KDM6A mutations promote acute cytoplasmic DNA release, DNA damage response and mitosis defects.

BACKGROUND: KDM6A, encoding a histone demethylase, is one of the top ten mutated epigenetic cancer genes. The effect of mutations on its structure and function are however poorly characterized. METHODS: Database search identified nonsense and missense mutations in the N-terminal TPR motifs and the C-terminal, catalytic JmjC domain, but also in the intrinsically disordered region connecting both these two well-structured domains. KDM6A variants with cancer-derived mutations were generated using site directed mutagenesis and fused to eGFP serving as an all-in-one affinity and fluorescence tag to study demethylase activity by an ELISA-based assay in vitro, apoptosis by FACS, complex assembly by Co-immunoprecipitation and localization by microscopy in urothelial cells and apoptosis by FACS. RESULTS: Independent of the mutation and demethylase activity, all KDM6A variants were detectable in the nucleus. Truncated KDM6A variants displayed changes in complex assemblies affecting (1) known interactions with the COMPASS complex component RBBP5 and (2) KDM6A-DNA associated assemblies with the nuclear protein Nucleophosmin. Some KDM6A variants induced a severe cellular phenotype characterized by multiple acute effects on nuclear integrity, namely, release of nuclear DNA into the cytoplasm, increased level of DNA damage indicators RAD51 and p-γH2A.X, and mitosis defects. These damaging effects were correlated with increased cell death. CONCLUSION: These observations reveal novel effects of pathogenic variants pointing at new specific functions of KDM6A variants. The underlying mechanisms and affected pathways have to be investigated in future research to understand how tumor cells cope with and benefit from KDM6A truncations.

[Report from the 9th symposium of the German Association for Bladder Cancer Research]. / Bericht vom 9. Symposium des Deutschen Forschungsverbunds Blasenkarzinom.

The annual symposium of the German Research Association for Bladder Carcinoma (DFBK) was organized on February 7th and 8th, 2020, in Düsseldorf. On the first day, eight international guest speakers invited by the DFBK and the Department of Urology of the Heinrich Heine University Düsseldorf presented the current state of research on bladder cancer (BC). Topics were genomic changes and molecular classification in non-muscle-invasive and muscle-invasive BC, prospects and limits of proteome technology in urine diagnostics, function of chromatin regulators in bladder carcinogenesis, cellular reactions to aneuploidy, organoid technology and biobanking, as well as novel aspects of immunotherapy for BC. The second day was dedicated to new results and ideas of the DFBK members on BC pathomechanisms, diagnostics and therapeutic approaches, and most importantly, discussions on the further development of collaborative projects. Additional information is available at http://www.forschungsverbund-blasenkarzinom.de.

Analysis of DNA methylation in single circulating tumor cells.

Direct analysis of circulating tumor cells (CTCs) can inform on molecular mechanisms underlying systemic spread. Here we investigated promoter methylation of three genes regulating epithelial-to-mesenchymal transition (EMT), a key mechanism enabling epithelial tumor cells to disseminate and metastasize. For this, we developed a single-cell protocol based on agarose-embedded bisulfite treatment, which allows investigating DNA methylation of multiple loci via a multiplex PCR (multiplexed-scAEBS). We established our assay for the simultaneous analysis of three EMT-associated genes miR-200c/141, miR-200b/a/429 and CDH1 in single cells. The assay was validated in solitary cells of GM14667, MDA-MB-231 and MCF-7 cell lines, achieving a DNA amplification efficiency of 70% with methylation patterns identical to the respective bulk DNA. Then we applied multiplexed-scAEBS to 159 single CTCs from 11 patients with metastatic breast and six with metastatic castration-resistant prostate cancer, isolated via CellSearch (EpCAMpos/CKpos/CD45neg/DAPIpos) and subsequent FACS sorting. In contrast to CD45pos white blood cells isolated and processed by the identical approach, we observed in the isolated CTCs methylation patterns resembling more those of epithelial-like cells. Methylation at the promoter of microRNA-200 family was significantly higher in prostate CTCs. Data from our single-cell analysis revealed an epigenetic heterogeneity among CTCs and indicates tumor-specific active epigenetic regulation of EMT-associated genes during blood-borne dissemination.

[Tumour-stroma interactions in urothelial cancer]. / Tumor-Stroma-Interaktionen im Harnblasenkarzinom.

