_______________________________________________ Chapter 2x

Regulation of Phosphoinositide 3-kinase Expression in Health and disease Klaartje Kok, Barbara Geering, Bart Vanhaesebroeck

Regulation of PI3K expression 

Abstract The biology and therapeutic potential of the phosphoinositide 3-kinase (PI3K) signalling axis have been the subject of intense investigation, yet little is known about the regulation of PI3K expression. Evidence is emerging for alterations in PI3K levels upon cellular stimulation with insulin and nuclear receptor ligands, and during various physiological and pathological processes, including differentiation, regeneration, hypertension and cancer. Recently identified mechanisms that control PI3K production include increased gene copy number in cancer, transcriptional regulation of the PIK3CA/p110α catalytic subunit by FOXO3a, NF-κB and p53, and of the regulatory subunits by STAT3, EBNA-2 and SREBP. In most instances, however, the impact of alterations in PI3K expression on PI3K signalling and disease remains to be established.

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Introduction The importance of the PI3K signalling axis in a wide variety of normal and pathological responses is now well established. Most studies to date have focused on the acute alterations in PI3K activity induced by cell stimulation, and its impact on early downstream signalling by protein effectors of the PI3K lipids. However, evidence is emerging for more sustained changes in PI3K gene expression under various conditions, leading to persistent alterations in PI3K activity. Here we summarise and discuss the current knowledge on PI3K tissue distribution, the emerging evidence for dynamic regulation of PI3K expression and its impact on the PI3K subunit ratios and PI3K signalling, as well as the underlying molecular mechanisms that modulate PI3K expression. A better understanding of the regulation of PI3K expression might help to find ways to control PI3K activity in disease conditions where PI3K expression is deregulated. The PI3K family PI3Ks (phosphoinositide 3-kinases) generate 3-phosphorylated phosphoinositide lipids which transmit intracellular signals through binding to various protein effectors (reviewed in [1]). Mammals have genes for 8 catalytic and 6 regulatory subunits (Table 1). The PI3Ks have been divided into 3 classes [1], of which the class I PI3Ks have been studied most extensively and which will be the focus of this review. The currently available information on expression and regulation of class II and III PI3Ks is summarised in Box 1.

Chromosomal localisation Class  Subunit  Protein  Gene namehuman mouse

IA  catalytic  p110α  PIK3CA 3q26.32  3 band A3     p110β  PIK3CB 3q22.3  9 band E3.3     p110δ  PIK3CD 1p36.22  4 band E1   regulatory (p85s)  p85α  PIK3R1 5q13.1  13 band D1     p55α  PIK3R1 5q13.1  13 band D1     p50α  PIK3R1 5q13.1  13 band D1     p85β  PIK3R2 19p13.11  8 band B3.3     p55γ  PIK3R3 1p34.1  4 band D1 IB  catalytic  p110γ  PIK3CG 7q22.3  12 band A3   regulatory  p101  PIK3R5 17p13.1  11 band B3     p84/p87  PIK3R6  17p13.1  11 band B3 II  catalytic   PI3K‐C2α  PIK3C2A 11p15.1  7 band F1     PI3K‐C2β  PIK3C2B 1q32.1  1 band E4     PI3K‐C2γ  PIK3C2G 12p12.3  6 band G2 III   catalytic  Vps34  PIK3C3 18q12.3  18 band B2   regulatory  Vps15  PIK3R4 3q22.1  9 band F1 Table 1. The catalytic and regulatory PI3K subunits in mammals 

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Regulation of PI3K expression 

Class I PI3Ks are heterodimeric enzymes consisting of a 110 kDa catalytic subunit (p110α, β, γ, δ) in complex with a regulatory subunit [1]. They are acutely activated by tyrosine kinase pathways and GPCRs (G protein-coupled receptors), leading to the generation of the PIP3 lipid (phosphatidylinositol-3,4,5-trisphosphate) [1]. Downstream PIP3 effectors include protein kinases, adaptor proteins and regulators of small GTPases, which mediate signalling cascades controlling growth, cell cycle progression and migration [1]. The class IA catalytic subunits (p110α, p110β and p110δ) are bound to a regulatory subunit (p85α, p85β, p55α, p55γ or p50α; collectively referred to as “p85s”) which has two SH2 (Src homology 2) domains which can link these enzymes to tyrosine kinase signalling pathways. By contrast, the class IB catalytic subunit p110γ binds one of two non-p85 regulatory subunits, called p101 [2] and p84 [3,4], and mediates PI3K activity upon GPCR stimulation. Also the class IA PI3K p110β has recently been shown to be activated by GPCR agonists [5]. PI3K tissue distribution Most PI3K subunits appear to have a broad tissue distribution, with the exception of p110γ [6,7] and p110δ [8,9], which are highly expressed in leukocytes (Table 2). At the moment, PI3K expression patterns have only been studied at low resolution using approaches that do not allow discrimination between the different cell types in a tissue. In addition, high quality antibodies to PI3K subunits, especially antibodies that are validated for use in immunohistochemistry, are not available. In the rare instances where immunohistochemistry on tissue sections was used to address PI3K expression, unexpected cellular distribution of PI3K isoforms within tissues was observed. For example, the expression of p110α which is generally thought to be ubiquitous and uniform in various tissues, appears to be markedly enriched in the non-proliferating tumour regions of ovarian cancer in vivo [10]. In normal tissue, the same applies to p85α /p55α /p50α and the class II PI3K isoforms PI3K-C2α and PI3K-C2β, which all were mainly detected in the fully differentiated cells, and not in the proliferating cells [11]. To gain further insight into tissue distribution of class IA PI3Ks, we have created reporter mice with a β-Gal-LacZ reporter gene inserted into endogenous p110 loci by homologous recombination. Such mice are available for p110α [12] and p110δ [13]. These mice have thus far been instrumental in documenting the broad tissue distribution of p110α and the more restricted tissue distribution of p110δ, namely in the hematopoietic and, unexpectedly, also in the nervous system [14]. Reporter mouse strains have also been made for p110γ as part of the generation of p110γ-deficient mice [7]. This allowed the first clear documentation of the high enrichment of p110γ expression in

