Prostate cancer is the second most common cause of cancer and the sixth leading cause of cancer death amongst men worldwide [
]. Approximately 15% of men diagnosed with prostate cancer will die because of advanced metastatic disease; the majority of whom have castration resistant disease; and many of these will have received one or more treatment options [
]. Publications by Tannock et al. and Petrylak et al. demonstrated that docetaxel improved survival for men with metastatic castration resistant prostate cancer (mCRPC) [
]. Despite new treatment options for prostate cancer, advanced disease still represents a challenge for treatment, and current treatment options for castration resistant disease offer limited survival advantage due to the development of resistance [
Resistance to docetaxel is poorly understood, and may be caused by a number of mechanisms. These mechanisms include: (1) the fact that prostate tumours are slow-growing and are unlikely to respond to drugs that are S-phase dependent [
]. However, recent clinical trial data combining hormone ablation and docetaxel in hormone and chemo-naïve patients demonstrated an 18 month median overall survival (OS) advantage in patients with high volume prostate cancer [
]. (2) Reduced intra cellular concentrations of cytotoxic drugs as a result of alterations in drug transporters, particularly P-glycoprotein [
]. (3) Tumour suppressor protein mutations, such as the loss of PTEN results in increased cellular proliferation and survival as well as activation of the phosphatidylinositol 3′-kinase (PI3K) signal transduction cascade [
]. This is mediated through altered expression of survival factors that inhibit the apoptotic cell death pathway [
], mediated in part by survival signalling pathways such as the activation of AKT. (4) Alterations in β-tublin isotypes which exhibit different kinetics of microtubule formation particularly isotypes III and IV correlate with docetaxel resistance in vitro [
]. However the identification and manipulation of these multiple mechanisms of resistance represents a significant challenge and targeting individual proteins may have little clinical impact. More recently, O’Neill et al. undertook to characterise docetaxel resistance in prostate cancer cell lines [
]. This study highlighted a complex interplay between changes in the expression of both pro- and anti-apoptotic proteins which ultimately contributed to docetaxel resistance.
In the context of advanced, metastatic castration and docetaxel resistant prostate cancer, one or many of these pathways may be involved in its development. We hypothesised that by understanding the central signalling pathways and transcription factors (TFs) which govern multiple downstream genes we could identify key transcription factors, that when manipulated would alter docetaxel resistance. This study was undertaken to expand our understanding of the mechanisms of resistance to Docetaxel using our previously described PC-3 docetaxel resistant model [
Our objectives were to identify TFs which could account for this resistant phenotype in a model of docetaxel resistance, to validate these TFs in tissue from men who have died from docetaxel resistant mCRPC, and to evaluate if functional manipulation of such TFs could alter response to docetaxel therapy.
Gene expression profiling has been shown to predict clinical outcomes of prostate cancer [
] but complex gene expression profiles are often difficult to manipulate. Targeting the TFs associated with this profile may represent a better therapeutic approach. This study predicted TFs associated with docetaxel-resistance based on transcriptomic data by utilising an innovative bioinformatics approach (CIA) and compared gene expression profiling of the PC3- Ag cells versus the docetaxel resistant cell lines D8 and D12. In line with recent transcriptomic studies by our group and others on castration-resistance [
], analysis of our gene chip data showed gene expression changes in cellular processes relevant to cancer progression. These included cell proliferation, apoptosis, cell growth, survival and senescence and cell death with 375 unique genes differentially expressed between the parental Ag and docetaxel resistant sublines D8 and D12. The focus on upstream TFs regulating the transcriptomic profile rather than the gene expression offered the most novel insights: where transcriptomic data of docetaxel resistant cell lines was combined with a database of TFBS to identify TFs associated with docetaxel-resistance. The utilisation of this approach generated a list of 9 TFs (Table
) predicted to be associated with docetaxel resistance in prostate cancer. Members of this list have previously been associated with prostate cancer, where decreased expression of ESR1 has been found to be particularly associated with hormone refractory disease [
], and PPARγ whose activity is regulated by direct binding of steroid and thyroid hormones, vitamins, lipid metabolites and xenobiotics and have been shown to participate in the development of the disease [
