Author Correction: The evolutionary history of lethal metastatic prostate cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The evolutionary history of lethal metastatic prostate cancer.

Cancers emerge from an ongoing Darwinian evolutionary process, often leading to multiple competing subclones within a single primary tumour. This evolutionary process culminates in the formation of metastases, which is the cause of 90% of cancer-related deaths. However, despite its clinical importance, little is known about the principles governing the dissemination of cancer cells to distant organs. Although the hypothesis that each metastasis originates from a single tumour cell is generally supported, recent studies using mouse models of cancer demonstrated the existence of polyclonal seeding from and interclonal cooperation between multiple subclones. Here we sought definitive evidence for the existence of polyclonal seeding in human malignancy and to establish the clonal relationship among different metastases in the context of androgen-deprived metastatic prostate cancer. Using whole-genome sequencing, we characterized multiple metastases arising from prostate tumours in ten patients. Integrated analyses of subclonal architecture revealed the patterns of metastatic spread in unprecedented detail. Metastasis-to-metastasis spread was found to be common, either through de novo monoclonal seeding of daughter metastases or, in five cases, through the transfer of multiple tumour clones between metastatic sites. Lesions affecting tumour suppressor genes usually occur as single events, whereas mutations in genes involved in androgen receptor signalling commonly involve multiple, convergent events in different metastases. Our results elucidate in detail the complex patterns of metastatic spread and further our understanding of the development of resistance to androgen-deprivation therapy in prostate cancer.

Constitutively active androgen receptor splice variants AR-V3, AR-V7 and AR-V9 are co-expressed in castration-resistant prostate cancer metastases.

BACKGROUND: A significant subset of prostate cancer (PC) patients with a castration-resistant form of the disease (CRPC) show primary resistance to androgen receptor (AR)-targeting drugs developed against CRPC. As one explanation could be the expression of constitutively active androgen receptor splice variants (AR-Vs), our current objectives were to study AR-Vs and other AR aberrations to better understand the emergence of CRPC. METHODS: We analysed specimens from different stages of prostate cancer by next-generation sequencing and immunohistochemistry. RESULTS: AR mutations and copy number variations were detected only in CRPC specimens. Genomic structural rearrangements of AR were observed in 5/30 metastatic CRPC patients, but they were not associated with expression of previously known AR-Vs. The predominant AR-Vs detected were AR-V3, AR-V7 and AR-V9, with the expression levels being significantly higher in CRPC cases compared to prostatectomy samples. Out of 25 CRPC metastases that expressed any AR variant, 17 cases harboured expression of all three of these AR-Vs. AR-V7 protein expression was highly heterogeneous and higher in CRPC compared to hormone-naïve tumours. CONCLUSIONS: AR-V3, AR-V7 and AR-V9 are co-expressed in CRPC metastases highlighting the fact that inhibiting AR function via regions common to all AR-Vs is likely to provide additional benefit to patients with CRPC.

Somatic MED12 mutations in prostate cancer and uterine leiomyomas promote tumorigenesis through distinct mechanisms.

BACKGROUND: Mediator is a multiprotein interface between eukaryotic gene-specific transcription factors and RNA polymerase II. Mutations in exon 2 of the gene encoding MED12, a key subunit of the regulatory kinase module in Mediator, are extremely frequent in uterine leiomyomas, breast fibroadenomas, and phyllodes tumors. These mutations disrupt kinase module interactions and lead to diminished Mediator-associated kinase activity. MED12 mutations in exon 26, resulting in a substitution of leucine 1224 to phenylalanine (L1224F), have been recurrently observed in prostate cancer. METHODS: To elucidate the molecular mechanisms leading to tumorigenesis in prostate cancer, we analyzed global interaction profiles of wild-type and L1224F mutant MED12 with quantitative affinity purification-mass spectrometry (AP-MS). Immunoprecipitation and kinase activity assay were used to further assess the interactions between Mediator complex subunits and kinase activity. The presence of L1224F mutation was analyzed in altogether 877 samples representing prostate hyperplasia, prostate cancer, and various tumor types in which somatic MED12 mutations have previously been observed. RESULTS: In contrast to N-terminal MED12 mutations observed in uterine leiomyomas, the L1224F mutation compromises neither the interaction of MED12 with kinase module subunits Cyclin C and CDK8/19 nor Mediator-associated CDK activity. Instead, the L1224F mutation was shown to affect interactions between MED12 and other Mediator components (MED1, MED13, MED13L, MED14, MED15, MED17, and MED24). Mutation screening revealed one mutation in a Finnish (Caucasian) prostate cancer patient, whereas no mutations in any other tumor type were observed. CONCLUSIONS: Specific somatic MED12 mutations in prostate cancer and uterine leiomyomas accumulate in two separate regions of the gene and promote tumorigenesis through clearly distinct mechanisms.

