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Prostate cancer is a leading cause of cancer death in men. Initially the disease is 'androgen-dependent' however it has the ability to switch to highly metastatic 'androgen-independent' states for which there are a number of proposed mechanisms. These mechanisms are potential targets for the development of new anti-cancer drugs. This review discusses the androgen receptor in prostate cancer and how the 'switch' to androgen-independence occurs. GPCR's have been proposed to have roles in cancer pathogenesis and progression. One key pathway of interest to prostate cancer is the bradykinin receptor signalling pathway involving the receptors B1 and B2.
Prostate cancer is one of the most commonly diagnosed cancers in the developed world and a leading cause of male cancer death . It accounts for more than 220,000 deaths annually worldwide . Normal prostate tissue and most prostate cancers are dependent on the steroid hormone androgen for both growth and survival .
The way in which prostate cancer is diagnosed has seen significant changes over recent years including the use of serum markers which has lead to the detection of more localised forms at the time of diagnosis . Radical prostatectomy or radiation is often an effective treatment for localised prostate cancers and both patient morbidity and mortality have been substantially reduced due to the development of surgical techniques. Initially, more advanced neoplasms respond to hormonal therapies such as inhibiting the androgen receptor, a process known as androgen ablation [2,4,5]. However, despite these advances in detection and treatment, more aggressive androgen-independent cancers eventually emerge and develop which are refractory to these conventional therapies and have an average survival time of less than two years. Despite the decreased levels of androgen, the androgen receptors continue to be expressed and functional .
Multiple studies have been carried out in order to identify the number of different mechanisms that are thought to contribute to the progression of prostate cancer from androgen-dependent to androgen-independent. In order to design appropriate and effective new therapeutics to target these more advanced hormonally refractive neoplasms it is crucial to identify and understand the mechanisms involved .
The Androgen Receptor:
The androgen receptor plays a critical role in the initiation, proliferation and progression of prostate cancers and crucially the response to therapy . The androgen receptor is a member of the nuclear receptor family or the steroid hormone superfamily of ligand-activated transcription factors. It contains a number of specific functional domains including a DNA-binding domain (DBD) that specifically recognises target DNA sequences, a ligand binding domain (LBD) that mediates the binding of high affinity ligands, a hinge domain and a N-terminal regulatory region [8,9].
The androgen receptor is encoded by eight exons, the first of which encodes the N-terminus region that comprises half of the molecule and contains repeated elements of polyglutamine and polyglycine. This amino terminus is an important site for interaction with co-regulators that alter the receptors transcriptional activity it is therefore responsible for mediation of most transcriptional activity. However, the mechanism remains unclear as to how the amino terminus contributes to gene regulation. An interaction between the LBD and the amino terminus has been suggested as an important interaction in modulating androgen receptor activity however due to lack of detailed structural analyses a clear understanding of the interaction has not been developed. The polyglutamine repeat segments vary in length in different individuals, ranging from 14 to 35 amino acids and these differences in length have been linked to modulation of androgen receptor activity. More aggressive cancer phenotypes, increased likelihood of recurrence and earlier age of onset are all associated with shorter length polyglutamine repeats [10-14].
The second and third exon encodes the DBD which is cysteine-rich and the most conserved region of the molecule. Androgen-response elements (ARE) are sequences usually found in the enhancer regions of androgen receptor regulated genes and the DBD is essential for recognising these sequences [13,15]. The distal part of exon 4 and exons 5-8 encode the C-terminal sequence which contains a second transcriptional activation region and the LBD. A hinge region that includes a nuclear translocation signal along with acetylation and phosphorylation sites bridges the LBD and DBD and is encoded by exon 4. The androgen receptor binds to heat shock proteins and is primarily localised in the cytoplasm when androgen is absent. When androgen binds to the LBD translocation into the nucleus occurs which is initiated by a cascade of events that alters androgen receptor conformation and ultimately causes dissociation of the receptor from the heat shock proteins [16,17].
