt, result-
`ing in exposure of its two main phosphorylation
`sites. Likewise, PH domains may mediate the Akt
`and PDK1 approach through their heterodimeriza-
`tion. PDK1, which is thought to be constituently
`active, phosphorylates Akt at T308, leading to
`stabilization of the activation loop in an active
`conformation. This model strongly resembles the
`general model for PTKs activation.12 T308 phos-
`phorylation is a requirement for the kinase acti-
`vation, but phosphorylation of the residue located
`at the hydrophobic C-terminal region is also re-
`quired to full activation of the kinase. This Akt S473
`kinase (PDK2) has not been identified as yet; how-
`ever, findings from several recent studies suggest a
`
`
`
`195195
`
`role for protein kinase integrin-linked kinase (ILK)
`in the activation process, but it remains unknown
`whether or not ILK directly phosphorylates Akt at
`S473.13–15 In a later step, active Akt is translocated,
`through an unknown mechanism, to the nucleus
`where many of its substrates are localized.16
`
`Cellular processes regulated by Akt
`
`Akt protein modulates the function of numerous
`substrates involved in the regulation of cell sur-
`vival, cell cycle progression and cellular growth
`(Table 1 and Fig. 1).
`
`survival
`
`Cell-death regulation
`Akt activation induces different cell
`mechanisms.17
`Akt inactivates by phosphorylation the proapop-
`totic factors Bad and procaspase-9, as well as the
`Forkhead family of transcription factors that induce
`the expression of proapoptotic factors such as Fas
`ligand. Moreover, very recently Akt activation
`has been related, in cancer cells, with increased
`resistance to apoptosis induced by TRAIL/APO-2L
`(TNF-Related Apoptosis-Inducing Ligand), a mem-
`ber of the TNF superfamily that has been shown to
`have selective antitumor activity.18–21
`On the other hand, Akt activates by phosphory-
`lation the transcription factor cyclic AMP response
`element-binding protein (CREB), and the IjB kinase
`(IKK), a positive regulator of NF-jB, both of them
`regulating the expression of genes with antiapop-
`totic activity.
`
`Cycle progression and cell growth
`Glycogen synthase kinase-3 (GSK3), phosphodis-
`terase-3B, mammalian
`target
`of
`rapamycin
`(mTOR), insulin receptor substrate-1 (IRS-1), the
`Forkhead family member FKHR, Cyclin-dependent
`kinase inhibitors p21CIP1=WAF1 and p27KIP1, and,
`possibly, Raf1, are all Akt targets involved in pro-
`tein synthesis, glycogen metabolism and cell cycle
`regulation.12
`A key mechanism for Akt modulation of specific
`substrate activity, such as b-catenin, p21, p27,
`Mdm2 or Forkhead transcription factors, involves
`the regulation of their cytoplasmic or nuclear lo-
`calization by phosphorylation. GSK3 inhibition by
`Akt prevents the phosphorylation of the cytoplas-
`mic signalling molecule b-catenin, which impedes
`its degradation; hence it is translocated to the
`nucleus. Once in the nucleus, b-catenin combines
`with different transcription factors, like TCF/LEF-
`1, to induce the expression of several genes, such
`as that of the Cyclin D1, which induces cell cycle
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`Akt-regulated cell processes
`Table 1
`Akt substrates
`
`Effect
`
`Survival
`Bad
`
`CREB
`Forkhead Family (Fkhr, Fkhrl1, Afx)
`
`IKK
`
`Procaspase-9
`Ask1
`Mdm2
`
`Cell growth and proliferation
`GSK3
`p21Cip=Waf1, p27Kip
`mTOR/FRAP
`TSC2
`
`Association of Bad with 14-3-3 proteins; suppression of Bad proapoptotic
`activity
`Increased transcription of CREB-regulated survival genes
`Association with 14-3-3 proteins: Nuclear exclusion and inhibition of
`transcription of proapoptotic genes
`Induction of NF-jB transcriptional activity; activation of transcription of
`survival genes
`Suppression of Caspase-9 induced apoptosis
`Inhibition of stress activated kinases
`Mdm2 nuclear entry; inhibition of p53 regulated processes
`
`Inhibition of GSK3 catalytic activity
`Nuclear exclusion; prevention of its antiproliferative activity
`Modulation of mRNA translation
`Modulation of mTOR activity
`
`progression via regulation of RB hyperphosphory-
`lation and inactivation. In a similar way, decreased
`Cyclin D1 phosphorylation by GSK3 promotes the
`stabilization of this protein.22 Akt phosphorylates
`p21 and inhibits its antiproliferative effects by re-
