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Are class II phosphoinositide 3-kinases potential targets for anticancer therapies?

Bulletin du Cancer. Volume 93, Number 5, 10053-8, Mai 2006, Electronic Journal of Oncology

Summary

Author(s) : Colin J Traer, Fiona M Foster, Siemon M Abraham, Michael J Fry , School of Biological Sciences, AMS Building, University of Reading, Whiteknights, PO Box 228, Reading RG6 6AJ, Berkshire, United Kingdom.

Summary : Of the three classes of true phosphoinositide (PI) 3-kinases, the class II subdivision, which consists of three isoforms, PI3K-C2α, PI3K-C2β and PI3K-C2γ, is the least well understood. There are a number of reasons for this. This class of PI 3-kinase was identified exclusively by PCR and homology cloning approaches and not on the basis of cellular function. Like class I PI 3-kinases, class II PI 3-kinases are activated by diverse receptor types. To complicate the elucidation of class II PI 3-kinase function further, their in vitro substrate specificity is intermediate between the receptor activated class I PI 3-kinases and the housekeeping class III PI 3-kinase. The class II PI 3-kinases are inhibited by the two commonly used PI 3-kinase family selective inhibitors, wortmannin and LY294002, and there are no widely available, specific inhibitors for the individual classes or isoforms. Here the current state of understanding of class II PI 3-kinase function is reviewed, followed by an appraisal as to whether there is enough evidence to suggest that pharmaceutical companies, who are currently targeting the class I PI 3-kinases in an attempt to generate anticancer agents, should also consider targeting the class II PI 3-kinases.

Keywords : phosphoinositide 3-kinase, phosphatidylinositol (3) phosphate, cell signalling, cell migration, intracellular trafficking, cancer

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ARTICLE

Auteur(s) : Colin J Traer, Fiona M Foster, Siemon M Abraham, Michael J Fry

School of Biological Sciences, AMS Building, University of Reading, Whiteknights, PO Box 228, Reading RG6 6AJ, Berkshire, United Kingdom

It is over twenty years since a lipid kinase activity was discovered associated with retroviral oncogene products and growth factor receptors, that later turned out to have a novel specificity for the 3’ position OH group of the inositol ring [1]. This kinase activity is now known to be attributable to a class Ia phosphoinositide (PI) 3-kinase and in recent years the evidence has grown for this class of PI3-kinase playing important roles in the establishment of key aspects of the cancer phenotype [2-4]. A class Ia PI3-kinase has been found as a chicken retroviral oncogene. The phosphatase, PTEN, an enzyme that dephosphorylates the major in vivo product of class I PI3-kinases, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P₃), is a widely inactivated tumour suppressor. Most recently cementing the role of class Ia PI3-kinases in human disease, mutations have been found in many human tumours in the PIK3CA gene, which encodes the catalytic subunit of the class Ia PI3-kinase, p110α [4]. This has lead to this class Ia PI3-kinase becoming a potentially important future therapeutic target and it has become the focus of many drug programmes to develop new small molecule inhibitors [5]. Other class Ia catalytic subunits (e.g., p110δ and p110γ) have also been identified as targets in other diseases.Over this same twenty year period, the human PI3-kinase family of enzymes has also grown in number and complexity and now encompasses eight different catalytic subunits with PI 3-kinase activity which have been organised into three classes (I–III) based on enzyme domain structure and substrate specificity (and an additional class IV subgroup with protein kinase, but not lipid kinase activity) [6, 7].The class I subfamily of PI3-kinases consists four catalytic subunits: three class Ia (protein-tyrosine kinase regulated) subunits p110α, 110β, and p110δ and a single class Ib isoform (G-protein coupled receptor (GPCR) regulated), p110γ [6, 8]. The main in vivo product of this class, as noted above, is phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P₃) [9]. The class I PI3-kinases control a range of cellular functions including cell proliferation, cell differentiation, cell survival and cell migration depending on the cell type [6, 8, 9]. There are three human class II PI3-kinase isoforms, PI3K-C2α, PI3K-C2β/HsC2-PI3K and PI3K-C2γ [6]. In vitro their preferred substrates are PtdIns and PtdIns(4)P resulting in the production of PtdIns(3)P and PtdIns(3,4)P₂. The roles of this class will be discussed in detail below. There is a single class III isoform in mammals, which is the homolog of the sole yeast PI3-kinases, the Vps34 protein. This enzyme is a PtdIns-specific PI3-kinase which makes PtdIns(3)P mainly on intracellular vesicles where it plays a role in the regulation of intracellular trafficking [6, 8–10].Routine investigation into potential roles of these PI3-kinases regularly involves the use of the structurally unrelated inhibitors, wortmannin and LY294002 [5, 6]. However, the lack of selectivity of these inhibitor for the different classes and isoforms of PI3-kinase has led to further confusion in the field as many researchers seem to equate the effects of these two inhibitors with class I enzymes alone. The sensitivity of the class II PI3-kinase members, PI3K-C2β and PI3K-C2γ, to wortmannin is in the low nM range ~ 2-30 nM [11-15] similar to that reported for human class I and class III PI3-kinases [5]. However, PI3K-C2α is refractory to wortmannin, with an IC₅₀ of around 420 nM [7, 16]. PI3K-C2α is also inhibited by higher concentrations of LY294002 (~ 20 μM) [16] than PI3K-C2β (~ 7 μM) or p110α (~ 1 μM) [11].