The events that cause the progression from androgen-dependent to androgen-independent prostate cancer are still not fully understood. It is possible that in castrate levels of androgen, androgen-dependent cells gain the ability to proliferate or it may be that castrate levels cause the outgrowth of a small population of cells that are androgen independent. It has been shown in analyses that the androgen receptor is still expressed in most androgen-independent tumours therefore suggesting that the androgen receptor pathway remains present and that there are alterations in its regulation [6,7]. A number of mechanisms may be used to explain activation of the androgen receptor in low levels of androgen: overexpression/amplification of androgen receptor; mutation; increased local androgen production; activation by non-steroid ligands; altered expression of co-activators; and proteolytic processing, the most recently proposed mechanism .
Mechanisms in the Switch from Androgen-dependence to Androgen-Independence:
Overexpression / amplification of androgen receptor
When the androgen receptor gene is amplified there is a substantial increased expression of the androgen receptor. Androgen receptor amplification is found in approximately 25-30% of androgen-independent tumours that occur after hormonal therapy . Androgen receptor signalling can be sustained in castrate levels of androgen because there is sufficient ligand binding due to the increase in abundance of the receptor. In progression from androgen dependence to independence, androgen receptor gene expression increases .
Androgen receptor mutations
Androgen-independent properties can develop when mutations occur in the androgen receptor gene. Mutations may lead to hypersensitivity of the receptor or broaden its ligand specificity and can be seen in all eight exons. Mutations seem to be rare in early stages with frequency increasing in recurring or more advanced tumours. 10-30% of patients treated with hormonal therapy who then developed androgen-independent tumours had androgen receptor mutations . LBD mutations can confer the potential for activation by other steroid molecules for example in the LNCaP cell line . It is suggested that anti-androgen therapy is a selective pressure that encourages the proliferation of cells with these mutations because it was found that these mutations occurred frequently in cancers treated with such therapy . Diverse transcriptional activity can arise from mutations in the androgen receptor such as loss of function, partial function, wildtype function and gain of function including part activation by a combination of two hormones and widened ligand specificity .
Increased local production of androgen by prostate cells
It has been suggested that androgen-independent properties may develop due to an increased production of active androgens locally in the prostate . After androgen ablation therapy serum and prostate analyses have been carried out and the results showed higher levels of dihydrotestosterone in tumour samples than in serum. This may indicate that prostate tumour cells can more effectively produce adequate levels of dihydrotestosterone, the more active form of testosterone, to induce androgen receptor signalling. This could be due to conversion from testosterone as a result of upregulation of the enzyme 5α-reductase. Another explanation is that androgenic hormone is endogenously produced rather than converted because in some tumours there was increased expression of genes for enzymes involved in the steroid biosynthetic pathway [25,26]. Targeting these enzymes involved in biosynthesis could therefore be a potential target for therapy.
Activation of androgen receptor by non-steroid ligands
Many studies have shown that non-steroid molecules such as growth factors and cytokines can activate the androgen receptor by initiation of a signalling cascade . Activation of the receptor can also occur in the absence of ligand by molecules such as neuropeptides and interleukins [28,29]. Both of these mechanisms include the MAPK and AKT pathways even though the initial signalling event is different. Evidence has shown an increase in neuropeptides in androgen ablation therapy supporting the suggestion that these molecules can induce androgen-independent tumour proliferation .
Altered expression of co-regulators
It has been proposed that changes in co-regulator levels is associated with androgen-independence and over 130 associated with the androgen receptor are known to exist, however physiological function of some are not clear . Many studies have been carried out on various co-regulators and key molecules and alterations in protein expression that are associated with androgen-independence have been identified but to date results have been conflicting and further analyses are required .
Studies have shown that translocation to the nucleus and androgen-independent functioning occurs when the LBD is removed from the androgen receptor. A recently proposed mechanism for this is the calcium dependent protease, calpain. This protease can cut out the LBD from the androgen receptor leaving the DBD and transactivation domain intact . Calpains are ubiquitously expressed however recent studies show that levels of calpain 2 mRNA are higher in invasive prostate tumours . An additional mutation, the duplication of exon 3, of the androgen receptor may increase the likelihood of calpain mediated proteolysis . Increased androgen receptor proteolysis occurs in higher levels of calpain which confers androgen-independence and proteolysis of other targets of this enzyme would also increase and alter other properties that promote tumour growth. Therefore inhibiting calpain activity can limit metastatic potential and is potential therapeutic target for some prostate cancers  .
