Androgen receptor phosphorylation in prostate cancer

Prostate cancer is the most common male cancer in the UK. Although incidence is increasing, prostate cancer mortality is decreasing, mainly owing to the over diagnosis of disease that would not have become clinically apparent during the patient’s lifetime. The gold-standard for prostate cancer diagnosis is transrectal ultrasound guided biopsy of the prostate. Whilst prostate biopsy can inform on diagnosis, it’s prognostic ultiltiy is poor. Currently clinicians lack pathological biomarkers to differentiate between patients with prostate cancer who have indolent disease that can be safely managed with surveillance strategies, and those who will go onto develop aggressive disease which requires early radical curative treatment. Phosphorylation of the androgen receptor has been extensively investigated in relation to prostate cancer development and progression. Androgen receptor phosphorylation has been shown to regulate cellular localisation, transcriptional activity, cell growth and sensitivity to androgens in prostate cancer. However, only a small number of studies have investigated the prognostic significance of androgen receptor phosphorylation, and only consider a limited number of serine residues in clinical specimens. The research presented in this thesis sought to investigate the prognostic and predictive significance of AR phosphorylation at serine 578 in hormone-naïve prostate cancer. It was hypothesised that pARS578 would be associated with poor outcomes in prostate cancer and may be utilised as a prognostic marker at diagnosis in prostate cancer and predict response to drug treatment with a PKC inhibitor. It was also hypothesised that PKC, the putative kinase for phosphorylation at serine 578, would be associated with poor outcomes and may offer a potential therapeutic target in prostate cancer. In the current study, the phosphorylation site of primary interest was serine 578. Scansite 2.0, an online kinase search tool, predicted that PKC is the putative kinase mediating phosphorylation at serine 578 on the androgen receptor. Phosphorylation of the androgen receptor at serine 578 has been linked with increased AR transcriptional activity, cell growth, nuclear cytoplasmic shuttling, modulation of other AR phosphorylation sites and DNA-repair mechanisms. The prognostic significance of androgen receptor phosphorylation at serine 81 was also investigated in this study. Serine 81 is phosphorylated in response to DHT via an alternative pathway to that of serine 578. Serine 81 phosphorylation is associated with increased androgen receptor transcriptional activity and increased cell growth in prostate cancer. It was therefore hypothesised that androgen receptor phosphorylation at serine 578 and serine 81 would be associated with poor outcome measures in prostate cancer. Immunohistochemical analysis was performed in a cohort of 105 hormone-naïve prostate cancer patients undergoing active surveillance, representing a cohort of patients with low-risk disease, as defined by current clinical markers such as PSA and Gleason score at diagnosis. Nuclear PKC expression was significantly associated with pARS578 expression in the clinical specimens, supporting the prediction of Scnasite 2.0 that PKC is the kinase responsible for phosphorylation of the AR at this site. High cytoplasmic expression of pARS81 was associated with decreased time to intervention (HR 2.76 (95% CI 1.1-7.3), p=0.032). There was no association between pARS578 and time to intervention in this cohort. Analysis of combined expression of both phosphorylation sites revealed an association between high dual expression of cytoplasmic pARS81 and cytoplasmic pARS578 and decreased time to treatment intervention (HR 2.35 (95% CI 1.2-4.6), p=0.031). These results suggest a synergistic prognostic effect when these two phosphorylation sites are combined and identifies a sub-population of low-risk prostate cancer patients who are at increased risk of disease progression. A second study was conducted to investigate if these results could be replicated in a cohort of prostate cancer patients with all stages of disease at diagnosis. Immunohistochemical analysis in 90 hormone-naïve prostate cancer patients found that high expression of nuclear pARS81 (HR 2.1 (95% CI 1.1 – 4.2), p=0.030), nuclear pARS578 (HR 2.24 (95% CI 1.0-4.9), p=0.036) and cytoplasmic pARS578 (HR 4.54 (95% CI 2.0-10.4), p=<0.001) was associated with decreased disease survival. Furthermore, high expression of cytoplasmic pARS578 was associated with decreased time to biochemical relapse (HR 2.1 (95% CI 1.0-4.2), p=0.034) and decreased disease-specific survival following biochemical relapse (HR 3.2 (95% CI 1.0-9.9), p=0.034). Dual expression of nuclear, cytoplasmic and total pARS81 and pARS578 were all associated with decreased-disease specific survival, suggesting that there is a sub-population of prostate cancer patients who may benefit from dual targeted therapy with androgen deprivation therapy and PKC inhibitors. A validation cohort of 243 hormone-naïve prostate cancer patients with all stages of disease was utilised to verify the results of the second cohort. Unfortunately, due to technical issues and time constraints, IHC could not be completed for the phosphorylation sites of interest in all patients. Despite this, high expression of cytoplasmic pARS578 was significantly associated with decreased time to biochemical relapse (HR 2.9 (95% CI 1.0-8.2), p=0.037) and trended towards an association with decreased overall survival (p=0.076). Interestingly, dual expression of high cytoplasmic pARS81 and cytoplasmic pARS578 was associated with decreased overall survival (HR 2.1 (95% CI 1.3-3.3) p=0.001) despite neither phosphorylation site independently predicting decreased overall survival. Lastly, a study to develop a technique for isolation, propagation and characterisation of primary prostate cancer cells from TRUS biopsy specimens was undertaken. Two primary prostate cell cultures were developed which were confirmed to have a malignant luminal epithelial cell phenotype with a functional AR using flow cytometry, RT-PCR and immunofluorescence. This technique is of high translational relevance, as it provides a model with potential to identify biomarkers to predict individual patient’s response to prostate cancer therapies. Overall these results suggest that androgen receptor phosphorylated at serine 81 and serine 578 are associated with poor outcomes in prostate cancer and are potential targets for new drug therapies. Additional studies are required to validate these results in a larger multi-centre cohort of prostate cancer patients before either of these phosphorylation sites can be utilised as a biomarker in clinical practice

