Abstract

The prostanoid thromboxane (TX)A₂ plays a fundamental role in vascular haemostasis and, more recently, is increasingly implicated in various neoplasms including in prostate, breast and bladder cancers, among others. In humans, TXA₂ signals through the TPα and TPβ isoforms of the T prostanoid receptor (TP), two structurally related receptors that display both common, over-lapping but also distinct, isoform-specific physiologic roles. Consistent with this, while TPα and TPβ are encoded by the same gene, the TBXA2R, they are differentially expressed due to their transcriptional regulation by distinct promoters where promoter (Prm) 1 regulates TPα expression and Prm3 regulates TPβ. While the clinical evidence for the role of the TXA₂-TP axis in neoplastic progression is increasing, few studies to date have investigated the role of the individual TPα/TPβ isoforms in human cancer or indeed in most other diseases in which the TXA₂-TP axis is implicated. Focusing on TPα, this review details the current understanding of the factors regulating its expression and transcriptional regulation through Prm1, including in prostate and breast cancers. Emphasis is placed on the trans-acting transcriptional regulators that bind to cis-elements within the core and upstream regulatory regions of Prm1 under basal conditions and in response to cellular differentiation. A particular focus is placed on the role of the tumour suppressor Wilms’ tumour 1 in the regulation of TPα expression through Prm1 in megakaryoblastic cells of vascular origin and in prostate and breast carcinoma cells. Collectively, this review details current knowledge of the factors determining regulation of the TXA₂-TPα axis and thereby provides a genetic basis for understanding the role of TXA₂ in the progression of certain human cancers.

Introduction

Thromboxane (TX)A₂, a prostanoid synthesized from arachidonic acid by the sequential enzymatic actions of cyclooxygenase (COX)-1/-2 and TXA synthase (TXS) predominantly in platelets and in monocytes/activated macrophages, plays an essential role in haemostasis regulating platelet aggregation and vascular tone (1). It also induces the constriction of various other types of smooth muscle (SM), including pulmonary, renal and prostate SM, and promotes vascular remodelling in response to endothelial injury contributing to neointimal hyperplasia and restenosis post-stenting (2–4). Accordingly, imbalances in the levels of TXA₂ or of its synthase (TXS) or of its receptor, the T prostanoid receptor (or, in short, the TP), are widely implicated in several cardiovascular, pulmonary, renal and prostate pathologies (1–3, 5). In humans and in other primates, the TP exists as two structurally related isoforms referred to as TPα and TPβ, which are identical for their N-terminal 328 amino acid residues but which differ exclusively in their intracellular C-terminal domains (6). While TPα and TPβ are encoded by the same TP gene, the TBXA2R (Figure 1), they are differentially expressed in several cell/tissue types being transcriptionally regulated by two different promoters referred to as Prm1 and Prm3, respectively, within the TBXA2R (7–10). Functionally, as members of the G-protein-coupled receptor (GPCR) superfamily, TPα and TPβ both primarily couple to Gαq-mediated phospholipase Cβ activation, raising intracellular calcium levels in response to inositol phosphate turnover, but also readily couple to Gα12-mediated RhoA and to extracellular signal-regulated protein kinase (ERK) activation (Figure 2) (6, 11–14). In contrast, TPα and TPβ undergo distinct mechanisms of agonist-induced homologous (15, 16) and heterologous (17–19) desensitization to differentially regulate their intracellular signalling. Most notably, signalling by TPα, but not by TPβ, is completely desensitized/inhibited by the counter-regulatory anti-platelet and vasodilatory agents prostacyclin/prostaglandin I₂ and nitric oxide, which is mediated by direct protein kinase (PK) A and PKG phosphorylation of TPα at Ser³²⁹ and Ser³³¹, respectively, the very first residues within its unique carboxyl-terminal tail domain of TPα and divergent from those of TPβ (13, 18). The conclusion from those studies is that TPα is the TP isoform essential for haemostasis/thrombosis, while the role of TPβ in this pathophysiologic process remains unclear (13, 18). Hence, TPα and TPβ have both shared and unique patterns of expression and function to mediate the (patho)physiologic actions of the potent autocrine/paracrine mediator TXA₂ in human health and disease.

Figure 1.

