A key to the development of improved pharmacological treatment strategies for cancer is an understanding of the integration of biochemical pathways involved in both tumorigenesis and cancer suppression. Furthermore, genetic markers that may predict the outcome of targeted pharmacological intervention in an individual are central to patient-focused treatment regimens rather than the traditional ‘one size fits all’ approach. Prostate cancer is a highly heterogenous disease in which a patient-tailored care program is a holy grail. This review will describe the evidence that demonstrates the integration of three established pathways: the tumour-suppressive TGF-β (transforming growth factor-β) pathway, the tumorigenic PI3K/Akt (phosphoinositide 3-kinase/protein kinase B) pathway and the tumour-suppressive PTEN (phosphatase and tensin homologue deleted on chromosome 10) pathway. It will discuss gene polymorphisms and somatic mutations in relevant genes and highlight novel pharmaceutical agents that target key points in these integrated pathways.

INTRODUCTION

Prostate cancer is a major public health problem in Western industrialized countries. It is the most commonly diagnosed non-cutaneous cancer in men and is the second leading cause of cancer death in men [1]. It is estimated that, in the United States alone, there will be 186295 new cases diagnosed in 2008, with at least 22000 prostate-cancer deaths (American Cancer Society; http://www.cancer.org). Significantly, prostate cancer is a disease of the aging male. Evidence of prostate cancer can be found in 15–30% of men over the age of 50 years, increasing to 60–70% of men by 80 years of age [1]. With aged populations increasing, particularly in Western countries, prostate cancer is set to be an even greater health burden. By 2011, as the 65-year-plus age group is predicted to increase by 30%, there will be a concomitant increase in prostate-cancer incidence [2].

Despite the statistics above, treatment options remain limited and have significant drawbacks. The most common treatment of advanced metastatic prostate cancer is to deprive tumours of androgen, either by surgical or chemical castration. This can lead to the unwanted side effects of lost libido and potency. Unfortunately, for most patients, the cancer becomes insensitive to the androgen ablation therapy, they relapse and inevitably die from hormone-refractory metastatic prostate cancer [3]. For organ-confined tumours, complete removal (radical prostatectomy) of the prostate is the most common intervention, with a 10-year survival rate of 60%. Unfortunately, the operation itself carries a 2% mortality rate, 70% of patients develop erectile dysfunction, 50% have urine leakage with 2–5% of patients being left incontinent [4].

As well as a need for improved treatment regimens, there is a desperate need for diagnostic, prognostic and risk-assessment screens for prostate cancer. A raised level of circulating PSA (prostate-specific antigen) is currently used to predict the presence of prostate cancer [5]. This screen, although indicative of the disease, is not diagnostic. A raised PSA can be caused by other, non-cancer-related, factors, it is not specific to the disease state, is unable to indicate risk of death [6], and clinical decisions cannot be informed using this biochemical index. Indeed, 84% of men with raised PSA who undergo radical prostatectomy do so without prospective health benefit [6].

Central to the development of prognostic and risk-assessment screens and improved pharmacological treatment strategies is an understanding of the integration of pathways known to be involved in prostate cancer pathophysiology. This review will: (1) describe commonly affected tumour-suppressive and tumorigenic pathways in prostate cancer and examine current evidence for integration of these pathways; (2) describe polymorphisms that exist in relevant genes; and (3) discuss novel pharmaceutical agents that target key points of these integrated pathways.

BIOCHEMICAL PATHWAYS KNOWN TO PLAY A ROLE IN PROSTATE CANCER

The TGF-β (transforming growth factor-β) pathway

Members of the TGF-β superfamily have roles in regulating cell proliferation, apoptosis, cell differentiation and cell migration [7]. TGF-β acts on normal prostate epithelial cells and some prostate-cancer cells to inhibit proliferation and induce apoptosis [8,9]. Indeed, re-activation of TGF-β signalling in an androgen-insensitive prostate-cancer cell line re-establishes TGF-β tumour-suppressive properties [10,11].

TGF-β signalling is activated on binding of the cytokine to its receptor, TGF-βRII (transforming-growth-factor-β receptor II) [12] (Figure 1). This is followed by the subsequent recruitment of TGF-βR1 (transforming-growth-factor-β receptor I) and the phosphorylation of TGF-βR1 serine and threonine residues [12] (Figure 1). Phosphorylation activates TGF-βR1 serine/threonine kinase activity and leads to a subsequent signal cascade via downstream Smad proteins [13] (Figure 1). R-Smad2 and R-Smad3 (receptor Smads 2 and 3) are anchored by the cytoskeletal protein filamin A, thereby facilitating their recruitment to the active TGF-βR complex and consequential phosphorylation [14] (Figure 1). R-Smad2 and R-Smad3 form heteromeric complexes with the cytosolic co-Smad 4, translocate to the nucleus and regulate gene expression [14] (Figure 1).

The TGF-β Pathway

Figure 1
The TGF-β Pathway

Step

graphic
: on binding of TGF-β to the receptor subunit TGF-βRII, the TGF-βRI receptor is recruited (Step
graphic
) to the complex and phosphorylated. Steps
graphic
and
graphic
: receptor Smads (R-Smad2/3) are translocated to the receptor complex, phosphorylated and form a heteromeric complex with cytosolic Smads (Smad4). Step
graphic
: the Smad complex translocates to the nucleus and binds to Smad-binding elements (SBE) to activate or repress TGF-β-responsive-gene expression.

Figure 1
The TGF-β Pathway

Step

graphic
: on binding of TGF-β to the receptor subunit TGF-βRII, the TGF-βRI receptor is recruited (Step
graphic
) to the complex and phosphorylated. Steps
graphic
and
graphic
: receptor Smads (R-Smad2/3) are translocated to the receptor complex, phosphorylated and form a heteromeric complex with cytosolic Smads (Smad4). Step
graphic
: the Smad complex translocates to the nucleus and binds to Smad-binding elements (SBE) to activate or repress TGF-β-responsive-gene expression.

The suppression of prostate-cancer cell proliferation by TGF-β is modulated by the up-regulation of the cyclin-dependent kinase inhibitors p21CIP1, p27KIP1 and p15INK4B [10,15] and down-regulation of c-Myc [16] (Figure 1). Promotion of apoptosis is mediated by induced expression of pro-apoptotic Bax, down-regulation of anti-apoptotic Bcl-2 and increased expression and activation of the effector caspase 1 [11] (Figure 1). Additional apoptotic cross-talk with the androgen receptor also occurs, depending on the mutational status of this protein.