Although the androgen receptor is still expressed in most androgen-independent tumours suggesting that it is alterations in the regulation of the receptor pathway that leads to androgen-independence, some androgen-independent cell lines have displayed no androgen receptor expression [36,37] In the context of introducing the androgen receptor into cells that are negative the receptor acts as a growth suppressor whereas usually tissue is dependent on the receptor for survival and there are various explanations for this. Continued signalling in cells that have bypassed the need for the androgen receptor can be detrimental to cell growth. This is evident in the androgen-dependent LNCaP cell line which grows at physiological concentrations of androgen but stops at high levels .
Some of the main mechanisms that can confer androgen-independence have been discussed and there are also other pathways that can play a role. These mechanisms may work synergistically to confer the androgen-independent properties however targeting an individual pathway may be sufficient to inhibit tumour growth .
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GPCR signalling in prostate cancer:
The guanosine phosphate binding (G) protein coupled receptor (GPCR) family, a family of extracellular signal regulators play an important role in the progression of androgen-independent prostate cancer. In prostate cancer cells and surrounding stromal tissue there is increased expression of some GPCRs and their ligands. Stimulation of GPCR induces proliferation via activation of the extracellular signal regulated kinase (ERK) pathway and also decreases apoptosis, therefore conferring a survival advantage to these cancer cells . It has been demonstrated in In vitro studies that growth factor induced PC3 cell proliferation requires ERK phosphorylation. The major role that ERK plays in prostate cancer spread is supported by the evidence that there are increased levels of activated ERK in advanced tumours [41,42] There is evidence to suggest that epidermal growth factor receptor has an intermediary role in GPCR mediated ERK activation . The mechanism of how this pathway is involved is unknown however it is thought that uncontrolled GPCR signalling may play a role in malignant growth partially via stimulation of epidermal growth factor receptor dependent mitogenic signalling pathways. There are many suggestions of crosstalk between androgen receptors and downstream effectors of GPCRs. Phosphorylation of the androgen receptor to confer androgen-independence is by tyrosine and serine-threonine kinases and these are regulated by GPCRs . Since GPCRs are probably primary transducers of proliferation and survival of androgen-independent prostate cancer then therapy aimed at targeting these receptors and their effectors is a promising potential. However systemic therapy will likely have serious side effects due to the ubiquitous nature of the GPCRs. With further knowledge and understanding of the GPCR signalling pathways involved in prostate cancer, specific targets for delivery of localised inhibitors may allow selective blockade of mitogenic signalling .
Bradykinin receptor signalling:
There is evidence to support the increased production of kinins in several types of cancer and it is thought that the ability of bradykinin to increase vascular permeability and stimulate growth may contribute to the behaviour of tumours. Novel bradykinin receptor antagonists have been shown to inhibit growth and induce apoptosis and have therefore been proposed as treatment for various cancers. Recent interest has emerged regarding the potential of bradykinin antagonists as chemotherapeutic agents .
Bradykinin is an endogenous nonapeptide that is a product of the kallikrein-kinin system . It activates bradykinin subtype 1 receptor (B1) and bradyknin subtype 2 receptor (B2) to exert various pathophysiological functions such as effects on vascular permeability and mitogenesis [46,47]. The B2 receptor is ubiquitously expressed and is predicted to control most of the physiological actions of bradykinin. However the B1 receptor shows marked up-regulation after cell injury, inflammation and stress but is usually expressed at very low levels in non-pathological conditions. Antagonists of both peptide and non-peptide origin have been discovered which have high affinity and selectivity for either B1 or B2. The receptors are rather different in sequence homology and their rank order of potency towards specific agonists and antagonists is therefore also very different. Bradykinin receptor antagonists have proposed to have potential therapeutic uses for pain, inflammation, respiratory disorders and stroke [48,49].
