Summary The lack of in vitro prostate cancer models that recapitulate the diversity of human prostate cancer has hampered progress in understanding disease pathogenesis and therapy response. Using a 3D “organoid” system, we report success in long-term culture of prostate cancer from biopsy specimens and circulating tumor cells. The first seven fully characterized organoid lines recapitulate the molecular diversity of prostate cancer subtypes, including TMPRSS2-ERG fusion, SPOP mutation, SPINK1 overexpression and CHD1 loss. Whole exome sequencing shows a low mutational burden, consistent with genomics studies, but with mutations in FOXA1 and PIK3R1, as well as of DNA repair and chromatin modifier pathways that have been reported in advanced disease. Loss of p53 and RB tumor suppressor pathway function are the most common feature shared across the organoid lines. The methodology described here should enable the generation of a large repertoire of patient-derived prostate cancer lines amenable to genetic and pharmacologic studies.

Summary The prostate gland consists of basal and luminal cells arranged as pseudo-stratified epithelium. In tissue recombination models, only basal cells reconstitute a complete prostate gland, yet murine lineage-tracing experiments show that luminal cells generate basal cells. It has remained challenging to address the molecular details of these transitions and whether they apply to humans, due to the lack of culture conditions that recapitulate prostate gland architecture. Here we describe a 3D culture system that supports long-term expansion of primary mouse and human prostate organoids, composed of fully differentiated CK5+ basal and CK8+ luminal cells. Organoids are genetically stable, reconstitute prostate glands in recombination assays and can be experimentally manipulated. Single human luminal and basal cells give rise to organoids, yet luminal cell-derived organoids more closely resemble prostate glands. These data support a luminal multilineage progenitor cell model for prostate tissue and establish a robust, scalable system for mechanistic studies.

We demonstrate that the androgen receptor (AR) regulates a transcriptional program of DNA repair genes that promotes prostate cancer radioresistance, providing a potential mechanism by which androgen deprivation therapy (ADT) synergizes with ionizing radiation (IR). Using a model of castration-resistant prostate cancer, we show that second-generation antiandrogen therapy results in downregulation of DNA repair genes. Next, we demonstrate that primary prostate cancers display a significant spectrum of AR transcriptional output which correlates with expression of a set of DNA repair genes. Employing RNA-seq and ChIP-seq, we define which of these DNA repair genes are both induced by androgen and represent direct AR targets. We establish that prostate cancer cells treated with IR plus androgen demonstrate enhanced DNA repair and decreased DNA damage and furthermore that antiandrogen treatment causes increased DNA damage and decreased clonogenic survival. Finally, we demonstrate that antiandrogen treatment results in decreased classical non-homologous end joining.

Studies of ETS-mediated prostate oncogenesis have been hampered by the lack of suitable experimental systems. Here we describe a new conditional mouse model which gives robust, homogenous ERG expression throughout the prostate. When combined with homozygous Pten loss, mice developed accelerated, highly penetrant invasive prostate cancer. In mouse prostate tissue, ERG significantly increased androgen receptor (AR) binding. Robust ERG-mediated transcriptional changes, observed only in the setting of Pten loss, included restoration of AR transcriptional outut and genes involved in cell death, migration, inflammation and angiogenesis. Similarly, ETV1 positively regulated AR cistrome and transcriptional output in ETV1-translocated, PTEN-deficient human prostate cancer cells. In two large clinical cohorts, ERG and ETV1 expression correlated with higher AR transcriptional output in PTEN-negative prostate cancer specimens. We propose that ETS factors cause prostate-specific transformation by altering the AR cistrome, priming the prostate epithelium to respond to aberrant upstream signals such as PTEN loss.

The E2F transcription factors are critical regulators of genes required for appropriate progression through the cell cycle, and in special circumstances they can also promote the expression of another class of genes that function in the apoptotic program. Since E2Fs can initiate both cell proliferation and cell death, it is not surprising that the pro-apoptotic capacity of these proteins is subject to complex regulation. Recent study has expanded our knowledge both of the factors influencing E2F-induced apoptosis, as well as downstream targets of E2F in this process.

Tumor development is dependent upon the inactivation of two key tumor-suppressor networks, p16Ink4a -cycD/cdk4-pRB-E2F and p19Arf -mdm2-p53, that regulate cellular proliferation and the tumor surveillance response. These networks are known to intersect with one another, but the mechanisms are poorly understood. Here, we show that E2F directly participates in the transcriptional control of Arf in both normal and transformed cells. This occurs in a manner that is significantly different from the regulation of classic E2F-responsive targets. In wild-type mouse embryonic fibroblasts (MEFs), the Arf promoter is occupied by E2F3 and not other E2F family members. In quiescent cells, this role is largely fulfilled by E2F3b, an E2F3 isoform whose function was previously undetermined. E2f3 loss is sufficient to derepress Arf, triggering activation of p53 and expression of p21 Cip1. Thus, E2F3 is a key repressor of the p19 Arf -p53 pathway in normal cells. Consistent with this notion, Arf mutation suppresses the activation of p53 and p21Cip1 in E2f3-deficient MEFs. Arf loss also rescues the known cell cycle re-entry defect of E2f3 −/− cells, and this correlates with restoration of appropriate activation of classic E2F-responsive genes. Our data also demonstrate a direct role for E2F in the oncogenic activation of Arf. Specifically, we observe recruitment of the endogenous activating E2Fs, E2F1, and E2F3a, to the Arf promoter. Thus, distinct E2F complexes directly contribute to the normal repression and oncogenic activation of Arf. We propose that monitoring of E2F levels and/or activity is a key component of Arf's ability to respond to inappropriate, but not normal cellular proliferation. The development of mammalian tumors is dependent upon the disruption of two key biological activities, the control of cellular proliferation and the apoptotic response (Hanahan and Weinberg 2000). Remarkably, the Ink4a/Arf locus encodes two distinct tumor-suppressor proteins, p16Ink4a and p19 Arf (p14 Arf in humans), that influence one or both of these processes Sherr 2001). p16 Ink4a is a core component of the cell cycle control machinery (Sherr and Roberts 1999). It controls the activity of the G 1 kinase, cyclinD · cdk4/6, and consequently, the phosphorylation status of the pocket protein family. This family includes the retinoblastoma protein (pRB) tumor suppressor and its relatives, p107 and p130. In the unphosphorylated state, the pocket proteins bind to the E2F family of transcription factors and prevent the expression of genes that are essential for entry into, and passage through the cell cycle (Trimarchi and Lees 2002). This inhibition occurs through two distinct mechanisms. pRB binds to the activating E2Fs, E2F1, E2F2, and E2F3a, and blocks their transcriptional activity. At the same time, the repressive E2Fs, E2F4, and E2F5 recruit p107 or p130 and their associated histone deactylases to E2F-responsive promoters. Under these conditions, the cell is blocked in G 0 /G 1 . Mitogenic signaling activates cell cycle re-entry by ...