BACKGROUND: The histopathological structure of malignant tumours involves two essential compartments - the tumour parenchyma with the actual transformed cells, and the supportive tumour stroma. The latter consists of specialized mesenchymal cells, such as fibroblasts, macrophages, lymphocytes and vascular cells, as well as of their secreted products, including components of the extracellular matrix, matrix modifying enzymes and numerous regulatory growth factors and cytokines. In consequence, the tumour stroma has the ability to influence virtually all aspects of tumour development and progression, including therapeutic response. AIM: In this article we review the current knowledge of tumor stroma interactions in urothelial carcinoma and present various experimental systems that are currently in use to unravel the biological basis of these heterotypic cell interactions. RESULTS: For urothelial carcinoma, an extensive tumour stroma is quite typical and markers of activated fibroblasts correlate significantly with clinical parameters of advanced disease. Another clinically important variable is provided by the stromal expression of syndecan-1. CONCLUSION: Integration of markers of activated stroma into clinical risk evaluation could aid to better stratification of urothelial bladder carcinoma patients. Elucidation of biological mechanisms underlying tumour-stroma interactions could provide new therapeutical targets.

[Epigenetics in urothelial cancer: Pathogenesis, improving diagnostics and developing novel treatment options]. / Epigenetik des Urothelkarzinoms : Pathogenese, verbesserte Diagnostik und neue Therapieansätze.

Urothelial carcinoma of the bladder is a common tumor for which improvements in diagnostic markers and new therapy approaches, in addition to or combined with standard chemotherapy, are urgently required. Epigenetic alterations could provide both novel diagnostic markers and therapeutic targets as they are emerging as crucial factors in the development and progression of this tumor type, likely contributing to altered differentiation and metastatic potential. These alterations affect DNA methylation, histone modifications, chromatin remodeling, long noncoding RNAs, and microRNAs. Factors involved in histone modifications and chromatin remodeling appear to be particularly frequently inactivated by mutations. Thus, histone-modifying enzymes may represent good targets for rational new therapeutic approaches, although thorough investigation of their complex functions is a prerequisite. DNA methylation changes and altered miRNA expression provide promising biomarkers for diagnosis and prognosis that need further validation in comprehensive and well-standardized studies.

Stem cells in the biology of normal urothelium and urothelial carcinoma.

Urothelium is a special type of stratified epithelium that lines the distal portion of the urinary tract. For a long time, basal urothelial cells have been suspected to include a population of urothelial stem cells. Recent experiments identifying label-retaining cells as well as lineage tracing analyses corroborate this notion. There are striking morphological and antigenic similarities between basal or differentiated urothelial cells and the corresponding cells in some urothelial carcinomas. In this respect, basal cell-specific markers provide good candidates to identify urothelial carcinoma stem cells, e.g. specific cytokeratins (CK5, CK14, CK17) or adhesion molecules (specific integrin subspecies, CD44). Common properties of the stem cells of normal urothelium and urothelial cancer have thus emerged. Both are characterized by a remarkable plasticity and both rely on reciprocal interactions with stromal fibroblasts. However, the stem cells of individual urothelial carcinomas appear to differ considerably and may contribute to the heterogeneity of this disease. The presence, quantity, and particular biological nature of urothelial carcinoma stem cells in each case may thus carry important clinical information that might allow a rationale stratification of urothelial cancer patients for treatment in the near future.

The role of c-FLIP splice variants in urothelial tumours.

Deregulation of apoptosis is common in cancer and is often caused by overexpression of anti-apoptotic proteins in tumour cells. One important regulator of apoptosis is the cellular FLICE-inhibitory protein (c-FLIP), which is overexpressed, for example, in melanoma and Hodgkin's lymphoma cells. Here, we addressed the question whether deregulated c-FLIP expression in urothelial carcinoma impinges on the ability of death ligands to induce apoptosis. In particular, we investigated the role of the c-FLIP splice variants c-FLIP(long) (c-FLIP(L)) and c-FLIP(short) (c-FLIP(S)), which can have opposing functions. We observed diminished expression of the c-FLIP(L) isoform in urothelial carcinoma tissues as well as in established carcinoma cell lines compared with normal urothelial tissues and cells, whereas c-FLIP(S) was unchanged. Overexpression and RNA interference studies in urothelial cell lines nevertheless demonstrated that c-FLIP remained a crucial factor conferring resistance towards induction of apoptosis by death ligands CD95L and TRAIL. Isoform-specific RNA interference showed c-FLIP(L) to be of particular importance. Thus, urothelial carcinoma cells appear to fine-tune c-FLIP expression to a level sufficient for protection against activation of apoptosis by the extrinsic pathway. Therefore, targeting c-FLIP, and especially the c-FLIP(L) isoform, may facilitate apoptosis-based therapies of bladder cancer in otherwise resistant tumours.