In prostate-cancer cells expressing wild-type androgen receptor, dihydrotestosterone antagonized the pro-apoptotic effects of TGF-β, but TGF-β still suppressed proliferation in these cells [16]. By contrast, in prostate-cancer cells expressing a constitutively activated mutant androgen receptor, in the absence of androgen, activated Smad4 induces translocation of androgen receptor to the nucleus alone [16]. In the presence of androgen, the apoptotic effects of TGF-β are enhanced in these cells [17]. In prostate cancer it is common to find down-regulation of TGF-βRs (rather than mutations in Smads as observed in other cancers, e.g., breast and colon cancers), particularly TGF-βRII, and this is associated with aggressive prostate cancer [18].

PI3K/Akt (phosphoinositide 3-kinase/protein kinase B) pathway

Akt-1, -2 and -3 are isoforms of the serine/threonine protein kinase B family [19]. Akt-1 and Akt-2 are expressed in most tissue types and contribute to increased cell proliferation, survival and glucose metabolism [20]. The role of Akt3 is less clear, but it appears to play a role in mitochondrial biogenesis [21] and is predominantly expressed in the brain [2224]. The oncogenic Akt pathway refers mainly to Akt-1 (for a review, see [24]).

Activation of the Akt pathway depends on the frequency and duration of PIP3 (phosphatidylinositol 3,4,5-trisphosphate) availability at the plasma membrane [25] (Figure 2). In fact, PIP3 provides the necessary platform for phosphorylation of Akt by 3-phosphoinositide-dependent kinases (PDKs; [25]). Conversion of PIP2 (phosphatidylinositol 4,5-bisphosphate) into PIP3 is catalysed by PI3K. This occurs on activation of a tyrosine kinase receptor such as via binding of IGF-1 (insulin-like growth factor-1) to IGF-R (insulin-like-growth-factor receptor) [25] (Figure 2). Activation of Akt by phosphorylation requires binding of Akt, PDK1 and PDK2 {now known as mTORC2 [mTOR (mammalian target of rapamycin) complex 2]} to PIP3 [25,26]. Of interest, PDK1 and mTORC2 phosphorylate Akt at Thr308 and Ser473 respectively [2123].

Activated Akt promotes cellular proliferation

Figure 2
Activated Akt promotes cellular proliferation

Steps

graphic
and
graphic
: activation of tyrosine kinase receptors (e.g. IGF-R) by their ligands (i.e. IGF-1) activates PI3K, which phosphorylates PIP2 to PIP3. Step
graphic
: PDK1 and PDK2 are recruited to PIP3 and activate Akt by phosphorylating it to pAkt (pAKT). pAkt blocks the anti-proliferative effects of glycogen synthase kinase-3β, but stimulates the oncogenic mTOR that promotes cell survival by increasing anti-apoptotic Bcl-2 and promotes cell proliferation via c-Myc and 4EBP (eIF4E-BP). Step
graphic
: the Akt pathway is modulated by the tumour suppressor PTEN, which dephosphorylates PIP3 to PIP2. PTEN also has tumour-suppressive actions through its interactions with p53. It promotes p53 activity by blocking MDM2 inactivation of p53. This promotes a feed-forward loop as p53 stimulates PTEN expression.

Figure 2
Activated Akt promotes cellular proliferation

Steps

graphic
and
graphic
: activation of tyrosine kinase receptors (e.g. IGF-R) by their ligands (i.e. IGF-1) activates PI3K, which phosphorylates PIP2 to PIP3. Step
graphic
: PDK1 and PDK2 are recruited to PIP3 and activate Akt by phosphorylating it to pAkt (pAKT). pAkt blocks the anti-proliferative effects of glycogen synthase kinase-3β, but stimulates the oncogenic mTOR that promotes cell survival by increasing anti-apoptotic Bcl-2 and promotes cell proliferation via c-Myc and 4EBP (eIF4E-BP). Step
graphic
: the Akt pathway is modulated by the tumour suppressor PTEN, which dephosphorylates PIP3 to PIP2. PTEN also has tumour-suppressive actions through its interactions with p53. It promotes p53 activity by blocking MDM2 inactivation of p53. This promotes a feed-forward loop as p53 stimulates PTEN expression.

pAkt (activated Akt, that is, phosphorylated Akt) results in a cascade involving various downstream signalling proteins [2527] (Figure 2). A high level of pAkt is correlated with poor prognosis of prostate cancer, whereas in normal prostate tissue, pAkt is undetectable [28]. Activated Akt inhibits GSK3β (glycogen synthase kinase 3β), which normally prevents up-regulation of cell proliferation due to increased cyclin D1 degradation (Figure 2). Also, inhibition of GSK3β is shown to spare free β-catenin from degradation in prostate-cancer cells [29]. β-Catenin normally acts as a bridge between the cytoskeleton and cadherin motifs in cell-to-cell junctions. Further, increased β-catenin can bind and stabilize cyclin D1 mRNA, which leads to enhanced expression which could promote G1-phase/S-phase progression [30].

Cyclin D1 expression marks progression of cells into the early G1 (growth) phase [31]. An excessive rate of cyclin D1 production promotes cell-cycle progression even in androgen- or serum-deprived cells, a trait of neoplastic transformation in the prostate [32]. The mechanism behind such involvement of cyclin D1 in cancer is due to its accumulation and the subsequent increase in cyclin/Cdk4 (cyclin-dependent kinase 4) complex that promote G1/S progression. On reaching appropriate levels, these complexes phosphorylate and cause the dissociation of the E2F transcription factor from the pRb (retinoblastoma protein) and herald progression of the cell cycle from G1 to S phase [31].

Another key effector downstream of Akt is mTOR [33,34] (Figure 2). It has been shown that mTOR is a serine/threonine kinase that plays a central role in protein translation, cell proliferation and evasion of apoptosis [33]. mTOR regulates both pro- and anti-apoptotic proteins in the Bcl-2 family, as well as c-myc, and 4EBPs [elF4E (eukaryotic initiation factor 4E)-binding proteins] [33]. Of interest, 4EBP regulates the availability of elF4E that is responsible for recruitment of mRNA for translation [35]. Up-regulation of mTOR has been observed in a variety of cancer cells [33]. Rapamycin and other inhibitors of mTOR reduce oncogenic transformation and inhibit angiogenesis and growth of metastatic tumours [36].