Sensitive circulating tumor DNA-based residual disease detection in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is one of the leading causes of cancer-related death in women worldwide, and is characterized by a high rate of recurrence after surgery and chemotherapy. We sought to implement a circulating tumor DNA (ctDNA)-based blood test for more accurate post-operative surveillance of this disease. We analyzed 264 plasma samples collected between June 2016 and September 2021 from 63 EOC patients using tumor-guided plasma cell-free DNA analysis to detect residual disease after treatment. Assay specificity was verified using cross-patient analysis of 1,195 control samples. ctDNA was detected in 51 of 55 (93%) samples at diagnosis, and 18 of 18 (100%) samples at progression. Positive ctDNA in the last on-treatment sample was associated with rapid progression (median 1.02 versus 3.38 yr, HR = 5.63, P < 0.001) and reduced overall survival (median 2.31 versus NR yr, HR = 8.22, P < 0.001) in patients with high-grade serous cancer. In the case of 12 patients, ctDNA assays detected progression earlier than standard surveillance, with a median lead time of 5.9 mo. To approach the physical limits of ctDNA detection, five patients were analyzed using ultra-sensitive assays interrogating 479-1,856 tumor mutations, capable of tracking ctDNA fractions down to 0.0004%. Our results demonstrate that ctDNA assays achieve high sensitivity and specificity in detecting post-operative residual disease in EOC.

AR and ERG drive the expression of prostate cancer specific long noncoding RNAs.

Long noncoding RNAs (lncRNAs) play pivotal roles in cancer development and progression, and some function in a highly cancer-specific manner. However, whether the cause of their expression is an outcome of a specific regulatory mechanism or nonspecific transcription induced by genome reorganization in cancer remains largely unknown. Here, we investigated a group of lncRNAs that we previously identified to be aberrantly expressed in prostate cancer (PC), called TPCATs. Our high-throughput real-time PCR experiments were integrated with publicly available RNA-seq and ChIP-seq data and revealed that the expression of a subset of TPCATs is driven by PC-specific transcription factors (TFs), especially androgen receptor (AR) and ETS-related gene (ERG). Our in vitro validations confirmed that AR and ERG regulated a subset of TPCATs, most notably for EPCART. Knockout of EPCART was found to reduce migration and proliferation of the PC cells in vitro. The high expression of EPCART and two other TPCATs (TPCAT-3-174133 and TPCAT-18-31849) were also associated with the biochemical recurrence of PC in prostatectomy patients and were independent prognostic markers. Our findings suggest that the expression of numerous PC-associated lncRNAs is driven by PC-specific mechanisms and not by random cellular events that occur during cancer development. Furthermore, we report three prospective prognostic markers for the early detection of advanced PC and show EPCART to be a functionally relevant lncRNA in PC.

Introducing PIONEER: a project to harness big data in prostate cancer research.