[Signal transduction in urothelial cancer: how exactly do we know the targets for targeted therapy?]. / Signaltransduktion im Urothelkarzinom : Wie genau kennen wir die Ziele für eine zielgerichtete Therapie?

Targeted therapies have helped to improve the quality of life and prolong the survival of many cancer patients. This progress is based on the growing understanding of cellular signal transduction pathways and regulatory systems in human cancers. In urothelial carcinoma, a number of specific alterations have been identified. These include mutations in FGFR3, HRAS, and PIK3CA leading to overactivity of MAPK and Akt signaling pathways especially in papillary tumors. In comparison, the RB1 and p53 regulatory systems that act more directly on cell cycle control are more commonly compromised in invasive carcinomas. Nevertheless, targeted therapies have shown little efficacy in the treatment of urothelial carcinoma so far, owing presumably to our incomplete knowledge of molecular changes affecting signal transduction pathways in this cancer type. Target genes of cancer pathways are regulated by epigenetic mechanisms, which determine their inducibility. Elucidating these control mechanisms could therefore prove important for developing targeted therapies for urothelial carcinoma.

DKC1 overexpression associated with prostate cancer progression.

BACKGROUND: Dyskerin encoded by the DKC1 gene is a predominantly nucleolar protein essential for the formation of pseudouridine in RNA and the telomerase RNA subunit hTR. Inherited mutations inactivating dyskerin cause dyskeratosis congenita, a syndrome with progeroid features characterised by skin defects and haematopoiesis failure, as well as cancer susceptibility. In this study, we report DKC1 overexpression in prostate cancers. METHODS: Expression of DKC1 was measured by quantitative RT-PCR in prostate cancer tissues in relation to hTR and the proliferation marker MKI67. Effects of dyskerin downregulation on proliferation, apoptosis and senescence of prostate cancer cell lines were determined. RESULTS: DKC1 was significantly overexpressed in prostate cancers, particularly in high-stage and recurring cases, correlating moderately with hTR and MKI67. Dyskerin downregulation in prostate carcinoma cell lines by siRNA diminished cell proliferation, but elicited neither apoptosis nor senescence. Apoptosis induction by TNF-alpha or tunicamycin was not enhanced. Long-term downregulation led predominantly to cell shrinking and loss of adhesion. INTERPRETATION: DKC1 upregulation in prostate cancers is common and likely to be necessary for extensive tumour growth. The phenotype of prostate carcinoma cell lines after dyskerin downregulation suggests that its most critical function is sustaining protein biosynthesis. Intriguingly, compromised function and overexpression of dyskerin can both contribute to cancer development.

Differential effects of Nucleostemin suppression on cell cycle arrest and apoptosis in the bladder cancer cell lines 5637 and SW1710.

OBJECTIVES: The Nucleostemin (NS) gene encodes a nucleolar protein enriched in adult and embryonic stem cells. NS is thought to regulate cancer cell proliferation, but the mechanisms involved are poorly understood. In this study, we have investigated the role of NS in bladder cancer. MATERIALS AND METHODS: Expression of NS was determined by quantitative reverse transcription-polymerase chain reaction in bladder carcinoma cell lines and in normal uro-epithelial cell cultures. We used an RNAi strategy to investigate the function of NS in two selected carcinoma cell lines. RESULTS: High NS expression was found in most bladder carcinoma cell lines and normal uro-epithelial cells. Knockdown of NS expression induced a severe decline in cell proliferation in 5637 and SW1710 cell lines, both with mutant p53. Apoptosis was more strongly enhanced in 5637 cells lacking RB1 than in SW1710 cells lacking p16(INK4A). Moreover, NS-siRNA-treated 5637 cells accumulated mainly in G(2)/M, whereas SW1710 cells arrested in G(0)/G(1). CONCLUSION: Our data indicate that NS expression is necessary for cell proliferation and evasion of apoptosis in bladder cancer cells, independent of its effect on p53. Also, we speculate that the precise effect of NS on cell cycle regulation may relate to functional status of RB1 and CDKN2A/p16(INK4A).