Class II PI 3-kinases

Expression

As noted earlier there are three human class II PI3-kinase isoforms, PI3K-C2α [16], PI3K-C2β/HsC2-PI3K [11, 17], and PI3K-C2γ [18]. Initial reports based on Northern blotting of limited numbers of tissue mRNAs suggested that both PI3K-C2α and PI3K-C2β are widely expressed [16,17, 19]. Subsequent studies at both the RNA and protein level would seem to confirm this, although they are not ubiquitous and their levels vary considerably between cell types [20]. Thus, as with the multiple class I p110 isoforms, the possibility exists that these two isoforms may share some overlapping redundant functions. PI3K-C2γ has a much more restricted expression profile. Most reviews to date have mistakenly reported it to be exclusively expressed in liver, but this is not the case. PI3K-C2γ can also be found in breast, prostate and salivary glands at similar levels to that found in liver, and can also be detected at lower levels in other tissues [18, 19].

Enzyme structure

In contrast to class I and III PI3-kinases, which both possess regulatory subunits, the class II PI3-kinases are large monomeric enzymes with extended N- and C-termini. The central core of class II PI3-kinases (comprising the Ras binding, C2-like, PIK and the core kinase domains) is structurally related to the class I enzymes, but lacks the N-terminal p85/p101 adaptor protein binding motifs ( (figure 1) ). Instead the class II PI3-kinases all possess an extended N-terminus, which has low sequence homology, and varies in structure, between the three isoforms, containing coiled-coil domains, proline-rich sequences and clathrin binding motifs. The extension at the C-terminus is common between all three class II PI3-kinase isoforms and contains two clearly delineated regulatory domains; a Phox homology (PX) domain [21] followed by a C2 [22] domain ( (figure 1) ). These PX and C2 domains are likely to play a role in membrane association. Both the N- and C-terminal extensions are hypothesised to be involved in the regulation of their activity [23].

Localization

Class II PI3-kinases have been reported to be present in a number of different cellular compartments, but appear to be predominantly localised to intracellular membranes, often being observed in punctate structures and perinuclear accumulation [11, 24, 25]. Only small amounts of these enzymes can be found associated with the plasma membrane [26]. Interestingly, both PI3K-C2α and PI3K-C2β have been reported to have nuclear associated forms, with PI3K-C2α reported to be found in nuclear speckles [27, 28]. The significance of the nuclear association/localisation remains to be determined.

Substrate specificity

Class II PI3-kinases can utilise PtdIns and PtdIns(4)P as substrates in vitro, but not PtdIns(4,5)P₂. In vivo the class II PI 3-kinases are likely to be tightly regulated and the in vivo substrates for these kinases remain somewhat controversial. Conflicting evidence exists which suggests that they may generate any of the four known 3’phosphorylated phosphoinositides [11, 15, 16, 26, 29, 30]. We, and others, have transient transfected PI3K-C2α and PI3K-C2β into cells, but have failed to observe changes in in vivo labelled lipids [11, 25, MJF, unpublished observations]. An assumption in much of the literature seems to have been that PtdIns(4)P was likely to be their major substrate based on the in vitro ability of class II PI3-kinases to generate PtdIns(3, 4)P₂ and on the possibly mistaken assumption that all PtdIns(3)P in cells is made by a class III PI3-kinase (as is the case in yeast). However, several recent studies point to PtdIns(3)P as an in vivo product of class II PI3-kinases [31, 32] with sites of production within the cell that are distinct from those that are accessible to class III PI3-kinase. This will be discussed further later.