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leukocytes [7]. Reporter mice such as these will also be useful to determine PI3K isoform expression with resolution at the single cell level in tissue sections. Expression profiles of PI3K isoforms can be found in several databases, including https://biogps.gnf.org/, USCS Genome Browser (http://genome.ucsc.edu/index.html), Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo), ArrayExpress (http://www.ebi.ac.uk/microarray-as/aer) and Oncomine (http://www.oncomine.org/). PI3K   Expression  Refs.  

p110α  ubiquitous   [8,15,16] 

p110β  ubiquitous   [8,16‐18] 

p110δ  highly enriched in leukocytes, moderate expression in neurons and  

cancer cell lines from various origin (including melanoma, breast,  

colon) low expression in most other cell types 

[8,9,14,16, 

19] 

p85α  ubiquitous, with lowest expression in skeletal muscle  [16,20,21] 

p55α  highest expression in brain and muscle, undetectable in most other 

tissues  

[21,22] 

p50α  high in liver, moderate expression in kidney and brain  [21,23] 

p85β  ubiquitous, with lowest expression in skeletal muscle  [16,20,21] 

p55γ  high mRNA expression in brain and testis, moderate mRNA expression 

in most tissues, not detectable in liver and muscle. Low protein 

expression in liver, muslce, fat, liver, spleen. 

[16,20,24] 

p110γ  high in leukocytes, moderate to low expression in most other tissues  [25‐29] 

p101  high in leukocytes  [3,4] 

p84 and 

p87 

high in leukocytes and heart   [3,4] 

PI3K‐C2α  ubiquitous, highest in heart, placenta, ovary  [11,30,31] 

PI3K‐C2β  ubiquitous, highest in thymus and placenta  [11,30,31] 

PI3K‐C2γ  high in liver and prostate, moderate expression in breast and salivary 

gland 

[11,32‐34] 

Vps34  ubiquitous   [35,36] 

Vps15  ubiquitous   [35,36] 

Table 2. Tissue distribution of the mammalian PI3K family members  

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Regulation of PI3K expression 

Dynamic regulation of the ratio of p85 to p110 subunits - a mechanism to regulate PI3K activity? Under basal conditions, class IA PI3Ks are thought to exist mainly as obligate heterodimers, with the regulatory and catalytic subunits constitutively bound to each other. This model is based on the notion that the p85 and p110 proteins bind each other extremely tightly, an interaction which can even withstand high concentrations of salt, urea or detergent [37,38]. Furthermore, both the catalytic [39] and the regulatory subunits [40,41] appear to be unstable as monomers. Together, this is expected to lead to the presence of equal amounts of p85 and p110 protein in cells. In addition, there appears to be a good correlation between the total mRNA and total protein levels for all the distinct p85s and p110s, at least in certain cell lines [16], suggesting that the same amounts of regulatory and catalytic subunits are generated under basal conditions. Some researchers have suggested that a constitutive excess of p85 over p110 exists under basal conditions (reviewed in [42]). This hypothesis was put forward to explain the paradoxical observation that p85α and p85β gene knockout mice exhibit enhanced insulin-induced PI3K signalling in insulin-sensitive tissues [43-46]. In wild-type mice, free p85 was thought to compete with p110-bound p85 for binding to signalling complexes, thereby decreasing PI3K lipid-dependent signalling. In p85 knockout cells, the level of free p85 was considered to be preferentially reduced, leading to improved access of the existing p85-p110 dimers to signalling complexes. As discussed recently [47], increasing evidence argues against the presence of excess p85, at least under basal, unstimulated conditions. However, the existence of equal amounts of p85 and p110 subunits under basal conditions does not exclude the possibility that p85 and/or p110 expression can be acutely upregulated in cells. Indeed, p85α mRNA and protein can be induced in diverse cell types upon cellular stimulation ([48-59]; Table 3). Such an increase in p85 might stabilise p110 proteins leading to an increase in p85–p110 heterodimers, possibly enhancing PI3K activity, as might be the case upon stimulation of nuclear receptor RAR (retinoic acid receptor) [56]. Alternatively, increased p85 upon cellular stimulation might exist 'p110-free' - be it temporarily, given that p85 appears to be unstable on its own [40,41]. Such p110-free p85 could be a mechanism for short-term inhibition of PI3K activity and/or might allow p110-independent biological activity of p85 (reviewed in [60]). At the moment, no solid evidence for 'p110-free' p85 is available. Studies to assess the protein expression levels of the whole spectrum of p85 and p110 isoforms would be required to gain insight into this question. Short-term upregulation of p85 levels seems to increase PI3K activity. Thus, increased p85 levels following cellular stimulation with retinoic acid [56], estradiol [61] or cannabinoid receptor type 1 (CB1) antagonist ribonamant [57] correlate with sustained