[Bladder carcinoma cell lines as models of the pathobiology of bladder cancer. Review of the literature and establishment of a new progression series]. / Harnblasenkarzinomzelllinien als Modellsysteme zur Pathobiologie des Harnblasenkarzinoms. Uberblick und Etablierung einer neuen Progressionsserie.

BACKGROUND: Tumour cell lines represent valuable preclinical models to decipher underlying biology and identify potential therapy targets and pharmacologically useful compounds. Approximately 50 human bladder cancer cell lines have been established to date, mainly from invasive and metastatic tumours. Two of these, namely T24 and 253J, were experimentally further developed into progression series. These models have provided important insights into later tumour progression events and metastatic dissemination. Only a few cell lines are available as models of non-invasive papillary bladder cancer and no progression series have yet been established. MATERIAL AND METHODS: During the course of establishing a doxorubicin-resistant variant cell line of the human papillary bladder carcinoma cell line BFTC-905, a unique cell colony was identified, apparently involving cells with divergent growth patterns. Subsequent subculturing yielded three daughter cell lines, BFTC-905-compact, BFTC-905-diffuse und BFTC-905-diffuse M. Their fundamental characterization included basic cell morphology, cell membrane expression of E-Cadherin, karyotype analysis, invasion and colony forming capacity in soft agar. The clonal origin of the newly established daughter cell lines was assessed by means of molecular genetic methods. RESULTS: We could identify important differences in multiple transformation related traits among the cell lines of the BFTC-905 progression series. Both diffuse cell lines (BFTC-905-diffuse und BFTC-905-diffuse M) differed from the BFTC-905-compact cell line by growing in a less organized,"diffuse" manner, which involved colonies of cells exhibiting apparently normal cell-to-cell adhesion as well as individual cells outside of them. This diminution of the cell-to-cell adhesion was accompanied by a corresponding decrease of membranous E-Cadherin. The BFTC-905-diffuse M cell line displayed a dramatic increase in the overall chromosome number, resulting in a hypertetraploid karyotype. At the same time, this cell line, as the only one in the progression series, acquired the ability to grow independent of anchorage in soft agar. All three cell lines remained noninvasive. Allelic distribution of highly polymorphic DNA-markers in the cell lines of the BFTC-905 progression series provided unequivocal evidence of their common origin. CONCLUSION: The newly established BFTC-905 progression series manifests two aspects of the early progression of non-invasive bladder carcinoma, not exhibited by any other progression series published so far, namely dynamic changes in the expression of E-Cadherin and a complex karyotypic evolution. It may thus contribute important insights into further understanding of the pathobiology of bladder cancer.

Disruption of the FA/BRCA pathway in bladder cancer.

Bladder carcinomas frequently show extensive deletions of chromosomes 9p and/or 9q, potentially including the loci of the Fanconi anemia (FA) genes FANCC and FANCG. FA is a rare recessive disease due to defects in anyone of 13 FANC genes manifesting with genetic instability and increased risk of neoplasia. FA cells are hypersensitive towards DNA crosslinking agents such as mitomycin C and cisplatin that are commonly employed in the chemotherapy of bladder cancers. These observations suggest the possibility of disruption of the FA/BRCA DNA repair pathway in bladder tumors. However, mutations in FANCC or FANCG could not be detected in any of 23 bladder carcinoma cell lines and ten surgical tumor specimens by LOH analysis or by FANCD2 immunoblotting assessing proficiency of the pathway. Only a single cell line, BFTC909, proved defective for FANCD2 monoubiquitination and was highly sensitive towards mitomycin C. This increased sensitivity was restored specifically by transfer of the FANCF gene. Sequencing of FANCF in BFTC909 failed to identify mutations, but methylation of cytosine residues in the FANCF promoter region was demonstrated by methylation-specific PCR, HpaII restriction and bisulfite DNA sequencing. Methylation-specific PCR uncovered only a single instance of FANCF promoter hypermethylation in surgical specimens of further 41 bladder carcinomas. These low proportions suggest that in contrast to other types of tumors silencing of FANCF is a rare event in bladder cancer and that an intact FA/BRCA pathway might be advantageous for tumor progression.

