phosphoinositide 3-kinase signalling pathways in ...

Tumori, 90: 2-8, 2004

PHOSPHOINOSITIDE 3-KINASE SIGNALLING PATHWAYS IN TUMOR PROGRESSION, INVASION AND ANGIOGENESIS Sharon Brader and Suzanne A Eccles Tumor Biology and Metastasis, Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, McElwain Laboratories, Belmont, Surrey, UK

Aims and background: The PI3 kinase signalling pathway is now accepted as being at least as important as the ras-MAP kinase pathway in cell survival and proliferation, and hence its potential role in cancer is of great interest1. The purpose of this review is briefly to examine evidence for an involvement of PI3K in human cancers, discuss the mechanisms by which its activation promotes tumor progression, and consider its utility as a novel target for anticancer therapy. Methods and study design: A Medline review of recent literature concerning the role of PI3 kinase in tumor progression – mechanisms of action and clinical implications. Results: Evidence is presented that misregulation of the PI3 kinase pathway is a feature of many common cancers, either by loss of the suppressor protein PTEN, or by constitutive activation of PI3 kinase isoforms or downstream elements such as AKT and mTOR. This activation potentiates not only cell sur-

vival and proliferation, but also cytoskeletal deformability and motility; key elements in tumor invasion. In addition the PI3K pathway is implicated in many aspects of angiogenesis, including upregulation of angiogenic cytokines due to tumor hypoxia or oncogene activation and endothelial cell responses to them. These cytokines signal though receptors such as VEGF-R, FGF-R and Tie-2 and potentiate processes essential for neoangiogenesis including cell proliferation, migration, differentiation into tubules and “invasion” of these capillary sprouts into extracellular matrix (ECM). Conclusions: A more complete understanding of the role of the PI3 kinase pathway in cancer will lead the way to the development of more potent and selective inhibitors which should be a useful adjunct to conventional therapies, potentially interfering with tumor progression at several pivotal points; in particular cell survival, invasion and angiogenesis.

Key words: angiogenesis, cancer, invasion, metastasis, motility, phosphoinositide 3-kinase.

The phosphoinositide 3-kinase (PI3K) family

PI3 kinases are enzymes which, when activated by a variety of cellular stimuli, phosphorylate inositol lipids to generate 3-phosphoinositides which are important second messengers in cell signalling pathways. Phosphorylated lipids specifically recruit many signalling proteins including serine-threonine kinases, protein tyrosine kinases and exchange factors that regulate G proteins. These proteins are normally present in the cytosol in an inactive form, but when localized to the membrane, they too become activated and are involved in assembly of signalling complexes and initiation of kinase cascades. PI3K are conserved from yeast to mammals with a high degree of homology, attesting to their importance in the physiology of all eukaryotic cells. Interest in them grew when they were found to be associated with two viral oncoproteins: polyoma virus middle T and pp60v-src and, when activated, to be able to prevent cell death (apoptosis). PI3 kinases are a large and complex family which are grouped into three subfamilies based on their primary structure, regulation and in vitro lipid substrate preferences. Class 1 PI3K are heterodimers comprised of a regulatory domain (p110) and a catalytic domain (generally referred to as p85, although some forms are smaller). They can be further classified into two structurally and functionally different subgroups, 1A and 1B

which are activated by cell surface receptor tyrosine kinases and G-protein coupled receptors respectively. The catalytic subunit of the class 1A group is encoded by three genes (α, β, δ) which have a very similar structure. The regulatory subunits are also the product of three different genes (α, β, γ) which produce further variants by alternative splicing. Class 1A catalytic subunits associate with p85, p50 and p55 adaptors; class 1B (p110γ) associates with a unique p101 adaptor protein. Class II enzymes are large (170-210 kDa) and primarily expressed in leukocytes as membrane-associated proteins containing characteristic C-terminal domains. They are capable of generating phosphatidylinositol 3phosphate and 3,4-bisphosphonate (class I enzymes additionally generate the -3,4,5 trisphosphate). The class III enzymes can only generate the monophosphate PtdIns (3)P. Since class 1A PI3Ks have been primarily implicated in tumor progression, the following discussion will focus on this subfamily. PI3K regulation

