Recent developments in prostate cancer biomarker research: therapeutic implications

Abstract

This review aims to present an overview of recent clinical trials targeting biomarkers in advanced prostate cancer. We searched ClinicalTrials.gov for early phase clinical trials on treatments of prostate cancer that have been recently completed, are ongoing or are actively recruiting participants. Drug targets and their mechanism of actions were assessed and summarized. Trials were categorized according to prostate cancer biomarkers that have potential as therapeutic targets. A total of 19 new therapeutic agents for the treatment of prostate cancer are included in this review. Trials are summarized according to the targeted biomarkers and are categorized into five therapeutic approaches: prostate cancer vaccine, epigenetic therapy, pro-apoptotic agents, prostate cancer antibodies and anti-angiogenesis approach. Some of the therapeutic agents reviewed showed promising results, warranting further investigation in late phase clinical trials. Recent novel prostate cancer biomarkers that made it through clinical trials and their relevance as drug targets are summarized. This review emphasizes the importance of specific prostate cancer biomarkers and their potentials as targets of the disease. Some clinical trials of targeted treatments in prostate cancer show promising results. Better understanding of disease mechanisms should potentially lead to more specific treatments for individual patients.

Introduction

Prostate cancer and PSA screening

Prostate cancer is among the most commonly diagnosed male disease and remains a leading cause of death in most Western countries, especially in elderly men [1–3]. More than half of all men diagnosed with cancer are over the age of 70 years, with prostate cancer constituting about 50% of cancers in this age group [4].

Rapid increases in incidence rates for prostate cancer in the past two decades have occurred in part due to the widespread use of screening since the 1980s, by serum prostate-specific antigen (PSA), a glycoprotein produced by the prostate gland [5–8]. Whether PSA screening and earlier detection of prostate cancer has an impact on the decline of mortality is still debated [9–12]. Recently, large scale studies from the US [13] and Europe [14] looking at correlation between PSA screening and prostate cancer mortality have been published. In the US, they found no significant decline in mortality rate between patients receiving annual PSA screening and digital rectal examination (DRE) and those receiving usual care which may sometimes include screening [13]. However a study from European countries found a weak association between increased PSA screening and mortality rate [14]. There is, nonetheless, an urgent need for better biomarkers to replace or work in conjunction with PSA screening, due to the high prevalence of false positive and false negative rates seen with PSA alone [8].

Biomarkers leading to new therapeutic approaches

The knowledge of biomarkers in cancer offers new hope for more specific therapies and provides important understanding of the factors influencing the aggressiveness of prostate cancer and potential new treatments [15]. Molecular genetic approaches examining tumour gene expression profiling using microarrays have been introduced in recent years and analysis using a small panel of genes of interest has the potential for use in clinical practice to allow better diagnosis and classification of the disease, provide information on how each individual patient may respond to treatments and lead to reduced toxicity and identification of new therapeutics [16, 17]. We will review a number of biomarkers in prostate cancer which have involved development of targeted therapies in animal and human trials. Figure 1 schematically summarizes the function of these various biomarkers. Table 1 summarizes these targets and ongoing clinical trials which will be discussed in the following section. Although, as mentioned above, PSA measurement has limitations in prostate cancer management, most studies we reviewed utilized PSA responses as an efficacy endpoint. Some studies also used the RECIST system (to evaluate response rates of tumour lesions) [18].

Figure 1.

Site of actions of drugs that are currently in clinical trials for treatment of prostate cancer and target biomarkers and their roles in prostate cancer. [Experimental therapeutics: RAD001, CCI779, mRNAs vaccines, MLN2704, 177-Lu-J591, YM155, HDAC inhibitors, G3139, AT-101, bevacizumab, sunitinib, imatinib and cancer vaccines are indicated as RED diamonds], lymphoid cells are indicated as B () for B lymphocyte; CTL () for cytotoxic T lymphocytes); M () for macrophage; T_H () for T helper cells and DC () for dendritic cells; indicates phosphorylation. (For others see ‘abbreviations and acronyms’ listing for definitions of targets under study

Table 1.

Current drugs in clinical trials for treatment of prostate cancer (PCa) and their targets (Source: clinical trial data and drug names are available on http://ClinicalTrial.gov; []= study reference number)

