Prostate Cancer: The Progress to Androgen-Independence and how Novel Treatments that Target GPCR's could Potentially Be a new Route of Treatment
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. Novel and potent bradykinin receptor antagonists derived from amphibian peptides have been identified and sequenced by research groups in the Queen's University labs to target these bradykinin receptors. These may prove to be a novel therapeutic approach to treatment of prostate cancer, a very promising and exciting prospect.
Abbreviations used: PC, prostate cancer; AR, androgen receptor; ERK, extracellular signal related kinase; BK, bradykinin; B1, bradykinin subtype 1 receptor; B2, bradykinin subtype 2 receptor; EGF, epidermal growth factor; MAPK, mitogen-activated protein kinase.
A Background to Prostate cancer:
PC 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 PCs are dependent on the steroid hormone androgen for both growth and survival .
The way in which PC 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 PCs 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]. Drugs used in androgen ablation therapy include gonadorelin analogues such as goserelin (Zoladex®), anti-androgens such as bicalutamide (Casodex®) and flutamide, and the gonadotrophin-releasing hormone antagonist degarelix (Firmagon®) . 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 are lethal. Despite the decreased levels of androgen, the androgen receptors continue to be expressed and functional . Docetaxel (Taxotere®) is the current recommended treatment for men with hormone refractory metastatic PC (REF).
Multiple studies have been carried out in order to identify the number of different mechanisms that are thought to contribute to the progression of PC 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 AR plays a critical role in the initiation, proliferation and progression of prostate cancers and crucially the response to therapy . It 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 [9,10].
The N-terminus region is an important site for interaction with co-regulators that alter the receptors transcriptional activity and an interaction between the LBD and the amino terminus has been suggested as an important interaction in modulating AR activity, however, an understanding of the these mechanisms is unclear. The region contains repeated elements of polyglutamine and polyglycine which vary in length in different individuals and these differences have been linked to modulation of AR activity. More aggressive cancer phenotypes, increased likelihood of recurrence and earlier age of onset are all associated with shorter length polyglutamine repeats [11-15]. The AR 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 AR conformation and ultimately causes dissociation of the receptor from the heat shock proteins [14,15].
The events that cause the progression from androgen-dependent to androgen-independent PC 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 result in the outgrowth of a small population of cells that are androgen independent. It has been shown in analyses that the AR 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 [7,8]. A number of mechanisms may be used to explain activation of the AR 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:
Amplification of the AR gene consequently causes a substantial increased expression of AR and receptor gene expression increases in progression from androgen dependence to independence. . AR signalling can be sustained in castrate levels of androgen because there is sufficient ligand binding due to the increase in abundance of the receptor. .
Mutations in the AR gene can cause androgen-independent properties such as hypersensitivity of the receptor or broadened ligand specificity. Mutations seem to be rare in early stages with frequency increasing in more advanced tumours . LBD mutations can confer the potential for activation by other steroid molecules for example in the LNCaP cell line . Diverse transcriptional activity can arise from mutations in the androgen receptor such as loss of function, partial function, wildtype function and gain of function .
It has been suggested that an increased production of active androgens locally in the prostate may cause androgen-independent properties . After androgen ablation therapy higher levels of dihydrotestosterone exist in tumour samples than in serum indicating that prostate tumour cells can more effectively produce adequate levels of dihydrotestosterone, the more active form of testosterone, to induce androgen receptor signalling [23, 24].
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 [26, 27]. Evidence has shown an increase in neuropeptides in androgen ablation therapy supporting the suggestion that these molecules can induce androgen-independent tumour proliferation .
It has been proposed that changes in co-regulator levels are associated with androgen-independence . Key molecules and alterations in protein expression that are associated with androgen-independence have been identified but further analyses are required .
Studies have shown that the calcium dependent protease calpain, which can cut out the LBD from the androgen receptor leaving the DBD and transactivation domain intact, may cause androgen-independence . Calpains are ubiquitously expressed however recent studies show higher levels in invasive prostate tumours . Another mutation of the androgen receptor may increase the likelihood of calpain mediated proteolysis .
There are a number of novel signalling pathways that are thought to be involved in the progression of prostate cancer. PC cells show increased expression of sonic hedgehog ligand which may activate a certain transcription factor leading to expression of various tumorigenic genes that cause sustained growth of PC cells. This activity is primarily seen in aggressive and metastatic PC. There is significant expression of several Wnt ligands in all types of PC cell lines and prostatic stromal cells and abnormal activation of the Wnt signalling cascade appears to contribute to PC progression by enhancing their tumorigenicity. Different Wnt ligands may have different roles in progression of PC. A number of neuropeptides also participate in the progression of PC such as bombesin, bradykinin, calcitonin, neurotensin and serotonin which act via GPCRs. It is suggested that these may promote progression to androgen-independence during hormone treatment and therefore blocking certain GPCR pathways with selective antagonists during anti-androgen therapy may inhibit differentiation induced by these neuropeptides .