`taining it within the cytoplasm.23 A similar mech-
`anism has been recently described for p27.24–26
`Following stimulation with growth factors, Mdm2 is
`phosphorylated by Akt and enters the nucleus,
`wherein it induces a decrease in both p53 levels
`and its transactivation. In the absence of the tumor
`suppressor p19/p14ARF, the complex Mdm2-p53
`leaves the nucleus and enters the cytoplasm,
`where p53 becomes degraded through an ubiqui-
`tine/proteasome-mediated process.27
`Finally, p70S6K phosphorylation by PDK1 and
`mTOR phosphorylation by Akt constitute processes
`showing a linkage between both signalling path-
`ways. In fact, it has recently been demonstrated
`that some of the transforming effects of PI3K/Akt
`promoting the cell cycle and growth are mediated
`by mTOR/p70S6K cascade: tuberous sclerosis (TSC)
`is an autosomal dominant disorder characterized
`by the formation of hamartomas in a wide range of
`human tissues. Mutation in either the TSC1 or TSC2
`tumor suppressor gene is responsible for both the
`familial and sporadic forms of this disease. Normal
`cellular functions of hamartin and tuberin, en-
`coded by the TSC1 and TSC2 tumor suppressor
`genes, are closely related to their direct interac-
`tions. Akt stimulates growth by phosphorylating the
`tuberous sclerosis complex 2 (TSC2) tumor sup-
`pressor and inhibiting formation of a TSC1:TSC2
`complex. This complex inhibits the p70 ribosomal
`protein S6 kinase 1 (an activator of translation) and
`activates the eukaryotic initiation factor 4E binding
`
`protein 1 (4E-BP1, an inhibitor of translational
`initiation). These functions of TSC1;TSC2 are
`mediated by inhibition of mTOR.28–32
`
`Characteristic cancer processes involving
`PI3K/Akt
`
`As proposed by Hanahan and Weinberg,33 most of
`genetic/epigenetic changes related to tumor phe-
`notype are representative of a finite succession
`of physiologic disturbances, which, collectively,
`cause the cell to become malignant. In this setting,
`as shown in Table 2, Akt-regulated signalling plays
`an outstanding role in numerous processes which
`are known to be characteristic of cancer.
`
`Alterations of PI3K/Akt pathway in
`human cancer
`
`PI3K gene alterations
`
`The major alteration described to occur in the gene
`PI3K is amplification. The gene PIK3C, which en-
`codes the p110a catalytic subunit of PI3K, is lo-
`cated in the chromosome 3q26, a region that is
`frequently amplified in several human cancers.
`Findings from recent studies have confirmed PIK3C
`amplification in ovarian34 and cervix35 cancer.
`
`Akt gene alterations
`
`No modifications or mutations in the akt gene have
`been found in mammals. Nevertheless, various
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`Table 2
`
`Akt implication in different processes characteristic of cancer
`
`Process
`
`Akt function
`
`Growth signal autonomy
`
`Insensitivity to antiproliferative signals
`
`Inhibition of apoptosis
`
`Unlimited replicative potential
`Angiogenesis
`Invasion and metastasis
`
`Akt overexpression or activation may lead to increased response to
`ambient levels of growth factors
`Induces nuclear entry of Mdm2, which leads to inhibition of p53
`regulated processes. Induces cytoplasmic localization of p21Cip=Waf1and
`p27Kip, promoting proliferation. Stabilizes Cyclin D1
`Inactivates the proapoptotic factors Bad and Procaspase-9. Activates
`IKK, activating the transcription of NF-jB regulated antiapoptotic
`genes. Inactivates Forkhead family transcription factors, inhibiting
`proapoptotic gene expression, such as Fas ligand
`Increases telomerase activity by phosphorylation of hTERT
`Pomotes angiogenesis through eNOS activation
`Contributes to invasiveness by inhibiting anoikis and stimulating MMP
`secretion
`
`studies have found akt amplifications in human
`cancers. The first research work describing akt as
`a potential human oncogene detected akt1 in a
`gastric carcinoma. Akt2 gene amplifications have
`been found in ovarian, pancreas, breast and
`tumors.36;37 Overall,
`such
`stomach malignant
`studies suggest that akt amplification, particularly
`that of akt2, may be a frequent event in different
`human cancers. Although one study has demon-
`strated increased Akt2 protein expression in pan-
`creas cancer with akt2 amplification,38 data about
`Akt levels and its activation in clinical materials
`are still scant.