Prostate Cancer Diagnosis and Treatment Enhancement Through the Power of Big Data in Europe (PIONEER) is a European network of excellence for big data in prostate cancer, consisting of 32 private and public stakeholders from 9 countries across Europe. Launched by the Innovative Medicines Initiative 2 and part of the Big Data for Better Outcomes Programme (BD4BO), the overarching goal of PIONEER is to provide high-quality evidence on prostate cancer management by unlocking the potential of big data. The project has identified critical evidence gaps in prostate cancer care, via a detailed prioritization exercise including all key stakeholders. By standardizing and integrating existing high-quality and multidisciplinary data sources from patients with prostate cancer across different stages of the disease, the resulting big data will be assembled into a single innovative data platform for research. Based on a unique set of methodologies, PIONEER aims to advance the field of prostate cancer care with a particular focus on improving prostate-cancer-related outcomes, health system efficiency by streamlining patient management, and the quality of health and social care delivered to all men with prostate cancer and their families worldwide.

Author Correction: Introducing PIONEER: a project to harness big data in prostate cancer research.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Integrative proteomics in prostate cancer uncovers robustness against genomic and transcriptomic aberrations during disease progression.

To understand functional consequences of genetic and transcriptional aberrations in prostate cancer, the proteomic changes during disease formation and progression need to be revealed. Here we report high-throughput mass spectrometry on clinical tissue samples of benign prostatic hyperplasia (BPH), untreated primary prostate cancer (PC) and castration resistant prostate cancer (CRPC). Each sample group shows a distinct protein profile. By integrative analysis we show that, especially in CRPC, gene copy number, DNA methylation, and RNA expression levels do not reliably predict proteomic changes. Instead, we uncover previously unrecognized molecular and pathway events, for example, several miRNA target correlations present at protein but not at mRNA level. Notably, we identify two metabolic shifts in the citric acid cycle (TCA cycle) during prostate cancer development and progression. Our proteogenomic analysis uncovers robustness against genomic and transcriptomic aberrations during prostate cancer progression, and significantly extends understanding of prostate cancer disease mechanisms.

Feasibility of Prostate PAXgene Fixation for Molecular Research and Diagnostic Surgical Pathology: Comparison of Matched Fresh Frozen, FFPE, and PFPE Tissues.

Advances in prostate cancer biology and diagnostics are dependent upon high-fidelity integration of clinical, histomorphologic, and molecular phenotypic findings. In this study, we compared fresh frozen, formalin-fixed paraffin-embedded (FFPE), and PAXgene-fixed paraffin-embedded (PFPE) tissue preparation methods in radical prostatectomy prostate tissue from 36 patients and performed a preliminary test of feasibility of using PFPE tissue in routine prostate surgical pathology diagnostic assessment. In addition to comparing histology, immunohistochemistry, and general measures of DNA and RNA integrity in each fixation method, we performed functional tests of DNA and RNA quality, including targeted Miseq RNA and DNA sequencing, and implemented methods to relate DNA and RNA yield and quality to quantified DNA and RNA picogram nuclear content in each tissue volume studied. Our results suggest that it is feasible to use PFPE tissue for routine robot-assisted laparoscopic prostatectomy surgical pathology diagnostics and immunohistochemistry, with the benefit of significantly improvedDNA and RNA quality and RNA picogram yield per nucleus as compared with FFPE tissue. For fresh frozen, FFPE, and PFPE tissues, respectively, the average Genomic Quality Numbers were 7.9, 3.2, and 6.2, average RNA Quality Numbers were 8.7, 2.6, and 6.3, average DNA picogram yields per nucleus were 0.41, 0.69, and 0.78, and average RNA picogram yields per nucleus were 1.40, 0.94, and 2.24. These findings suggest that where DNA and/or RNA analysis of tissue is required, and when tissue size is small, PFPE may provide important advantages over FFPE. The results also suggest several interesting nuances including potential avenues to improve RNA quality in FFPE tissues and confirm recent suggestions that some DNA sequence artifacts associated with FFPE can be avoided.

The expression of AURKA is androgen regulated in castration-resistant prostate cancer.