Activation of Akt also helps to evade apoptosis directly by phosphorylation of the pro-apoptotic protein Bad [35] (Figure 2). On detection of DNA damage or ATP depletion, Bad induces cytochrome c release from the mitochondrion [37]. Cytochrome c assembles a catalytic complex, the apoptosome, that converts pro-caspase into active caspase enzymes and initiates subsequent apoptosis [37]. Previous studies have demonstrated that pAkt inhibits apoptosis by phosphorylation of Bad at Ser136 to maintain mitochondrial integrity, and this has been suggested to prevent cytochrome c release [37].

PTEN (phosphatase and tensin homologue deleted on chromosome 10)

PTEN is inactivated or deleted in many cancers [35,38] including prostate, where up to 50% of tumours display loss of PTEN [39]. Re-expression of normal PTEN in prostate-cancer cell lines causes morphological changes associated with anoikis [40] and apoptosis [41]. Significantly, targeted deletion of the murine homologue, Pten, in tentative prostate stem cells causes increased numbers of this cell population and development of prostate cancer in mice, with a pathological progression similar to that of human prostate cancer [42]. Many factors influence PTEN activity, including localization, protein–protein interactions and post-translational modification (for a review, see [43]). Transcriptional regulation of PTEN and its physiological relevance is not yet clear. However, regulation of PTEN expression by p53 [44] and TGF-β [45] have been described.

The major tumour-suppressive activity of PTEN is due to its phosphatidylinositol phosphate phosphatase activity [38] (Figure 2). Although PTEN can dephosphorylate PIPs (namely both PIP2 and PIP3), it is PIP3 that is its most important substrate [46]. Formation of PIP3 from PIP2 by PI3K heralds the signalling cascade mediated by Akt (Figure 2). Dephosphorylation of PIP3 by PTEN thus modulates this signalling and has opposite effects on cell proliferation and survival to PI3K [38,47] (Figure 2).

Interaction of PTEN with p53 also plays a significant role in tumour suppression [44] (Figure 2). Deletion of p53 and Pten accelerates prostate tumorigenesis in mice [48], whereas in the Pten-null mouse, p53 expression is increased and induces senescence. A feedback loop between p53 and PTEN thus exists [44]. Although PTEN appears to suppress p53 expression, it promotes p53 activity [49,50]. PTEN inactivation leads to increased expression of the p53 repressor MDM2 (murine double minute-2) and Akt-mediated activation [49]. In addition, there is nuclear translocation of MDM2, where it sequesters and acts to repress p53 activity [49]. In fact, PTEN negatively regulates the P1 MDM2 promoter through its phosphatase activity in a p53-independent manner [51]. The first promoter (P1) of MDM2 yields mRNA with exon 2 spliced out (L-MDM2), giving rise to the p90MDM2 isoform [52]. The second promoter (P2) of MDM2 yields mRNA with exon 1 spliced out (S-MDM2), leading to the shorter p76MDM2 isoform which cannot bind p53 [52]. Although p90MDM2 is down-regulated by PTEN, p76MDM2 is unaffected by PTEN levels, effectively leading to an increased ratio of p76MDM2 to p90MDM2 [51]. As p76MDM2 antagonizes the ability of p90MDM2 to degrade p53 [52], this alteration in the ratios of the two MDM2 isoforms may be one mechanism by which PTEN promotes p53 activity.

Furthermore, nuclear PTEN interacts directly with p53 to enhance p53-mediated cell-cycle arrest and apoptosis in prostate-cancer cells [50]. Recent studies have found that two common missense mutations of PTEN, namely K289E and K13E, are associated with hereditary and spontaneous cancer respectively and lead to a defect in PTEN nuclear import [53]. In fact, both K289 (Lys289) and K13 (Lys13) are ubiquitin-targeted residues on PTEN, suggesting that ubiquitination plays an important role in PTEN nuclear shuttling. Although PTEN mono-ubiquitination increases its transfer to the nucleus, PTEN poly-ubiquitination prevents nuclear import and promotes its degradation by the proteasome [53]. Hence the tumour-suppressive effects of PTEN appear to be mediated by its localization, which is in turn regulated by ubiquitination.

INTEGRATION OF THE Akt, PTEN AND TGF-β PATHWAYS: EVIDENCE OF POTENTIAL LINKS

It is well established that IGF-1 plays a significant role in prostate cancer [54,55]. In fact, the oncogenic activity of IGF-1 is conferred via activation of the potent inhibitor of apoptosis, Akt (Figure 2). As well as blocking the intrinsic and extrinsic pathways of apoptosis to promote cell survival, Akt also promotes cell survival by suppression of TGF-βRI activation of Smad3 in prostate epithelium [56] (Figure 3a). Suppression of TGF-β signalling by IGF-1 is reversed by both PTEN and the PI3K inhibitor LY29004 [56].

Integration of Akt, TGF-β and PTEN through PI3K

Figure 3
Integration of Akt, TGF-β and PTEN through PI3K

(a) TGF-β signalling is inhibited by pAkt (pAKT) via mTOR-mediated suppression of Smad3. (b) PTEN modulates IGF-1 signalling by suppression of IGFBP-2 and by p53 suppression of IGF-R. PI3K activation stimulates IGFBP-2 expression, which enhances IGF-1 activation of the IGF-R. PTEN blocks this activity by antagonizing PI3K activity and by increasing p53 activity, which in turn suppresses expression of IGF-R.

Figure 3
Integration of Akt, TGF-β and PTEN through PI3K

(a) TGF-β signalling is inhibited by pAkt (pAKT) via mTOR-mediated suppression of Smad3. (b) PTEN modulates IGF-1 signalling by suppression of IGFBP-2 and by p53 suppression of IGF-R. PI3K activation stimulates IGFBP-2 expression, which enhances IGF-1 activation of the IGF-R. PTEN blocks this activity by antagonizing PI3K activity and by increasing p53 activity, which in turn suppresses expression of IGF-R.

PTEN also modulates IGF-1 signalling by suppressing IGFBP-2 (IGF-1-binding protein 2) expression [57] (Figure 3b). IGFBP-2 enhances IGF-1 binding with its receptor [58]. Elevated levels of IGFBP-2 in PTEN mutant tumours suggests that it may play a functional role in enhancing the oncogenic activity of IGF-1 [57]. Furthermore, PTEN might also influence this pathway by increasing p53 activity, as p53 down-regulates expression of IGF-R (Figure 3b). This possible interaction requires experimental verification.

