Androgen receptor phosphorylation in prostate diseases

Prostatic diseases are common; benign prostate hyperplasia (BPH) is almost ubiquitous in elderly men and 899,000 men were diagnosed with prostate cancer worldwide in 2008. The incidence of both is increasing and expected to continue to rise. Therefore, prostatic diseases represent a considerable economic burden, but there are currently no reliable markers available to accurately differentiate indolent from aggressive disease nor to predict who will benefit from treatment for either BPH or prostate cancer. This results in over and under-treatment of both diseases with consequent patient related morbidity and mortality. The molecular mechanisms underlying the natural history of prostatic diseases remain elusive. It is accepted that prostate cell growth and survival are exquisitely dependent upon activation of the androgen receptor (AR) by androgens. Following ligand binding, AR undergoes further phosphorylation at serine residues, which inhibit proteolytic degradation, stabilise AR and influence AR transactivation. It is therefore plausible that alterations in AR phosphorylation may drive prostatic disease progression. However, few studies have explored the significance of AR phosphorylation, or the kinases driving AR serine phosphorylation in the clinical setting. The over-riding objective of this study was to establish the clinical relevance of AR serine phosphorylation status in prostate tissue in both BPH and prostate cancer. The specific aims of the current study were: • To firstly establish and validate a panel of AR phosphospecific antibodies. • To evaluate site specific AR serine phosphorylation expression levels in prostate cancer and BPH patient cohorts, with full clinical data and follow-up. • To investigate the expression of candidate kinases mediating such phosphorylation. This involved establishing tissue banks with linked comprehensive clinical databases, and utilising this tissue to establish AR phosphorylation expression profiles for each patient. Six AR phosphospecific antibodies (pARS81, pARS94, pARS213, pARS515, pARS578, pARS650) were verified using peptide competition assays and western blotting. Cdk1, ERK1/2, Akt and PKC were identified as putative kinases mediating AR phosphorylation using the online kinase search tool Scansite 2.0. Immunohistochemistry was performed on hormone naïve diagnostic prostate cancer tissue relating to 90 patients. High expression levels of AR phosphorylation at serine sites 81, 515 and 578 were each associated with a poorer clinical outcome. Following cox regression analysis, cytoplasmic pARS515 expression (p=0.038, HR 4.5 (95% CI 1.1–20.6)) and pARS81 nuclear expression (p=0.030, HR 0.033 95% CI 0.002-0.721) were independently associated with shorter time to biochemical relapse and shorter disease specific survival respectively. Cdk1 and/or pCdk1161 were significantly associated with pARS81 and pARS515 as predicted by Scansite 2.0. Similarly, nuclear PKC expression was significantly associated with pARS578 expression both in the cytoplasm and the nucleus. In patients with PSA at diagnosis ≤20ng/ml, high cytoplasmic pARS515 expression was associated with significantly shorter time to biochemical relapse (p=0.019). This translated into significantly shorter disease-specific survival (p<0.001, 10y survival 38.1% vs 100%). Prostate cancer patients with a low serum PSA level at diagnosis may be suitable for delayed radical treatment via active surveillance. An investigation was therefore undertaken in 51 prostate cancer patients treated by active surveillance. Active surveillance is a deferred radical treatment approach which provides a potential solution to the problem of over treatment as a result of over-diagnosis. However some patients harbour occult aggressive disease and delay in treatment may result in disease progression and failure of radical therapy. Although none of the individual AR serine phosphorylation sites were associated with clinical outcome measures on univariate analysis, high expression of total AR in the cytoplasm (p=0.021, HR 4.6 (95% CI 1.3-16.8)) and presence of perineural invasion in the tumour specimen (p=0.003, HR 8.6 (95% CI 2.1-35.7)) were deemed independent with regards to shorter time to treatment intervention in a cox regression analysis. Validation of the results seen in the first active surveillance prostate cancer cohort was undertaken in a second prospectively collected cohort consisting of 84 active surveillance patients. The results in the first cohort were not replicated in the second. Although cytoplasmic pARS81 was associated with time to intervention (p=0.032) and pARS515 expression trended towards an association (p=0.072), an increase in patient numbers in both cohorts may have provided more reliable results. However even with the numbers available in contrast to the first active surveillance cohort, but in line with the pilot prostate cancer cohort, Cdk1 was associated with pARS515 expression, and pCdk1161 trended towards an association. BPH is also an androgen driven disease dependent upon the AR. Previous research into predictive and prognostic markers in BPH is scant. Therefore a comprehensive analysis of clinical and novel pathological factors, including markers of inflammation, was performed in 336 BPH patients. Following this a complete panel of AR serine phosphorylation sites, and associated kinases, was analysed with reference to clinical outcome measures in the BPH cohort. Low expression levels of total AR and AR phosphorylated at Ser-81, 515 and 650 were associated with poorer clinical outcomes. Low expression of smooth muscle pARS515 (p=0.029, HR 0.31 (95% CI 0.10-0.94)) and older age (p=0.004, HR 5.13 (95% CI 1.43-18.41)) were deemed independent on cox regression analysis with regards to shorter time to postoperative acute urinary retention (AUR). Furthermore, low expression of pARS515 in the smooth muscle was associated with increased incidence of postoperative AUR in patients over 70 years old (25.1% vs 2.8% at 10 years following transurethral resection of prostate (TUR)), (p=0.002, HR 0.20 (95% CI 0.06-0.62)). This may have important clinical implications in postoperative counselling of these patients. In addition it may influence the decision to commence early postoperative medical treatment (with 5-alpha-reductase inhibitors and/or alpha blockers) on a prophylactic basis in these patients. Cytoplasmic pARS650 expression (p=0.010, HR 0.50 (95% CI 0.29-0.86)) and PSA at diagnosis (p=0.018, HR 1.89 (95% CI 1.11-3.16)) were independently associated with time to failure of surgical intervention. Furthermore, low expression of pARS650 in the cytoplasm was associated with increased failure of surgical intervention in patients with PSA ≥4ng/ml at diagnosis (45.5% vs 13% at 5 years post TUR), (p=0.026, HR 0.52 (95% CI 0.29-0.93)). This comprehensive study on immunohistochemical expression of site specific AR serine phosphorylation and associated kinases fills a gap in the current literature. It has demonstrated the clinical significance of AR serine phosphorylation in prostate cancer and BPH and uncovered potentially exciting new avenues for future investigation. Site specific serine phosphorylation of the AR may serve as a prognostic and predictive biomarker in prostatic disease and has potential as a future target for therapeutic intervention