Structural organization of the TBXA2R gene. Panel A: In humans, a single TBXA2R gene, located on Chr19p13.3, encodes both the TPα and the TPβ isoforms of the T prostanoid receptor (the TP), where promoter (Prm) 1 exclusively regulates (more...)

Figure 2.

Summary of the main signalling cascades regulated by TPα and TPβ. 1 (Black): Both TPα and TPβ primarily couple to Gαq-mediated phospholipase (PL)Cβ activation, leading to the generation of IP₃ and mobilization (more...)

The role of thromboxane in cancer

In addition to its prominent role within the vasculature, there is growing evidence highlighting a central role for TXA₂ in human cancers (20, 21). In recent years, evidence supporting this hypothesis has been strengthened by several longitudinal studies showing the prophylactic benefits of long-term daily use of Aspirin in reducing the risk of many prevalent cancers, predominantly gastrointestinal but also breast, lung and prostate cancers (PCa), with numerous clinical trials completed or underway testing the benefits of Aspirin and other COX-1/-2 inhibitors in chemoprevention (22–29). While those longitudinal studies do not specify which COX-1/-2-derived prostanoid metabolite(s) is actually lowered by Aspirin to account for its prophylactic benefits in cancer risk reduction, recent reports strongly suggest that some/many of its anti-cancer effects may be due to its ability to inhibit TXA₂ generation, as stated a prostanoid more typically associated with thrombosis and cardiovascular disease (20, 21). Indeed, it has long been known that platelets, the main source of TXA₂ and key target of Aspirin, play a key role in cancer progression promoting cancer cell metastasis, immune evasion and extravasation (30). Furthermore, increased levels of TXA₂ and expression of its synthase and its T prostanoid receptor, the TP, occur in a number of prevalent cancers including, for example, strongly correlating with bladder (31), prostate (32, 33), colorectal (34, 35) and non-small-cell lung cancer (36). Mechanistically, the role of TXA₂ in neoplastic progression is at least partly explained by the ability of the TXA₂-TP axis to regulate key mitogenic/ERK- and RhoA-mediated signalling cascades that contribute to tumour development and metastasis (12, 14) and also by its ability to regulate local inflammation and immunity (37–42), including within the tumour (Figure 2; summary of TXA₂-TP signalling). Hence, aside from its regulation of ERK- and RhoA-mediated processes (Figure 2) (12, 14), TXA₂ is a potent proinflammatory and immune-modulatory agent being abundantly produced in monocytes/activated macrophages and promotes monocyte chemoattractant protein-1 expression in tumours, recruiting tumour-associated macrophages, and negatively regulates the interaction between T-cells and dendritic cells, a process essential for adaptive/acquired immunity (38, 39, 43, 44). Moreover, TXA₂ is critical for early B-cell development, also with implications for its role in tumour-infiltrating B-cells (42, 45). Hence, due to its role in tumour growth and metastasis combined with its ability to regulate local inflammation and immunity, the TXA₂-TP axis can impact at multiple levels within the tumour environment.

Genome-wide association studies also reveal that certain single nucleotide polymorphisms within the TXS gene (the TBXAS1) may predispose individuals to breast cancer (46), while inhibition of TXS activity enhances apoptosis of lung carcinoma A549 cells in vitro, implicating a role for TXA₂ also in tumour cell survival (36). In the prostate, an increased expression of TXS and the TPα/TPβ isoforms directly correlate with the tumour Gleason score and pathologic stage (32, 33, 47), where expression of both TXS and the TP is mainly found in areas of perineural invasion, a recognized mechanism by which PCa cells invade the prostatic capsule and metastasize to other tissues (20, 32). Significantly in the context of PCa, through detailed mechanistic studies, we recently discovered that both the TPα and the TPβ isoforms directly interact with and regulate signalling by protein kinase C-related kinase/protein kinase novel (PRK/PKN) (48), a family of 3 AGC kinases that act immediately downstream of phosphatidylinositol 3′kinases, and are strongly, yet differentially, implicated in several cancers (49–51) and in B-cell development (52). Indeed, in addition to acting as Rho GTPase effectors, activation of the PRKs (e.g., PRK1) in response to androgen receptor (AR) signalling within the prostate catalyses phosphorylation of histone (H)3 at Thr11 (H3pThr11) which, in turn, serves as a specific epigenetic marker, and gatekeeper, of androgen-induced chromatin remodelling and transcriptional activation (48, 53–55). Hence, owing to their ability to regulate RhoA-/C-mediated responses, including metastatic processes, combined with their epigenetic priming of tumour cells, members of the PRK family are key chemotherapeutic targets particularly in castrate-resistant prostate cancer, the metastatic lethal form of PCa that occurs following androgen deprivation therapy (53, 55, 56).