[Tumour stem cells--a new concept in tumour biology]. / Tumorstammzellen--ein neues Konzept in der Tumorbiologie.

There is growing evidence that tumours display a hierarchy similar to normal tissues. Accordingly, a small population of tumour stem cells is supposed to perpetuate tumour growth. These cells renew themselves and are highly tumourigenic upon injection into immunocompromised animals, yielding tumours largely identical to those from which they were derived. Tumour stem cells exhibit a high degree of chemoresistance due to slow cycling and constitutive expression of multiple members of the ATP-BINDING CASSETTE (ABC) family of transporters. An increasing number of tumours with tumour stem cells has been identified by now, including breast, brain, colon and prostate cancers, as well as leukemias. Detailed characterization of tumour stem cells may contribute to a better understanding of the underlying biology of the respective tumours and lead to novel curative therapies.

Concomitant down-regulation of SPRY1 and SPRY2 in prostate carcinoma.

Sprouty proteins encoded by the SPRY genes act as modulators and feedback inhibitors of signalling by epidermal growth factor (EGF) and fibroblast growth factor (FGF). Overactivity of EGF and FGF signalling common in prostate cancer might therefore be exacerbated by Sprouty down-regulation. Indeed, down-regulation of SPRY1 and SPRY2 expression has been independently reported. We found both genes modestly down-regulated by microarray expression analysis of microdissected prostate cancers and by quantitative RT-PCR in macrodissected specimens compared with benign tissues. Importantly, the decreases paralleled each other and expression levels of both genes were significantly lower in cancers that recurred within the average follow-up period of 32 months. In contrast to a previous report, no hypermethylation was found to accompany down-regulation of SPRY2 in cancer tissues and cell lines. We additionally investigated the expression of an SPRY1 alternative transcript presumed to be specific for fetal tissues and found its expression moderately well correlated with expression of the standard transcript through diverse tissues and cell lines. The present study confirms and extends previous reports by demonstrating concomitant down-regulation and a significant association with recurrence of SPRY genes.

Methylation of endogenous human retroelements in health and disease.

Retroelements constitute approximately 45% of the human genome. Long interspersed nuclear element (LINE) autonomous retrotransposons are predominantly represented by LINE-1, nonautonomous small interspersed nuclear elements (SINEs) are primarily represented by ALUs, and LTR retrotransposons by several families of human endogenous retroviruses (HERVs). The vast majority of LINE and HERV elements are densely methylated in normal somatic cells and contained in inactive chromatin. Methylation and chromatin structure together ensure a stable equilibrium between retroelements and their host. Hypomethylation and expression in developing germ cells opens a "window of opportunity" for retrotransposition and recombination that contribute to human evolution, but also inherited disease. In somatic cells, the presence of retroelements may be exploited to organize the genome into active and inactive regions, to separate domains and functional regions within one chromatin domain, to suppress transcriptional noise, and to regulate transcript stability. Retroelements, particularly ALUs, may also fulfill physiological roles during responses to stress and infections. Reactivation and hypomethylation of LINEs and HERVs may be important in the pathophysiology of cancer and various autoimmune diseases, contributing to chromosomal instability and chronically aberrant immune responses. The emerging insights into the pathophysiological importance of endogenous retroelements accentuate the gaps in our knowledge of how these elements are controlled in normal developing and mature cells.

Epigenetics of prostate cancer: beyond DNA methylation.