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phosphorylation of Akt in cells. Moreover, insulin treatment of cells that express elevated p85 as a consequence of stimulation with nuclear receptors PPAR (peroxisome proliferator-activated receptor) and RAR, gives rise to increased in vitro PI3K activity in p85-immunoprecipitates [50,51]. Similarly, also class IA catalytic subunit expression can be increased upon cellular stimulation (e.g. by poly-2-hydroxyethyl-methacrylate (in keratinocyte differentiation [62]), by 3-isobutyl-1-methylxanthine and dexamethasone (in fibroblast differentiation [63]) and by the CB1 antagonist ribonamant [57]; Table 3). In the only case investigated, the observed increase in p110α appeared to be accompanied by a concomitant increase in p85α protein resulting in increased phosphorylation of Akt in cells [57]. At present, no data are available for physiological conditions where 'p85-free' p110 exists. Regulation of class I PI3K gene expression p110α p110α, encoded by PIK3CA, shows a broad tissue distribution in embryonic [15] and adult tissue [8,15,16]. Recently, three studies have begun to characterise the PIK3CA promoter, indicating that p110α expression can be positively controlled by FOXO3a (forkhead box O3a) [64] and NF-κB (nuclear factor-kappa B) [10] and negatively by p53 [65]. The human PIK3CA locus gives rise to two alternative transcripts, by splicing of one of two distinct 5' untranslated exons [exon -1a (80 bp) or exon -1b (49 bp); Figure 1] onto the ATG-containing exon (defined as exon 1), with the untranslated exons positioned about 50 kb upstream of the translation start site [64,65]. FOXO3a directly induces p110α expression via binding to FOXO3a-responsive elements approximately 792 and 225 bp 5’ upstream of exon -1b [64]. Given that the PI3K-Akt signalling axis inhibits FOXO3a activity (by mediating its cytoplasmic sequestration), p110α has the potential to negatively regulate its own gene expression. NF-κB binds approximately 700 bp 5’ upstream of exon -1b, and TNF-α (tumour necrosis factor alpha) stimulation can increase binding of NF-κB to the PIK3CA promoter and augment p110α mRNA expression in ovarian cancer cells [10]. A p53 binding site is present approximately 140 bp 5’ upstream of exon -1b, leading to PIK3CA transcriptional inhibition [65]. Indeed, p53 overexpression reduces p110α protein levels, whereas suppression of p53 expression increased p110α mRNA and protein levels. These data are in line with earlier reports of negative regulation of PIK3CA expression by p53 [66].

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Regulation of PI3K expression 

FOXO3a

(‐225 bp)

ATG428 bp80 bp49 bp

50.2 kb304 bp

exon ‐1b exon ‐1a exon 1

NF‐κB(~ ‐700 bp)

FOXO3a p53

(‐140 bp)

PIK3CA, human

translated exonuntranslated exonbinding leads to increased p110α transcriptionbinding leads to decreased p110α transcription

chromosome 3, 180,348,661, human genome assembly 17 (May 2004). Nucleotide 1 in exon ‐1b is set as position 1 (i.e. 180,348,661)

Figure  1.  5’  UTR  and  first  translated  exon  of  human  PIK3CA,  and  position  of  potential transcription factor binding sites. Graphic representation showing the two untranslated exons (exon ‐1b and exon ‐1a) and exon 1 which contains the ATG translation start site as identified by Astanehe  et  al.  [65]  and  Hui  et  al.  [64],  and  the  positions  of  the  binding  sites  for  p53  and FOXO3a. Also  shown  is  the putative NF‐κB binding  site  as  identified by  Yang  et  al.  [10].  The positions of  transcription  factor binding  sites are expressed  relative  to  the  first nucleotide of exon ‐1b.  NF-κB, FOXO3a and p53 are controlled by many different stimuli and agonists, often in an acute manner. It is therefore somewhat surprising that p110α expression has not been reported to undergo acute changes upon cellular stimulation that engages these transcription factors. Rapid changes in p110α expression have been observed following stimulation of L6 musculoskeletal cells with the CB1 antagonist rimonabant [57]. It is not clear at present whether any of the transcription factors mentioned above is implicated in this induction of p110α. As early as 5 h following rimonabant stimulation, p85α and p110α protein expression were increased and remained elevated for at least 72 h. PI3K activity was increased within minutes of stimulation and remained elevated for at least 24 h post-stimulation. These observations suggest that a stimulus-induced increase in PI3K expression can lead to sustained high PI3K activity. There are several examples of persistent alterations in p110α expression. The levels of p110α mRNA [67] as well p85α, p55α and p50α mRNA [68] were increased for up to two weeks following motor nerve injury. During cell differentiation (terminal differentiation of human keratinocytes in vitro), p110α mRNA and protein levels (and p110β mRNA) were transiently upregulated, with maximal mRNA increase at day 9, while phosphorylation of Akt was reduced, possibly also due to the increased levels of PTEN found under those conditions [62]. In cancer, increases in PIK3CA gene copy number and/or mutational activation of p110α [69,70] are prevalent (Table 4). In cervical, ovarian and gastric carcinoma, increased PIK3CA gene copy number is associated with increased p110α transcription, translation and total PI3K activity [71-73].