Drugs	Targets	Mode of action	Stage of development (Trial phase)	Efficacy PSA response	Anti-tumour response (RECIST [18])	Dose limiting toxicities (DLT)	Treatment modality	Intent of treatment	Ref.	Clinical trials
GVAX® vaccine	GM-CSF	• Prolonged immunity against PCa cells via DC differentiation and proliferation • Specific to PCa cells	I, II	Phase I/II • Overall median PSA DT increased compared with pre-treatment [35]• 8/19 (42%) patients had stable PSA during treatment period[35]• 16/21(76%) patients had decreased PSA post-treatments [34]• 42/80 (52%) patients that had decrease PSA concentrations had longer survivals [33]	Phase I/II • T-cell and B-cell stimulations [34, 35]• 28/80 patients had SD[33]	DLT not reached in any trials	Single/combination (ADT)	Palliation	[33–35]	[154, 155]
RNActive®-derived PCa vaccine (CV9103)	PSA, PSMA, PSCA and STEAP	• Direct anti-tumour activity via delivery of mRNA encoding prostate-specific Ag	I, II	N/A	N/A	N/A	Single	Palliation	[37]	[38, 39]
IL-2-IFN-γ-secreting tumour vaccine	IL-2 and IFN- γ	• Specific anti-tumour responses • Enhancing Ag-presenting cells and T-cells responses to tumour Ags	I, II	Phase I [41]• 2/6 patients had <50% PSA decline • 2/6 patients had PSA stabilization Phase I/II [40]• 22/30 patients had increased PSA DT compared with pre-treatment • 12/30 had plateau PSA for a period of 12 weeks	Phase I [41]• Immunogenic Ag PSGR-1, STEAP, PSA, PRAME (5/6 patients) • Immunogenic Ag PSCA, PSMA and EpCAM (3/6 patients) Phase I/II [40]• Immunogenic Ags including survivin, PSCA, PSMA, PSA, STEAP, PAP and Ep-2H Both trials had T-cell response against PCa Ag (but was not correlated with PSA response)	DLT is not reached in any trials	Single	Palliation	[40, 41]	[36]
Adenovirus-mediated IL-12 gene delivery and PSMA-pulsed PBMC with recombinant IL-12	IL-12 gene	• Increase tumour cells susceptibility to cytotoxic T cells	I	N/A	N/A	N/A	Single	Palliation	[42]	[51, 52]
Vorinostat (SAHA), panobinostat, belinostat (PXD101), valproic acid and romidepsin (FK228)	HDACs	• Epigenetic therapy • Prevention of gene modification promoting cancer survival	I, II	Vorinostat Phase I Single agent [66, 68]• Trials of combined solid and haematological malignancies • PSA concentrations were not determined Panobinostat Phase I Combination [71]All patients receiving panobinostat alone had PSA progression	Vorinostat Phase I Single agent [66, 68]• PCa patients did not have anti-tumour response Panobinostat Phase I Combination [71]• 2/7 patients receiving panobinostat + docetaxel had PR	Vorinostat Phase I Single agent [66, 68]• Grade 4 leukopenia • Grade ¾ thrombocytopenia • Grade 4 neutropenia Panobinostat Phase I Combination [71]• Grade 3 neutropenia • Grade 3 dyspnea	Single/Combination (chemothera-py, ADT, isotretinoin and bevacizumab)	Palliation	[66–71, 156]	[53–60, 157, 158]
Cont'd				• 5/8 patients receiving panobinostat+ docetaxel had ≥50% PSA decline Phase Ib Combination [69]• 10/18 patients had decline PSA (7 patients <50% decline) Belinostat Phase I Single agent [67]• PSA concentrations not measured	(Including 1 patient who had ≥50% PSA decline • 4/7 patients had receiving panobinostat + docetaxel had SD Phase Ib Combination [69]• 2/13 patients had PR • 6/13 patients had SD Belinostat Phase I Single agent [67]• 18/46 patients had SD • 12/24 (MTD) patients had SD	Phase Ib Combination [69]• Grade 4 bradycardia • Grade 4 neutropenia Belinostat Phase I Single agent [67]• Grade 3 fatigue • Grade 3 atrial fibrillation
				Romidepsin Phase II Single agent [70]• 2/35 had ≥50% PSA decline PSA response rate of 5.7%	Romidepsin Phase II Single agent [70]2/35 patients had PR	• Grade 3 diarrhoea • Grade 2 nausea/vomiting Romidepsin Phase II Single agent [70]DLT was not reached due to early trial closure
Oblimersen sodium (G3139) and R-(-)-gossypol acetic acid (AT-101)	Bcl-2	• Targeting anti-apoptotic Bcl-2 protein • Increase susceptibility of cancer cells to cytotoxic drugs and radiotherapy	I, II	Oblimersen Combination • Contradicting results on PSA responses [79, 80]• 14/27 patients had PSA response (6 patients had ≥80% PSA decline)[79]• PSA decline of ≥30% was not reached [80]	Oblimersen Combination • Contradicting results[79, 80]• 4/12 had PR [79]• Docetaxel-oblimersen and docetaxel alone showed similar clinical responses [80]	Oblimersen • Grade ¾ neutropenia • Grade ¾ leukopenia • Grade ¾ fatigue	Combination (Chemotherapy and ADT)	Palliation	[79, 80, 84, 85]	[78, 83, 159, 160]
				AT101 Single agent [84]• 2/23 (8%) patients had ≥50% decline in PSA concentrations Combination [85]• 14/20 (70%) patients had ≥50% decline in PSA concentrations	AT101 Single agent [84]• 2/19 patients SD Combination [85]• 50% PR levels	AT101 • Grade 3 small intestinal obstruction • Grade 3 gastrointestinal toxicities
YM155	Survivin	• Pro-apoptotic agents • Block apoptosis inhibitor protein, survivin	I, II	• 2/9 (22%) PCa patients had decline PSA concentrations	• Only PSA concentrations were determined for PCa patients	• Increase creatinine concentrations • Grade ¾ neutropenia • Grade 3 thrombocytopenia	Single/Combination (Chemotherapy)	Palliation	[89]	[90, 91]
MLN2704 and 177Lu-J591	PSMA	• PCa Ab • Ab-directed cytotoxic drug and radioisotopes	I, II	MLN2704 Single agent [94]• 2/23 (8%) with PSA decline of ≥50%	MLN2704 Single agent [94]• 40% SD	MLN2704 • Uncomplicated febrile neutropenia	Single/Combination (177Lu-J591 with chemotherapy and ADT)	Palliation	[93, 94, 100, 101]	[95–99, 161, 162]
Cont'd				177Lu-J591 Single agent • 2/29 had 70–80% decline in PSA concentrations[101]• 6/29 had PSA stabilization [101]• 4/35 had ≥50% PSA decline [100]• 16/35 had PSA stabilization [100]	177Lu-J591 Single agent 2/12 patients objective responses[101]	177Lu-J591 • Grade 4 thrombocytopenia • Grade 4 neutropenia
Bevacizumab (Avastin)	VEGF	• Anti-angiogenesis	I, II	Combination • 11/20 (55%) patients has major PSA response	Combination • 2/8 patients had SD • 3/8 patients had PR • 3/8 patients had PD	• Grade ¾ neutropenia • Grade ¾ thrombocytopenia	Single/Combination (Chemotherapy, ADT, mTOR inhibitor)	Palliation and prevention	[146]	[111, 112, 142, 147, 148, 163–165]
Imatinib mesylate (Gleevec®)	PDGFR	• Anti-angiogenesis	I, II	Single agent • 1/19 patients had ≥50% decline and 2/19 had a decline of <50% [118]• Median PSA DT (16/19 patients) were not different pre- and post-treatments [118]	Single agent • 11/20 patients withdrew from study (most due to imatinib-related toxicity) [118]• 6/20 patients had PD [118]	Single agent • Trial was stopped due to toxicity [118]• Grade ¾ neutropenia • Grade 4 dyspnoea	Combination (Chemotherapy and ADT)	Palliation	[118, 120, 166, 167]	[121–123]
				• 9/16 patients had PSA stabilization [120]• 5/16 patients had PSA progression [120]Combination • PSA changes in imatinib-docetaxel group were similar to docetaxel-placebo group [165]	Combination No apparent benefit when combined with docetaxel [165]	• Grade 3 dyspepsia • Grade 3 rash • Grade 3 diarrhoea • Grade 4 haematuria Combination • 5/89 patients had sigmoid diverticular perforations [165]• 3 deaths related to therapy; pneumonitis, peritonitis and diverticular perforation [165]
Sunitinib malate (SU011248)	VEGFR and PDGFR	• Anti-angiogenesis	I, II	• Single agent 8/34 patients had ≥30% PSA decline (2 patients had ≥50% decline) • 15/34 (44%) patients had PSA stabilization	Single agent • 1/34 patients had PR • 18/34 patients had SD • 10/34 patients had PD	• Grade ¾ neutropenia • Grade ¾ leukopenia • Grade ¾ hypertension • Grade ¾ diarrhoea and fatigue	Single/Combination (Chemotherapy, ADT, RT)	Palliation and prevention	[124]	[127–129, 149, 168]
Everolimus (RAD001) and temsirolimus (CCI-779)	mTOR	• Anti-angiogenesis • Interrupt cancer survival signalling	I, II	Single agent • 1/13 (7.5%) patients had PSA decline of 50%	Single agent • 4/13 patients had SD • 1/13 patients had PR	N/A	Single/Combination (Chemotherapy and bevazicumab)	Palliation and prevention	[141]	[111, 142, 144, 152, 153]