Class II PI 3-kinase activation

Like class I enzymes, class II PI3-kinases are linked to diverse receptor-mediated signalling processes. A growing number of growth factors (e.g., EGF, PDGF) [33, 34], stem cell factor (SCF) [35], insulin [36–38], chemokines [29], and cytokines, leptin and tumour necrosis factor α (TNFα) [39] have been shown to induce an increase in the activity of either PI3K-C2α or PI3K-C2β in a variety of cell types. Some stimuli such as insulin have been shown to activate both isoforms [36-38]. Integrin activation has also been shown to activate class II PI3-kinases [14, 40]. Aggregation of platelets caused by activation of integrin α_iibβ₃ and the consequent binding of fibrinogen resulted in an increase in PI3K-C2β activity, which was associated with a transient increase in PtdIns(3)P levels [14]. In migrating vascular smooth muscle cells, the integrin α_vβ₃ activated PI3K-C2α [40]. Stimulation of THP1 cells with the GPCR agonist, MCP1, resulted in a rapid activation of PI3K-C2α activity, which subsequently declined to basal levels within 30 seconds [29]. Turner et al. (1998) also demonstrated that this MCP1 activation of PI3K-C2α activity was pertussis toxin sensitive and was temporally correlated with an increase in PtdIns(3,4,5)P₃ levels and a sustained increase in PtdIns(3,4)P₂ levels [29]. More recently lysophosphatidic acid, acting via a GPCR, has been shown to activate PI3K-C2β in HeLa cells and was linked to the production of PtdIns(3)P at the plasma membrane [31].

Class I PI3-kinases have been well characterised in regards to their activation mechanism, by both receptor protein-tyrosine kinases (class Ia) and GPCRs (class Ib). However, little is currently known about the mechanism by which class II PI3-kinases are activated by receptors. Both PI3K-C2α and PI3K-C2β have been shown to have clathrin-interacting motifs, and the binding of clathrin can activate both of these isoforms in vitro [30, 41, 42]. As many of the receptor types that have been linked to class II PI3-kinase activation are also recruited into clathrin-coated pits soon after their activation, this may provide the missing link. Evidence also exists for more direct interactions between class II PI3-kinases and receptors. The interaction between PI3K-C2β and the EGFR was reported to be mediated by the adaptor protein, Grb2, and proline-rich sequences in the N-terminus of PI3K-C2β [34]. Class II PI3-kinase association with other receptors has been reported, but the molecular basis has yet to be examined.

Normal functions of class II PI 3-kinases

Signalling

As noted above, class II PI3-kinase are activated by diverse receptors involved in cell signalling cascades. However, as the products of the class II PI3-kinases remain unclear, so too are their potential downstream effectors and their ability to activate cell signalling pathways. The in vitro ability of class II PI3-kinases to generate PtdIns(3,4)P₂ has been used to hypothesise that they may activate known PI 3-kinase signalling targets such as protein kinase B (PKB) [17]. During an investigation of PI3-kinase signalling in small lung cell carcinomas (SCLC), PI3K-C2β was found to be highly expressed and was shown to contribute, along with class I PI3-kinases, to PKB activation [35]. Using PI3K-C2β antisense oligonucleotides, and a dominative negative PI3K-C2β construct, Arcaro and co-workers disrupted the EGF-dependent phosphorylation of PKB, but not the ERK, GSK-3β, or S6 pathways, and also disrupted SCLC growth in response to SCF [35].

Trafficking

PI3K-C2α has been shown to associate with clathrin, and also localised to the TGN [24, 25, 30]. These subcellular locations would be ideal for PI3K-C2α to generate 3-PIs that can modulate trafficking events. Despite the substantial evidence that class II PI3-kinases are localised to trafficking compartments scant work has been carried out to study any functional role they may play [30]. Brief investigation of PI3K-C2α in clathrin-dependent trafficking suggested a potential role in transferrin endocytosis, and the localisation of LAMP2 (lysosome associated membrane protein 2) and the M6P (mannose-6-phosphate) receptor [30]. As class II PI3-kinases are activated by a wide range of receptors, and can generate PtdIns(3)P, they would be placed in an ideal position to down-regulate cell signalling through the endocytosis of cell receptors [33, 43-45]. With the recent reports that PI3K-C2β is also activated by clathrin [42], further work is clearly required to determine the roles these two kinases may play in receptor trafficking following stimulation.

