The catalytic subunit of DNA-PK regulates transcription and splicing of AR in advanced prostate cancer.

Aberrant androgen receptor (AR) signaling drives prostate cancer (PC), and it is a key therapeutic target. Although initially effective, the generation of alternatively spliced AR variants (AR-Vs) compromises efficacy of treatments. In contrast to full-length AR (AR-FL), AR-Vs constitutively activate androgenic signaling and are refractory to the current repertoire of AR-targeting therapies, which together drive disease progression. There is an unmet clinical need, therefore, to develop more durable PC therapies that can attenuate AR-V function. Exploiting the requirement of coregulatory proteins for AR-V function has the capacity to furnish tractable routes for attenuating persistent oncogenic AR signaling in advanced PC. DNA-PKcs regulates AR-FL transcriptional activity and is upregulated in both early and advanced PC. We hypothesized that DNA-PKcs is critical for AR-V function. Using a proximity biotinylation approach, we demonstrated that the DNA-PK holoenzyme is part of the AR-V7 interactome and is a key regulator of AR-V-mediated transcription and cell growth in models of advanced PC. Crucially, we provide evidence that DNA-PKcs controls global splicing and, via RBMX, regulates the maturation of AR-V and AR-FL transcripts. Ultimately, our data indicate that targeting DNA-PKcs attenuates AR-V signaling and provide evidence that DNA-PKcs blockade is an effective therapeutic option in advanced AR-V-positive patients with PC.

Reducing the Frequency of Follow-up Cystoscopy in Low-grade pTa Non-muscle-invasive Bladder Cancer Using the ADXBLADDER Biomarker.

BACKGROUND: Non-muscle-invasive bladder cancer (NMIBC) is one of the most expensive cancers owing to frequent follow-up cystoscopies for detection of recurrence. OBJECTIVE: To assess if the noninvasive ADXBLADDER urine test could permit a less intensive surveillance schedule for patients with low-grade (LG) pTa tumor without carcinoma in situ (CIS) at the previous diagnosis. DESIGN, SETTING, AND PARTICIPANTS: In a prospective, double-blind, multicenter study, 629 patients underwent follow-up cystoscopy, transurethral resection of bladder tumor/biopsy of suspect lesions, and ADXBLADDER testing. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Diagnostic test accuracy and decision curve analysis were used to evaluate the impact of ADXBLADDER on decision-making on whether to perform follow-up cystoscopy. The primary endpoint was the negative predictive value (NPV) of ADXBLADDER for detection of high-grade and/or CIS (HG/CIS) recurrence and its impact on reducing unnecessary cystoscopies. RESULTS AND LIMITATIONS: ADXBLADDER had sensitivity of 66.7% (95% confidence interval [CI] 34.9-90.1%) and an NPV of 99.15% (95% CI 97.8-99.8%) for detection of HG/CIS recurrence. The probability of HG/CIS recurrence was 5.0% for ADXBLADDER-positive patients and 0.85% for ADXBLADDER-negative patients. For HG/CIS recurrence threshold probabilities between 0.85% and 5.0%, ADXBLADDER yields a net benefit with omission of cystoscopy for ADXBLADDER-negative patients. The corresponding net reduction in unnecessary cystoscopies ranges from 11 to 62 per 100 patients. CONCLUSIONS: Patients with LG pTa tumor at the previous diagnosis, for which the risk of HG/CIS recurrence is low and the ADXBLADDER NPV for ruling out HG/CIS recurrence is 99.15%, are ideally suited for a less intensive, personalized follow-up surveillance strategy using ADXBLADDER, with omission of cystoscopy for ADXBLADDER-negative patients. PATIENT SUMMARY: ADXBLADDER is a urine test that can predict the probability of recurrence of bladder cancer. Patients diagnosed with low-grade cancer confined to the bladder mucosa are ideally suited for less intensive follow-up using this test, which could reduce unnecessary cystoscopy procedures for those with a negative result, potentially improve quality of life, and reduce overall health care costs.

Comparison of the performances of the ADXBLADDER test and urinary cytology in the follow-up of non-muscle-invasive bladder cancer: a blinded prospective multicentric study.