Bradykinin is an autocrine growth factor in prostate cancer . The B1 receptor is not expressed in benign human prostate tissues but there is specific expression in malignant lesions and prostatic intraepithelial neoplasia. The B2 receptor is expressed in both benign and malignant tumours. Therefore B2 signalling inhibition would likely disrupt normal functioning of all cells and exert toxic side effects and so is considered of limited clinical value. Androgen-independent PC3 cells have been used to show that specific inhibition of B1 receptor signalling reduces mitogenic and survival signalling as well as cell growth and metastasis. A target for treatment of advanced prostate cancer may therefore be targeted blocking of B1 receptors with antagonists .
Bradykinin Receptor Antagonists:
Academic centres and pharmaceutical companies have been developing bradykinin antagonists for more than 20 years and over this time there has been a shift in work from the B2 to the B1 antagonists. However, only a small number have reached clinical trials despite the large numbers that have been discovered and the only bradykinin receptor antagonist to have ever reached the market is the decapeptide B2 antagonist Icatibant (HOE-140) and this was in 2008, nearly 20 years after its discovery. Possible reasons for this disappointment in success may be that in vitro results are not translated in vivo or even good activity in certain animal receptors may not be displayed in the human receptor. Also, some of the compounds exhibit poor pharmacokinetic profiles but some companies have targeted conditions in which oral administration is not a requirement . For a successful product there must be a balance between bioavailability and patient acceptability.
BRADYKININ RECEPTOR ANTAGONISTS AS A POTENTIAL TREATMENT FOR PROSTATE CANCER:
The discovery of a bradykinin receptor complex in prostate cancer cells:
It has more recently been proposed that for the proliferation of androgen-independent prostate cancer PC3 cells it is critical for direct cross-talk between the B1 and B2 receptors and hence the occurrence of these B1- B2 complexes increases under pathological conditions. Receptor internalization properties support suggestions of the existence of these complexes in prostate cells. Following direct stimulation of either the B1 or B2 receptor, B1 internalises into intracellular compartments and also blockage of B2 with selective antagonist inhibits B1 internalization . It had been thought that GPCRs function as monomeric signalling units, however growing evidence indicates that the receptors, through direct interactions with identical or non-identical family members, form higher oligomeric structures . It is still unclear what the underlying mechanism in oligomerization is the specific role it has on GPCR function.
Studies have shown that stimulation of the receptors with their selective agonists induced an increase in the cell proliferation rate. However, the ability of one receptor to induce cell growth was inhibited when the cells were pre-incubated with the antagonist of the opposite receptor. These findings suggest that both B1 and B2 receptors are present in PC3 cells and in order to promote cell growth they communicate with each other in some way . It was previously known that ERK pathway activation was required for bradykinin induced PC3 cell growth so it was investigated to see if ERK activation itself required the cross-talk between the bradykinin receptors . Results showed that blockage of B1 halted ERK activation by both B1 and B2 receptors and the same outcome occurred upon blockage of B2. Levels of ERK 2 did not change with different treatments and no additive affect was seen when both agonists or antagonists were simultaneously added, indicating that observed changes were due to variations in ERK activation rather than expression levels. It is known that signal transduction is modulated at many levels such as ligand binding and efficacy and potency of binding by the formation of receptor complexes. One study has confirmed receptor specificity and indicated that the consequences of cross-talk between B1 and B2 occur at a point distal to ligand binding since it showed that agonists and antagonists of B1 do not bind to the active site of B2. It has been suggested that the point of interaction between B1 and B2 is at receptor coupling to the G-protein since following receptor blockage there is inhibition of phospholipase C-mediated inositol phosphate formation. Also proposed is that the role of the unactivated receptor is to enable efficient coupling of the activated receptor to the G-protein since blockage of one receptor impaired inositol phosphate formation by the other. Selectively targeting these B1-B2 complexes may introduce new treatments of advanced prostate cancer .