Cell survival

Interestingly it has been recently suggested that depletion of PI3K-C2α causes apoptotic cell death in the CHO-IR cell line based on an increase in DNA fragmentation [46]. The research by Kang and co-workers utilised short sense and antisense DNA oligonucleotides with the sequence of a fragment of human PI3K-C2α. Although the sequences used were not identical to either the mouse or rat orthologs of PI3K-C2α, the oligonucleotides did appear to deplete the level of PI3K-C2α in the hamster CHO-IR cell line [46]. Given the apparently broad specificity of these oligonucleotides, the relevance of this work is unclear as an examination of the sequences used show good homology with a number of mRNA species including potential cell cycle regulators such as PAK2, PAK3 and the PHD finger protein 2. Work in our laboratory has found little effect following knockdown of either PI3K-C2α or PI3K-C2β with isoform specific siRNAs on cell survival of HeLa cells (CJT, FMF and MJF, unpublished observations). Clearly more work is needed in additional cell types to clarify this issue.

Migration

The ability of class II PI3-kinases to regulate aspects of trafficking and signalling might affect a range of basic cell functions, including cell adhesion and migration. Class II PI3-kinases are activated by integrins in platelets and smooth muscle cells [14, 40], and this would position them for a role in cell adhesion and migration. Recent work we have carried out with collaborators using class II PI3-kinase specific siRNAs, showed that PI3K-C2β, but not PI3K-C2α, has a role downstream of LPA in regulating cell migration in HeLa cells [31]. Further evidence supporting for a role for a PI3K-C2β/PtdIns(3)P/cdc42 signalling pathway in cell migration in HEK293 cells has come from Domin and co-workers [32]. The lack of a role for PI3K-C2α in LPA stimulated cell migration may be due to specific migration pathways in some cell types not involving PI3K-C2α, but requiring PI3K-C2β [31]. A recent study showing that PI3K-C2α can affect cell contraction through a Rho-dependent mechanism suggests that this isoform might also contribute to migration in other cell types [47]. The potential role of class II PI3-kinases in intracellular trafficking and signalling may also involve the trafficking and sorting of the integrins and other adhesion proteins around the cell. Other activators of class II PI3-kinases, such as EGF and MCP-1, are also known chemoattractants [29, 33, 48]. It would be interesting to see whether class II PI 3-kinases are involved in the chemotaxis processes induced by other stimuli. Given that EGF stimulation is known to cause the association of both PI3K-C2α and PI3K-C2β with the EGFR [33], the potential role of PI3K-C2α in migration could be compared to that of PI3K-C2β to determine if this function is specific to PI3K-C2β.

Class II PI3-kinases and cancer

As noted earlier, a number of roles for class I PI3-kinases in cancer have been established [3, 4], but little has been done to determine whether the class II PI3-kinases might also play a role [2](table 1)( Table 1 ). This is clearly an important issue given the fact that class II PI3-kinase can produce PtdIns(3,4)P₂ (at least in vitro). More credence is given to this possible role from the report that both class I and class II PI3-kinase may contribute to the activation of PKB, a known oncogene in its own right, and a kinase clearly linked to cell survival [35]. So what evidence, if any, exists to implicate class II PI3-kinases as potential anticancer target proteins.

A single published study suggests that over expression of PI3K-C2β in colonic epithelial cells, but not Vps34 (the other PtdIns(3)P producing PI3-kinase), led to in vitro transformation, with cells forming colonies in soft agar, and foci on cell monolayers, albeit somewhat less efficiently than observed with the class I enzyme p110α [49]. Our experience however is that over expression of PI3K-C2β in NIH 3T3 fibroblasts had no transforming effects that could be discerned (MJF, unpublished observations). However, we have observed PI3K-C2β to be expressed at high levels in a number of epithelial cell derived cancers relative to the normal epithelial compartment (SMA and MJF, data not shown).

A close survey of the literature reveals that class II PI3-kinases, in particular PI3K-C2β, are suggested to be up-regulated in a range of cancers by microarray analysis [50-52]. PI3K-C2β expression was found to be increased in mixed lineage leukaemias (MLL) [49]. In a subtype of acute myeloid leukaemia (AML), not associated with MLL translocations, PI3K-C2β expression was also upregulated [51]. In patients with an increased PI3K-C2β expression, the protein tyrosine kinase receptor, FLT3, and Bcl2 genes were also found to show increased expression. These gene products were suggested to form a signalling pathway, which promoted growth in these cases of AML [51]. PI3K-C2β expression is not only elevated in leukaemias. The PI3K-C2β gene (PIK3C2B) is located to the 1q32 amplicon found in some cases of glioblastomas [53]. The major amplification target of the amplicon in malignant gliomas is the p53 regulator, MDM4, although it was suggested that the amplification of neighbouring genes including PI3K-C2β might provide further growth advantage [54]. Interestingly another co-amplified gene is PEPP3 (phosphoinositol-3-phosphate-binding PH-domain protein-3), a potential downstream target of PI3K-C2β produced PtdIns(3)P. The significance of this co-amplification is unclear. PI3K-C2β mRNA has also been shown to be upregulated in invasive intraductal papillary mucinous neoplasms, a type of pancreatic cancer [52]. PI3K-C2β expression was once more found to be increased along with other proteins with which it may form a signalling network, notably CXCR4 (chemokine (C-X-C motif) receptor 4), which is an activator of PI3K-C2β (FMF and MJF unpublished observations) and is linked with metastasis in a number of tissues.
Table 1 Overview of the PI 3-kinase family. This table provides a guide to possible expression, subcellular localisation, substrates and functions of members of the PI 3-kinase family that possess lipid kinase activity but due to the wealth of data on this family is not exhaustive