OBJECTIVE: To compare directly the performance of the ADXBLADDER test with that of cytology in the detection of non-muscle-invasive bladder cancer (NMIBC) recurrences. BACKGROUND: ADXBLADDER is a urine test based on the detection of MCM5, a DNA licensing factor expressed in all cells capable of dividing. Expression is usually restricted to the basal stem cell compartment; however, in malignancy, MCM5-expressing cells can be found throughout the epithelium. Detection of MCM5 in urine sediment can be indicative of the presence of a bladder tumour. PATIENTS AND METHODS: A multicentre prospective, blinded study was carried out from August 2017 and July 2019 at 21 European Union centres, 14 of which collected matching cytology data. Urine was collected from patients prior to cystoscopy. Urine cytology and ADXBLADDER were performed and compared to the diagnosis obtained by cystoscopy. The performance of cytology and ADXBLADDER were then compared. RESULTS: The overall performance of ADXBLADDER demonstrated a sensitivity of 51.9%, a specificity of 66.4%, and a negative predictive value (NPV) of 92%. The sensitivity of ADXBLADDER for low- and high-grade recurrences was 44.1% and 58.8%, respectively. By contrast, cytology sensitivity was 16.7%, specificity was 98% and NPV was 90.7%. Cytology sensitivity for both low- and high-grade disease was 17.6%. CONCLUSIONS: ADXBLADDER detection of both low- and high-grade NMIBC recurrence is superior to that of cytology, with ADXBLADDER able to exclude the presence of high-grade recurrence in 97.8% of cases compared to 97.1% with cytology. These results show that ADXBLADDER has promise as a more reliable alternative to urine cytology in the follow-up of NMIBC.

IKBKE activity enhances AR levels in advanced prostate cancer via modulation of the Hippo pathway.

Resistance to androgen receptor (AR) targeting therapeutics in prostate cancer (PC) is a significant clinical problem. Mechanisms by which this is accomplished include AR amplification and expression of AR splice variants, demonstrating that AR remains a key therapeutic target in advanced disease. For the first time we show that IKBKE drives AR signalling in advanced PC. Significant inhibition of AR regulated gene expression was observed upon siRNA-mediated IKBKE depletion or pharmacological inhibition due to inhibited AR gene expression in multiple cell line models including a LNCaP derivative cell line resistant to the anti-androgen, enzalutamide (LNCaP-EnzR). Phenotypically, this resulted in significant inhibition of proliferation, migration and colony forming ability suggesting that targeting IKBKE could circumvent resistance to AR targeting therapies. Indeed, pharmacological inhibition in the CWR22Rv1 xenograft mouse model reduced tumour size and enhanced survival. Critically, this was validated in patient-derived explants where enzymatic inactivation of IKBKE reduced cell proliferation and AR expression. Mechanistically, we provide evidence that IKBKE regulates AR levels via Hippo pathway inhibition to reduce c-MYC levels at cis-regulatory elements within the AR gene. Thus, IKBKE is a therapeutic target in advanced PC suggesting repurposing of clinically tested IKBKE inhibitors could be beneficial to castrate resistant PC patients.

Diagnostic Accuracy of MCM5 for the Detection of Recurrence in Nonmuscle Invasive Bladder Cancer Followup: A Blinded, Prospective Cohort, Multicenter European Study.

PURPOSE: Detection of MCM5 containing cells in urine has been shown to be indicative of the presence of a bladder tumor on primary diagnosis. In this study we evaluate diagnostic performance of ADXBLADDER in patients undergoing cystoscopic surveillance in nonmuscle invasive bladder cancer followup. MATERIALS AND METHODS: A multicenter prospective blinded study was performed at 21 European centers with patients undergoing cystoscopy for nonmuscle invasive bladder cancer surveillance, diagnosed in the preceding 2 years. Urine was collected from all eligible patients and ADXBLADDER-MCM5 testing was performed. Performance characteristics were calculated by comparing MCM5 results to the outcome of cystoscopy plus pathological assessment. RESULTS: Of 1,431 eligible patients enrolled 127 were diagnosed with a bladder cancer recurrence. The overall sensitivity for the ADXBLADDER-MCM5 test in detecting bladder cancer recurrence was 44.9% (95% CI 36.1-54) with a 75.6% sensitivity for nonpTaLG tumors (95% CI 59.7-87.6). Specificity was 71.1% (95% CI 68.5-73.5). The overall negative predictive value was 93% (95% CI 91.2-94.5). However, ADXBLADDER was able to rule out the presence of a nonpTaLG recurrent tumor with a negative predictive value of 99.0% (95% CI 98.2-99.5). No statistically significant differences in the performance of ADXBLADDER were observed as a result of age or sex. CONCLUSIONS: This large blinded prospective study demonstrates that in the followup of patients with nonmuscle invasive bladder cancer ADXBLADDER is able to exclude the presence of the most aggressive tumors with a negative predictive value of 99%. These results indicate that ADXBLADDER could be incorporated in the followup strategy of nonmuscle invasive bladder cancer.