Although second generation endocrine therapies have significantly improved survival, castration-resistant prostate cancer (CRPC) cells are eventually able to escape available hormonal treatments due to reactivation of androgen receptor (AR) signaling. Identification of novel, non-classical and druggable AR-target genes may provide new approaches to treat CRPC. Our previous analyses suggested that Aurora kinase A (AURKA) is regulated by androgens in prostate cancer cells that express high levels of AR. Here, we provide further evidence that AURKA is significantly overexpressed in AR-positive CRPC samples carrying amplification of AR gene and/or expressing AR in high levels. We also demonstrate androgen-induced AR binding in the intronic region of AURKA. The expression of AURKA is increased upon androgen stimulation in LNCaP-ARhi cells that express high levels of AR. The growth of the cells was also significantly inhibited by an AURKA specific inhibitor, alisertib (MLN8237). Together, these findings suggest that the expression of AURKA is regulated by androgen in prostate cancer cells that highly express AR, emphasizing its potential as a therapeutic target in patients with CRPC.

Androgen Receptor Deregulation Drives Bromodomain-Mediated Chromatin Alterations in Prostate Cancer.

Global changes in chromatin accessibility may drive cancer progression by reprogramming transcription factor (TF) binding. In addition, histone acetylation readers such as bromodomain-containing protein 4 (BRD4) have been shown to associate with these TFs and contribute to aggressive cancers including prostate cancer (PC). Here, we show that chromatin accessibility defines castration-resistant prostate cancer (CRPC). We show that the deregulation of androgen receptor (AR) expression is a driver of chromatin relaxation and that AR/androgen-regulated bromodomain-containing proteins (BRDs) mediate this effect. We also report that BRDs are overexpressed in CRPCs and that ATAD2 and BRD2 have prognostic value. Finally, we developed gene stratification signature (BROMO-10) for bromodomain response and PC prognostication, to inform current and future trials with drugs targeting these processes. Our findings provide a compelling rational for combination therapy targeting bromodomains in selected patients in which BRD-mediated TF binding is enhanced or modified as cancer progresses.

Measuring the Expression of microRNAs Regulated by Androgens.

The discovery of microRNAs (miRNAs) provided yet another mechanism of gene expression regulation. miRNAs have recently been also implicated in many diseases, including prostate cancer (PC). As PC is a highly androgen-dependent disease, extensive effort has been invested to identify the miRNAs that are androgen regulated. However, relatively few of them have been shown to be directly androgen regulated in PC. In this chapter we introduce the commonly used techniques to study the androgen regulation of miRNAs. The most cost-effective tool to profile global miRNA expression is microarray-based hybridization, whereas real-time quantitative reverse transcription PCR (qRT-PCR) is commonly used for the study of individual miRNAs.

Integrated clinical, whole-genome, and transcriptome analysis of multisampled lethal metastatic prostate cancer.

We report the first combined analysis of whole-genome sequence, detailed clinical history, and transcriptome sequence of multiple prostate cancer metastases in a single patient (A21). Whole-genome and transcriptome sequence was obtained from nine anatomically separate metastases, and targeted DNA sequencing was performed in cancerous and noncancerous foci within the primary tumor specimen removed 5 yr before death. Transcriptome analysis revealed increased expression of androgen receptor (AR)-regulated genes in liver metastases that harbored an AR p.L702H mutation, suggesting a dominant effect by the mutation despite being present in only one of an estimated 16 copies per cell. The metastases harbored several alterations to the PI3K/AKT pathway, including a clonal truncal mutation in PIK3CG and present in all metastatic sites studied. The list of truncal genomic alterations shared by all metastases included homozygous deletion of TP53, hemizygous deletion of RB1 and CHD1, and amplification of FGFR1. If the patient were treated today, given this knowledge, the use of second-generation androgen-directed therapies, cessation of glucocorticoid administration, and therapeutic inhibition of the PI3K/AKT pathway or FGFR1 receptor could provide personalized benefit. Three previously unreported truncal clonal missense mutations (ABCC4 p.R891L, ALDH9A1 p.W89R, and ASNA1 p.P75R) were expressed at the RNA level and assessed as druggable. The truncal status of mutations may be critical for effective actionability and merit further study. Our findings suggest that a large set of deeply analyzed cases could serve as a powerful guide to more effective prostate cancer basic science and personalized cancer medicine clinical trials.