Class	Catalytic subunit	Expression	In vitro substrates	Likely in vivo substrate	Adapter/regulatory subunits	Localisation	Regulation	Functions
Ia	p110α	Ubiquitous?	PtdIns		p85α, p85β	Cytosol (resting), PM (stimulated), Nuclear	PTK, GPCR, Ras	Cell proliferation, Cell differentiation, Cell survival, Cell migration, Chemotaxis, Phagocytosis.
p110β	Widely expressed, but not ubiquitous.	PtdIns(4)P	PtdIns(4,5)P₂	p55^PIK/p55γ
p110δ	Whole blood, thymus, breast.	PtdIns(4,5)P₂
Ib	p110γ	Whole blood, thymus, heart, endothelium	PtdIns PtdIns(4)P PtdIns(4,5)P₂	PtdIns(4,5)P₂	p101 p84/p87PIKAP	Cytosol (resting), PM (stimulated), Nuclear	GPCR, Heterotrimeric G Proteins, Ras	GPCR signalling, Cell migration, Chemotaxis.
II	PI3K-C2α	Widely expressed, but not ubiquitous.	PtdIns PtdIns(4)P	PtdIns	?	TGN, Clathrin-coated vesicles, Nuclear speckles	PTK, GPCR, Integrins, Clathrin	Vascular smooth muscle contraction, Priming of neurosecretory granule exocytosis, Insulin signalling, Clathrin-mediated membrane trafficking.
II	PI3K-C2β	Widely expressed, but not ubiquitous. High in thymus and epithelia.	PtdIns PtdIns(4)P	PtdIns	?	Intracellular membranes, Nucleus	PTK, GPCR, Integrins, Clathrin	Cell migration, Cell proliferation? PTK receptor signalling.
II	PI3K-C2γ	Liver, Breast, Prostate	PtdIns PtdIns(4)P	PtdIns?	?	Golgi?	?	?
III	hsVps34	Ubiquitous	PtdIns	PtdIns	p150 (hsVps15) Beclin 1 (autophagy)	Endosomes Nucleus?	Constitutive?	Protein and vesicular trafficking and sorting, Autophagy, Phagocytosis?

Conclusions

After 10 years with only slow progress as to their possible functions, the class II PI3-kinases seem finally to have come of age with clear roles emerging in cell signalling, receptor trafficking and cell migration and at least some indication that their in vivo product is likely to be PtdIns(3)P. It is easy to see how if class II PI3-kinases are linked to these functions how they could contribute to the transformed phenotype. Western blotting of cancer cell line and tissue extracts and microarray analysis would seem to suggest that at least PI3K-C2β expression is fairly frequently enhanced in many cancer types. Together this would seem to suggest that if therapeutic inhibitors against class I PI3-kinases show some activity against class II PI3-kinase family members also this may be beneficial. However a final note of caution. It should be noted that the expression of the PI3K-C2γ isoform, which exhibits a narrower expression pattern that the other two members of this class, was observed to be reduced to non-detectable levels in breast cancer tissue samples and in breast cancer cell lines when compared to normal breast tissue/cell lines [M. Rozycka and MJF, unpublished observations]. This result together with the links described earlier between PI3K-C2α and PI3K-C2β and vesicle trafficking pathways that may down regulate receptor signalling suggests that more work on this class of PI3-kinases is really needed to substantiate the above assessment.

Acknowledgements

FMF, SMA and CJT were funded by the following grants awarded to MJF; FMF, BBSRC (grant number 45/C14421), SMA, Cancer Research UK (grant number C1469/A2603), CJT, University of Reading RETF PhD studentship.

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