RECIST, Response Evaluation Criteria in Solid Tumours criteria [18]; CR, (complete response) = disappearance of all target lesions; PR, (partial response) = 30% decrease in the sum of the longest diameter of target lesions; PD, (progressive disease) = 20% increase in the sum of the longest diameter of target lesions; SD, (stable disease) = small changes that do not meet above criteria.

Current clinical trials in prostate cancer biomarker targeting

Prostate cancer vaccines

Cancer vaccines stimulate systemic anti-tumour immune responses which may provide reduced toxicities compared with traditional chemotherapy. Patients who develop an androgen-independent prostate cancer after radiation therapy or hormone ablation therapy and those who have metastatic disease at the time of diagnosis are much less likely to be cured. Therefore it is crucial to identify markers early in the disease process that might be able to distinguish between indolent and aggressive cancers [19–22]. The presence of markers that are prostate-specific and prostate cancer-specific [23] such as PSA, prostate specific membrane antigen (PSMA) (PSCA), and early prostate cancer antigen (EPCA) makes them potential candidates for cancer vaccines.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) vaccine or GVAX® is a promising approach for prostate cancer [24]. This vaccine involves genetically modified irradiated prostate cancer cells expressing the cytokine, GM-CSF which is a known mediator of immune system activation [25]. In mice, irradiated tumour cells expressing GM-CSF need CD4+ and CD8+ T-cells for immune activation and result in a long lasting and specific immune response [26]. In 2000, successful phase I/II trials using dendritic cells (DC) with encoding fusion proteins of prostatic acid phosphatase (PAP) and GM-CSF in hormone-refractory prostate cancer patients demonstrated anti-PAP immune responses in all patients. Almost half of participants involved had a decline in PSA concentrations, with no major adverse effects [27]. The mechanism of action of this immunotherapy involves DC (adaptive) immune responses to tumour cell antigens, thus promoting differentiation of bone marrow-derived progenitors into DCs at the local injection site [28]. GM-CSF can reverse tumour tolerance and produce anti-tumour responses by initiating bone marrow-derived progenitor differentiation into DCs and DC proliferation [24, 28, 29].1

Hege et al. [28] have summarized clinical trials that combined immunotherapy with other treatments to enhance the effectiveness of anti-tumour activity and this includes the use of CD40 and Toll-like receptors (TLRs) for DC activation, anti-CTLA4 and anti-CD25 antibodies. These approaches attempt to inhibit down-modulation of T-cell responses, vascular endothelial growth factor (VEGF) blockade to prevent inhibitory effects of the VEGF receptor, and interferon (IFN)-α for promotion of immunomodulatory responses. Furthermore, chemotherapy, for example with docetaxel, a cytotoxic anti-microtubule agent and anti-androgen therapy, has been combined with cancer vaccines in clinical trials to see if these more traditional treatments can enhance anti-tumour responses [30, 31].