Indeed, our research shows that TPα-/TPβ-mediated PRK1 activation not only leads to histone H3 threonine 11 phosphorylation in response to TXA₂ but can also cooperate with the AR to enhance the androgen-induced chromatin remodelling (H3pThr11) and transcriptional activation (48). Collectively, these studies raise the exciting possibility that TXA₂, through its ability to directly regulate PRK-induced H3pThr11, may be a strong epigenetic regulator, thereby adding to the range of possible mechanisms, whereby the Aspirin-target TXA₂ may influence the neoplastic growth. Added to this complexity, we recently established that the TPα and TPβ isoforms differentially associate with and regulate signalling by the other individual members of the PRKs (PRK1/PKNα, PRK2/PKNγ, PRK3/PKNβ) (57). Furthermore, consistent with our previous studies involving PRK1 (48), siRNA disruption of PRK1 and PRK2, but not PRK3, expression eliminates TP-mediated cancer cell responses (proliferation, anchorage-independent growth, migration) and H3pThr11 phosphorylation in the prostate carcinoma PC-3 cell line (57). Identification of a direct, functional interaction of both TPα and TPβ with the PRKs provides yet another molecular link accounting for the role of TXA₂ in tumour progression, particularly in prostate and other cancers in which the TXA₂-TP and PRKs are increasingly implicated. Critically, as stated, it suggests that the TXA₂-TP axis may serve as an epigenetic regulator, adding to the range of possible mechanisms whereby the Aspirin-target TXA₂ may influence neoplastic growth.

Factors determining transcriptional regulation of TPα in platelet progenitor megakaryoblastic cells

Collectively, these and numerous other studies provide significant mechanistic insights into the role of TXA₂ and of the TPs (TPα/TPβ) in cancer progression. However, with only limited exceptions (20, 31), few of those studies investigated the roles of the individual TPα or TPβ isoforms or examined their transcriptional regulation in cancer. To address this and focussing on TPα, the predominant isoform expressed in most cell/tissue types (10), we recently examined its expression in prostate and breast cancer and identified a key role for the tumour suppressor gene product Wilms’ tumour (WT)1 in its transcriptional regulation (58). Prior to presenting and discussing these findings, it is first relevant to review knowledge on the transcriptional regulation of TPα in the haematopoietic system where most data and insight is available.

As stated, while TPα and TPβ are encoded by the same TBXA2R gene (Figure 1), they are differentially expressed being regulated by distinct promoters, whereby promoter (Prm)1 exclusively regulates TPα expression and Prm3 regulates TPβ (7–9, 59, 60). Through initial studies carried out in the platelet progenitor megakaryoblastic human erythroleukaemia (HEL) 92.1.7 and K562 cell lineages (9, 60, 61), the transcription factors Sp1 (stimulating protein 1), early growth response 1 (Egr1) and NF-E2 were identified as the key trans-acting factors that bind to the ‘core promoter region’ of Prm1 to drive basal expression of TPα mRNA (9). In addition, several functional upstream activator regions (UARs; UAR1 and UAR2) and upstream repressor regions (URRs; URR1, URR2 and RR3, where repressor region 3 specifically lies within the core promoter; Figure 1) were identified within Prm1 (9). While GATA-1, Ets-1, Ets-2 and Oct-1 were identified as the main trans-acting transcription factors that regulate the UARs in the megakaryoblastic lineages, the tumour suppressor gene product WT1 was found to bind to several GC-enriched consensus cis-elements within the repressor regions (URR1, URR2 and RR3) to repress Prm1, maintaining TPα expression at relatively low levels when cells were cultured under basal conditions (60). However, following differentiation of the pluripotent megakaryoblastic cell lineages to the platelet phenotype, it was established that TPα expression was strongly upregulated, and this occurred through a complex transcriptional mechanism involving coordinated: (i) alleviation of TPα/Prm1 repression by WT1 by displacement of its binding to its consensus GC-enriched cis-acting elements within the URRs of Prm1, (ii) induction of TPα expression by binding of the transcriptional activator Egr1 to the same cis-acting GC elements within Prm1, followed by (iii) sustained upregulated expression of TPα through binding of Sp1 also to the same cis-acting GC elements within Prm1 (Figure 3) (61). Hence, WT1 plays a central role in repressing TPα expression by binding to multiple cis-acting elements within the repressor regions (URR1, URR2 and RR3) of Prm1, maintaining TPα expression at low levels under basal/non-differentiated conditions. However, in response to cellular differentiation, WT1 repression is lifted in favour of sequential high-affinity binding of the transcriptional activators Egr1 followed by Sp1 to the same cis-acting GC elements within Prm1 to induce (by Egr1) and maintain (by Sp1) high levels of TPα expression following differentiation (Figure 3) (61).