Drug screening of biopsy-derived multicellular spheroids using microfluidic technology

Performing drug screening, of physiologically relevant three-dimensional (3D) tumor models, for personalized treatment remains challenging, due to the small amount of tissue available from most biopsies. New microfluidic technologies, enabling greater control over cell positioning and fluid behavior at the micro-scale, allow extensive testing of anticancer agents on human tumor tissue preparations in 3D and offer new solutions for the development of anticancer compounds and personalized medicine. We have developed a microfluidic platform for extensive drug screening of tumor biopsies in a cost-effective manner and validated the system with tumor prostate patient samples. As a typical drug screening assay, up to 22 drug concentration-response curves could be generated from a single biopsy, within a time frame of up to 4 weeks. Biopsy tissue, grown as a heterogeneous co-culture from the primary sample, was prepared as cancer-cell enriched multicellular spheroids, cultured for 3 to 5 days prior to the application of a panel of standard-of-care drugs for prostate cancer. Readouts were obtained via bright-field and epifluorescence microscopy. The microfluidic platform was designed to be operated entirely without the need of external fluid actuation, with the microfluidic network capable of generating long-lasting, stable and repeatable drug concentration gradients across arrays of 240 spheroids. Outcomes were generated as 8-point drug concentration response curves per device, with each drug concentration tested on at least 24 spheroids. In-house developed software was used to analyze bright-field and fluorescent images to provide readouts of spheroid growth and viability, as well as information on drug penetration and drug efficacy over time. Following platform and assay validation using cancer cell lines, proof-of-concept screening was performed on prostate biopsies from 2 different patients. Results showed that biopsy-derived spheroids were more resistant to treatment than LNCaP spheroids, a prostate cancer cell line. For one biopsy, spheroids were sensitive to docetaxel, but resistant to enzalutamide, despite the presence of intact androgen receptors. This preliminary data outlines how this technology could become a useful tool to investigate patient-specific drug resistance and to test novel anticancer agents in a cost-effective manner, based on maximized screening of human tumor tissue in a 3D format