`
`Further PI3K/Akt pathway alterations
`
`Ligand-dependent activation of protein tyrosine
`kinase receptors, receptors coupled with proteins
`G or integrins results in PI3K activation. Such ac-
`tivation may also occur independently of the re-
`ceptor, as
`is
`the case in cells expressing
`constitutively active Ras.39;40 As surface receptors
`are overexpressed or permanently active in many
`human cancers,12
`their downstream signalling
`pathways are also active. One important example
`is the RPTK ErbB2/HER2/Neu, which is overex-
`pressed due to gene amplification in a large num-
`ber of breast cancers and other types of human
`malignant tumors.41 ErbB2 is an orphan receptor
`that has no specific ligands, which acts as a partner
`for dimerization of other ErbB family members.
`ErbB2-formed heterodimers are potent triggers for
`multiple signalling pathways involved in cell pro-
`liferation, invasion and survival.42 When ErbB2 is
`overexpressed,
`it constitutively associates
`to
`ErbB3. ErbB2–ErbB3 dimers, in turn, are potent
`
`triggers for the PI3K/Akt pathway. In fact, tumor
`cells expressing ErbB2 show constitutive Akt ac-
`tivity.43 Thus, recent findings suggest a major role
`for PI3K/Akt pathway in stimulation of prolifera-
`tion and survival in overexpressing ErbB2 cells.44
`Further examples of PI3K/Akt pathway activation
`are mentioned below in relation to therapeutic
`implications.
`Downstream of PI3K are other molecules regu-
`lating this pathway, such as PI-3,4,5-P3 phosphata-
`ses. PTEN (Phosphatase and tensin homologue
`deleted on chromosome 10; also referred to as
`MMAC1, mutated in multiple advanced cancers 1) is
`a phosphatase with dual activity on lipids and pro-
`teins. It was originally identified as a tumor-sup-
`pressor gene. Its main physiologic lipid substrate is
`PI(3,4,5)P3, i.e., the PI3K product. PTEN dep-
`hosphorylates PI(3,4,5)P3 at 30 inositol position and,
`in this way, acts as a negative regulator for PI3K-
`induced signalling. Studies on PTEN overexpression
`in different cell lines suggest that it acts as a tumor
`suppressor by inhibiting cell growth45 and enhancing
`cellular sensitivity for apoptosis and anoikis, the
`latter being a particular type of apoptosis induced
`in epithelial cells due to alterations in integrin-
`extracellular matrix interaction.46 PTEN is fre-
`quently mutated in advanced stages of several
`human tumors, notably in glioblastoma and endo-
`metrial and prostate cancers. In addition, PTEN
`mutations in germ cell lines result in the rare he-
`reditary syndrome referred to as Cowden’s disease,
`which is associated with a higher risk for develop-
`ment of malignant tumors, notably breast cancer.47
`In summary, PTEN negatively regulates Akt acti-
`vation through PI(3,4,5)P3 dephosphorylation;
`thereby, PTEN activity loss leads to permanent
`PI3K/Akt pathway activation.
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`
`Therapeutic implications and emergent
`drugs
`
`Therapeutic implications
`
`To date, tumor resistance to antineoplastic drugs
`has been attributed to increased drug export from
`the cell or enhanced detoxification mechanisms.
`Despite correlations between drug export and de-
`toxification and resistance, it is increasingly rec-
`ognized that these mechanisms cannot account by
`themselves for tumor cell resistance to antineo-
`plastic agents. Currently, we know that chemo-
`therapy agents kill tumor cells by inducing apoptosis
`and there is increasing evidence that the exploita-
`tion of survival pathways, which may have consid-
`erably contributed to the disease onset, may also be
`important in the development of chemotherapy
`drug resistance.48 Survival signals induced by sev-
`eral receptors are mediated mainly by PI3K/Akt,
`hence this pathway may play a major role in drug
`resistance appearance. In fact, there is convincing
`evidence from recent research suggesting that
`PI3K/Akt pathway activation is related to tumor cell
`resistance to both chemotherapy and radiation.