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Interestingly, immunohistochemistry studies on ovarian cancer sections indicate that p110α levels in the cancer cells are the highest in the non-proliferating regions (as assessed by co-staining with the cell proliferation marker Ki-67), such as in the under-vascularised tumour areas. Interestingly, the proliferating cancer cells expressed relatively low amounts of p110α [10]. p110β This PI3K isoform appears to have a broad tissue distribution, as assessed by Northern and Western blotting across a range of embryonic and adult mouse tissues [8,16-18]. A detailed analysis of the PIK3CB promoter has not been performed, yet in silico analysis of single nucleotide polymorphisms in the putative PIK3CB promoter region (as part of a search for functional polymorphisms associated with the genetics of insulin resistance) revealed the presence of a C>T variant (rs361072; C>T at -359) which could disrupt a putative GATA-binding motif [74]. The T variant decreases GATA transcription factor binding and reduces the transcriptional activity of p110β promoter constructs in vitro and in cell lines. The C variant thus allows higher p110β expression and Akt activation than the T variant [74]. This difference could help to explain why, among obese children, homozygous C/C individuals have a trend for milder insulin resistance than T/T obese patients. The distribution of rs361072 alleles was comparable in obese and non-obese cohorts. p110β mRNA and protein levels are increased upon differentiation of 3T3-L1 mouse embryonic fibroblasts into adipocytes [63] and p110β mRNA is increased upon terminal ex vivo differentiation of human primary keratinocytes (as was shown for p110α in the same study [62]). p110β (and p110δ) protein expression is also upregulated in aorta tissue extracts under conditions of hypertension [75] and in cancer, with PIK3CB gene amplification in primary ovarian tumours [76] and thyroid cancer [77], and increased p110β mRNA expression in prostate cancer [78] and neuroblastoma [79] (Table 4). p110δ p110δ is highly expressed in leukocytes [8,9,16], present at intermediate levels in neurons [14] and found at low levels in most other cell types [8,19], including endothelial cells [80,81]. p110δ has been implicated in endothelial transmigration of leukocytes during inflammation [81] but it is not clear whether this is due to expression of p110δ in leukocytes, endothelial cells or both, although some evidence for endothelial p110δ in leukocyte transmigration has been provided [81]. Other studies on the role of p110δ in blood vessels are also difficult to interpret. Indeed, p110δ expression has been reported to

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Regulation of PI3K expression 

be upregulated in arteries of hypertensive rats [75,82,83], with immunohistochemistry indicating that p110δ is located in the smooth muscle layer of the arteries [82,83]. However, in these studies, vessel homogenates were used to monitor p110δ expression, which does not allow to exclude that increases in p110δ expression are related to increased leukocyte infiltration. p110δ appears to be expressed at similar levels in transformed and non-transformed leukocytes. In acute myeloid leukaemia cells, p110δ is the only class I PI3K isoform that is consistently detected, with the levels of the other class I PI3K being extremely variable [84,85]. p110δ is expressed at moderate levels in some cancer cells of non-leukocyte origin such as melanoma, breast and colon, often with large differences in expression levels in cell lines of the same tissue origin [19], for reasons that are unclear. Increased p110δ mRNA expression without an increase in gene copy number was found in glioblastoma [86]. Both p110δ mRNA and protein levels were shown to be increased in neuroblastoma [79] (Table 4). p110γ This PI3K isoform is highly enriched in leukocytes [6,7] but is also found at lower levels in other cell types, including cardiomyocytes [27,28,87], endothelial cells [81], pancreatic islets [88,89] and smooth muscle cells [90]. Keratinocytes were reported to express high levels of p110γ [29] but others have not been able to confirm this observation (Matthias Wymann and Sabine Werner, personal communication). A putative promoter region in the p110γ gene PIK3CG has been identified and contains several consensus binding sites for leukocyte-specific transcription factors, including c/EBPβ (CCAAT/enhancer binding protein beta) and GATA-1 [91]. Some evidence for a selective upregulation of p110g amongst the PI3K isoforms has been presented. Indeed, treatment of U937 leukaemia cells with ATRA (all-trans retinoic acid), which acts via the RARs and induces cell differentiation, results in an increase in p110γ mRNA and protein levels within 8 h of stimulation, without alterations in the protein levels of p110β, p110δ, p85, or p101 [92] (Table 3). Expression of the BCR-ABL fusion oncoprotein in leukocytes has also been reported to induce p110γ expression at the mRNA and protein level, leading to increased PI3K activity, without affecting p110α, p110β or p110δ mRNA and protein levels [93]. In cancer, increased PIK3CG gene copy number has been documented in ovarian cancer [76] (Table 4). p85α Studies across a panel of mouse and rat tissues indicate that p85α is expressed at moderate to high levels in most tissues, and low levels in skeletal muscle [16,20,21].

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Tables 3 and 4 summarise the conditions of altered p85α expression under various conditions of stimulation, and in cancer. p85α expression in insulin signalling and diabetes: an unclear link p85α mRNA levels are rapidly and significantly induced upon insulin stimulation of human muscle derived from healthy donors [58]. This observation, together with the metabolic phenotype (increased insulin sensitivity) of p85α and p85β knockout mice, led several groups to monitor the expression and activity of PI3K pathway components in insulin resistance in humans [52,53,55,94-97] and in mouse models [48,54,98,99]. As described in more detail below, it has not been possible to correlate diabetic conditions with alterations in p85 expression and/or PI3K activity. Although PI3K is a key effector in insulin signalling [12,100], it is possible that factors other than PI3K itself impact on the PI3K signalling axis in diabetes. Indeed, expression and/or phosphorylation of the insulin receptor and insulin receptor substrate have been found to be reduced in insulin-resistant cells compared to control cells [52,53,55,94-96,101], suggesting that PI3K signalling could be reduced due to decreased PI3K recruitment to the plasma membrane [94]. In other words, the primary defect in insulin-resistant cells might not arise from changes in the levels of PI3K itself, but rather as a consequence of defects in upstream signalling pathways. Under basal conditions, human p85α mRNA and protein levels have been found to be unchanged [52,53,55,59,97] or increased [94,97] in primary human skeletal and adipose tissue of diabetic patients, and unchanged [96] or increased [95] in insulin-sensitive tissue of women with gestational diabetes mellitus. In line with these variable results, PI3K activity upon insulin stimulation (assessed by in vitro kinase assay or Akt phosphorylation) was found to be unchanged [94] or reduced [94,97] in insulin-sensitive cells of diabetic patients as compared to control samples. In mouse models of insulin resistance, such as transgenic overexpression of growth hormone or treatment of wild-type mice with GH, the levels of p85α [48,54,98,99] and total p85 [54,99] protein were increased in skeletal muscle and adipose tissue, with unaltered levels of p110 in one of the studies (as analysed by anti-pan-p110 antibody) [98]. Similar to what was observed in diabetic patients, data on PI3K signalling in this mouse model were inconsistent in the different studies. Indeed, under basal conditions in GH-overexpressing muscle cells, in vitro PI3K activity was either unchanged (as assessed in IRS-1 immunoprecipitates) [48] or increased (as assessed in phosphotyrosine immunoprecipitates) [99]. Upon insulin stimulation, however, PI3K activity was always found to be decreased in insulin-sensitive tissue of GH-overexpressing mice, compared to controls [48,98,99].