An early phase I trial of eight prostate cancer patients involved treatment with autologous cancer vaccine prepared from the patients' own prostate tumours taken during prostatectomy, which were then irradiated and engineered to express GM-CSF [32]. This trial evaluated patients' T- and B-cell immune responses at the injection sites, hypersensitivity against the un-transduced autologous tumour cells and antibodies against tumour antigens in the serum. There were increased immune responses at the vaccination site including CD4±, CD8± T-cells, T helper cells, macrophages and DCs. Three out of eight patients had antibodies specific to prostate cancer antigens. Clinical trial phase I/II studies using allogeneic human GM-CSF gene transduced irradiated prostate tumour cell lines administered via intradermal injection have demonstrated early evidence of safety and clinical activity, showing dose–response immune responses and good tolerance in patients with metastatic hormone-refractory prostate cancer [33–35].

RNActive®-Derived Prostate Cancer Vaccine, CV9103. is another promising immunotherapy vaccine for prostate cancer which derives from mRNA encoding prostate-specific antigens which have been modified to maintain stability (PSA, PSMA, PSCA and six-transmembrane epithelial antigen of the prostate [STEAP]) [36]. The mechanism of action involves stimulation of cytotoxic T lymphocytes by gene therapy vehicle mRNAs to respond against PSA-, PSMA-, PSCA- and STEAP-expressing prostate tumour cells [37]. The use of naked mRNA vectors has been recently developed and engineered to overcome the issue of instability of mRNA leading to rapid digestion within the body. This mRNA technology is now being tested in prostate cancer and non-small cell lung cancer. Current open clinical trials phase I/IIa and II are now recruiting participants with evidence of hormone refractory prostate cancer showing increasing concentrations of PSA despite hormone deprivation therapy, using CV9103 delivery via multiple intradermal injections [38, 39].

Interleukin-2 (IL-2)-interferon-gamma (IFN-γ)-secreting allogenic tumour vaccine derived from allogeneic prostate cancer cell lines with recombinant human IL-2 and IFN-γ have been involved in early clinical trials via multiple intradermal injections [36, 40, 41] and have shown prostate cancer antigen-specific T-cell responses (reactivity against peptides PSMA, PAP, survivin, PCTA and PSA), but assessment of effectiveness of this combination of the two cytokines requires further investigation.

Pre-clinical data using adult bone marrow cells as a delivery vehicle for Il-12 genes showed satisfactory anti-metastatic effects (and prolonged survival in a mouse model with metastatic prostate cancer, with elevated levels of CD4+ and CD8+ T-cells [42, 43]. Adenovirus pre-infected with Il-12 into the transgenic adenocarcinoma of the mouse prostate (TRAMP) model of prostate cancer also results in tumour-specific immune responses and anti-tumour activity [44, 45]. An orthotopic mouse model of prostate cancer treated with Il-12 recombinant adenoviral vector transduced macrophages survived longer than control treated mice [46]. Although, specific anti-tumour and anti-metastatic activity was seen, phase I trials involving other cancers such as advanced digestive tumours, ovarian cancer, renal cell cancer and metastatic melanoma, whilst encouraging in terms of drug safety, did not show anti-tumour activity [47–50]. A current phase I trial of Il-12 for patients with recurrent non-metastatic prostate cancer after radiation therapy is now ongoing [51]. A phase II trial involving recombinant Il-12 transduced into PSMA-pulsed autologous peripheral blood mononuclear cells (PBMC) in metastatic prostate cancer is also ongoing [52].

Epigenetic therapy

Early phase clinical trials for histone deacetylase (HDAC) inhibitors in a wide range of solid tumours including prostate cancer have involved suberoylanilide hydroxamic acid (SAHA) or vorinostat [53, 54], LBH589 (panobinostat) [55, 56], PXD101 (belinostat) [57], valproic acid [58, 59] and FK228 (romidepsin) [60]. The mechanism of action is via direct inhibition of acetylation or methylation of histones affecting cancer-related gene expression and may also be via an action through non-histone proteins such as p53, heat shock protein 90 (Hsp90) and the androgen receptor, which leads to activation of transcription of certain genes [61–65]. These early phase trials showed satisfactory tolerance, with results examining serum PSA and acetylated histones (measurement of accumulated acetylated histones) across various solid tumour patients and provided good rationale to proceed to further clinical trials. Some of these trials recruited a wide range of patients with solid tumours, and anti-tumour activities were measured via clinical examinations that were analysed as partial response, complete response or stable disease [66–68]. PSA concentrations were measured for those trials that involved castrate-resistant prostate cancer patients and the reduction in PSA concentrations of more that 50% from baseline level was considered to have major anti-tumour activity [69–71]. Measurements of acetylated histones were carried out for all trials via either Western blotting, immunohistochemistry or enzyme immunoassay (ELISA). Other effects of the drugs i.e. pro-apoptitic activities were measured via concurrent monitoring of apoptotic markers in the blood.