`It has been recently reported that a correlation
`exists between Her2/Neu overexpression and Akt
`activation in breast cancer, and Akt-induced sig-
`nalling has been suggested to increase stress-
`induced apoptosis resistance in cells overexpress-
`ing Her-2/Neu.49 Regarding Her2/Neu, there is a
`clear-cut relationship between its overexpression
`and increased antineoplastic drug resistance.
`Findings from a recent study suggest a relationship
`between Her2-induced Akt activation and in-
`creased tumor cell resistance to chemotherapy
`drug-induced apoptosis via Mdm2 phosphoryla-
`tion.23 Moreover, it has been suggested that acti-
`vation of Akt1 by Her2/PI3K plays an important role
`in mediating multidrug resistance in human breast
`cancer cells and that Akt may therefore be a novel
`molecular target for therapies that would improve
`the outcome of patients with breast cancer.50
`In ovarian cancer, aberrant Akt expression or
`activation in different cell lines has been described
`to be able to confer paclitaxel resistance.51 It has
`also been reported that PI3K inhibition increases
`paclitaxel efficiency in in vivo and in vitro ovarian
`cancer models.52 Moreover, it has been shown that
`integrin-mediated protection to paclitaxel- and
`vincristine-induced apoptosis
`is dependant on
`PI3K/Akt signalling pathway activation.53
`In a recent study on cell lines from non-small
`cell lung cancer (NSCLC), it has also been observed
`that PI3K/Akt pathway constitutive activation
`
`J.A.F. Vara et al.
`
`promotes cell survival, and its genetic or pharma-
`cological modulation alters the response to dif-
`ferent therapeutic approaches.54 In addition, one
`very recently published article describes that Akt is
`a central mediator in the suppression of anoikis and
`modulation of chemotherapy-induced apoptosis in
`NSCLC cells, and therefore proposes Akt as a
`promising target for small molecule inhibitors to
`shift the apoptotic threshold in cancer cells after
`treatment with standard chemotherapy.55
`In pancreatic cancer cell lines, PI3K/Akt path-
`way has been found to be implicated in chemo-
`therapy drug
`resistance,
`and the potential
`therapeutic benefit of PI3K inhibitors combined
`with gemcitabine for pancreas cancer treatment
`have been described.56
`In bladder cancer PTEN overexpression has been
`shown to be able to induce tumor cell growth
`suppression and enhance sensitivity to doxorubicin.
`Therefore, it has now been recognized that PTEN-
`regulated pathways may be therapeutic targets for
`bladder cancer treatment.57
`On the other hand, results from a recent study
`indicate that Akt overexpression/activation is a
`frequent event in human colon cancer, being less
`common in colon tumors with microsatellite in-
`stability. This study suggests that apoptosis inhi-
`bition during sporadic colon cancer carcinogenic
`process can be attributed, at least partially, to
`Akt.58 Moreover, it has been reported that Akt
`phosphorylation status in human colon carcinoma
`correlates with cell proliferation and apoptosis in-
`hibition, as well as with different clinicopathologic
`parameters like invasion grade, vessel infiltration,
`metastases to lymphatic nodes, and stage.59
`Finally, an interesting research work has shown
`that PI3K/Akt pathway activation, evaluated by
`measuring Akt phosphorylation by means of immu-
`nohistochemistry techniques, may be a marker in
`predicting response to radiation therapy in head and
`neck cancer. Findings from this study suggest that
`EGFR-PI3K signalling may lead to radiation resistance
`and that PI3K may constitute a therapeutic target.60
`
`New drugs
`
`As we have seen above, the PI3K/Akt/PTEN pathway
`has become an attractive target for drug develop-
`ment as such agents might inhibit proliferation, and
`reverse the repression of apoptosis and the resis-
`tance to cytotoxic therapy in cancer cells. Inhibitors
`of proteins that are involved in several PI3K/Akt
`signalling pathways have been under development
`for some time, and some have now entered clinical
`trials. These include inhibitors that target both
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`upstream regulators of PI3K/Akt, such as growth
`factor receptors, and downstream effectors, such
`as the components of the mTOR pathway.
`Used alone or in combination with existing
`therapies, these inhibitors of the PI3K/Akt pathway
`will be developed to exploit activation of the PI3K/
`Akt pathway within cancer cells and/or enhance
`the efficacy of existing chemotherapeutic agents.