Loss of signal transducer and activator of transcription 1 is associated with prostate cancer recurrence

STAT1 loss has previously been implicated in cell line studies to modify prostate cancer cell growth and survival, however the clinical significance of this has not previously been established. This study investigated if STAT1 loss was associated with patient outcome measures and the phenotypic consequence of STAT1 silencing. STAT1 expression was assessed in two patient cohorts with localised (n = 78) and advanced prostate cancer at initial diagnosis (n = 39) by immunohistochemistry (IHC). Impact of STAT1 silencing on prostate cancer cells lines was assessed using Cell Death detection ELISA, TLDA gene signature apoptosis arrays, WST-1 assay, xCELLigence system, clonogenic assay, and wound healing assay. In the localised patient cohort, low expression of STAT1 was associated with shorter time to disease recurrence (3.8 vs 7.3 years, P = 0.02) and disease specific survival (6.6 vs 9.3 years, P = 0.05). In the advanced patient cohort, low expression was associated with shorter time to disease recurrence (2.0 vs 3.9 years, P = 0.001). When STAT1 was silenced in PC3 cells (AR negative) and LNCaP cells (AR positive) silencing did not influence levels of apoptosis in either cell line and had little effect on cell viability in the LNCaP cells. In contrast, STAT1 silencing in the PC3 cells resulted in a pronounced increase in cell viability (WST-1 assay: mock silenced vs STAT1 silenced, P &lt; 0.001), clonagenicity (clonogenic assay: mock silenced vs STAT1 silenced, P &lt; 0.001), and migration (wound healing: mock silenced vs STAT1 silenced, P &lt; 0.001). In conclusion, loss of STAT1 may promote prostate cancer recurrence in AR negative patients via increasing cell viability