Figure 3.

Proposed model for PMA-mediated increases in Prm1 activity. Panels A-E: Proposed model for PMA induction of Prm1/TPα mRNA transcriptional regulation in HEL92.1.7 and K562 megakaryoblastic cells. In resting cells, WT1 binds in a cooperative manner (more...)

In addition to its recognized role in normal and aberrant haematopoiesis (62), WT1 was initially described as a tumour suppressor in Wilms’ tumour (WT), a rare form of renal cancer (63–65), but can also play an oncogenic role in certain cancers (66–69). Considering the recognized role of WT1 in WT of the kidney and in other cancers while also acting as a key transcriptional repressor/regulator of TPα expression in megakaryoblastic HEL and K562 cell lineages combined with the increasing awareness of the role of the TXA₂-TP axis in neoplastic progression, we recently investigated the possible regulation of TPα/Prm1 by WT1 in prostate and breast cancer, including in the model prostate PC3 and breast MCF-7 [a model oestrogen receptor/oestrogen receptor (ER)-positive breast cancer cell line] and MDA-MB-231 (a model oestrogen receptor/ER, progesterone receptor/PR and Her2/neu triple-negative breast cancer cell line) carcinoma cell lines, respectively. In brief and consistent with the findings in the megakaryoblastic lineages, it was established that WT1 can repress Prm1-directed TPα expression in both the prostate and the breast cancer lineages. Overall, as elaborated upon in detail later in this chapter, the study provided a comprehensive molecular analyses of the factors regulating the TPα expression through Prm1 in the prostate and breast and suggested that aberrant regulation by/or dysfunction of the tumour suppressors WT1, along with hypermethylated in cancer (HIC) 1, may account at least, in part, for the increased association of TXA₂/TP signalling with certain prostate and breast cancers and, potentially, in other cancers in which TXA₂, WT1 and/or HIC1 are implicated (58). The reader is referred to the original study for full details on the role of HIC1 in its regulation of TPα/Prm1 expression (58), while this communication will mainly focus on the role of WT1.

Role of Wilms’ tumour 1 in regulating TPα expression in prostate and breast cancer

The WT1 gene encodes a zinc finger transcription factor critical for development of the genitourinary, haematopoietic and central nervous systems (62, 70). The finding that mutations within the WT1 gene are a leading cause of the childhood renal cancer WT first led to the suggestion that it might serve as a tumour suppressor (64). However, the fact that non-mutated/wild-type WT1 is also abundantly expressed in a variety of cancers, including cancers of the breast (71), oesophagus (72) and pancreas (73), indicated that WT1 might also play an oncogenic role.