The PI3 kinase class 1A enzyme family is activated by binding of ligands such as insulin, platelet derived growth factor (PDGF), heregulins (HRGs) and vascular endothelial growth factor (VEGF) to their respective tyrosine kinase receptors. Receptors can also indirectly activate PI3K via RAS, which can bind to and activate the

Correspondence to: Dr Suzanne A Eccles, Tumor Biology and Metastasis, Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, McElwain Laboratories, Belmont, Cotswold Rd, Belmont, Sutton, Surrey SM2 5NG, UK. Tel +44-2087224210; fax +44-2087224134; e-mail [email protected] Received August 27, 2003; accepted September 9, 2003.

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PI3K AND TUMOR PROGRESSION

p110 subunit. Ligand-dependent recruitment of PI3K subunits catalyses the phosphorylation of the inositol ring of phosphatidylinositol (PtdIns) lipids to produce second messenger PtdIns(4,5)P2 (PIP2) which is itself an important PI3K substrate and is converted to PtdIns(3,4,5)P3 (PIP3). These inositol lipid products regulate a diverse set of signalling proteins downstream of PI3K. PIP3 is tightly regulated by phosphatases (PTEN, SHIP1 and SHIP2). PTEN reverts PIP3 back to PIP2, preventing downstream signaling, but the SHIP phosphatases convert PIP3 to PtdIns (3,4)P2 which can still function as a second messenger, suggesting that PTEN has primary responsibility for controlling the mitogenic effects of phosphoinositides. PTEN is a tumor suppressor protein with phosphatase activity against both lipid and protein substrates, although PIP3 is thought to be its main target (reviewed in Vazquez et al.2). Signaling downstream of PI3K Signaling molecules with pleckstrin homology (PH) domains such as AKT/PKB and PDKs bind directly to PIP2 and PIP3. This proximity in the plasma membrane allows activation of AKT which then propagates further signaling cascades. AKT is a 57 KDa serine/threonine kinase which, when translocated to the membrane, undergoes a conformational change to expose an activation loop. For full activation, phosphorylation is required at two sites. This is mediated by PH domain-containing protein kinases PDK1 at Thr308 and PDK2 at Ser473. The activity of PDK1 is also regulated by PI3K. There are three members of the serine/threonine kinase AKT family (AKT1, AKT2 and AKT3). AKT-1 was originally identified as a homologue of the retroviral oncogene v-Akt. Once active, AKT phosphorylates proteins on serine and threonine residues resulting in activation or inhibition of a multiplicity of downstream substrates. These have been linked to different functions associated with PI3 kinase activation (Figure 1). PI3K pathway misregulation in cancer PI3K is tightly regulated in normal tissues, but it is estimated to be constitutively active in up to 50% of human cancers. Alterations in the PI3K signalling cascade include overexpression or upregulation of PI3K or AKT isoforms and inactivation/silencing of PTEN; all of these processes result in hyperactivation of the pathway. Table 1 gives examples of human cancers where the PI3K pathway has been shown to be aberrantly upregulated. In several cases it seems that these changes are associated with advanced disease, loss of hormone (or drug) responsiveness and poor prognosis, suggesting functions in more than simply cell proliferation/survival, which are also features of benign disease. Experimental evidence for a role of PI3K in tumor progression Several pieces of evidence attest to the importance of PI3-AKT in oncogenesis and tumor progression. These

Figure 1 - Signalling pathways downstream of PI3K resulting in inhibition of apoptosis, increased cell survival, metabolic activity and protein synthesis; cytoskeletal rearrangements and upregulated motility/chemotaxis.

include experiments which show that over-expression or constitutive activation of these enzymes results in cell transformation, that the activity of certain viruses and oncogenes is PI3K pathway dependent, and that inTable 1 - Examples of human cancer where the PI3K pathway has been shown to be abenantly upregulated Cancer type