Right ventricular free wall longitudinal strain is independently associated with mortality in mechanically ventilated patients with COVID-19

Background: Right ventricular (RV) dysfunction has been commonly reported in patients with Coronavirus disease 2019 (COVID-19), and is associated with mortality in mixed cohorts of patients requiring and not requiring invasive mechanical ventilation (IMV). Using RV-speckle tracking echocardiography (STE) strain analysis, we aimed to identify the prevalence of RV dysfunction (diagnosed by abnormal RV-STE) in patients with COVID-19 that are exclusively undergoing IMV, and assess association between RV dysfunction and 30 day mortality. We performed a prospective multicentre study across 10 ICUs in Scotland from 2/9/20 to 22/3/21. One-hundred-and-four echocardiography scans were obtained from adult patients at a single timepoint between 48 h after intubation, and day 14 of intensive care unit admission. We analysed RV-STE using RV free-wall longitudinal strain (RVFWLS), with an abnormal cutoff of  &gt; −20%. We performed survival analysis using Kaplan–Meier, log rank, and multivariate cox-regression (prespecified covariates were age, gender, ethnicity, severity of illness, and time since intubation). Results: Ninety-four/one-hundred-and-four (90.4%) scans had images adequate for RVFWLS. Mean RVFWLS was −23.0% (5.2), 27/94 (28.7%) of patients had abnormal RVFWLS. Univariate analysis with Kaplan–Meier plot and log-rank demonstrated that patients with abnormal RVFWLS have a significant association with 30-day mortality (p = 0.047). Multivariate cox-regression demonstrated that abnormal RVFWLS is independently associated with 30-day mortality (Hazard-Ratio 2.22 [1.14–4.39], p = 0.020). Conclusions: Abnormal RVFWLS (&gt; −20%) is independently associated with 30-day mortality in patients with COVID-19 undergoing IMV. Strategies to prevent RV dysfunction, and treatment when identified by RVFWLS, may be of therapeutic benefit to these patients. Trial Registration: Retrospectively registered 21st Feb 2021. ClinicalTrials.gov Identifier: NCT04764032

Cardiac biomarkers and right ventricular dysfunction are independently associated with one year mortality in patients with COVID-19 receiving mechanical ventilation: a prospective cohort study

Background: The cardiac biomarkers N-terminal pro-brain natriuretic peptide (NT-proBNP) and troponin frequently are raised in patients with acute COVID-19. As a secondary analysis of the Right Ventricular Dysfunction in Ventilated Patients With COVID-19 study, we sought to determine the association between raised cardiac biomarkers and 1-year mortality in patients with COVID-19 receiving invasive mechanical ventilation (IMV). As an exploratory investigation, we combined point-of-care echocardiography and cardiac biomarker analyses to determine whether the biomarker signal represented a global or regional cardiac injury. Study Question: Are abnormal cardiac biomarker levels associated with 1-year mortality in patients with COVID-19 requiring IMV? Study Design and Methods: In this prospective cardiac biomarker and echocardiography study in patients with COVID-19 across 10 ICUs in the west of Scotland, patients underwent contemporaneous cardiac biomarker testing with point-of-care echocardiography between days 2 and 14 after intubation. Survival analyses was performed using univariable log-rank and multivariable Cox regression. Results: One hundred twenty-one patients were recruited between September 2, 2020, and March 22, 2021. At 1 year, 57.6% of patients (68 of 118) had died. Patients with abnormal NT-proBNP levels and patients with abnormal troponin levels showed a 1-year mortality incidence of 71.4% (50 of 70) and 80.4% (45 of 56), respectively. Both abnormal NT-proBNP and abnormal troponin levels were associated with 1-year mortality (P &lt; .001 for both). Abnormal troponin level was associated with subjective right ventricular dysfunction (RVD; P = .003), and no association with subjective left ventricular dysfunction was found (P = .342). On multivariable analysis, abnormal NT-proBNP level, abnormal troponin level, and subjective RVD were associated independently with 1-year mortality (hazard ratios, 2.82 [95% CI, 1.19-6.67], 2.84 [95% CI, 1.44-5.62], and 2.09 [95% CI, 1.07-4.07], respectively). Interpretation: Abnormal NT-proBNP level, abnormal troponin level, and subjective RVD are associated independently with 1-year mortality in patients with COVID-19 receiving IMV. Cardiac biomarker testing and point-of-care echocardiography are available readily during ICU admission and may identify a group of patients who are at very high risk of poor outcomes