Epigenetic mechanisms permit the stable inheritance of cellular properties without changes in DNA sequence or amount. In prostate carcinoma, epigenetic mechanisms are essential for development and progression, complementing, amplifying and diversifying genetic alterations. DNA hypermethylation affects at least 30 individual genes, while repetitive sequences including retrotransposons and selected genes become hypomethylated. Hypermethylation of several genes occurs in a coordinate manner early in carcinogenesis and can be exploited for cancer detection, whereas hypomethylation and further hypermethylation events are associated with progression. DNA methylation alterations interact with changes in chromatin proteins. Prominent alterations at this level include altered patterns of histone modification, increased expression of the EZH2 polycomb histone methyltransferase, and changes in transcriptional corepressors and coactivators. These changes may make prostate carcinoma particularly susceptible to drugs targeting chromatin and DNA modifications. They relate to crucial alterations in a network of transcription factors comprising ETS family proteins, the androgen receptor, NKX3.1, KLF, and HOXB13 homeobox proteins. This network controls differentiation and proliferation of prostate epithelial cells integrating signals from hormones, growth factors and cell adhesion proteins that are likewise distorted in prostate cancer. As a consequence, prostate carcinoma cells appear to be locked into an aberrant state, characterized by continued proliferation of largely differentiated cells. Accordingly, stem cell characteristics of prostate cancer cells appear to be secondarily acquired. The aberrant differentiation state of prostate carcinoma cells also results in distorted mutual interactions between epithelial and stromal cells in the tumor that promote tumor growth, invasion, and metastasis.

Effects of WNT/beta-catenin pathway activation on signaling through T-cell factor and androgen receptor in prostate cancer cell lines.

Dysregulation of the WNT/beta-catenin pathway is thought to contribute to prostate cancer progression. Mutations of beta-catenin occurring in 5-7% of advanced prostate cancers may act by stimulating TCF-dependent and/or androgen receptor (AR)-dependent transcription. Using a reporter gene approach we found overexpressed mutated beta-catenin to enhance AR-regulated probasin-promoter activity in the AR-positive prostate cancer cell line 22Rv1, particularly at low androgen levels. In 22Rv1 cells mutated beta-catenin was able to stimulate TCF-dependent transcription but was unable to do so in LNCaP cells where it activates the AR. Since beta-catenin mutations are rare in vivo, we studied further possible routes of WNT-pathway modulation. Higher concentrations of LiCl, a GSK3beta-inhibitor, were required to activate TCF-dependent rather than AR-dependent reporter constructs. In 22Rv1 overexpression of E-cadherin repressed androgen-dependent transcription, but did not inhibit transcription of TCF-dependent reporter genes as in bladder cancer cell lines. Interestingly, Wnt-3a stimulated proliferation selectively in the AR-positive prostate cancer cell lines 22Rv1 and LNCaP, even though TCF-dependent reporter gene transcription was not induced in LNCaP cells. In summary, the data from our study support the idea that activation of WNT/beta-catenin signaling in AR-positive prostate cancer cells may predominantly act through AR-dependent mechanisms rather than classical TCF-dependent mechanisms.

Coordinate hypermethylation at specific genes in prostate carcinoma precedes LINE-1 hypomethylation.

In prostate carcinoma (PCa) increased DNA methylation ('hypermethylation') occurs at specific genes such as GSTP1. Nevertheless, overall methylation can be decreased ('hypomethylation') because methylation of repetitive sequences like LINE-1 retrotransposons is diminished. We analysed DNA from 113 PCa and 36 noncancerous prostate tissues for LINE-1 hypomethylation by a sensitive Southern technique and for hypermethylation at eight loci by methylation-specific PCR. Hypermethylation frequencies for GSTP1, RARB2, RASSF1A, and APC in carcinoma tissues were each >70%, strongly correlating with each other (P<10(-6)). Hypermethylation at each locus was significantly different between tumour and normal tissues (10(-11)82% of PCas. PCa may fall into three classes, that is, with few DNA methylation changes, with frequent hypermethylation, or with additional LINE-1 hypomethylation.

[Microarrays]. / MicroarraysTechnik und Potenzial beim Prostatakarzinom.

Microarrays allow a simultaneous gene expression analysis of thousands of genes, providing an expression profile of the specimen investigated. Thus, this procedure is well suited to characterize the complex genetic alterations of malignant tumors. Using unsupervised hierarchical cluster analysis, characteristic expression profiles for individuals, organs and tissues, as well as for different cell types, can be identified. Molecular signatures have been observed in tumors compared to normal tissue, for different tumor stages, risk groups or response therapy. In prostate cancer, many tumor-specific gene expression alterations have been identified. Among these, the cell surface protease hepsin and alpha-methyl-acryl-CoA-racemase might gain importance as diagnostic tools. Moreover, gene expression profiles were identified which are associated with advanced tumor stage, poor differentiation or progress after radical prostatectomy. Increased expression of enzymes of steroid biosynthesis and the androgen receptor appears to be part of the molecular signature of hormone refractory prostate cancer.