At the integration crossroads: the metastasis suppressor, NDRG-1 (N-myc downstream regulated gene-1)

Of interest, p53 provides another potential arm of integration in the PTEN and PI3K/Akt pathways. p53 has been suggested to increase the expression of the tumour suppressor gene NDRG-1 [59,60]. However, other studies have shown no relationship between p53 and NDRG-1 expression [61], and these conflicting observations could reflect differences in cell types and incubation conditions. Significantly, PTEN and NDRG-1 are both repressed in poorly differentiated prostate tumours, whereas higher levels of expression appear to correlate with increased survival [62,63]. These latter authors demonstrated that forced expression of PTEN up-regulated NDRG-1 expression in the prostate-cancer cell lines PC-3 and DU-145 [63]. That study also showed that the effect was mediated via the phosphatase activity of PTEN, which directly decreases PIP3, leading to suppression of the PI3K/Akt pathway [63]. It is therefore hypothesized that Akt activation acts to inhibit NDRG-1 expression, whereas PTEN antagonizes this effect through its ability to convert PIP3 into PIP2 (Figure 4a). As NDRG-1 has been found to suppress cell proliferation and metastasis in prostate cancer [62,64], its inhibition by the PI3K/Akt pathway may be another step towards oncogenesis. As such, the possible role of Akt suppression of NDRG-1 in prostate cancer demands further analysis.

TGF-β, Akt and PTEN integration via NDRG-1 and AA

Figure 4
TGF-β, Akt and PTEN integration via NDRG-1 and AA

(a) Integration via NDRG-1 and HIF1-α. It is hypothesized that NDRG-1 expression is suppressed by pAkt (pAKT). PTEN antagonizes this effect by conversion of PIP3 into PIP2. NDRG-1 expression is also increased by HIF1-α, the transcriptional activity of which is induced by PTEN, but inhibited by FOXO3a. HIF1-α induces TGF-β expression, which suppresses HIF1-α transcriptional activity via FOXO3a in a negative-feedback loop. pAkt suppresses p27 directly and indirectly via suppression of FOXO3a. (b) AA synthesis stimulates PI3K/Akt. Suppression of Akt phosphorylation by PTEN is countered by the formation of free AA from phospholipids by cPLA2-α). Free AA increases phosphorylation of Akt. Furthermore, AA is converted into eicosanoids by COX and LOX. Eicosanoids also increase Akt phosphorylation, promoting cell proliferation and reducing apoptosis.

Figure 4
TGF-β, Akt and PTEN integration via NDRG-1 and AA

(a) Integration via NDRG-1 and HIF1-α. It is hypothesized that NDRG-1 expression is suppressed by pAkt (pAKT). PTEN antagonizes this effect by conversion of PIP3 into PIP2. NDRG-1 expression is also increased by HIF1-α, the transcriptional activity of which is induced by PTEN, but inhibited by FOXO3a. HIF1-α induces TGF-β expression, which suppresses HIF1-α transcriptional activity via FOXO3a in a negative-feedback loop. pAkt suppresses p27 directly and indirectly via suppression of FOXO3a. (b) AA synthesis stimulates PI3K/Akt. Suppression of Akt phosphorylation by PTEN is countered by the formation of free AA from phospholipids by cPLA2-α). Free AA increases phosphorylation of Akt. Furthermore, AA is converted into eicosanoids by COX and LOX. Eicosanoids also increase Akt phosphorylation, promoting cell proliferation and reducing apoptosis.

Although HIF1-α (hypoxia-inducible factor 1-α) is normally implicated in angiogenesis through the increased expression of its target gene, namely that coding for VEGF1 (vascular endothelial growth factor 1) [65], HIF1-α also appears to have some role in tumour suppression, as it induces expression of NDRG-1 [66]. Up-regulation of NDRG-1 expression following hypoxia or iron depletion is driven, in part, by HIF1-α [61]. HIF1-α has also been shown to increase TGF-β expression in the prostate under hypoxia [67]. Treatment of hepatocytes with the classical iron chelator DFO (desferrioxamine) increases TGF-β expression, leading to increased levels of the cyclin-dependent kinase inhibitor p27kip1 and G1-phase arrest [68]. Furthermore, increased p27kip1 expression can be blocked by treatment with an anti-TGF-β antibody, demonstrating an autocrine activation of TGF-β1 causing tumour suppression.

Interestingly, PTEN and PI3K/Akt have negative and positive roles on HIF1-α respectively in prostate cancer cell lines [69] (Figure 4a). This positive activity is associated with angiogenesis and induction of VEGF1 [69]. PTEN acts to activate the transcriptional activity of HIF1-α, whereas Akt-mediated suppression of the forkhead transcription factor FOXO3a decreases HIF1-α transcriptional activity [70]. Typically, a counter-effect is mediated by PIP3, where Akt may stimulate activity of HIF1-α indirectly by inhibition of FOXO3a [70] (Figure 4a).

Clearly, interactions of these pathways concerning HIF1-α and NDRG-1 are complex and probably reflect the contradictory tumour-suppressive versus tumorigenic activity described for both HIF1-α and TGF-β. Furthermore, the activity of these molecular pathways may also depend on the genetic profile of different cancer cells, whereby different mutations exist in key regulators such as p53, K-ras, Smad4 and others. Indeed, the function of NDRG-1 has been shown to be pleiotropic, with different molecular targets in various cancer cell types [64,66]. Hence, these interactions demand further exploration.

Pathway integration: arachidonic acid and eicosanoids are positive regulators of Akt signalling

Several studies have indicated that AA (arachidonic acid) can increase phosphorylation of Akt. In VSMCs (vascular smooth-muscle cells), AA mimics the effect of angiotensin II in stimulating VSMC mitogenesis via eicosanoids, reactive oxygen species and, subsequently, PI3K/Akt [7173]. Inhibition of either PI3K or Akt blocked the effects of AA on thymidine incorporation in VSMCs [71]. However, Gorin et al. [74] have shown that AA metabolism is not necessary, that is, AA alone is sufficient for Akt activation in VSMCs. Furthermore, the same investigators demonstrated that activation of Akt by AA is not necessarily PI3K-dependent [74]. In pancreatic-cancer cells, AA/eicosanoids stimulated PI3K/Akt signalling via inactivation of PTEN [75]. Oxidation of PTEN by AA metabolism decreased PTEN activity, resulting in elevated PIP3 levels and increased signalling through Akt and its downstream targets (Figure 4b).