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Regulation of PI3K expression 

Induction of p85α expression by nuclear receptors: a way to transiently increase PI3K activity? An increase in p85α expression, leading to a concomitant increase in PI3K activity, also occurs upon stimulation of various nuclear receptors, including members of the PPAR, RAR and estrogen receptor (ER) families. Some of these studies originated from investigations into the mechanism of action of the PPARγ ligand Rosiglitazone, known to increase insulin sensitivity and used in the treatment of diabetes [102]. Upon ligand binding, PPARs translocate to the nucleus where they interact with 9-cis-RA-bound RXR (retinoid X receptor) to induce gene transcription. In human adipocytes, which express high levels of PPARγ, p85α is one of the genes induced by Rosiglitazone [49,50]. p85α mRNA was also induced by the RXR activator LG1069, and further enhanced by co-treatment with Rosiglitazone. These data indicate that the increase in p85α expression is mediated by the PPARγ–RXR heterodimer in these cells [49,50]. Rosiglitazone increased p85α mRNA and protein expression without affecting p110α or p110β mRNA levels (the effects on the levels of p85β, p55γ or p110δ mRNA and on protein expression of the PI3K subunits were not investigated [50]). Of greater importance in glucose uptake than adipocytes are muscle cells, and these cells are therefore a more relevant cellular context to study the impact of anti-diabetics. Muscle cells, however, express low levels of PPARγ and indeed, do not show p85α induction upon Rosiglitazone treatment [51]. p85α mRNA and protein are induced in these cells upon activation of PPARα and the PPARα–RXR heterodimer instead [51]. Thus, p85α is a target of PPAR-RXR nuclear receptors. This increased p85α expression by PPAR ligands corresponds with increased PI3K activity upon insulin stimulation, as measured by by in vitro PI3K activity assays in p85α immunoprecipitates. [50,51]. It remains unclear if this effect is due to an increase in p85α alone or whether there is concomitant increase in overall p110 levels. RAR activation can also induce p85 transcription. In F9 mouse embryocarcinoma cells, retinoic acid (RA) transiently induced p85α expression, correlating with a biphasic activation of PI3K and Akt [56]. The increase in PI3K activity is likely due to an increased level of the p85α–p110α heterodimer, as shown by immunoprecipitation with anti-p110α antibody. Interestingly, increased p110 protein levels did not correlate with p110α and p110β mRNA levels, which were not significantly changed upon RA treatment. These data suggest that p110 protein expression might be regulated post-translationally under these conditions, i.e. by stabilisation through p85 binding. Furthermore, the regulation of p85 expression by RA might be tissue-specific, given that treatment of U937 leukaemia cells with ATRA (which induces p110γ expression) does not increase p85 expression [50,51].

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p85α expression is also subject to ER stimulation [61]. In several cell types, stimulation with estradiol leads to an induction of PI3K activity, ranging from minutes [103,104] to hours [105]. The immediate increase in PI3K activity upon estradiol stimulation is likely due to a direct interaction between ERα and p85α [103,104], leading to increased PI3K recruitment to the plasma membrane. Yet, in MCF-7 breast cancer cells, Akt phosphorylation was increased up to 72 h after estradiol stimulation and is accompanied by an increase in p85 protein expression [61], suggesting that enhanced p85α expression might result in increased PI3K activity. This effect is achieved through the ERα receptor, and not the ERβ receptor [61]. p85β Based on studies using Northern blotting, the p85β isoform has the highest expression in brain, intermediate expression levels in liver and spleen and is nearly undetectable in muscle cells [16,20,21]. Absolute protein quantification approaches using mass spectrometry, however, revealed high levels of p85β in all tissues analysed, within the same range as those of p85α [16]. Previously, p85α was believed to be more abundant than p85β, a conclusion mainly based on the more severe phenotype of the p85α gene knockout mice (embryonic or perinatal lethality) [44,106] compared to the p85β knockout mice, which are viable and fertile [46]. Furthermore, analysis of PI3K protein expression suggested that p85β contributes only 10% of the total regulatory subunits in mouse embryo fibroblasts [107,108]. The reasons for these inconsistencies between different studies on the ratios of endogenous p85α to p85β protein levels [16,108] are not clear, but might be related to differences in the PI3K antibodies used.