Alterations in PI3 kinase pathway

Ovarian

Amplification of p110α gene PI3K p85α mutation Elevated AKT1 kinase activity AKT2 amplification PTEN mutation Loss of PTEN heterozygosity and silencing of remaining alleles PIK3CA associated with VEGF expression, microvessel density and invasion

Ref 3 4 5 6 7 8 9

Cervical

Amplification of p110α gene

10

Colorectal

Overexpression of PI3K class 1a protein PI3K p85α mutation PTEN mutations in tumors with microsatellite instability

11 4

Breast

12

Elevated AKT1 kinase activity AKT2 amplification Loss of PTEN heterozygosity AKT3 mRNA overexpression and high enzyme activity in ER negative cancers

14

Pancreatic

AKT2 amplification

15

Glioblastoma

PTEN mutation in 70% of advanced tumors PTEN mutation in high grade tumors

7, 16 17

Melanoma

PTEN mutation PTEN silencing

7, 18 19

Prostate

AKT1 amplification PTEN mutation Loss of PTEN heterozygosity and silencing of remaining alleles

20

Leukemia

PTEN activation

21

Lymphoma

PTEN inactivation

22

Gastric

AKT1 amplification Activation of PI3K via erbB-3 associated with de-differentiation

23

PTEN inactivation

25

Lung

5 6 13

5 7

4

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hibition of signalling by pharmacological or molecular means can inhibit tumor cell proliferation in vitro and in vivo. In addition, transgenic mice bearing polyoma virus middle T (PymT) antigen under the control of the mouse mammary tumor virus promoter develop multiple metastatic mammary tumors, whereas PymT mutants unable to signal via PI3K only develop hyperplastic, highly apoptotic lesions26. Mechanisms of action The biological effects of PI3K activation relevant to cancer progression can be broadly categorized into those relating to: • tumor growth (cell proliferation, apoptosis, senescence; • angiogenesis (production of, and response to angiogenic cytokines); • metastasis (cytoskeletal plasticity, cell adhesion, cell motility, invasion). The first set of functions has been extensively reviewed27 and therefore will only be briefly summarised, with more attention being given to functions related to angiogenesis and metastasis, features associated with progressive disease. Cell survival and proliferation

Cancer cells have devised mechanisms to inhibit apoptosis and increase their chances of survival. This is particularly important in anoikis, a specialized form of programmed cell death which normal epithelial cells undergo when they are deprived of attachment to (and survival signals from) physiological substrates. Metastatic carcinoma cells are able to over-ride this restraint during dissemination. One of the consequences of PI3K or AKT activation is engagement of an antiapoptotic pathway. This involves a variety of substrates downstream of AKT that are inhibited or activated to prevent apoptosis. For example, AKT prevents release of cytochrome c from mitochondria and inactivates forehead (FKHR) transcription factors preventing their nuclear translocation and subsequent activation of downstream pro-apoptotic proteins, including BIM and FAS ligands. AKT phosphorylates and inactivates a prodeath protease, caspase 9, and the anti-apoptotic factor BAD. AKT via Iκκ induces nuclear translocation of the survival protein NF-κB and MDM2 and targets the tumor suppressor gene p53 for degradation by the proteasome28. AKT also has several effects on the cell cycle machinery, acting to potentate proliferation. Cyclin D1 is important in the G1/S checkpoint and AKT is involved in preventing cyclin D1 degradation by regulating levels of its kinase GSKβ. AKT also influences levels of CDK inhibitors such as KIP1 and p21/WAF1 via FKHR allowing the cell to progress through the cell cycle.