The AA pathway contributes to prostate-cancer progression via modulation of prostate-cancer cell proliferation, apoptosis, angiogenesis and metastasis [76]. Growing evidence suggests that the AA pathway can stimulate PI3K/Akt in prostate-cancer cells (Figure 4b). Incubation of AA with prostate-cancer cells causes increased prostaglandin E2 synthesis, followed by induction of PI3K-mediated Akt activation, which then leads to increased cell proliferation [77]. Inhibition of the eicosanoid-producing enzyme COX-2 (cyclo-oxygenase-2), has been shown to induce apoptosis in both androgen-responsive LNCaP and androgen-unresponsive PC-3 cells by blocking Akt phosphorylation [78] and by down-regulation of cyclin D1 [79]. As PTEN is non-functional in LNCaP (mutation) and PC-3 (deletion) prostate- cancer cell lines, these results suggest that AA/eicosanoids can stimulate PI3K/Akt in the absence of PTEN. Considering that 30–40% of solid tumours have constitutively activated PI3K/Akt signalling [80], identification and characterization of a regulator of Akt activation in the absence of PTEN is of high significance. Since several studies examining regulation of PI3K/Akt by AA or eicosanoids were conducted with pharmacological inhibition of AA or eicosanoid production, this suggests that AA and eicosanoids have a stimulatory action on PI3K/Akt signalling [7779]. Further studies are needed to map the signalling cascade by which AA/eicosanoids stimulate PI3K/Akt activation.

Pathway integration: role of the cell–cell adhesion molecule, E-cadherin

E-cadherin is a cell–cell adhesion molecule that is down-regulated in many tumours, including prostate cancer [81] (Figure 5a). TGF-β has also been shown to stimulate E-cadherin expression in prostate-cancer cells treated with an mTOR inhibitor [82]. E-cadherin is anchored to the cell membrane by the bridging protein β-catenin bound to the actin cytoskeleton (Figure 5a). Disorganization of cytoskeletal actin filaments is a fundamental event in the development of a cancer cell phenotype and is associated with loss of E-cadherin from the cell membrane [83].

(a) Integration of PTEN, TGF-β and Akt regulates cytoskeletal organization to modulate cell signalling and cell adhesion, and (b) Ras/ERK activation switching TGF-β from tumour suppressor to tumour promoter via Smad-dependent and -independent integration of PTEN and Akt

Figure 5
(a) Integration of PTEN, TGF-β and Akt regulates cytoskeletal organization to modulate cell signalling and cell adhesion, and (b) Ras/ERK activation switching TGF-β from tumour suppressor to tumour promoter via Smad-dependent and -independent integration of PTEN and Akt

(a) Formation of actin stress fibres to form a stable cytoskeleton requires binding of AAPs to actin filaments. Expression of AAPs is stimulated by TGF-β signalling. In turn, Smad translocation to the TGF-β complex requires a stable cytoskeleton. Up-regulation of PTEN expression by TGF-β suppresses pAkt formation, blocking its inhibition of the degradation of free β-catenin (a promoter of cell cycle progression). Actin stress fibres bind free β-catenin to form a bridge between the cytoskeleton and the cell–cell adhesion molecule E-cadherin. TGF-β up-regulates expression of E-cadherin to suppress cancer-cell metastasis. (b) Normal Smad-dependent TGF-β signalling occurs in cells without constitutive activation of the RAS/ERK to promote PTEN and E-cadherin expression, resulting in suppression of cell migration and proliferation. In cells with activated Ras/ERK, TGF-β is Smad-independent, resulting in a switch to PTEN and E-cadherin suppression, thus promoting cell proliferation and migration. If these interactions do occur, then suppression of both PTEN and E-cadherin would lead to increased levels of free β-catenin and pAkt that also favour increased cellular proliferation.

Figure 5
(a) Integration of PTEN, TGF-β and Akt regulates cytoskeletal organization to modulate cell signalling and cell adhesion, and (b) Ras/ERK activation switching TGF-β from tumour suppressor to tumour promoter via Smad-dependent and -independent integration of PTEN and Akt

(a) Formation of actin stress fibres to form a stable cytoskeleton requires binding of AAPs to actin filaments. Expression of AAPs is stimulated by TGF-β signalling. In turn, Smad translocation to the TGF-β complex requires a stable cytoskeleton. Up-regulation of PTEN expression by TGF-β suppresses pAkt formation, blocking its inhibition of the degradation of free β-catenin (a promoter of cell cycle progression). Actin stress fibres bind free β-catenin to form a bridge between the cytoskeleton and the cell–cell adhesion molecule E-cadherin. TGF-β up-regulates expression of E-cadherin to suppress cancer-cell metastasis. (b) Normal Smad-dependent TGF-β signalling occurs in cells without constitutive activation of the RAS/ERK to promote PTEN and E-cadherin expression, resulting in suppression of cell migration and proliferation. In cells with activated Ras/ERK, TGF-β is Smad-independent, resulting in a switch to PTEN and E-cadherin suppression, thus promoting cell proliferation and migration. If these interactions do occur, then suppression of both PTEN and E-cadherin would lead to increased levels of free β-catenin and pAkt that also favour increased cellular proliferation.

Several AAPs (actin-associated proteins) bind and cross-link actin filaments to increase filament rigidity, protect against depolarization and bundle filaments into stress fibres [84]. Prolonged incubation of cells with TGF-β results in the expression of the AAPs, tropomyosin [85] and transgelin [86], which promote the formation of stress fibres (Figure 5a). In this way, both PTEN and TGF-β act to suppress metastasis in the normal cell. Consequently, loss of either PTEN or TGF-β signalling will promote a less organized cytoskeleton. It is hypothesized that the consequential disorganization of the cytoskeleton will lead to a greater amount of free β-catenin, protected from degradation by PI3K/Akt signalling [29] and promotion of cell-cycle progression (increased cyclin D1 mRNA levels) [30] (Figure 5a).

It is noteworthy that NDRG-1 stimulates E-cadherin expression in some cell types [87] but not in others [66]. In addition, NDRG-1 has also been associated with cytoskeletal organization, where it was found to interact with microtubules during mitosis, whereas its inhibition led to the disappearance of the α-tubulin protein in normal human epithelial cells [88]. This suggests that there may be significant interaction between PTEN and TGF-β signalling with NDRG-1 to maintain cytoskeletal structure. Such an interaction has yet to be demonstrated experimentally in normal and neoplastic cells.