Clinical trials of PXD101 and SAHA showed good tolerance, with a maximum tolerated dose determined to be 1000 mg m⁻² day⁻¹ and 400 mg day⁻¹, respectively, and both trials showed dose-dependent histone hyperacetylation [66–68]. These trials in patients with advanced solid tumours (and haematological malignancies for SAHA trials) showed some anti-tumour activity (i.e. tumour regression, stable disease and tumour related pain) but this was not evident in patients with advanced prostate cancer. Trials of panobinostat showed impressive anti-tumour effects with more than 50% of patients receiving panobinostat in combination with docetaxel or docetaxel/prednisone having ≥50% decline in PSA concentrations, in contrast to those receiving panobinostat alone, where almost all patients had progressive disease despite dose-related increase histone acetylation [69, 71]. While these trials obtained valuable information on drug safety and dose-limiting toxicities, correlation between levels of acetylated histones and disease progression could not be established. This may suggest the existence of a subpopulation of prostate cancer cells that do not respond to particular HDAC inhibition and therefore implicates narrowing of a selective group of responsive patients within chemo-naive patients.

The exact mechanism by which HDAC inhibitors induces tumour differentiation, cell cycle arrest and enhancing tumour cell sensitivity to chemotherapy is yet to be elucidated. Therefore each HDAC inhibitor may have different effects on prostate cancer [72]. Some of the trials mentioned above resulted in withdrawal of patients from the studies due to ongoing toxicities i.e. gastrointestinal toxic effects including nausea, diarrhoea and vomiting. These early trials suggest that the use of HDAC inhibitors in combination with other therapies may minimize drug-related toxicities. Careful selection of patients to enter the trials and drugs to be used for advanced prostate cancer in the clinical trials may therefore give more information regarding risk-benefit assessment of these compounds.

Pro-apoptotic agents Bcl-2 modulators

Ursolic acid, a pentacyclic triterpenoid compound [73, 74] and the CXC chemokine receptor-4 (CXCR4) antagonist, CTCE-9908 [75], down-regulate Bcl-2 resulting in induction of apoptosis. However, recent findings from Nariculam et al. [76] demonstrate that Bcl-2 and p53, although overexpressed in localized prostate cancer, were not associated with clinical outcomes. Nevertheless, Bcl-2 is a potential target and has been assessed in clinical trials. For example, oblimersen sodium (G3139, Bcl-2 antisense oligonucleotide) therapy targets the Bcl-2 initiation codon region of Bcl-2 mRNA and down-regulates mRNA expression [77, 78]. A trial of G3139 in combination with docetaxel suggested an encouraging correlation between steady-state concentrations and PSA decline, with no serious toxicities reported [79]. However, another trial treating patients with oblimersen-docetaxel combination showed increased toxicities (40% had major toxic events compared with 22% in the docetaxel group) and did not incur better outcomes than docetaxel treatment alone [80]. A more recent study using microarray to evaluate the effects of G3139 and the effects of Bcl-2 silencing via small interfering RNA (siRNA) on gene profiling of PC-3 cell lines has found that the apoptotic effect of G3139 was a result of an off-target response, via up-regulation of growth-inhibitory proteins, rather than of Bcl-2 silencing alone (both treatments showed similar Bcl-2 knock down) [77]. This is important as treatment with Bcl-2 siRNA alone in metastatic prostate cancer cell lines did not induce apoptosis as seen with cells treated with G3139 [77]. Furthermore, although there were some anti-tumour responses as a result of targeting Bcl-2, several issues still need to be addressed. Marked variation in patients' responses to different concentrations of oblimersen was a major factor in defining effective doses for maximal response. Thus assessment of specific mechanisms by which oblimersen induces apoptosis will need to be evaluated to fully understand and predict patients' responses and clinical adverse effects.

Another drug that is in clinical trials targeting Bcl-2 is the small molecule inhibitor AT-101 [81–83]. In contrast to G3139, AT-101 targets the Bcl-2 homologous (BH) region 3 of Bcl-2 and when given alone, there was satisfactory tolerance in most castrate-resistance prostate cancer patients, with some gastrointestinal toxicities leading to reduction in dosage [84]. Preliminary results of phase I/II trials using a combination of AT-101, docetaxel and prednisone showed 70% (14/20) of prostate cancer patients with a ≥50% PSA decline and 54% (6/11 patients with measurable disease) having partial responses by RECIST criteria (Response Evaluation Criteria in Solid Tumors) [18], thus warranting further assessment of this agent [85]. AT-101 may be a better candidate to target Bcl-2 as combination with traditional docetaxel did not result in increased toxicities and these trials were given a more usual dose regimen of 75 mg m⁻² docetaxel unlike G3139 trials where docetaxel had to be reduced (75 to 60 and then to 45 mg m⁻²) due to ongoing toxicities [80, 85].

Survivin modulators

Survivin is a member of the inhibitors of apoptosis (IAPs) gene family and its expression is seen during fetal development and not in normal, terminally differentiated adult human tissues [86]. However, survivin over-expression is seen in various adenocarcinomas including prostate [86]. Moreover, increased expression is associated with disease progression after radical prostatectomy [87]. Pre-clinical data have demonstrated that YM155, a small molecule survivin suppressant, promoted apoptosis in vitro and in an in vivo xenograft mouse model, using hormone-refractory prostate cancer cell lines and that this apoptotic effect was not significantly related to other IAPs or Bcl-2 related proteins [88]. YM155 has now been in early clinical trials and one single-agent trial in various advanced cancers showed some anti-tumour activity (two out of nine prostate cancer patients had a decline in PSA concentrations) [89]. Other recently completed early phase trials according to ClinicalTrials.gov (results unavailable) are either YM155 single agent in advanced cancers or YM155 in combination with docetaxel in hormone refractory prostate cancer patients [90, 91].