Novel factors associated with docetaxel resistance in prostate cancer included: (1) SRF which is known to be involved with cancer development and progression and its role in castration resistance was previously outlined by our group [
]. (2) BRN5, a pou domain TF of which very little is known, and (3) TR2 and TR4; members of the orphan nuclear receptor family, for which activation or deactivation involves an intricate interplay of different structural classes of endogenous ligands such as the heterodimeric receptors that partner with the retinoid X receptor and bind retinoids and vitamin D [
]. In support of our findings, in recent months Chen et al. demonstrated that TR4 enhances the chemo-resistance of docetaxel in CRPC, and that it may serve as a biomarker to determine the prognosis of docetaxel-based therapy [
The dataset and TF list identified by our study represents a useful resource for future studies on docetaxel-resistance with valuable targets to be explored, as resistance is complex and the mechanisms underlying it multifarious [
]. For the purpose of validating this study we chose to further investigate the functional significance of SRF. SRF is expressed in mature soft tissues such as lung, liver and prostate and has been noted to be dysregulated in a number of malignant tissues such as prostate, breast, gastric and liver carcinoma [
]. In primary gastric cancers- high SRF correlates with a more invasive cancer phenotype and high SRF acts as an independent risk factor of short disease free survival [
]. SRF has been associated with prostate cancer development and progression [
], and our group have previously studied its role in the development of castration resistance [
]. SRF has also recently been associated with androgen receptor (AR) hypersensitivity; where a negative feedback loop exists between SRF expression and AR transcriptional activity in the setting of castrate-resistant prostate cancer [
]. This study gave us the opportunity to expand our understanding of SRF’s role in docetaxel resistance, in the context of AR negative and docetaxel resistant PC-3 cells, and clinical tissues from castrate and docetaxel resistant prostate cancer.
The treatment of men with mCRPC has seen a large number of changes since 2004. Prior to 2004, men who failed primary androgen deprivation were then treated palliatively. Since 2010 the therapeutic armamentarium has increased, but median survival of mCRPC in the post-docetaxel setting is 15-18months [
]. This has led to calls for biomarkers of treatment response and a deeper understanding of the tumour heterogeneity and molecular biology underlying the disease [
]. Previous studies have demonstrated that SRF is associated with Gleason grade and extracapsular extension [
], poor post-operative outcome [
], and castration resistance [
]. To our knowledge, this study is the first to characterise the role of SRF in docetaxel-castration resistant prostate cancer. We found that nuclear tissue expression of SRF is significantly dysregulated in bone metastases of men with mCRPC in the post-docetaxel setting; such that low SRF expression is associated with significantly longer time to bone metastasis. Our research group and others have previously reported that SRF nuclear positivity is associated with higher Gleason score in primary prostate cancer tissues [
] and castrate-resistant TURPs [
] suggesting that SRF may play a role in prostate cancer progression. Additionally our group has demonstrated an association between SRF nuclear positivity and castration-resistant TURPs, with 95% of castrate-resistant TURPs showing nuclear positivity for SRF [
]. In our study of prostate cancer metastases to bone and soft tissue in men with advanced disease, approximately 40% displayed SRF nuclear positivity. In this cohort of men with mCDRPC, a negative association between SRF nuclear expression in bone metastases and survival from time of diagnosis with (1) prostate cancer (2) diagnosis with CRPC and (3) diagnosis with first bone metastasis was seen, which was independent from the number of metastatic sites. No significant association was noted between SRF and survival times in those men with mCRPC who had not been treated with Docetaxel. This finding demonstrates that with disease progression from localised prostate cancer, castration resistance and bone metastases; patients’ survival was inversely correlated with nuclear SRF expression in the context of docetaxel resistance.
Our group has also recently demonstrated that SRF has a negative association with the androgen receptor in CRPC and SRF is involved in the development of castration resistance [
]. In this cohort of men with mCRPC, the median difference in duration of androgen ablation between those subsequently classified as “high SRF and “low SRF” was 4.3 years (
= 0.000019). These findings suggest that those who have higher SRF are likely to have had more aggressive/adaptive disease, having evolved resistance to castration significantly sooner (by 4.3 years). Our data demonstrates a non-significant trend amongst those with SRF and duration of docetaxel therapy; with those with high SRF having received docetaxel for a shorter duration (median 0.166 years) compared to those with a low SRF (median duration 1.05 years).