Ras/ERK (extracellular-signal-regulated kinase) and their roles in the integrated Akt, PTEN and TGF-β pathways

TGF-β has been shown to both suppress and induce PTEN expression, depending on the Ras/ERK status (Figure 5b). When the Ras/ERK pathway is activated, as it is frequently in androgeninsensitive prostate cancer [89], it facilitates TGF-β suppression of PTEN via the Smad4-independent signalling pathway. However, when constitutive Ras/ERK is blocked, TGF-β induces PTEN expression through its classical Smad-dependent pathway and stimulates a tumour-suppressive response [90]. Such constitutive activation of Ras could be partly responsible for the switch from tumour-suppressive activity of TGF-β in normal and early-stage neoplasia, to its oncogenic activity seen in many advanced cancers, where it promotes tissue invasion. Similarly, block of Akt suppression of TGF-β (Figure 3a) by inhibition of mTOR (re-)activates the tumour-suppressive activity of TGF-β in prostate-cancer cells [82]. Such a switch is echoed in pancreatic-carcinoma cells, where both activated Ras and PI3K cause TGF-β to down-regulate E-cadherin expression through a Smad-independent pathway [83] (Figure 5b).

The androgen receptor: a key integration element of the PTEN and TGF-β pathways

An important integration of PTEN and TGF-β occurs in the prostate through the androgen receptor (Figure 6). Zhu et al. [17] have demonstrated a differential effect of normal androgen receptor and mutant androgen receptor on TGF-β-mediated apoptosis. In these studies, the mutant form enhanced TGF-β-induced apoptosis, whereas the wild-type androgen receptor inhibited this process [17]. Interestingly, TGF-β still inhibited proliferation of these latter cells [17]. PTEN has been shown to bind androgen receptor and suppress its activity in human prostate-cancer cells [91]. Therefore it is possible, in PTEN-silenced tumours, that TGF-β tumour suppression is antagonized by inappropriate androgen receptor activity. As such, this is an important hypothesis and demands experimental verification. Indeed, it is thought that PTEN loss is a key event in progression to androgen-independent prostate cancer [92,93]

PTEN suppresses androgen antagonism of TGF-β tumour suppression

Figure 6
PTEN suppresses androgen antagonism of TGF-β tumour suppression

Activation of the wild-type androgen receptor inhibits TGF-β induction of pro-apoptotic Bax and suppression of anti-apoptotic Bcl-2. PTEN can bind the androgen receptor, suppressing its inhibition of the pro-apoptotic effects of TGF-β, in addition to inhibiting pAkt (pAKT)-induced expression of Bcl-2.

Figure 6
PTEN suppresses androgen antagonism of TGF-β tumour suppression

Activation of the wild-type androgen receptor inhibits TGF-β induction of pro-apoptotic Bax and suppression of anti-apoptotic Bcl-2. PTEN can bind the androgen receptor, suppressing its inhibition of the pro-apoptotic effects of TGF-β, in addition to inhibiting pAkt (pAKT)-induced expression of Bcl-2.

POLYMORPHISMS AND SOMATIC MUTATIONS IN HUMAN PTEN, Akt AND TGF-β PATHWAYS

Mutations in the PI3K/Akt pathway probably occur more frequently than in other signalling pathways in cancer [94]. Until recently, Akt-1 did not itself appear to be subject to mutation. It is now evident that in a small proportion of cancers a recurrent activating mutation occurs (Akt-1E17K; [95,96]). However, neither this nor other mutations in Akt-1 have been described in prostate cancer (Table 1). In contrast, inactivating mutations of PTEN [45] and activating mutations of phosphoinositide 3-kinase class 1 α-polypeptide [97] are common in the dysregulation of the PI3K/Akt pathway (Table 1). The literature on somatic mutations in the PTEN gene in human tumour DNA is rather extensive and includes prostate cancer very prominently (for a review, see [93]; Table 1). In fact, 13% of all late-stage tumours display somatic mutations, 33% exhibit loss of heterozygosity and 27–60% exhibit loss of expression (for a review, see [98]).

Table 1
Human SNPs and somatic mutations in the TGF-β, PI3K/AKT and PTEN pathways

The range of known frequencies in all cancers are given where determined, as well as reported ranges in prostate cancer. SNPs were identified from the NCBI SNP database (http://www.ncbi.nlm.nih.gov) on the l8 July 2008. Abbreviation used: ND, none to date.

Frequency of somatic mutations (%) in:
GeneChromosomeTotal exonic SNPsTotal SNPs in gene regionAll cancersProstate cancerReference(s)
AKT-1 14 17 273 2.4 ND [127
NDRG-1 393 ND ND – 
PTEN 10 477 3–42 13–60 [98,101,128
cPLA2G2A 10 82 ND ND – 
cPLA2G4A 1047 ND ND – 
TGF-β1 19 10 171 17–68 ND [101
TGF-βR1 313 1–82 0–25 [101
TGF-βRII 13 632 3–86 12.5 [101
SMAD2 18 412 2–10 ND [101
SMAD3 15 751 38 (gastric) ND [101
SMAD4 18 141 2–56 30 [101
Frequency of somatic mutations (%) in:
GeneChromosomeTotal exonic SNPsTotal SNPs in gene regionAll cancersProstate cancerReference(s)
AKT-1 14 17 273 2.4 ND [127
NDRG-1 393 ND ND – 
PTEN 10 477 3–42 13–60 [98,101,128
cPLA2G2A 10 82 ND ND – 
cPLA2G4A 1047 ND ND – 
TGF-β1 19 10 171 17–68 ND [101
TGF-βR1 313 1–82 0–25 [101
TGF-βRII 13 632 3–86 12.5 [101
SMAD2 18 412 2–10 ND [101
SMAD3 15 751 38 (gastric) ND [101
SMAD4 18 141 2–56 30 [101

The current (as of l8 July 2008) NCBI SNP (National Center for Biotechnology Information Single Nucleotide Polymorphism) database lists six polymorphisms in the human PTEN gene (Table 1). Similarly, the database lists a total of 19 polymorphisms in the human Akt gene (18 SNPs and one frameshift; Table 1). To date, there appear to be no reports on the role of these 25 polymorphisms in constitutional DNA in prostate cancer. This may be a fertile area for future investigations. However, we note that some of these SNPs have already been investigated in other cancers [99,100]. Finally, many somatic mutations have been reported in the human TGF-β pathway to be associated with cancer (for a review, see [101]). All those reported in prostate cancer occur in factors associated with signal transduction, such as the TGFβRII and Smad4 genes (Table 1).

Future studies on SNPs may be useful in at least two separate ways: (1) by establishing the contribution of constitutional (also known as ‘germline’) SNPs to susceptibility, for example in the Akt, NDRG-1, TGF-β and PTEN genes (Figures 1–3 and Table 1) and (2) by developing strategies for personalized tumour treatment based on a patient's individual genetic background, for example with the drugs described below that act at different points in these integrated pathways. Similarly, somatic mutations could be useful in targeting treatment as well.