In addition to acting as a regulator of transcription, WT1 can also play a role in post-transcriptional regulation, including in RNA splicing (74, 75), and also in translation (76). Such diverse functions are likely due to fact that WT1 protein exists as multiple isoforms that arise owing to differential splicing and/or the use of multiple translational initiation sites within the WT1 gene (77). Of the most prevalent WT1 isoforms, the best characterized are the variants that differ due to the presence or absence (+/–) of exon 5 and +/–KTS (Lys-Thr-Ser) sequences (Figure 4). More specifically, differential splicing at these two sites yields four different isoforms, namely (+/+), (+/–), (–/+) and (–/–), each of which differ in respect of exon 5 and KTS sequences, respectively. While the -KTS isoforms can act as transcriptional repressors or activators, the +KTS isoforms do not readily bind DNA and, therefore, are less active in the process of transcription [reviewed in reference (62)]. WT1 has four Kruppel-like C₂H₂ fingers within its C-terminal region that share similarity with those of the aforementioned Egr1, another prominent member of the zinc finger family of transcription factors. While the zinc finger domain of WT1 can facilitate its DNA at the consensus Egr1 DNA-binding site (consensus sequence 5′GCG(G/T)GGGCG3′), the binding affinity of WT1 for the Egr1 consensus site is significantly less than that of Egr1 itself (78). In addition to binding to WT1 and/or Egr1 cis-acting elements, WT1 can also bind to another motif termed the Wilms’ tumour element (consensus sequence, 5′GCGTGGGAGT3′) (79). Hence, depending on the cellular context and/or on the particular promoter, WT1 can therefore serve as a transcriptional repressor or activator. This is exemplified in the case of c-Myc where over-expression of WT1 in K562, a HEL cell line, and in breast cancer cells activates the c-Myc promoter (80) but WT1 represses the c-Myc promoter in HeLa cells (81). WT1 can also complex with other DNA-binding trans-acting co-factors, such as p53 (82), and with certain co-activators or co-repressors, such as CBP (83) or BASP1 (84), respectively, to regulate transcription. It is the identity of these co-factor-binding partners that determines whether WT1 serves as an activator or repressor during transcription. In the case of the TBXA2R, the overwhelming evidence is that WT1 predominantly acts as a transcriptional repressor by binding to multiple GC-enriched cis-acting elements within Prm1 to suppress TPα expression in pluripotent megakaryoblastic cells but that in response to cellular differentiation, this repression is lifted coinciding with the increased expression and binding of Egr1 to the same GC elements to induce TPα expression (Figure 3) (9, 60, 61). In turn, following Egr1-mediated induction, subsequent binding of the constitutive Sp1 to the same cis-elements within Prm1 maintains the expression of TPα at high levels in the fully differentiated state (61). The known increased affinity of Egr1 relative to that of WT1 for binding to the same cis-acting elements accounts for why Egr1 can displace WT1 binding when progressing from transcriptional repression by WT1 to induction by Egr1 as exemplified by the coordinated regulation of TPα/Prm1 during cell differentiation (Figure 3) (61, 78).

Figure 4.

Schematic representation of Wilms’ tumour (WT) 1 isoforms. WT1, encoded by the tumour suppressor gene WT1, can exist as multiple isoforms depending on the translational start site and the inclusion/exclusion of amino acids encoded by exon 5 (encodes (more...)

In our recent studies investigating the expression and the transcriptional regulation of TPα through Prm1 in prostate and breast cancer, immunohistochemical analysis confirmed that the expression of TPα correlated with increasing prostate and breast tissue tumour grade (Figure 5), while stimulation of the prostate (PC3) and breast (MCF-7 and MDA-MB-231) carcinoma cell lines with the TXA₂ mimetic U46619 increased both cell proliferation and migration (58). Collectively, these data provided further evidence of a role for the TXA₂-TP signalling axis in prostate and breast cancer progression. In order to identify the factors regulating TPα in the prostate and breast through Prm1, genetic-based reporter analyses confirmed that the repressor regions, designated URR1, URR2 and RR3 and previously identified within Prm1, are functional in the prostate and breast carcinoma lineages. Furthermore, in each of the prostate and breast carcinoma lineages studied, over-expression of WT1 repressed TPα mRNA and Prm1-directed reporter gene expression, while chromatin immunoprecipitation analysis confirmed that WT1 binds in vivo to each of the consensus GC-enriched cis-elements within the repressor regions of Prm1. Furthermore, in the prostate and breast cellular systems, it was established that the tumour suppressor HIC1 represses TPα mRNA expression through its binding to a functional cis-element, referred to as the HIC1(b) element, within Prm1 in PC3 and MCF-7 cells, while a second HIC1 element, referred to as HIC1(a), was identified within Prm1 through bioinformatics analyses but was not found to be functional (58).