`
`Inhibitors targeting pathways upstream of
`PI3K/Akt
`
`Even in tumors in which PI3K/Akt pathway is not
`mutationally activated, this cascade might well be
`stimulated by aberrant activation of upstream
`signalling pathways. The components of the regu-
`latory system for PI3K/AKt that have proved most
`amenable to therapeutic intervention are the
`growth-factor-receptor tyrosine kinases, in partic-
`ular, the EGFR and its close relative ERBB2.
`Several approaches have been used to target the
`ERBB family, but by far the progress which has been
`made the most is in two areas: small-molecule ty-
`rosine kinase inhibitors61;62 and humanized anti-
`bodies against the receptor extracellular domains.63
`In general, antibodies bind to the extracellular do-
`main of the receptors, inhibiting their activation by
`ligand, and promoting receptor internalization and
`downregulation, whereas small molecules compet-
`itively inhibit ATP binding to the receptor, thereby
`hindering autophosphorylation and kinase activa-
`tion. Antibodies with intact Fc binding domains
`might also induce antibody-mediated cellular cyto-
`toxicity against the tumor cells.64
`Preclinical models indicate that EGFR expression
`is required, although the degree of expression
`above an undefined threshold does not predict
`sensitivity to EGFR inhibitors.65–69
`At present, the most advanced of the newer
`therapies in clinical development are anti-receptor
`monoclonal antibodies IMC-C225 (cetuximab, Erbi-
`tux; Imclone), and the reversible small-molecule
`inhibitors of EGFR, ZD1839 (gefitinib,
`Iressa;
`AstraZeneca) and OSI-774 (erlotinib,Tarceva; OSI
`Pharmaceuticals) (Table 3).
`ZD1839 has been through several Phase I trials,
`with the most common toxic side effect being skin
`rash and diarrhoea. Four Phase II trials have also
`been completed. Promising single-agent clinical
`antitumor activity has been reported in advanced
`NSCLC, head and neck cancer and prostate carci-
`noma.70 No effect was seen against metastatic
`renal-cell carcinoma. Phase III randomized trials of
`ZD1839, in combination with gemcitabine and cis-
`platin or with paclitaxel and carboplatin, for the
`
`treatment of NSCLC proved disappointing, given
`the expectations from earlier trials, leading to re-
`evaluation of treatment regimens.
`OSI-774 is similar to ZD1839 in its pharmacolog-
`ical characteristics, with around a tenfold lower
`IC50 against EGFR kinase activity in vitro.71 The
`two drugs had similar toxicities in Phase I studies,
`whereas Phase II trials using OSI-774 as a single
`agent produced promising results against NSCLC,
`ovarian, and head and neck cancer. Other EGFR-
`directed small-molecule tyrosine kinase inhibitors
`in early stage trials include PKI116 (Novartis),
`GW2016 (GlaxoSmithKline), EKB-569 (Genetics In-
`stitute/Wyeth–Ayerst) and CI-1033 (Pfizer).
`The other important class of therapeutic agents
`directed against ERBB-family receptors are hu-
`manized monoclonal antibodies. The most ad-
`vanced of this drug type against EGFR is a chimeric
`antibody – IMC-C225 – which is developed by Im-
`Clone Systems and Bristol-Myers Squibb (Cetux-
`imab, Erbitux). Phase I trials showed that the drug
`was reasonably well tolerated,72 and a Phase II trial
`showed promising results in advanced colorectal
`carcinoma. Unfortunately, widely reported regu-
`latory difficulties have delayed further develop-
`ment of this potentially important drug.
`The other
`important anti-receptor drug is
`trastuzumab (Herceptin), which was developed by
`Genentech. This humanized monoclonal antibody
`against ERBB2 has proved to be effective against
`breast carcinomas in which ERBB2 is highly ex-
`pressed – that is, 20–30% of cases of metastatic
`breast cancer. In Phase III trials, it extended the
`median survival of patients from 20.3 months to
`25.1 months.73 Despite some problems with cardiac
`toxicities, trastuzumab has now been licensed in
`the United States and elsewhere as a single agent
`for treating metastatic breast cancer with ERBB2
`overexpression.
`
`Inhibitors targeting Ras
`The covalent attachment of the farnesyl isoprenoid
`group to the HRas, KRas and NRas proteins is es-
`sential for the biological activity of Ras. Therefore,
`it was an obvious target for the design of new ra-
`tional therapies against the Ras pathway.
`A large number of highly effective farnesyl-
`transferase inhibitors (FTIs) have been identified.