NOVEL PHARMACOLOGICAL AGENTS FOR PROSTATE CANCER TREATMENT

As already discussed, prostate cancer remains an important clinical problem, with current therapies being far from adequate. Hence, it is essential to develop new therapeutic approaches. In the present review we have discussed the integration of the tumoursuppressive PTEN and TGF-β pathways as well as the tumorigenic PI3K/Akt pathway. In this section we will describe potential treatments that target key points of these integrated pathways. First, this will include the development of iron chelators that act on the expression of a number of molecular targets, including the aforementioned molecules NDRG-1, cyclin D1 and TGF-β. Secondly, we will examine the suppression of Akt by cPLA2-α (cytosolic phospholipase A2-α) inhibitors.

Targeting iron and NDRG-1 for inhibition of tumour growth

Iron is essential for life, as it is involved in critical processes such as the rate-limiting step in DNA synthesis catalysed by RR (ribonucleotide reductase) (for a review, see [102]). Without iron, cells cannot proceed from the G1-phase to the S-phase of the cell cycle [31]. Studies using chelators such as DFO have shown that iron deprivation results in cell-cycle arrest [103,104]. Hence, all cells require iron, and tumour cells have a high demand, as reflected by a marked increase in transferrin receptor 1 expression, making them more sensitive to iron deprivation than normal cells [105].

Studies with classical iron chelators such as DFO led to the generalization that, to induce inhibition of cancer-cell proliferation, systemic iron depletion of an organism would be necessary [104,105]. Clearly, this would not be useful for the treatment of cancer patients who often suffer the anaemia of chronic disease [106]. Moreover, the development of novel chelators that show marked anti-tumour activity and do not induce whole-body iron depletion [101,107,108] have led to a paradigm shift in the development of these agents for cancer treatment [109]. In this case, the generation of an intracellular redox-active iron complex without tumour iron depletion is of importance [107,109,110]. These new compounds include chelators of the DpT class such as Dp44mT (di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone) and related ligands [107,108].

Interestingly, Dp44mT is highly active against a wide range of cultured tumour cells in vitro, including prostate-cancer lines, and also in vivo using a panel of human tumour xenografts [108]. Importantly, little or no toxicity was seen in normal tissues at optimal doses. The reason for this specificity could be the wellknown differences in iron metabolism between normal and neoplastic cells [104]. This includes, in cancer cells, the high iron uptake rate from transferrin and the greater activity of the iron-containing enzyme of DNA synthesis, RR, compared with normal cells [108].

A series of studies have shown that the high potency of iron chelators at inhibiting tumour growth is due to their effects on multiple targets [61,111,112], in addition to the classical target, RR [104]. These include the ability of chelators to: (i) increase expression of the iron-regulated metastasis suppressor NDRG-1 [61,64]; (ii) prevent iron uptake from transferrin in vitro [107]; (iii) increase iron efflux from cells[107]; and (iv) affect the expression of molecules involved in cell cycle progression that can lead to apoptosis (e.g. p21WAF1/Cip1, cyclin D1; [107,111,112]).

Of greatest relevance to the integrated pathways discussed previously are studies that have identified up-regulation of NDRG-1 by iron depletion [61]. These experiments, and those of others [113], showed that the metastasis- and growth-suppressor gene NDRG-1 was up-regulated at the mRNA and protein levels by iron depletion. The increased expression of NDRG-1 following iron depletion could also be reversed by incubation with iron donors, which restored cellular iron levels [61]. The regulation of NDRG-1 mRNA was not mediated by the iron-regulatory protein–iron responsive element system that mediates the regulation of the transferrin receptor 1, ferritin and other iron-regulated molecules [114]. However, when actinomycin D was used at high concentrations (4 μM) to inhibit transcription, it prevented the ability of chelators to up-regulate NDRG-1, suggesting transcriptional up-regulation following iron depletion [61]. This was mediated by HIF1-α-independent and -dependent mechanisms [61]. Of relevance to this pathway, it is known that cellular iron depletion prevents the activity of prolyl hydroxylase, which requires iron in its active site for hydroxylating HIF1-α for proteasomal degradation [104].

Another pathway by which iron chelators up-regulate NDRG-1 occurs through EGR-1 (early response gene 1) [115]. The transcription factor coded by this gene was found to increase NDRG-1 expression by binding to the EGR-1/transcription factor SP-1 overlapping binding sites on the NDRG-1 promoter in response to DFO in some cells [115]. Interestingly, EGR-1 is a direct regulator of TGF-β1, PTEN and p53, playing an important role in the interaction of these pathways to maintain the normal cell phenotype [116]. Paradoxically, in prostate cancer, where PTEN and p53 mutations are particularly common, EGR-1 promoted oncogenesis via its effects on TGF-β1 and fibronectin [116]. These findings further illustrate the complex nature of the TGF-β, PTEN and PI3K/Akt pathways, suggesting that molecules such as NDRG-1 play an important role in their interaction and balance. Clearly, the ability of a chelator to markedly up-regulate this gene could be vital in terms of designing therapies to inhibit metastasis and tumour growth. Significant recent studies have shown that NDRG-1 up-regulation plays a critical role in preventing metastatic spread in prostate cancer and other tumours [61,63,117]. Thus this molecule is an important target for iron chelators.

In summary, the drug-induced up-regulation of molecules such as the metastasis suppressor NDRG-1, which is at the crossroads of the TGF-β/Akt and PTEN pathways, is a potential new therapeutic target that deserves further assessment.

SUPPRESSION OF Akt BY cPLA2-α INHIBITORS

Considering other molecular targets, studies have progressed to examine the effect of PLA2-α inhibitors that suppress Akt [76]. The family of PLA2 can be classified into four classes on the basis of their nucleotide and amino acid sequence homology (for a review, see [118]). Many forms of PLA2 are differentially expressed in a tissue-, species- and/or genotype-specific manner (for a review, see [118]). Most research has been conducted on the cPLA2-α, the cytosolic PLA2-α, as it is the only family member that cleaves AA selectively from membrane phospholipids [119]. In contrast, secretory PLA2, for example, cleaves fatty acids at the sn-2 position non-selectively [119]. Phosphorylation of cPLA2 by mitogen-activated protein kinase orientates the enzyme on membranes for productive and sustained phospholipid hydrolysis [119].