A novel CRISPR-engineered prostate cancer cell line defines the AR-V transcriptome and identifies PARP inhibitor sensitivities.

Resistance to androgen receptor (AR)-targeted therapies in prostate cancer (PC) is a major clinical problem. A key mechanism of treatment resistance in advanced PC is the generation of alternatively spliced forms of the AR termed AR variants (AR-Vs) that are refractory to targeted agents and drive tumour progression. Our understanding of how AR-Vs function is limited due to difficulties in distinguishing their discriminate activities from full-length AR (FL-AR). Here we report the development of a novel CRISPR-derived cell line which is a derivative of CWR22Rv1 cells, called CWR22Rv1-AR-EK, that has lost expression of FL-AR, but retains all endogenous AR-Vs. From this, we show that AR-Vs act unhindered by loss of FL-AR to drive cell growth and expression of androgenic genes. Global transcriptomics demonstrate that AR-Vs drive expression of a cohort of DNA damage response genes and depletion of AR-Vs sensitises cells to ionising radiation. Moreover, we demonstrate that AR-Vs interact with PARP1 and PARP2 and are dependent upon their catalytic function for transcriptional activation. Importantly, PARP blockade compromises expression of AR-V-target genes and reduces growth of CRPC cell lines suggesting a synthetic lethality relationship between AR-Vs and PARP, advocating the use of PARP inhibitors in AR-V positive PC.

Ex vivo Culture and Lentiviral Transduction of Benign Prostatic Hyperplasia (BPH) Samples.

To assess oncogenic potential, classical transformation assays are based on cell line models. However, cell line based models do not reflect the complexity of human tissues. We thus developed an inducible expression system for gene expression in ex vivo human tissues, which maintain native tissue architecture, such as epithelia and stroma. To validate the system, we transduced and expressed known tumor suppressors (p53, p33ING1b), oncoproteins (RasV12, p47ING3), or controls (empty vector, YFP) in ex vivo prostate tissues, then assessed proliferation by immunohistochemistry of markers (H3S10phos). Herein, we describe how to generate lentiviral vectors and particules, successfully transduce human prostate tissues, induce exogenous gene expression, and assess cellular proliferation.

Molecular mechanism of the TP53-MDM2-AR-AKT signalling network regulation by USP12.

The TP53-MDM2-AR-AKT signalling network plays a critical role in the development and progression of prostate cancer. However, the molecular mechanisms regulating this signalling network are not completely defined. By conducting transcriptome analysis, denaturing immunoprecipitations and immunopathology, we demonstrate that the TP53-MDM2-AR-AKT cross-talk is regulated by the deubiquitinating enzyme USP12 in prostate cancer. Our findings explain why USP12 is one of the 12 most commonly overexpressed cancer-associated genes located near an amplified super-enhancer. We find that USP12 deubiquitinates MDM2 and AR, which in turn controls the levels of the TP53 tumour suppressor and AR oncogene in prostate cancer. Consequently, USP12 levels are predictive not only of cancer development but also of patient's therapy resistance, relapse and survival. Therefore, our findings suggest that USP12 could serve as a promising therapeutic target in currently incurable castrate-resistant prostate cancer.

Ubiquitin-specific protease 12 interacting partners Uaf-1 and WDR20 are potential therapeutic targets in prostate cancer.

UNLABELLED: The androgen receptor (AR) is a key transcription factor in the initiation and progression of prostate cancer (PC) and is a major therapeutic target for the treatment of advanced disease. Unfortunately, current therapies are not curative for castration resistant PC and a better understanding of AR regulation could identify novel therapeutic targets and biomarkers to aid treatment of this disease. The AR is known to be regulated by a number of post-translational modifications and we have recently identified the deubiquitinating enzyme Usp12 as a positive regulator of AR. We determined that Usp12 deubiquitinates the AR resulting in elevated receptor stability and activity. Furthermore, Usp12 silencing was shown to reduce proliferation of PC cells.Usp12 is known to require the co-factors Uaf-1 and WDR20 for catalytic activity. In this report we focus further on the role of Uaf-1 and WDR20 in Usp12 regulation and investigate if these co-factors are also required for controlling AR activity. Firstly, we confirm the presence of the Usp12/Uaf-1/WDR20 complex in PC cells and demonstrate the importance of Uaf-1 and WDR20 for Usp12 stabilisation. Consequently, we show that individual silencing of either Uaf-1 or WDR20 is sufficient to abrogate the activity of the Usp12 complex and down-regulate AR-mediated transcription via receptor destabilisation resulting in increased apoptosis and decreased colony forming ability of PC cells. Moreover, expression of both Uaf-1 and WDR20 is higher in PC tissue compared to benign controls. Overall these results highlight the potential importance of the Usp12/Uaf-1/WDR20 complex in AR regulation and PC progression. HIGHLIGHTS: • Androgen receptor is a key transcriptional regulator in prostate cancer • Usp12/Uaf-1/WDR20 complex plays a crucial role in androgen receptor stability and activity • Destabilising an individual Usp12/Uaf-1/WDR20 complex member reduces the protein levels of the whole complex and diminishes androgen receptor activity • Protein levels of all members of the Usp12/Uaf-1/WDR20 complex are significantly increased in PC.