Prostate cancer antibodies

Immunoconjugates consisting of a humanized monoclonal Ab which is directed against prostate-specific membrane antigen (PSMA) have been investigated in prostate cancer [92]. One of the current drugs in clinical trials is MLN2704. MLN2704 is an immunoconjugate consisting of humanized monoclonal Ab directed against PSMA (named MLN591 Ab) which was linked to a maytansinoid (DM1). DM1, a potent microtubule-depolymerizing drug, is an analogue of maytansine, a naturally occurring ansa macrolide [93]. The monoclonal antibody moiety of MLN2704 binds to tumour cells expressing PSMA and is then internalized into the tumour cell, where the DM1 maytansinoid moiety binds to tubulin and inhibits tubulin polymerization and microtubule assembly, resulting in a disruption of microtubule activity, cell division and cell death. Pre-clinical data showed MLN2704 efficiency in anti-tumour activity in a mouse xenograft model, in a dose- and schedule-dependent manner [93]. Early phase I/II trials using MLN2704 showed acceptable safety (no antibody responses to either MLN2704, MLN591 or DM1) with minor grade toxicities such as fatigue and headache with only 1/23 patients reaching dose-limiting toxicity of uncomplicated febrile neutropenia, but neuropathy was observed in 35% of patients [94]. The efficacy of MLN2704 was measured by PSA concentrations and tumour regression. Two patients sustained ≥50% decline in PSA concentrations compared with baseline and six other patients treated at doses ≥156 mg m⁻² sustained stable PSA concentrations for up to 86 days. Of 10 assessable patients, four had stable disease up to a dose of 343 mg m⁻² and one patient receiving 264 mg m⁻² had a partial response. This trial provided useful information regarding the dosage and immunogenic responses to the drug. Further trials are ongoing using MLN2704 alone in progressive metastatic prostate cancer patients [95–97].

Radiolabelled monoclonal Ab HuJ591-GS (177Lu-J591) derived from J591, an immunoglobulin G (IgG) monoclonal Ab targeting the extracellular domain of PSMA (tagged with radionuclide lutetium-177) is currently under phase I/II clinical trials [98, 99]. However some results from earlier phase I trials determined dose-limiting toxicity (including grade 4 neutropenia, severe thrombocytopenia, and other severe non-haematologic toxicities), In another trial, some patients had more than 70–80% decline in PSA concentrations which lasted up to 3–8 months and there was a strong correlation between PSA concentrations and measurable disease responses [100, 101]. Both trials did not show anti-immunogenic responses to the drugs.

These trials warrant the potential further use of PSMA as a biomarker for targeted treatments in prostate cancer, suggesting efficacy together with safety and lack of immunogenic responses to the PSMA antibodies, foreshadowing the need for further clinical assessment.

Anti-angiogenesis approaches

Vascular endothelial growth factor (VEGF) is currently being targeted as a treatment for cancer together with traditional cancer treatments. Expression levels of VEGF, an angiogenic stimulator, are elevated in prostate cancer and PIN and found to be down-regulated by androgen deprivation, resulting in changes in patterns of vascularization [102]. In addition, Ferrer et al. [103] found that there was no or diminished VEGF and IL-8 expression in BPH and normal prostate tissue, whilst expression was also reduced in higher grades of prostate cancer. Overexpression of angiogenic factors in malignant tissues, particularly VEGF is proposed to be mediated largely by hypoxia-inducible factor-1 alpha (HIF-1 α), a transcription factor [104]. As a response to oxygen deprivation (hypoxia), HIF-1 heterodimer complex is formed and consists of HIF-1α and HIF-1β transcription factors which in turn regulate activation of angiogenesis to help restore oxygen homeostasis [105]. HIF-1α is found to be overexpressed in prostate cancer specimens compared with BPH, but this overexpression is not associated with progression of the disease [106, 107]. HIF-1α expression is not seen in normal prostate tissues [107, 108]. Targeting of hypoxia-induced angiogenesis with HDAC inhibitors that inhibit HIF-1α activity and hence reduce expression of VEGF (and basic fibroblast growth factor [bFGF]) are currently being investigated in prostate cancer [105]. Other drugs targeting VEGF in phase I and II clinical trials are bevacizumab, a humanized anti-VEGF antibody, which has been approved for treatments of other cancers such as metastatic breast cancer, non-small-cell lung cancer, glioblastoma, renal cell carcinoma and colorectal cancers [58, 109, 110]. Bevacizumab in combination with docetaxel is well tolerated and approximately 50% of patients had decreased concentrations of PSA in a phase II clinical trial for metastatic hormone refractory prostate cancer [111, 112].