Figure 5.

Analysis of TPα expression in the prostate and breast. Panels A and B: Immunohistochemical analysis of prostate and breast tissue (benign and increasing tumour grade) screened with an affinity-purified anti-TPα antibody (200× magnification; (more...)

Among the many cell-specific differences we observed in the transcriptional activity of Prm1 in the prostate and breast carcinoma-derived cell types, it was noteworthy that the UAR1/UAR2 repressor regions within Prm1, previously identified in the megakaryoblastic HEL92.1.7 and K562 cell lines (9) where they are regulated by GATA-1, Ets-1, Ets-2 and Oct-1, were not found to be functionally active in the prostate PC3 or breast MCF-7 lineages (Table 1). These observations pointed to clear cell-/tissue-specific differences in the regulation of TPα expression through Prm1 in the haematopoietic system versus prostate and/or breast tissues. Moreover, as summarized in Table 1, our analysis of Prm1 in the prostate and breast carcinoma lineages revealed additional cell-specific differences in the upstream regulatory regions. Included in this is a prostate-specific Novel URR identified in PC3 cells, a novel UAR1 in PC3 and MCF-7 cells and an additional Novel UAR2 in PC3, MCF-7 and MDA-MB-231 cells (58). Detailed bioinformatic analysis by us revealed a putative oestrogen response element (ERE) within the Novel URR (–7962 to –7859; Table 1), suggesting that this region might function as a binding site for the oestrogen and/or ARs (85). The finding that oestrogen production increases in men with age, mainly due to aromatase conversion of androgens to oestrogens, indicates that like androgens (86), oestrogens may also play a role in PC progression (87, 88). Whether the ERE within the Novel URR of Prm1 regulates the TPα expression in the prostate remains to be investigated.

Table 1.

Activator and repressor regions within Prm1

With regard to the Novel UAR1 (–7504 to –6848; Table 1) identified in both the prostate PC3 and the ER-positive breast MCF-7 lines, but not in the triple-negative breast cancer MDA-MB-231 cell line, several putative cis-acting binding elements were identified through bioinformatics, including multiple EREs that lie in close proximity to each other. While remaining speculative, binding of the ER to some or all of these adjacent EREs may explain why the Novel UAR1 repressor region is functional in both the ER-positive PC3 and the MCF-7 lines but not active in the ER-negative MDA-MB-231 cell line.

In terms of the novel activator regions identified within Prm1 and confirmed to be transcriptionally active in all prostate and breast cell lineages examined, the Novel UAR2 located between –6648 and –6492 of Prm1 was of particular note (Table 1). Specifically, similar to that found in all other repressor regions (UAR1, UAR2 and RR3) within Prm1, putative binding elements for Egr1 and stimulating protein (Sp) 1 were also identified within this novel prostate-/breast-specific Novel UAR2 region of Prm1. As stated, our previous studies in differentiated megakaryoblastic HEL and K562 lineages established that both the inducible Egr1 and the constitutive Sp1 factors bind to multiple GC-consensus WT1 cis-elements within the UAR1, UAR2 and RR3 repressor regions of Prm1 to strongly up-regulate the TPα mRNA expression (9, 60, 61). Hence, it is indeed possible, if not likely, that the expression of TPα through Prm1 in the prostate and breast may be subject to a similar type of complex transcriptional regulation involving occupancy of the common/shared and Novel prostate-/breast-specific GC-enriched cis-acting elements that can act as consensus binding sites for WT1, Egr1 and/or Sp1, where temporal occupancy may possibly be determined by the (patho)physiologic setting. Clarity on this matter remains to be experimentally determined and is necessary to shed further light on the transcriptional regulation of TPα in both the normal and the malignant prostate/breast tissue and potentially in other tissues in which the TXA₂-TP axis is implicated. Critically, given that Egr1 serves as a master regulator in several key aspects of prostate and breast cancer progression (89, 90), investigation of the interplay between WT1 and Egr1 in the regulation of TPα expression through Prm1 within the TBXA2R as a function of tumour grade merits detailed investigation.