No data are currently available regarding p85β regulation under physiological conditions. Increased PIK3R2 gene copy number has been documented in ovarian cancer, without an associated increase in p85β mRNA expression [76] (Table 3).

Recent findings show that p85β expression is subject to post-transcriptional regulation by the noncoding RNA miRNA-126, which post-transcriptionally downregulates p85β mRNA expression [109]. Likewise, miR-29 miRNA family members have been shown to directly suppress p85α expression [110]. Whereas the effect of miR-29 miRNA on p85β has not been reported, it has been shown that miRNA-126 does not affect p85α [109]. Loss-of-expression of miRNA-126 in colon cancer is thought to contribute to PI3K-Akt pathway activation, possibly by increasing p85β-p110 levels. In endothelial cells, however, increased p85β expression was put forward as a negative regulator of PI3K action, contributing to loss of blood vessel integrity [111]. Such a difference in outcome (up- versus downregulation of PI3K activity) could be

37 

Regulation of PI3K expression 

related to whether p110 levels change concomitantly with changes in the levels of p85β, which could be cell type-specific. p55α and p50α Like p85α, p55α and p50α are encoded by the PIK3R1 locus. p55α and p50α each possess their own promoters [112,113] and thus are not p85α splice variants as originally speculated [21,23]. Northern blot analysis of rat tissues revealed that p55α is expressed mainly in brain and muscle and is undetectable in most other tissues [21,22], whereas p50α has the highest expression in liver, kidney and brain [21,23]. This distribution pattern could indicate tissue-specific functions of these regulatory subunits. p55α and p50α are transcriptional targets of STAT3 (signal transducer and activator of transcription 3) during mammary gland involution in vivo [112]. STAT3 directly binds the p55α and the p50α promoter (and not to the p85α promoter), leading to upregulation of their expression. Such regulation was not observed in embryonic stem cells, indicating tissue-specific regulation of p50α and p55α expression. The EBNA-2 transcription factor of Epstein-Barr virus can selectively induce p55α expression in the EREB2.5 human B-lymphoblastoid cell line, without alterations in the protein expression of p85α, p55α and p55γ and the class IA p110 catalytic subunits [114]. Expression of p55α is essential for proliferation of EREB2.5 cells, and RNAi-mediated selective knock-down of p55α was accompanied by increased apoptosis [114]. In response to injury of motor neurons in rats, increased expression of mRNA and protein of p55α and to a lesser extent of p50α, lasting up to 28 days has been observed [68]. In a separate study, also p110α was increased upon motor nerve injury [67]. p55α and p50α mRNA levels are increased in skeletal muscle cells upon stimulation with insulin, as was shown for p85a [59]. This study further indicated that PI3K activity itself might regulate transcription of the regulatory subunits by a positive feedback loop, given that treatment of these cells with the PI3K inhibitor LY294002 downregulated the levels of p85α, p55α and p50α mRNA. p55γ PIK3R3 mutant mice have not been reported and the function of p55γ is unknown. Using Northern blotting analysis across a panel of mouse [24] and rat [20] tissues, p55γ was shown to be expressed at high levels in brain and testis, at intermediate levels in several other tissues such as heart and lung, and is undetectable in liver and skeletal muscle. However, protein quantification by mass spectrometry revealed very low levels of p55γ protein in murine muscle, liver, fat, brain and spleen [16]. Good quality antibodies to p55γ are not available, and its tissue expression at the protein level has not been assessed.

38 

Chapter 2 

PIK3R3 is a direct transcriptional target for sterol-regulatory element binding proteins (SREBPs), which are transcription factors known to regulate genes controlling lipid metabolism. SREBP is ubiquitously expressed as a precursor protein that is processed into active SREBP upon several metabolic stimuli. SREBP-1 activation by stimulation of human fibroblasts with sterols, insulin and platelet derived growth factor triggers p55γ mRNA and protein upregulation [115]. In vivo SREBP activation (by feeding mice with high fat diet) induces p55γ expression in murine liver, which usually expresses low amounts of p55γ [115]. The processing of SREBP itself is dependent on PI3K activity, since inhibition of PI3Ks with LY294002 blocks this activation [116]. Significant DNA copy number gain and mRNA upregulation of PIK3R3 (quantitative PCR in ovarian cell lines) have been documented in ovarian cancer [76]. In the same study, p55γ mRNA up-regulation was also observed in liver, prostate, and breast cancer (Table 4). p101 and p84 p101 and p84 are the regulatory subunits for p110γ. p101 and p84 are encoded by 2 separate genes (located next to each other on chromosome 17), and are highly expressed in leukocytes [3,4]. Northern and Western blotting and RT-PCR analysis showed that p84 is also highly expressed in the heart [4]. p101 expression remains unchanged upon treatment with ATRA or BCR-ABL expression, which both induce p110γ expression [92,93]. The impact of these stimuli on p84 expression has not been investigated. Concluding remarks Under basal, i.e. unstimulated conditions, the expression of the PI3K regulatory and catalytic subunits seems to be mainly under transcriptional control, with mRNA levels correlating well with protein levels, leading to equal amounts of p85 and p110 protein in cells [16]. Stimuli and transcription factors that induce PI3K gene expression have recently been identified. Some stimuli (such as a CB1 antagonist and estradiol) can induce an immediate, initial increase in PI3K activity due to receptor-mediated PI3K activation, as well as a sustained increase in PI3K activity as a result of increased PI3K subunit expression. It is not clear at present how generic this mechanism of PI3K upregulation by PI3K expression will be, given that the long-term impact of most stimuli on PI3K gene expression has not been investigated. In order to understand the functional impact of changes in PI3K subunit alterations, it will be essential to assess more comprehensively the expression levels of PI3K isoforms as well as the impact on cellular PI3K activity. Quantitation of PI3K lipid products, especially in primary tissues, remains a formidable technical challenge

39 

Regulation of PI3K expression 

[117,118]. It is anticipated that new developments in analytical techniques (such as mass spectrometry [119]) will most likely be critical to move this field forward. In addition, the community should invest in generating high quality antibody reagents against PI3Ks, to enable better monitoring of PI3K expression alterations in health and disease. This is anticipated to be conducive for effective therapeutic interference with PI3K signalling.