Platelet derived growth factor receptor (PDGFR) and platelet derived growth factor (PDGF)

PDGFR and PDGF are frequently expressed in primary and metastatic prostate cancer [113, 114] and PDGF A and its receptor PDGFR alpha are expressed in PIN, a likely precursor of prostate cancer [115]. This suggests that inhibition of PDGFR may be of therapeutic benefit to advanced prostate cancer patients. Imatinib mesylate is a potent inhibitor of PDGFR and has been approved by the FDA for the treatment of chronic myelogenous leukaemia and metastatic gastrointestinal stromal cancers [116]. Treatment with imatinib in an experimental prostate cancer mouse model was better than paclitaxel alone in reducing bone metastases but the anti-tumour effect was strongest with the combination of both [117]. However, several phase II trials evaluating imatinib mesylate alone did not show decline in PSA concentrations and, with one study, severe side effects (grade 3 and 4 haematological toxicities, neutropenia and lymphopenia, or recurrent grade 1 and 2 non-haematological toxicities) had lead to early closure [118–120], although there are several trials that are underway [121–123]. Another drug that inhibits both PDGFR and VEGFR is sunitinib malate, a multiple receptor tyrosine kinase inhibitor [124], which is currently an approved treatment for stomach cancer and gastrointestinal stromal cancer [125]. A phase II trial with only sunitinib given to two groups of patients (docetaxel-resistant prostate cancer patients and patients with no prior treatment) showed no significant decline in PSA levels in the majority of patients but serum angiogenesis biomarkers i.e. VEGFR and PDGFaa, a member of PDGF family, confirmed effects of sunitinib malate upon angiogenesis factors [124]. Even though this trial did not reach the expected PSA responses (confirmed ≥50% decline in PSA concentrations from baseline), an interesting point from this trial was that sunitinib affected PSA changes in both group of patients similarly (only one patient per group had a major PSA response and there were similar numbers of patients with stable PSA for up to 12 weeks post-treatment). Another trial targeting metastatic castration-resistant prostate cancer patients with prior docetaxel treatment demonstrated declines in PSA in 30% of the patients, but half of the participants discontinued the drug due to severe grade 3–4 toxicities of fatigue, anorexia, nausea and leukopenia and there were two deaths deemed to be related to the study [126]. Actively recruiting phase II clinical trials are ongoing for sunitinib malate with either combination with taxotere (docetaxel/prednisone) [127], hormone ablation therapy [128] or radiation therapy [129].

mTOR (mammalian target of rapamycin)

mTOR is a protein kinase that regulates cell growth, cell proliferation, cell motility, cell survival and protein synthesis and mTOR pathway proteins are overexpressed in primary prostate cancer compared with high-grade PIN and BPH tissues [130], expression being correlated with aggressiveness of the disease [131]. The loss of phosphatase and tensin homolog (PTEN) tumour suppressor gene indicating progression towards the malignant form leads to activation of the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B family)/mTOR signalling pathway [132]. PTEN is correlated with increased in mTOR signalling pathway markers [131] and oncogenic Akt activation in prostate cancer [133]. In addition, PTEN expression is often lost where Bcl-2 is often overexpressed as the disease progresses [134, 135]. One study demonstrated that resistance to apoptosis via an mTOR pathway inhibitor can be a response mediated by HIF-1α regulation and Bcl-2 up-regulation, suggesting a combination of a mTOR inhibitor and Bcl-2 inhibitor to prevent mTOR inhibitor resistance may be useful [136]. Pre-clinical data suggest a combination of mTOR inhibitor and chemotherapy may inhibit growth of prostate cancer and prolong survival in a xenograft model [137]. Also, a combination of a mTOR inhibitor and anti-androgen therapy may benefit some patients as there is evidence that androgen receptor stimulation promotes mTOR activation but only at a concentration of up to ∼1 nmol l⁻¹[138] and androgen deprivation has no effect on mTOR activation in PTEN-deficient mice [139]. In vivo experiments using an androgen-independent metastatic cell line in a xenograft mouse model suggested that inhibition of growth via mTOR inhibitor was a result of re-population of PTEN-deficient cancer cells [137]. Pre-clinical data of RAD001 (everolimus) treatment in mice implanted with androgen-independent cell lines with mutated PTEN resulted in reduction of bone metastases and serum PSA concentrations and the result was enhanced with a combination of RAD001, docetaxel and zoledronic acid [140]. A pilot study using rapamycin (sirolimus) in 13 hormone-refractory prostate cancer patients resulted in some patients (1/13) with a 50% PSA decline and stable disease (4/13), with mean survival time after treatments being nearly 24 months [141]. mTOR inhibitors, everolimus and temsirolimus are now in phase II clinical trials in patients with castration-resistant metastatic prostate cancer [111, 142–145].

Discussion

Treatment for hormone-refractory prostate cancer includes docetaxel-based chemotherapy regimens. However there are very limited choices of treatment if there is an inadequate response to this approach. The majority of clinical trials reviewed focused on hormone-refractory prostate cancer patients. However more clinical trials recruiting patients prior to any treatment may also be valuable in studying initial responses of cancer cells to each drug.

The present review focuses on new agents administered in conjunction with traditional systemic therapies. A number of novel drugs, mentioned in this review, offer more specific treatments targeting either prostate cancer cell surface proteins (CV9103, MLN2704 and 177Lu-J591) or introducing irradiated tumour cells (GVAX and IL-2/IFNγ tumour vaccine) which may lead to activation of the host immune system. The obvious benefits of targeted therapeutics would be the potential for minimization of adverse effects and hence better quality of life for patients. Although cancer treatment vaccines may be beneficial for prostate cancer patients, cancer preventive vaccines are yet to be developed.