Half of all prostate cancers are caused by the TMPRSS2–ERG genefusion, which enables androgens to drive expression of the normally silent E26 transformation-specific (ETS) transcription factor ERG in prostate cells1,2. Recent genomic landscape studies of such cancers3–8 have reported recurrent point mutations and focal deletions of another ETS member, the ETS2 repressor factor ERF9. Here we show these ERF mutations cause decreased protein stability and mostly occur in tumours without ERG upregulation. ERF loss recapitulates the morphological and phenotypic features of ERG gain in normal mouse prostate cells, including expansion of the androgen receptor transcriptional repertoire, and ERF has tumour suppressor activity in the same genetic background of Pten loss that yields oncogenic activity by ERG. In the more common scenario of ERG upregulation, chromatin immunoprecipitation followed by sequencing indicates that ERG inhibits the ability of ERF to bind DNA at consensus ETS sites both in normal and in cancerous prostate cells. Consistent with a competition model, ERF overexpression blocks ERG-dependent tumour growth, and ERF loss rescues TMPRSS2–ERG-positive prostate cancer cells from ERG dependency. Collectively, these data provide evidence that the oncogenicity of ERG is mediated, in part, by competition with ERF and they raise the larger question of whether other gain-of-function oncogenic transcription factors might also inactivate endogenous tumour suppressors.

Ste5 is essential for pheromone response and binds components of a mitogen-activated protein kinase (MAPK) cascade: Ste11 (MEKK), Ste7 (MEK), and Fus3 (MAPK). Pheromone stimulation releases G␤␥ (Ste4-Ste18), which recruits Ste5 and Ste20 (p21-activated kinase) to the plasma membrane, activating the MAPK cascade. A RING-H2 domain in Ste5 (residues 177-229) negatively regulates Ste5 function and mediates its interaction with G␤␥. Ste5(C177A C180A), carrying a mutated RING-H2 domain, cannot complement a ste5⌬ mutation, yet supports mating even in ste4⌬ ste5⌬ cells when artificially dimerized by fusion to glutathione S-transferase (GST). In contrast, wild-type Ste5 fused to GST permits mating of ste5⌬ cells, but does not allow mating of ste4⌬ ste5⌬ cells. This differential behavior provided the basis of a genetic selection for STE5 gain-of-function mutations. MATa ste4⌬ ste5⌬ cells expressing Ste5-GST were mutagenized chemically and plasmids conferring the capacity to mate were selected. Three independent single-substitution mutations were isolated. These constitutive STE5 alleles induce cell cycle arrest, transcriptional activation, and morphological changes normally triggered by pheromone, even when G␤␥ is absent. The first, Ste5(C226Y), alters the seventh conserved position in the RING-H2 motif, confirming that perturbation of this domain constitutively activates Ste5 function. The second, Ste5(P44L), lies upstream of a basic segment, whereas the third, Ste5(S770K), is situated within an acidic segment in a region that contacts Ste7. None of the mutations increased the affinity of Ste5 for Ste11, Ste7, or Fus3. However, the positions of these novel-activating mutations suggested that, in normal Ste5, the N terminus may interact with the C terminus. Indeed, in vitro, GST-Ste5(1-518) was able to associate specifically with radiolabeled Ste5(520-917). Furthermore, both the P44L and S770K mutations enhanced binding of full-length Ste5 to GST-Ste5(1-518), whereas they did not affect Ste5 dimerization. Thus, binding of G␤␥ to the RING-H2 domain may induce a conformational change that promotes association of the N-and C-terminal ends of Ste5, stimulating activation of the MAPK cascade by optimizing orientation of the bound kinases and/or by increasing their accessibility to Ste20-dependent phosphorylation (or both). In accord with this model, the novel Ste5 mutants copurified with Ste7 and Fus3 in their activated state and their activation required Ste20. INTRODUCTIONThe pheromone response pathway of the yeast Saccharomyces cerevisiae has provided a system for elucidating mechanisms that convert an extracellular signal into both a morphological response and a change in the pattern of gene expression (reviewed in Bardwell et al., 1994;Leberer et al., 1997a). Mating of haploid cells (MATa and MAT␣) requires the action of peptide pheromones: MATa cells secrete a-factor, and MAT␣ cells secrete ␣-factor. The cell surface receptors for these peptides (Ste2 in MATa cells binds ␣-factor, and Ste3 in MAT␣ cells binds a-factor) a...