40 

Chapter 2 

41 

Box 1: Class II and III PI3Ks 

 

The  Class  II  PI3Ks, which  comprise  the  PI3K‐C2α,  PI3K‐C2β  and  PIK3C2γ  isoforms  (Table  1), 

preferentially phosphorylate phosphatidylinositol to produce phosphatidylinositol‐3‐phosphate. 

The importance of class II PI3Ks in cell signalling and biology, relative to that of class I PI3Ks, is 

not clear at the moment (reviewed in [123,124]). 

  Based on Northern blot analysis of human  tissues, PI3K‐C2α  [31] and PI3K‐C2β  [30] are 

thought to be ubiquitously expressed. PI3K‐C2γ is present in most tissues but enriched in liver, 

breast,  prostate  and  salivary  glands  [32‐34,125].  A  more  recent  study,  however,  using 

immunohistochemical techniques on normal human tissues,  indicated that PI3K‐C2α and PI3K‐

C2β are not ubiquitously distributed, but are restricted to fully differentiated cells (rather than 

proliferating  cells)  of  epithelial  or  mesenchymal  origin.  Smooth  muscle,  endothelial  and 

glomerular epithelium cells only expressed PI3K‐C2α, whereas neurons expressed PI3K‐C2β [11]. 

All class II PI3K enzymes were further detected in macrophages [11]. 

  PI3K‐C2α and PI3K‐C2β expression is elevated in a large number of human small cell lung 

cancer  cell  lines  when  compared  to  normal  lung  epithelial  cells  [126].  PIK3C2B  gene 

amplification  and mRNA  overexpression  has  further  been  documented  in  a  small  fraction  of 

glioblastoma  [86]  (Table  4).  Comparative  genomic  hybridization  in  89  human  ovarian  cancer 

specimens  revealed  increased  gene  copy  number  of  PIK3C2B  in  ovarian  tumours.  mRNA 

expression of PI3Ks was not determined on the same samples but using public micro‐array data 

sets that included 2 ovarian cancer studies. In this data set PI3K‐C2β mRNA expression was not 

significantly increased [76]. 

  Class  III PI3K. vps34, the sole class  III PI3K, exists as a heterodimer bound  to the vps15 

regulatory  subunit  (formerly  called  p150  in  mammals).  Vps34  has  been  implicated  in 

endocytosis, autophagy and nutrient signalling [127]. Northern blot analysis indicates that vps34 

and vps15 are both ubiquitously expressed  [35,36]. Their expression  levels are not known  to 

undergo dynamic changes. 

Chapter 2, Table 3 

  cell type/tissue  species    stimulus/process mRNA/ Protein 

p110α  p110β  p110δ  p85α  p55α  p50α  p85β  p55γ  p110γ  p101  ref. 

3T3‐L1 embryonic fibroblasts  mouse    differentiation into adipocytes  Protein  =  ↑                  [63] mRNA  ↑      ↑  ↑  ↑  =  =     

motor neurons in vivo  rat    regeneration after injury Protein          ↑           

[67,68]  

mRNA  ↑  ↑                 primary keratinocytes in vitro  human    differentiation 

Protein  ↑                   [62] 

aorta in vivo (homogenate)  rat    hypertension, DOCA induced  Protein  =  =  ↑  =          =    [75] aorta in vivo (homogenate)  rat    hypertension, L‐NNA induced  Protein  =  ↑  ↑  =          =    [75] aorta in vivo (homogenate)  rat    hypertension, spontaneous  Protein  =    ↑  =          =    [82] resistance artery in vivo (homogenate)

rat    hypertension, DOCA induced  Protein  =  =  ↑           =    [83] 

variou

s stim

uli/processes 

L6 skeletal muscle cell line  rat   Treatment with Cannabinoid receptor type 1 antagonist SR141716 (PKA dependent) 

Protein  ↑     ↑             [57] 

  cell type/tissue species  receptor stimulus/process p110α p110β  p110δ p85α p55α p50α p85β p55γ p110γ p101 ref.mRNA                  ↑  

U937 myelomonocytic cells  human  RAR  ATRA  Protein             =  =  = ↑ = 

[92]  

mRNA                   ↑ PPARα  Wy‐14643 

Protein                     =PPARβ  L‐165041  mRNA                     =PPARγ  15ΔPGJ2  mRNA                     =PPARγ  Rosiglitazone  mRNA                     =

mRNA                   ↑ RXR  9‐cis‐RA 

Protein                   ↑ mRNA        ↑            

primary muscle cells  

human 

PPARα/RXR  Wy‐14643/9‐cis‐RA Protein        ↑            

[51]  

mRNA  =  =    ↑            primary adipocytes  human 

PPARγ or PPARγ/RXR 

Rosiglitazone or Rosglitazone/LG1069  Protein        ↑            

[49,50] 

mRNA  =  =    ↑            =

nuclear recep

tors 

F9 embryocarcinoma cells   mouse  RARγ/RXR  9‐cis‐RA Protein        ↑            

[56] 