S BRADER, SA ECCLES

Angiogenesis

Angiogenesis can be considered to have both afferent (induction) and efferent (response) elements (Figure 2). The former includes the production of angiogenic cytokines by tumor and/or host cells due to oncogenic activation or hypoxia, and the latter the functional responses of vascular and lymphatic endothelial cells to these stimuli, which include cell proliferation, migration, “invasion” of the ECM and differentiation into new capillaries. Interestingly, many of the signalling pathways and processes used by activated endothelial cells mimic those used by invading tumor cells, and the PI3 kinase pathway plays a key role in both. Induction of angiogenic cytokines (“afferent” arm)

In many cancers, vascular endothelial growth factor(s) (VEGFs) are the most powerful and selective angiogenic cytokines. VEGF transcription is induced by hypoxia through activation of the PI3 kinase pathway and hypoxia-inducible factor alpha (HIF1α)29. Loss of PTEN upregulates this afferent angiogenic pathway, and reintroduction of PTEN into prostate carcinoma cell lines decreased VEGF production and their angiogenic potential) 30 . Similarly, the PI3 kinase inhibitor LY294002 reduced both constitutive and hypoxia-inducible levels of VEGF in ovarian carcinoma cells9. Induction of angiogenic cytokines such as VEGF and IL-8 by a variety of growth factors including PDGF, EGF, HGF and HRG is PI3 kinase dependent29,31-33. Endothelial cell responses to angiogenic cytokines (“efferent” arm)

PI3 kinase is downstream of the major VEGF receptors in endothelial cells, VEGFR-1 (Flt-1) and VEGFR2 (KDR/Flk-1)34,35 and this pathway mediates many of their functional responses. VEGF is an important survival factor in newly formed vasculature, and this process is mediated via PI3 kinase/AKT and induction of bcl-2. It has been reported that VEGF activation of Oncogenic TKR VEGF receptors

Proliferation Migration

Hypoxis

Proteolysis

Tie 2

Matrix invasion FGF receptors

A) Release of angiogenic cytokines

Differentiation

B) Functional responses of endothelial cells

Figure 2 - Angiogenic process. A) Production and release of angiogenic cytokines (eg, VEGF, bFGF and angiopoietins by tumor and host cells in response to hypoxia and/or the presence of activated oncogenic signalling pathways. These cytokines bind to cognate receptors on endothelial cells (VEGF-Rs, FGF-Rs, Tie-2) and induce B) a variety of functional response which collectively contribute to neoangiogenesis. The PI3K pathway has been implicated in all components of this phenomenon.

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PI3k is mediated via focal adhesion kinase (FAK)36, and both are implicated in VEGF-mediated endothelial cell migration. PI3 kinase is also involved in endothelial cell survival and migration mediated by angiopoietin 137. Recently it has been shown that topotecan inhibits both VEGF- and bFGF-induced endothelial cell migration via downregulation of the PI3k/AKT pathway38. In addition, a third important angiogenic mediator, nitric oxide synthase (eNOS), has also been shown to be an AKT substrate39. Thus PI3 kinase signal transduction is implicated in multiple key angiogenic pathways at both afferent and efferent levels, and may therefore represent an excellent pivotal point for therapeutic intervention. This has been exemplified by the finding that the mTOR inhibitor rapamycin can reduce both a) induction of VEGF and b) endothelial cell proliferation and tube formation in response to this cytokine, resulting in significant inhibition of angiogenesis, tumor growth and metastasis in vivo40. Cell migration and invasion

The molecular mechanism of tumor invasion is a complex multi-stage process. For cancer cells to invade, they must penetrate the surrounding tissue by releasing proteases to degrade and remodel the extracellular matrix (ECM). They then move randomly on the ECM or directionally in response to chemotactic gradients by a variety of mechanisms which may include transient changes in substrate adhesion. This allows the leading edge of a cell to project actin protrusions called lamellipodia and filopodia41 and to break existing ECM contacts at their trailing edge, so that the cell can move forward42. A protease-independent amoeboid-like movement has also recently been described43. Protease production PI3K has been shown to be involved in the secretion of urokinase plasminogen activator-1, which degrades plasminogen in the ECM and which is important for both tumor cell invasion and vascular smooth muscle cell migration44. Secretion of a second key protease, matrix-metalloproteinase 9 (MMP-9), which degrades collagen IV, a major component of basement membrane, is also regulated by PI3K45. Recently, the PI3KAkt-mTOR pathway has been shown to play a central role in IGF-1R mediated tumor cell invasion via co-ordinated upregulation of MMP-2 expression and its activator MT1-MMP46. Adhesion Integrins are the major family of cell surface receptors that mediate attachment to the ECM. They are composed of α and β subunits that heterodimerize to form receptors with different but overlapping specificity for matrix proteins such as fibronectin, laminin, collagens etc. PI3K regulates integrin-dependent cell motility by modulating integrin responses42. For example, PI3K is important for EGF and HRGβ stimulated breast carci-