This transition of SRF expression levels from primary to metastatic tissues, castration resistance and docetaxel therapy, amongst other factors, may explain the findings of a phase III randomised controlled trial. CHAARTED randomized men with newly diagnosed metastatic prostate cancer to ADT alone or ADT plus 6 cycles of docetaxel [
]. In this castration sensitive group, Sweeney et al. described a median OS of 57.6 months in the ADT plus docetaxel group, versus 44 months median OS in the ADT alone group (
= 0.003). This survival benefit contrasts sharply with docetaxel therapy in the castration resistant setting where median survival was 18.9 months in the docetaxel q 3 weekly group, versus 16.5 months median overall survival in the mitoxanthrone group (
= 0.009) [
]. Nuclear SRF expression is associated with castration resistance [
], and nuclear positivity is associated with shorter survival from castration resistance [
], and this study has demonstrated that high SRF expression after docetaxel therapy is correlated with a shorter survival. SRF and other factors likely represent a marker of disease progression; a common denominator or a waypoint in the pathway through which docetaxel and androgen ablation therapies exert their therapeutic effect in prostate cancer (so that men receiving combination therapy in CHAARTED who have progressive disease, are likely to express high levels of SRF in their primary tumour and bone metastases.
The finding that nuclear expression of SRF in soft tissue metastases does not correlate with survival from diagnosis with prostate cancer, castration resistance or first bone metastasis is likely due to a combination of factors including the heterogeneity of prostate cancer metastases, features unique to the respective microenvironments as opposed to just differential bioavailability of docetaxel in various tissue types. This distinction of microenvironmental factors from bioavailability in bone is made as Brubaker et al. have shown in in-vivo models of prostate cancer that docetaxel at a dose which effectively inhibits growth of subcutaneous tumours did not show any effect on the tumours in bone [
]. Meanwhile, Van Der Veldt et al. demonstrated adequate bioavailability of docetaxel in vertebrae in cancer patients, which was comparable to the bioavailability of docetaxel in lung tissues of these patients [
]. This differential effect of docetaxel in different tissue types, may in part be explained by SRF; SRF is associated with mesodermal formation; the embryonic germ layer from which bone and skeletal muscle is derived, in contrast with the endodermal origin of lung, liver and lymph nodes. The relationship of SRF to the origin of the tissues combined with our finding that high SRF in bone metastases is associated with shorter survival supports the role of SRF as a marker of docetaxel resistance, while the differential relationship between nuclear SRF expressivity in bone and soft tissues suggests SRF has a mechanistic role in bone metastasis.
Immuno-histochemical characterisation of a man’s disease necessitates a biopsy specimen. Although this is not the current standard of care for prostate cancer patients, biopsy of new lesions in other malignancies has led to treatment adjustments being carried out in as few as one in seven patients [
]. Indeed in the context of prostate cancer, despite its multifocal and multi-clonal heterogeneity, most distant metastases from different anatomic sites in the same patient share the majority of genetic alterations [
]. As there is an increased risk of bone fracture amongst this population, where Melton et al. noted that 58% of men with castration resistant prostate cancer sustain at least 1 pathologic fracture [
], fixation of such fractures could represent one suitable time-point to obtain a biopsy for immuno-histochemical analysis. Surgery has remained the dominant modality by which solid cancers have been sampled for such analyses, and some note that metastatic tissue is often inaccessible and the purity and yield of biopsy samples are low [
]. More recently though, work by the Michigan Oncology Sequencing Project (MI-ONCOSEQ) [
], Hong et al. in Melbourne [
], and Van Allen et al.[
] have successfully demonstrated that with improved techniques and tools, the vehicle by which metastatic tissue will be obtained for a model of personalised medicine is image-guided percutaneous biopsy.
In order to investigate the functional role of SRF we undertook SRF siRNA knockdown experiments and demonstrated significant reversal in resistance to docetaxel in our PC-3 model of docetaxel resistant prostate cancer. Previous studies by Prencipe et al. have demonstrated that in a LNCaP model of castration resistance, that SRF inhibition impacts upon cell death and proliferation [
]. As mentioned earlier, studies of the role of SRF in prostate cancer are limited. However Taylor et al. have demonstrated that SRF inhibition leads to integrin activation and trafficking, and so reduces migration of neutrophils in response to inflammation in both in-vivo and in-vitro studies [
]. Knockout of SRF reduces Enigma; a LIM domain protein which has been shown to be highly expressed in bone metastases and may function as an oncoprotein [
]. Coupled together these findings further suggest that SRF may play a role in progression of prostate cancer, and maybe an amenable therapeutic target for manipulation at various disease stages [
There are limitations to the present study. Because the functional work was performed in a single cell line it is difficult to make absolute assumptions about the generalizability of the finding that resistance to docetaxel can be overcome by inhibiting SRF in men with advanced metastatic prostate cancer. Nevertheless, in a personalised medicine approach where each man’s disease is appropriately profiled, SRF inhibition may form part of an appropriate therapeutic pathway.