It has been known for a decade that eicosanoid synthesis rates are increased in prostate-cancer tissue when compared with benign hyperplastic lesions [120], suggesting an increased flux of AA through the COX and LOX (lipoxygenase) pathways (Figure 4b). Previously, studies have found an absence of the endogenous cPLA2 inhibitor ANX1 (annexin A1) and ANX2 (annexin A2), in prostate cancer [121]. For example, ANX2 is expressed in the membrane of ductal and basal epithelial cells in normal prostate [122]. In benign prostatic hyperplasia, ANX2 is expressed in the same location as normal prostate, with comparable intensity [122]. In contrast, in about two-thirds of the likely precursor of prostate cancer, namely prostatic intra-epithelial neoplasia, there was loss of ANX2, suggesting that silencing of ANX2 could be an early event [121]. It was found that there was no gross deletion of the ANX2 gene and that hypermethylation of the ANX2 promoter could be responsible for the gene silencing [118,122].

Release of AA from membrane phospholipids in response to epidermal growth factor and related growth-factor-receptor activation appears to be an obligatory step in mitogenesis as well as in motility and cytoskeletal rearrangement [123]. Hughes-Fulford et al. [124] have demonstrated that linoleic acid (a precursor of AA), AA and prostaglandin E2 stimulate prostate tumour growth and alter gene expression in human prostate carcinoma PC-3 cells. In mice, diets rich in linoleic acid (i.e. corn oil), markedly stimulate the growth of human prostate cancer xenografts [125]. These studies suggest a stimulatory effect of dietary ω–6 fatty acid on prostate-cancer cell growth.

In light of the above and considering the role of AA in stimulating Akt phosphorylation [7779] (Figure 4a), inhibition of AA metabolism has become an attractive new target for treating prostate cancer. On the basis of the knowledge that PLA2 is responsible for the production of AA and lysophospholipids, which could be the rate-limiting step in eicosanoid synthesis, PLA2 has the potential to be a target for the treatment of prostate cancer. In view of the role of AA signalling in promoting mutagenesis, mitosis, angiogenesis and metastasis [126129], it can be reasoned that a better outcome may be achieved with PLA2 inhibitor(s) than with a COX or LOX inhibitor alone. This is because the latter approaches suppress the production of prostaglandins or 5-hydroxyeicosatetraenoic acid only, whereas blockade of PLA2 enzymes is expected to prevent substrate supply to both pathways. For the same reason it is expected that inhibition of PLA2 will not have the problem of COX-2-selective inhibitors, namely an increased risk of thrombosis due to the sparing effect on COX-1. This is because blocking the cPLA2-α enzyme will decrease substrate supply to all eicosanoid-producing enzymes.

CONCLUSIONS

The many points of possible interaction highlighted in this review of the current literature on TGF-β, PI3K/Akt and PTEN pathways demonstrate that there are potentially several key biochemical integrations in tumour-cell biology that could be targeted by novel pharmaceutical approaches. Indeed, two new pharmacological approaches of iron chelation and PLA2-α inhibitors might exploit these pathways that are commonly disrupted in prostate and many other cancers. Undoubtedly, further analysis of our hypothesized points of interaction between these pathways and their roles in normal and neoplastic cells will result in advances in the treatment of prostate cancer and possibly other belligerent tumours.

We thank Professor Juergen Reichardt (School of Medical Sciences, Bosch Institute, University of Sydney, Sydney, Australia) for his valued input during the initial drafting of this review. Dr Danuta S. Kalinowski (Department of Pathology and Bosch Institute, University of Sydney, Sydney, Australia), Dr Helena Mangs (Basic and Clinical Genomics Laboratory, University of Sydney, Sydney, Australia) and Dr Katie Dixon (Discipline of Physiology and Bosch Institute, University of Sydney, Sydney, Australia) are sincerely acknowledged for carefully examining the manuscript prior to its submission. We very much appreciate the excellent help of Dr Y. Suryo Rahmanto (Iron Metabolism and Chelation Program, Department of Pathology, University of Sydney, Sydney, Australia) in preparing the Endnote reference file.

Abbreviations

     
  • AA

    arachidonic acid

  •  
  • AAP

    actin-associated protein

  •  
  • ANX

    annexin

  •  
  • Cdk4

    cyclin-dependent kinase 4

  •  
  • cPLA2

    cytosolic phospholipase A2

  •  
  • DFO

    desferrioxamine

  •  
  • Dp44mT

    di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone

  •  
  • 4EBPs

    elF4E (eukaryotic initiation factor 4E)-binding proteins

  •  
  • EGR-1

    early response gene 1

  •  
  • ERK

    extracellular-signal-regulated kinase

  •  
  • FOXO3a

    forkhead transcription factor

  •  
  • GSK3β

    glycogen synthase kinase 3β

  •  
  • HIF1-α

    hypoxia-inducible factor-1α

  •  
  • IGF-1

    insulin-like growth factor 1

  •  
  • IGFBP-2

    IGF-1-binding protein 2

  •  
  • IGF-R

    insulin-like-growth-factor receptor

  •  
  • LOX

    lipoxygenase

  •  
  • MDM2

    murine double minute-2

  •  
  • mTOR

    mammalian target of rapamycin

  •  
  • mTORC2

    mTOR complex 2

  •  
  • NDRG-1

    N-myc downstream regulated gene-1

  •  
  • pAkt

    activated (phosphorylated) Akt

  •  
  • PDK

    3-phosphoinositide-dependent kinase

  •  
  • PIP2

    phosphatidylinositol 4,5-bisphosphate

  •  
  • PIP3

    phosphatidylinositol 3,4,5-trisphosphate

  •  
  • PI3K

    phosphoinositide 3-kinase

  •  
  • PLA2

    phospholipase A2

  •  
  • pRb

    retinoblastoma protein

  •  
  • PSA

    prostate-specific antigen

  •  
  • PTEN

    phosphatase and tensin homologue deleted on chromosome 10

  •  
  • RR

    ribonucleotide reductase

  •  
  • R-Smad2 and R-Smad3

    receptor Smads 2 and 3

  •  
  • SNP

    single nucleotide polymorphism

  •  
  • TGF-β

    transforming growth factor-β

  •  
  • TGF-βRI

    transforming-growth-factor-β receptor I

  •  
  • TGF-βRII

    transforming-growth-factor-β receptor II

  •  
  • VEGF1

    vascular endothelial growth factor 1

  •  
  • VSMC

    vascular smooth-muscle cell

FUNDING

D. R. R. thanks the National Health and Medical Research Council of Australia for a Project Grant and Fellowship support and the Australian Research Council for a Discovery Grant. Z. K. thanks the Australian Rotary Health Research Fund (Dural Rotary Club) and the Cancer Institute of New South Wales for Ph.D. Scholarships.

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