FUS/TLS is a novel mediator of androgen-dependent cell-cycle progression and prostate cancer growth.

Progression of prostate cancer is highly dependent upon the androgen receptor pathway, such that knowledge of androgen-regulated proteins is vital to understand and combat this disease. Using a proteomic screen, we found the RNA-binding protein FUS/TLS (Fused in Ewing's Sarcoma/Translocated in Liposarcoma) to be downregulated in response to androgen. FUS has recently been shown to be recruited by noncoding RNAs to the regulatory regions of target genes such as cyclin D1, in which it represses transcription by disrupting complex formation. Here we show that FUS has some characteristics of a putative tumor suppressor, as its overexpression promoted growth inhibition and apoptosis of prostate cancer cells, whereas its knockdown increased cell proliferation. This effect was reproducible in vivo, such that increasing FUS levels in tumor xenografts led to dramatic tumor regression. Furthermore, FUS promoted conditions that favored cell-cycle arrest by reducing the levels of proliferative factors such as cyclin D1 and Cdk6 and by increasing levels of the antiproliferative Cdk inhibitor p27. Immunohistochemical analysis revealed that FUS expression is inversely correlated with Gleason grade, demonstrating that patients with high levels of FUS survived longer and were less likely to have bone metastases, suggesting that loss of FUS expression may contribute to cancer progression. Taken together, our results address the question of how androgens regulate cell-cycle progression, by demonstrating that FUS is a key link between androgen receptor signaling and cell-cycle progression in prostate cancer.

Regulation of the androgen receptor by SET9-mediated methylation.

The androgen receptor (AR) is a member of the nuclear hormone receptor family of transcription factors that plays a critical role in regulating expression of genes involved in prostate development and transformation. Upon hormone binding, the AR associates with numerous co-regulator proteins that regulate the activation status of target genes via flux to the post-translational modification status of histones and the receptor. Here we show that the AR interacts with and is directly methylated by the histone methyltransferase enzyme SET9. Methylation of the AR on lysine 632 is necessary for enhancing transcriptional activity of the receptor by facilitating both inter-domain communication between the N- and C-termini and recruitment to androgen-target genes. We also show that SET9 is pro-proliferative and anti-apoptotic in prostate cancer cells and demonstrates up-regulated nuclear expression in prostate cancer tissue. In all, our date indicate a new mechanism of AR regulation that may be therapeutically exploitable for prostate cancer treatment.

Human PIRH2 enhances androgen receptor signaling through inhibition of histone deacetylase 1 and is overexpressed in prostate cancer.

The androgen receptor (AR) is a hormone-dependent transcription factor critically involved in human prostate carcinogenesis. Optimal transcriptional control of androgen-responsive genes by AR may require complex interaction among multiple coregulatory proteins. We have previously shown that the AR coregulator TIP60 can interact with human PIRH2 (hPIRH2). In this study, we uncover important new functional role(s) for hPIRH2 in AR signaling: (i) hPIRH2 interacts with AR and enhances AR-mediated transcription with a dynamic pattern of recruitment to androgen response elements in the prostate-specific antigen (PSA) gene; (ii) hPIRH2 interacts with the AR corepressor HDAC1, leading to reduced HDAC1 protein levels and inhibition of transcriptional repression; (iii) hPIRH2 is required for optimal PSA expression; and (iv) hPIRH2 is involved in prostate cancer cell proliferation. In addition, overexpression of hPIRH2 protein was detected in 73 of 82 (89%) resected prostate cancers, with a strong correlation between increased hPIRH2 expression and aggressive disease, as signified by high Gleason sum scores and the presence of metastatic disease (P = <0.0001 and 0.0004, respectively). Collectively, our data establish hPIRH2 as a key modulator of AR function, opening a new direction for targeted therapy in aggressive human prostate cancer.