Anti-angiogenic treatments are one of the most pursued areas in cancer treatments, regulatory approval occurring with bevacizumab (breast cancer, glioblastoma, renal cell carcinoma and colorectal cancer), imatinib mesylate (chronic myelogenous leukaemia and metastatic gastrointestinal stromal cancer) and sunitinib mesylate (stomach cancer and gastrointestinal stromal cancer). In prostate cancer, a trial of bevacizumab as second-line treatment to docetaxel although showing good PSA responses with overall PSA concentrations declining by up to 65% (7/20 patients had major PSA response, which included four patients who were prior non-responders to docetaxel), a contribution by bevacizumab could not be confirmed due to the non-randomized nature of this trial [146]. The results of randomized control trials with patients receiving either docetaxel-based treatment or hormone therapy with and without bevacizumab are not yet fully available or the trials are ongoing [147, 148]. Trials of imatinib mesylate did not yield good responses in prostate cancer and a trial was terminated due to severe toxicities after single-drug treatments [118]. Despite these trial results, there are a number of ongoing clinical trials combining imatinib mesylate and docetaxel-based therapies [121–123]. VEGF may correlate with the development of prostate cancer but HIF-1α, a main regulator of VEGF, does not. Also androgen deprivation may also diminish VEGF expression and thus it would be irrelevant to target VEGF in hormone-refractory prostate cancer patients. Similarly, PDGFR and PDGF are expressed in PIN, primary and metastatic prostate cancer but do not correlate with the prognosis of the disease. In the case of biomarkers which may associate with the development of disease (VEGF, VEGFR), targeting these biomarkers prior to subjecting prostate cancer to any treatment (which several early phase trials are currently undertaking) or prior to evident metastatic disease may be more pertinent as a preventive approach [128, 148–151].

Targeting mTOR does appear to be a more promising approach as there is consistent in vitro evidence of expression of mTOR pathway proteins leading to activation of mTOR signalling, which correlates with the aggressiveness of prostate cancer. Pre-clinical data and a pilot study were also encouraging where treatments of single agent mTOR inhibitors initiated anti-tumour responses. Several trials of temrolimus and everolimus are now ongoing and are being tested in a neoadjuvant setting prior to prostatectomy or hormone therapy [143, 152, 153]. These drugs are also used in combination with bevacizumab in patients with advanced cancer, which may allow targeting of different sub-populations of cancer cells in castrate-resistance patients with metastases.

New generations of drugs targeting prostate cancer biomarkers are novel approaches aiming at different populations of prostate cancer cells that may be resistant or become resistant to traditional therapies. The majority of clinical trials concentrated on finding new agents for treatment of advanced disease in which treatments are currently limited to taxane-based chemotherapy. However, some agents can be applied as preventive treatments and are now being tested in a neoadjuvant setting. From this review, we can appreciate how finding potential biomarkers and understanding their role in cancer developmental processes can lead to promising new treatment targets for prostate cancer.

Conclusions

In prostate cancer, the main treatments are radical prostatectomy, radiotherapy and systemic therapies (hormone-deprivation therapy and chemotherapy). Prostate cancer is known to be a chronic disease and better detection allowing long-term management will improve quality of life of patients. With increasing awareness of the existence of different phenotypes of prostate cancer cell populations, personalized therapy is going to be the future of cancer treatment. By taking advantage of our increasing knowledge of prostate cancer initiation, progression and biomarkers associated with progression of disease, optimal regimens of novel therapeutic agents are now being tested in early clinical trials and will hopefully pave the way toward more targeted and individualized therapy for prostate cancer.

Glossary

Abbreviations and acronyms

177Lu

radionuclide lutetium-177

Ab

antibody

ADT

androgen deprivation therapy

Ag(s)

antigen(s)

Akt/PKB

protein kinase B

AR

androgen receptor

Bcl-2

apoptosis regulator protein

CTL

cytotoxic T lymphocyte

DC

dendritic cell

DLT

dose-limiting toxicity

DM1

maytansinoid

Ep-2H

Ep-CAM-derived peptides Ep-2H

EpCAM

epithelial cell adhesion molecule

GF(s)

growth factor(s)

GLUT1

Glucose Transporter 1

GM-CSF

granulocyte-macrophage colonystimulating factor

HDACs

histone deacetylases

HIF-1α

hypoxia-inducible factor-1 alpha

IFN-γ

interferon gamma

IL-2

interleukin 2

IL-12

interleukin 12

MTD

maximum tolerated dose

mTOR

mammalian target of rapamycin

N/A

data not available

PBMC

peripheral blood mononuclear cell

PCa

prostate cancer

PDGF

platelet derived growth factor

PDGFR

platelet derived growth factor receptor

PI3K

phosphoinositide 3 kinase

PRAME

preferentially expressed antigen in melanoma

PSA

prostate specific antigen

PSA DT

prostate specific antigen doubling time

PSCA

prostate stem cell antigen

PSGR-1

prostate specific G-protein coupled receptor-1

PSMA

prostate specific membrane antigen

PTEN

phosphatase and tensin homolog protein

Pts

patients

Raf

proto-oncogene serine/threonine-protein kinase

Ras

a protein superfamily of small GTPases.

RT

radiation therapy

SAHA

suberoylanilide hydroxamic acid

STEAP

six-transmembrane epithelial antigen of the prostate

TGF-α

transforming growth factor alpha

T_H

T helper cell

TSC ½

Tuberous sclerosis protein 1 and 2

VEGF

vascular endothelial growth factor

VEGFR

vascular endothelial growth factor receptor

Competing Interests

There are no competing interests to declare.

Sujitra Detchokul was a recipient of a Medical & Scientific Kidney & Urology related Research Biomedical Research Scholarship from Kidney Health Australia and a University of Melbourne Fee Remission Scholarship from The University of Melbourne. This work was supported by the Austin Hospital Medical Research Foundation and the Sir Edward Dunlop Medical Research Foundation.