Inflammatory infiltration is associated with AR expression and poor prognosis in hormone naïve prostate cancer

Background: Tumor microenvironment inflammatory infiltration is proposed as a protumorigenic mechanism for prostate cancer with proinflammatory cytokines stimulating androgen receptor (AR) activity. However, association with patient prognosis remains unclear. This study derives an inflammatory gene signature associated with AR expression and investigates CD3+ and CD8+ T‐lymphocyte infiltration association with AR and prognosis. Methods: Gene profiling of inflammatory related genes was performed on 71 prostate biopsies. Immunohistochemistry on 243 hormone‐naïve prostate cancers was performed for CD3, CD8, AR, and phosphorylated AR tumor expression. Results: Multiple proinflammatory genes were differentially expressed in association with high AR expression compared with low AR expression including PI3KCA and MAKP8 (adjusted P &lt; .05). High CD3+ and high CD8+ infiltration associated with reduced cancer‐specific survival (P = .018 and P = .020, respectively). High CD3+ infiltration correlated with high tumor cytoplasmic AR expression and if assessed together, they associated with reduced cancer‐specific and 5‐year survival from 90% to 56% (P = .000179). High CD8+ cytotoxic infiltration associated with high androgen‐independent tumor nuclear AR serine 213 phosphorylation (correlation coefficient = 0.227; P = .003) and when assessed together associated with poor clinico‐pathological features including perineural invasion (P = .001). Multiple genes involved in proinflammatory signaling pathways are upregulated in high AR expressing prostate samples. Conclusion: T‐lymphocyte infiltration in hormone‐naïve disease associates with androgen‐independent driven disease and provides possible therapeutic targets to reduce transformation from hormone‐naïve to castrate‐resistant disease

Drug screening of biopsy-derived spheroids using a self-generated microfluidic concentration gradient

Abstract Performing drug screening of tissue derived from cancer patient biopsies using physiologically relevant 3D tumour models presents challenges due to the limited amount of available cell material. Here, we present a microfluidic platform that enables drug screening of cancer cell-enriched multicellular spheroids derived from tumour biopsies, allowing extensive anticancer compound screening prior to treatment. This technology was validated using cell lines and then used to screen primary human prostate cancer cells, grown in 3D as a heterogeneous culture from biopsy-derived tissue. The technology enabled the formation of repeatable drug concentration gradients across an array of spheroids without external fluid actuation, delivering simultaneously a range of drug concentrations to multiple sized spheroids, as well as replicates for each concentration. As proof-of-concept screening, spheroids were generated from two patient biopsies and a panel of standard-of-care compounds for prostate cancer were tested. Brightfield and fluorescence images were analysed to provide readouts of spheroid growth and health, as well as drug efficacy over time. Overall, this technology could prove a useful tool for personalised medicine and future drug development, with the potential to provide cost- and time-reduction in the healthcare delivery

Selective targeting of NaV1.7 via inhibition of the CRMP2-Ubc9 interaction reduces pain in rodents

The voltage-gated sodium NaV1.7 channel, critical for sensing pain, has been actively targeted by drug developers; however, there are currently no effective and safe therapies targeting NaV1.7. Here, we tested whether a different approach, indirect NaV1.7 regulation, could have antinociceptive effects in preclinical models. We found that preventing addition of small ubiquitin-like modifier (SUMO) on the NaV1.7-interacting cytosolic collapsin response mediator protein 2 (CRMP2) blocked NaV1.7 functions and had antinociceptive effects in rodents. In silico targeting of the SUMOylation site in CRMP2 (Lys374) identified >200 hits, of which compound 194 exhibited selective in vitro and ex vivo NaV1.7 engagement. Orally administered 194 was not only antinociceptive in preclinical models of acute and chronic pain but also demonstrated synergy alongside other analgesics-without eliciting addiction, rewarding properties, or neurotoxicity. Analgesia conferred by 194 was opioid receptor dependent. Our results demonstrate that 194 is a first-in-class protein-protein inhibitor that capitalizes on CRMP2-NaV1.7 regulation to deliver safe analgesia in rodents