CIP2A is a candidate therapeutic target in clinically challenging prostate cancer cell populations.

Residual androgen receptor (AR)-signaling and presence of cancer stem-like cells (SCs) are the two emerging paradigms for clinically challenging castration-resistant prostate cancer (CRPC). Therefore, identification of AR-target proteins that are also overexpressed in the cancer SC population would be an attractive therapeutic approach.Our analysis of over three hundred clinical samples and patient-derived prostate epithelial cultures (PPECs), revealed Cancerous inhibitor of protein phosphatase 2A (CIP2A) as one such target. CIP2A is significantly overexpressed in both hormone-naïve prostate cancer (HN-PC) and CRPC patients . CIP2A is also overexpressed, by 3- and 30-fold, in HN-PC and CRPC SCs respectively. In vivo binding of the AR to the intronic region of CIP2A and its functionality in the AR-moderate and AR-high expressing LNCaP cell-model systems is also demonstrated. Further, we show that AR positively regulates CIP2A expression, both at the mRNA and protein level. Finally, CIP2A depletion reduced cell viability and colony forming efficiency of AR-independent PPECs as well as AR-responsive LNCaP cells, in which anchorage-independent growth is also impaired.These findings identify CIP2A as a common denominator for AR-signaling and cancer SC functionality, highlighting its potential therapeutic significance in the most clinically challenging prostate pathology: castration-resistant prostate cancer.

Transcriptome Sequencing Reveals PCAT5 as a Novel ERG-Regulated Long Noncoding RNA in Prostate Cancer.

Castration-resistant prostate cancers (CRPC) that arise after the failure of androgen-blocking therapies cause most of the deaths from prostate cancer, intensifying the need to fully understand CRPC pathophysiology. In this study, we characterized the transcriptomic differences between untreated prostate cancer and locally recurrent CRPC. Here, we report the identification of 145 previously unannotated intergenic long noncoding RNA transcripts (lncRNA) or isoforms that are associated with prostate cancer or CRPC. Of the one third of these transcripts that were specific for CRPC, we defined a novel lncRNA termed PCAT5 as a regulatory target for the transcription factor ERG, which is activated in approximately 50% of human prostate cancer. Genome-wide expression analysis of a PCAT5-positive prostate cancer after PCAT5 silencing highlighted alterations in cell proliferation pathways. Strikingly, an in vitro validation of these alterations revealed a complex integrated phenotype affecting cell growth, migration, invasion, colony-forming potential, and apoptosis. Our findings reveal a key molecular determinant of differences between prostate cancer and CRPC at the level of the transcriptome. Furthermore, they establish PCAT5 as a novel oncogenic lncRNA in ERG-positive prostate cancers, with implications for defining CRPC biomarkers and new therapeutic interventions.

Recurrent SKIL-activating rearrangements in ETS-negative prostate cancer.

Prostate cancer is the third most common cause of male cancer death in developed countries, and one of the most comprehensively characterized human cancers. Roughly 60% of prostate cancers harbor gene fusions that juxtapose ETS-family transcription factors with androgen regulated promoters. A second subtype, characterized by SPINK1 overexpression, accounts for 15% of prostate cancers. Here we report the discovery of a new prostate cancer subtype characterized by rearrangements juxtaposing the SMAD inhibitor SKIL with androgen regulated promoters, leading to increased SKIL expression. SKIL fusions were found in 6 of 540 (1.1%) prostate cancers and 1 of 27 (3.7%) cell lines and xenografts. 6 of 7 SKIL-positive cancers were negative for ETS overexpression, suggesting mutual exclusivity with ETS fusions. SKIL knockdown led to growth arrest in PC-3 and LNCaP cell line models of prostate cancer, and its overexpression led to increased invasiveness in RWPE-1 cells. The role of SKIL as a prostate cancer oncogene lends support to recent studies on the role of TGF-ß signaling as a rate-limiting step in prostate cancer progression. Our findings highlight SKIL as an oncogene and potential therapeutic target in 1-2% of prostate cancers, amounting to an estimated 10,000 cancer diagnoses per year worldwide.