GPCR signalling in prostate cancer:
The guanosine phosphate binding (G) protein coupled receptor (GPCR) family, are extracellular signal regulators that play an important role in the progression of androgen-independent PC. In PC cells and surrounding stromal tissue there is increased expression of some GPCRs and their ligands such as bradykinin, bombesin and endothelin-1. Stimulation of GPCR induces proliferation via activation of the extracellular signal regulated kinase (ERK) pathway and also inhibits 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 [38,39] 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 EGF receptor dependent mitogenic signalling pathways. There are many suggestions of crosstalk between ARs and downstream effectors of GPCRs. Phosphorylation of the AR 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 has promising potential. However systemic therapy will likely have serious side effects due to the ubiquitous nature of the GPCRs (REF??). 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 a 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 B1 and B2 to exert various pathophysiological functions such as effects on vascular permeability and mitogenesis [43, 44]. 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 [45, 46].
The B1 receptor is not expressed in benign human prostate tissues but there is significant expression in malignant lesions and prostatic intraepithelial neoplasia. 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 (REF??). A target for treatment of advanced prostate cancer may therefore be targeted blocking of B1 receptors with antagonists. The B2 receptor is expressed in both benign and malignant tumours. It had been proposed that B2 signalling inhibition would likely disrupt normal functioning of all cells and exert toxic side effects and therefore may be of limited clinical value . However, induction of cellular proliferation via ERK by B2 receptors in normal human prostate stromal cells was demonstrated which increased the potential of B2 as a target for intervention .
Bradykinin Receptor Antagonists:
Academic centres and pharmaceutical companies have been developing bradykinin antagonists for more than twenty 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:
PC cells are neuroendocrine in origin and have receptors for autocrine growth factors such as bradykinin, which when stimulated promoted cancer growth and progression. PC becomes more neuroendocrine in nature after it escapes hormonal control. Neuropeptide antagonists have been investigated as potential therapy for lung and prostate cancers, however, the first developed compound displayed low potency and a short life in vivo (REF?). Bradykinin antagonists were then developed that showed more promise. These bradykinin antagonists were first developed for small cell lung cancer but have now been applied both in vitro and in vivo in PC experiments. Studies have shown that potency is often higher in vivo than in vitro and this may be due to the fact that bradykinin acts as a cancer growth stimulant in various ways, it stimulates angiogenesis, it facilitates tumour invasion by increasing tissue permeation and also stimulates growth directly. The use of bradykinin antagonists synergistically with standard anti-cancer agents is an exciting new approach to cancer therapy .
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 androgen-dependent PC3 cells since direct stimulation of either receptor appears to result in internalisation of the other receptor . 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 has been suggested that dimerization of GPCRs may control signal generation and termination. Indications that GPCR hetero-dimerization is a common process offers a potential for more selective medications since GPCRs are already the most targeted proteins in the development of small molecule therapeutics drugs.
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 either 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 . There are three properties of GPCR function that present hetero-dimerisation at a potential pharmacological target. Hetero-dimers can create very distinct signals, some ligands are selective for hetero-dimers and tissue selectivity may be achieved via hetero-dimers. It is likely that further research will be carried out to determine the molecular basis and functional consequences of GPCR heterodimerisation.
Amphibians have specialised glands that release compounds as a defence and protective mechanism and it has been known for some time that amphibian skin secretions contain peptides with intriguing biological properties . These peptides can generally be classed as antimicrobial or neuroendocrine such as bradykinin, bombesin and gastrin-releasing peptide . They are highly potent for various reasons, for example, they must be capable of deterring predators and since they are delivered orally to the predator they must be stable in digestive enzymes making them a potential source of orally stable treatments. Bradykinin mediates a variety of physiological phenomenon such as vasodilation, hypotension, smooth muscle contraction, pain and inflammation. It also plays a role in respiratory disorders and angioedema and therefore bradykinin and its antagonists such as the novel B2 antagonist helokinestatin are very valuable peptides with many uses [54, 55]. Although a large number of bradykinin related peptides from amphibian skin secretions have been identified and structurally characterized, it is only relatively recently that encoding has occurred which involves molecular cloning technology after construction of a cDNA library. A lot of investigation still remains because there are thousands of species of frogs and toads which produce several peptides, all of which are unique. However, amphibian populations are rapidly declining and so preservation of species and habitat is important for therapeutic leads .