`These were shown to efficiently inhibit the farn-
`esylation of HRas in cells in culture, which led to
`high expectations of being effective against the
`20% of human tumors that have activating muta-
`tions in Ras genes. Unfortunately, this early po-
`tential has not been realized. The mode of action
`of FTIs has become increasingly unclear, and the
`initial spectacular successes that were achieved in
`
`Roxane Labs., Inc.
`Exhibit 1023
`Page 007
`
`

`
`200
`
`J.A.F. Vara et al.
`
`Table 3
`
`New drugs targeting PI3K/Akt related molecules
`
`Target
`
`Drug
`
`Class
`
`Development stage
`
`Upstream targets
`EGFR
`
`Antibody
`
`Antibody
`Antibody
`
`Antibody
`
`IMC-C225 cetuximab (Erbitux;
`Imclone)
`ABX-EGF (Abgenix)
`EMD 72000 (Merck KgaA
`Darmstadt)
`RH3 (York Medical
`Bioscience Inc.)
`MDX-447 (Medarex/Merck KgaA) Antibody
`ZD1839 gefitinib (Iressa;
`Kinase inhibitor
`AstraZeneca)
`OSI-774 erlotinib (Tarceva;
`OSI-Pharmaceuticals)
`PKI116 (Novartis)
`CI-1033/PD183805 (Pfizer)
`EKB-569 (Wyeth–Ayerst)
`GW2016/572016
`(GlaxoSmithKline)
`
`Kinase inhibitor
`
`Kinase inhibitor
`Kinase inhibitor
`Kinase inhibitor
`Kinase inhibitor
`
`Phase III
`
`Phase II
`Phase I
`
`Phase II
`
`Phase I
`Phase III
`
`Phase III
`
`Phase II
`Phase I
`Phase I
`Phase I
`
`HER-2/Neu
`
`BCR-ABL/PDGFR/c-Kit
`
`PDK1
`
`Ras
`
`Specific targets
`PI3K
`
`Dowstream targets
`mTOR
`
`Trastuzumab (Herceptin;
`Genentech)
`MDX-210 (Medarex/Novartis)
`2C4 (Genentech)
`17-AAG (Kosan)
`
`Imatinib (STI571/Gleevec;
`Novartis)
`
`Antibody
`
`Registered
`
`Antibody
`Antibody
`HSP90 inhibitor
`
`Phase I
`Phase I
`Phase I
`
`Kinase inhibitor
`
`Registered
`
`UCN-01 (Kyowa Hakko Kogyo)
`
`Staurosporine analogue
`
`Phase I/II
`
`Phase II
`ISIS 2503 (Isis Pharmaceuticals) Antisense oligonucleotide
`R115777 (Johnson and Johnson) Farnesyl transferase inhibitor Phase II/III
`SCH66336 (Schering-Plough)
`Farnesyl transferase inhibitor Phase II
`BMS214662 (Bristol-Myers
`Farnesyl transferase inhibitor Phase I
`Squibb)
`
`Wortmannin
`LY294002
`
`PI3K Inhibitor
`PI3K Inhibitor
`
`CCI-779 (Wyeth)
`
`RAD001 (Novartis)
`
`Rapamycin/sirolimus (Wyeth)
`
`Inhibits mTOR kinase by
`binding to FKBP12
`Inhibits mTOR kinase by
`binding to FKBP12
`
`Inhibits mTOR kinase by
`binding to FKBP12
`
`Preclinical
`Preclinical
`
`Phase II
`
`Phase I as a cancer
`therapeutic Phase II/III
`as an immunosuppressant
`Registered as an
`immunosuppressant
`
`mouse models have not been repeated in human
`patients.
`Despite uncertainty about their mechanism of
`function, FTIs do have marked effects on the
`
`growth and survival of some tumor cell lines in
`vitro and on xenografts in nude mice, although not
`necessarily those expressing activated Ras. The
`effects of FTIs in these pre-clinical systems have
`
`Roxane Labs., Inc.
`Exhibit 1023
`Page 008
`
`

`
`Akt signalling and cancer
`
`
`
`201201
`
`recently been reviewed extensively.74;75 These
`data have prompted the launch of several clinical
`trials using FTIs, even though their real target is
`still controversial.
`A different approach to the therapeutic tar-
`geting of the Ras pathways is to inhibit the ex-
`pression of Ras using short antisense synthetic
`oligonucleotides that are specific for sequences in
`the mRNAs for these proteins.76 Early trials in
`nude m