Androgen receptor phosphorylation at serine 308 and serine 791 predicts enhanced survival in castrate resistant prostate cancer patients

We previously reported that AR phosphorylation at serine 213 was associated with poor outcome and may contribute to prostate cancer development and progression. This study investigates if specific AR phosphorylation sites have differing roles in the progression of hormone naïve prostate cancer (HNPC) to castrate resistant disease (CRPC). A panel of phosphospecific antibodies were employed to study AR phosphorylation in 84 matched HNPC and CRPC tumours. Immunohistochemistry measured Androgen receptor expression phosphorylated at serine residues 94 (pAR&lt;sub&gt;94&lt;/sub&gt;), 308 (pAR&lt;sub&gt;308&lt;/sub&gt;), 650(pAR&lt;sub&gt;650&lt;/sub&gt;) and 791(pAR&lt;sub&gt;791&lt;/sub&gt;). No correlations with clinical parameters were observed for pAR&lt;sub&gt;94&lt;/sub&gt; or pAR&lt;sub&gt;650&lt;/sub&gt; in HNPC or CRPC tumours. In contrast to our previous observation with serine 213, high pAR&lt;sub&gt;308&lt;/sub&gt; is significantly associated with a longer time to disease specific death (p= 0.011) and high pAR&lt;sub&gt;791&lt;/sub&gt; expression significantly associated with a longer time to disease recurrence (p= 0.018) in HNPC tumours and longer time to death from disease recurrence (p= 0.040) in CRPC tumours. This observation in CRPC tumours was attenuated in high apoptotic tumours (p= 0.022) and low proliferating tumours (p= 0.004). These results demonstrate that understanding the differing roles of AR phosphorylation is necessary before this can be exploited as a target for castrate resistant prostate cancer

Study protocol for COVID-RV: a multicentre prospective observational cohort study of right ventricular dysfunction in ventilated patients with COVID-19

Introduction: COVID-19 can cause severe acute respiratory failure requiring management in intensive care unit with invasive ventilation and a 40% mortality rate. Cardiovascular manifestations are common and studies have shown an increase in right ventricular (RV) dysfunction associated with mortality. These studies, however, comprise heterogeneous patient groups with few requiring invasive ventilation. This study will investigate the prevalence and prognostic significance of RV dysfunction in ventilated patients with COVID-19 which may lead to targeted interventions to improve patient outcomes. Methods and analysis: This prospective multicentre observational cohort study will perform transthoracic echocardiography (TTE) in 150 patients with COVID-19 requiring invasive ventilation for more than 48 hours. RV dysfunction will be defined as TTE evidence of RV dilatation along with the presence of septal flattening. Baseline demographics, disease severity data and clinical information relating to proposed aetiological mechanisms of RV dysfunction (acute respiratory distress syndrome (ARDS), disordered coagulation, direct myocardial injury and ventilation) will be collected and analysed. Primary outcome measures include the prevalence of RV dysfunction and its association with 30-day mortality. Exploratory outcome measures will investigate the association of the proposed aetiological mechanisms of RV dysfunction to the primary outcomes. Prevalence of RV dysfunction will be determined along with 95% Clopper-Pearson CIs and 30-day survival will be analysed using logistic regression adjusting for patient demographics, phase of disease and baseline severity of illness. The role of potential aetiological factors (ARDS, disordered coagulation, direct myocardial injury and ventilation) in relation to the primary outcomes will be analysed using logistic regression. Ethics and dissemination: Approval was gained from Scotland A Research Ethics Committee (REC reference 20/SS/0059). Findings will be disseminated by various methods including webinars, international presentations and publication in peer-reviewed journals