Conclusions and future perspectives

The prostanoid TXA₂ plays a central role in haemostasis and is widely implicated in a range of cardiovascular, renal, pulmonary and prostate diseases (1–3, 5). In humans, TXA₂ signals through TPα and TPβ, two structurally related TP isoforms that display both common, over-lapping and isoform-specific physiologic roles (6). TPα and TPβ are encoded by the same gene, the TBXA2R, but are differentially expressed being regulated by distinct promoters where Prm1 regulates TPα expression, mainly involving NF-E2, Sp1, GATA-1, Ets-1, WT-1/Egr1, and Prm3 regulates TPβ, involving cFos/cJun and Oct-1/-2 (7–9, 59, 60). While the clinical evidence for the role of the TXA₂-TP axis in neoplastic progression is increasing, few studies to date have investigated the role of the individual TPα/TPβ isoforms in human cancer or indeed in most other diseases in which TXA₂ is implicated (20, 31). Focussing on TPα, we investigated its expression and transcriptional regulation through Prm1 in prostate and breast cancer and established that the tumour suppressor protein WT1 plays a key role in regulating its expression in both tissue types. Critically, it was established that WT1 can repress TPα expression through binding to multiple GC-consensus cis-elements within previously recognized regulatory regions within Prm1, while several other prostate- and/or breast-specific novel regulatory regions were identified accounting for cell-/tissue-specific regulation of TPα. Taken together with previous studies in platelet progenitor megakaryoblastic lineages (9, 60, 61), the findings provide a strong genetic basis for understanding the diverse physiological roles played by TXA₂-TP axis within the vasculature and other systems, including potentially in the progression of certain human cancers. These studies may also provide a mechanistic basis accounting, at least in part, for the prophylactic benefits of Aspirin in reducing certain cancer risks by lowering the overall TXA₂ levels.

WT1 has recently emerged as an important target in immune-therapy approaches to treat certain cancers, and ongoing clinical trials involving the WT peptide 1 vaccination are proving positive in reducing tumour growth in breast and lung cancers, in leukaemia and, more recently, in glioblastoma (91–93). As the TXA₂-TP axis has been implicated in the development of prostate and breast cancer (20, 33, 47, 94, 95), the findings herein suggest that aberrant WT1 regulation of TPα expression may contribute to such cancers and potentially to WT itself. In addition, bearing in mind that the TPα and TPβ isoforms display a number of important functional similarities but also differences in terms of their signalling (6) and regulation including in certain cancers (96, 97), coupled with the fact that they are regulated by distinct promoters within the TBXA2R gene, it will be of considerable interest to investigate the expression and transcriptional regulation of TPβ in human cancers, in particular in prostate and breast cancer.

Abbreviations

ADT, androgen deprivation therapy; AR, androgen receptor; ChIP, chromatin immunoprecipitation; COX, cyclooxygenase/prostaglandin G2/H2 synthase; CRPC, castrate-resistant prostate cancer; C-tail, carboxyl-terminal tail; Egr1, early growth response 1; ER, oestrogen receptor; ERE, oestrogen response element; ERK, extracellular signal-regulated protein kinase; GPCR, G-protein-coupled receptor; HEL, human erythroleukaemia; H3Thr11, histone H3 threonine 11; IHC, immunohistochemistry; PCa, prostate cancer; PDK-1, 3-phosphoinositide-dependent protein kinase-1; PG, prostaglandin; PI3′K, phosphatidylinositol 3′kinase; PK, protein kinase; PRK, protein kinase C-related kinase; RR, repressor region; SDM, site-directed mutagenesis; Sp1, stimulating protein 1; TP, thromboxane prostanoid receptor; TX, thromboxane; TXS, TXA synthase; UAR, upstream activator region; URR, upstream repressor region; WT1, Wilms’ tumour 1.

Conflict of Interest

The author declares no potential conflicts of interest with respect to research, authorship and/or publication of this article.

Acknowledgements

This work was funded by an Irish Cancer Society Research Award, co-funded by the Health Research Board Ireland and the Movember Foundation (Grant no. PCI12Kin/MCRG2012/3) and by the Programme for Research in Third Level Institutions (PRTLI; MolCellBiol) and the European Regional Development Fund. The author (BTK) wishes to acknowledge the many colleagues who contributed to the research cited in this communication, in particular Drs AnneMarie M. Gannon, Garret L. Keating, Eamon P. Mulvaney and Helen M. Reid.

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