 

transcription 

  cell type/tissue  species  stimulus/process    p110α  p110β  p110δ  p85α  p55α  p50α  p85β  p55γ  p110γ  p101  ref. factor 

       mRNA      =  =  ↑  ↑  [112] Mammary gland tissue in vivo  mouse  STAT3  involution 

Protein  =  =  =  =     ↓  ↑ ↑ ↓    [114] EREB2.5 B‐lymphoblasts  human  EBNA‐2    Protein  =  =  =  =    =  =     ↑ 

mRNA                   ↑ - AG01518 foreskin fibroblasts  - liver tissue in vivo 

human mouse 

SREBP ‐ PDGF (fibroblasts) ‐ high carbohydrate diet (liver)  Protein                   ↑ 

[115] 

non‐tumorigenic ovarian surface epithelial (OSE) cell

human  p53 stimulus unknown ‐ binding to region 5’ of exon ‐1b of PIK3CA

proteinmRNA ↓                  

[65] 

Ovarian cancer xenograft model 

human  NF‐κB  TNF‐α  mRNA  ↑                  [10] 

mRNA  ↑                  

transcription factors 

[64] K562 chronic myeloid leukaemia cell line 

stimulus unknown ‐ binding to region 5’ of exon ‐1b of PIK3CA 

human  FOXO3a                  Protein  ↑

  cell type/tissue species    stimulus/process p110α p110β  p110δ p85α p55α p50α p85β p55γ p110γ p101 ref.muscle tissue in vivo  human    insulin  mRNA  =  =            [52,55,58 ↑  ↑  ↑ adipocytes in vivo human    insulin  mRNA                [52,55,59↑   ↑   ↑  muscle tissue in vivo human    hypocaloric diet  mRNA                   ↑   [53] 

muscle cells  mouse       GH‐transgene expression  Protein  =  = ↑              [54,98,99

mRNA                   ↑ 

insulin

 

[48] adipose cells  mouse   

       GH‐transgene expression 

           Protein ↑ 

 Table 3. Regulation of class I PI3K isoform expression. =: unaltered levles; ↑: Increased levels; ↑ : increased expression correlates with a reduction in PI3K activity  or  Akt  phosphorylation;  DOCA:  deoxycorticosterone  acetate‐salt;  ↑  increase  in  expression  correlates with  increase  in  in  vitro  PI3K  activity  and reduction  in Akt phosphorylation; L‐NNA: Nω‐nitro‐L‐arginine; ↑:  increased  levels are associated with an  increase  in PI3K activity/Akt phosphorylation. For the increase in p85 expression after ligand binding to nuclear receptors, this increase in PI3K activity is measured upon stimulation with insulin; ↓: decreased levels; PDGF: platelet derived growth factor; ↓: decreased levels correlate with a decrease in PI3K activity 

43 

Chapter 2, Table 4 

   p110α 

PIK3CA 

p110β 

PIK3CB 

p110δ 

PIK3CD 

p110γ 

PIK3CG 

PI3K‐C2β 

PIIK3C2B 

p85α 

PIK3R1 

p85β 

PIK3R2 

p55γ 

PIK3R3 Refs 

ovarian  

carcinoma 

• ↑ GCN (19/26) 

• ↑GCN & ↑mRNA (13/14) 

• ↑GCN & ↑PI3K activity (3/3) 

• ↑GCN (21/89) 

• ↑GCN  

(24/89) 

  • ↑GCN  

(23/89) 

• ↑GCN  

(36/89) 

  • ↑GCN  

(31/89) 

• ↑GCN (19/89) 

• ↑mRNA,  

(18 cancer vs 16 non‐cancer) 

[72], 

[76]; 

Neuro‐ 

blastoma 

• ↑mRNA  (4/8) 

 

• ↑mRNA  

(6/8) 

 

• ↑mRNA (11/20) 

• ↑protein & ↑mRNA  (10/12) 

    • ↑mRNA  

(10/19) 

    [79] 

prostate 

carcinoma 

  • ↑mRNA  

(14/30) 

      • ↑mRNA  

(10/30) 

    [78] 

HNSCC  • ↑GCN (12/33) 

• ↑mRNA (16/33) 

• No correlation ↑GCN & ↑mRNA  

• ↑GCN (85/218) 

              [120], 

[121] 

lung  

cancers 

• ↑GCN (44/132) 

• ↑pAkt (nuclear) (36/215) 

• ↑pAkt (cytoplasmic) (26/215)  

               [122] 

thyroid  

cancer 

• ↑GCN (33/110) 

• ↑pAkt  (18/24) 

• ↑GCN (n=41/97) 

• ↑pAkt (n=13/17) 

            [77] 

Glio‐ 

blastoma 

         • ↑mRNA (6/95)    • ↑GCN (6/103) 

• ↑GCN & ↑mRNA (n =4/94) 

[86] 

cervical  

carcinoma 

• ↑GCN (42/55) 

• ↑GCN, ↑protein & ↑PI3K  

act,ivity (3/3) 

               [71] 

gastric  

carcinoma 

• ↑GCN (29/70) 

• ↑GCN correlates with ↑mRNA 

• ↑GCN & ↑pAkt  (8/9). 

               [73] 

Table 4. Altered expression of PI3K isoforms in cancer. GCN: Gene copy number. (19/26): number berween brackets indicate the number of positive samples per number of samples tested. HNSCC: Head and neck squamous cell carcinoma. NB: the references are listed for each cell in the same row. NB2: these data are mostly derived from studies using primary human tumours but data using cell lines are included as well.  

Chapter 2 

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