noma adhesion and migration driven by up-regulated β1 integrin function47,42. In breast carcinoma cells α6β4 integrin activates PI3K to promote lamellae formation and invasion42,48. Integrin clustering also activates focal adhesion kinase (FAK) resulting in PI3K co-activation. Other adhesion molecules involving PI3K signalling include integrin-linked kinase (ILK), which interacts with the cytoplasmic domains of integrins β1 and β3, whose activity is regulated in a PI3K dependent manner49. Cell interactions with collagen matrices also depend on PI3K.50 Overexpression of PTEN can also inhibit tumor cell migration. It has been shown to regulate dynamic cell surface interactions that involve integrins, FAK and the cytoskeleton51. Motility PI3K is recognized to be involved in initiating actin cytoskeletal rearrangements, cell polarization and cell migration in many cell types. During this process, PI3K is translocated from the cytosol to the cytoskeleton via interaction with additional proteins such as Tyk-2 and src. Small GTP binding proteins, cdc42 and Rac1, are involved in cell motility and actin cytoskeletal deformability. These are regulated by PI3K, in some cases independently of AKT52. Lamellipodia extension requires p110α to activate Rac53 or cdc42, leading to activation of PAK1 kinase, which phosphorylates LIM kinase, thus acting on cofilin (involved in depolymerisation of actin in response to EGF). 54PDGF-induced cell migration on collagen signals through p85α via the cdc42 pathway to promote filopodia formation55. Integrinlinked kinase has been implicated not only in AKT activation, but also in phosphorylation of GSK3β. This provides links with the β-catenin/APC pathway and hence further possible roles in cell deformability and motility. The accumulated evidence clearly demonstrates that many types of tumor cells and also endothelial cells utilize PI3K signaling for motility, chemotaxis and invasion of the ECM. PI3 kinase inhibitors

A number of naturally occurring compounds exist that inhibit PI3K, for example wortmannin and demethoxyviridin56 originally isolated from soil bacteria, and a bioflavinoid, quercetin. A chromenone analogue of quercetin, LY294002, was developed in 1994 and was found to be three times more active against PI3K57. However, wortmannin is unstable and also inhibits DNA-PK58,59; LY294002 is of low potency and inhibits casein kinase 260 at doses similar to those required for PI3K inhibition. These agents are far from ideal for in vivo use and have not been developed clinically. However, despite problems of specificity, potency and pharmacokinetic properties, they have proved useful for exploring possible PI3K functions in tumor cells. More recent gene “knock out” and “knock down” technologies have added to our knowledge, although these also are not without difficulties since loss of one subunit of the PI3K

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signalling complex (eg, P110α) may lead to aberrant responses due to intracellular accumulation of its catalytic partner, compensatory mechanisms, or loss of viability of the manipulated cells or animals. Although these inhibitory strategies are currently imperfect, in vivo data indicate that PI3K pathway inhibition may be an effective therapeutic treatment. LY294002 and wortmannin treatment have shown tumor growth inhibition in a variety of cancer models including ovarian61 colon62 pancreatic63 and small cell lung cancer64 and the mTOR inhibitor rapamycin (described earlier) also showed therapeutic potential in a

S BRADER, SA ECCLES

metastatic tumor model40. Recently, combination studies showed that PI3K inhibition increased the efficacy of conventional chemotherapy. This approach may overcome the toxicity seen with PI3K inhibitor treatment alone as lower doses are required when given in combination with cytotoxic drugs (reviewed in Kip et al65). There are many initiatives currently underway aiming to develop more potent, selective, or isoform-selective inhibitors of PI3K, AKT and downstream effectors. Several series of new, selective PI3K inhibitors have been disclosed in the patent literature although these are not yet available commercially6-68.

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