Identification of multipotent luminal progenitor cells in human prostate organoid cultures.

The prostate gland consists of basal and luminal cells arranged as pseudostratified 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.

Organoid cultures derived from patients with advanced prostate cancer.

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 in 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.

Glucocorticoid receptor confers resistance to antiandrogens by bypassing androgen receptor blockade.

The treatment of advanced prostate cancer has been transformed by novel antiandrogen therapies such as enzalutamide. Here, we identify induction of glucocorticoid receptor (GR) expression as a common feature of drug-resistant tumors in a credentialed preclinical model, a finding also confirmed in patient samples. GR substituted for the androgen receptor (AR) to activate a similar but distinguishable set of target genes and was necessary for maintenance of the resistant phenotype. The GR agonist dexamethasone was sufficient to confer enzalutamide resistance, whereas a GR antagonist restored sensitivity. Acute AR inhibition resulted in GR upregulation in a subset of prostate cancer cells due to relief of AR-mediated feedback repression of GR expression. These findings establish a mechanism of escape from AR blockade through expansion of cells primed to drive AR target genes via an alternative nuclear receptor upon drug exposure.

ERF mutations reveal a balance of ETS factors controlling prostate oncogenesis.

Half of all prostate cancers are caused by the TMPRSS2-ERG gene-fusion, which enables androgens to drive expression of the normally silent E26 transformation-specific (ETS) transcription factor ERG in prostate cells. Recent genomic landscape studies of such cancers have reported recurrent point mutations and focal deletions of another ETS member, the ETS2 repressor factor ERF. 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.

MAGI-2 scaffold protein is critical for kidney barrier function.

MAGUK Inverted 2 (MAGI-2) is a PTEN-interacting scaffold protein implicated in cancer on the basis of rare, recurrent genomic translocations and deletions in various tumors. In the renal glomerulus, MAGI-2 is exclusively expressed in podocytes, specialized cells forming part of the glomerular filter, where it interacts with the slit diaphragm protein nephrin. To further explore MAGI-2 function, we generated Magi-2-KO mice through homologous recombination by targeting an exon common to all three alternative splice variants. Magi-2 null mice presented with progressive proteinuria as early as 2 wk postnatally, which coincided with loss of nephrin expression in the glomeruli. Magi-2-null kidneys revealed diffuse podocyte foot process effacement and focal podocyte hypertrophy by 3 wk of age, as well as progressive podocyte loss. By 5.5 wk, coinciding with a near-complete loss of podocytes, Magi-2-null mice developed diffuse glomerular extracapillary epithelial cell proliferations, and died of renal failure by 3 mo of age. As confirmed by immunohistochemical analysis, the proliferative cell populations in glomerular lesions were exclusively composed of activated parietal epithelial cells (PECs). Our results reveal that MAGI-2 is required for the integrity of the kidney filter and podocyte survival. Moreover, we demonstrate that PECs can be activated to form glomerular lesions resembling a noninflammatory glomerulopathy with extensive extracapillary proliferation, sometimes resembling crescents, following rapid and severe podocyte loss.

Applying 89Zr-Transferrin To Study the Pharmacology of Inhibitors to BET Bromodomain Containing Proteins.

Chromatin modifying proteins are attractive drug targets in oncology, given the fundamental reliance of cancer on altered transcriptional activity. Multiple transcription factors can be impacted downstream of primary target inhibition, thus making it challenging to understand the driving mechanism of action of pharmacologic inhibition of chromatin modifying proteins. This in turn makes it difficult to identify biomarkers predictive of response and pharmacodynamic tools to optimize drug dosing. In this report, we show that (89)Zr-transferrin, an imaging tool we developed to measure MYC activity in cancer, can be used to identify cancer models that respond to broad spectrum inhibitors of transcription primarily due to MYC inhibition. As a proof of concept, we studied inhibitors of BET bromodomain containing proteins, as they can impart antitumor effects in a MYC dependent or independent fashion. In vitro, we show that transferrin receptor biology is inhibited in multiple MYC positive models of prostate cancer and double hit lymphoma when MYC biology is impacted. Moreover, we show that bromodomain inhibition in one lymphoma model results in transferrin receptor expression changes large enough to be quantified with (89)Zr-transferrin and positron emission tomography (PET) in vivo. Collectively, these data further underscore the diagnostic utility of the relationship between MYC and transferrin in oncology, and provide the rationale to incorporate transferrin-based PET into early clinical trials with bromodomain inhibitors for the treatment of solid tumors.

ETV1 is a lineage survival factor that cooperates with KIT in gastrointestinal stromal tumours.

Gastrointestinal stromal tumour (GIST) is the most common human sarcoma and is primarily defined by activating mutations in the KIT or PDGFRA receptor tyrosine kinases. KIT is highly expressed in interstitial cells of Cajal (ICCs)-the presumed cell of origin for GIST-as well as in haematopoietic stem cells, melanocytes, mast cells and germ cells. Yet, families harbouring germline activating KIT mutations and mice with knock-in Kit mutations almost exclusively develop ICC hyperplasia and GIST, suggesting that the cellular context is important for KIT to mediate oncogenesis. Here we show that the ETS family member ETV1 is highly expressed in the subtypes of ICCs sensitive to oncogenic KIT mediated transformation, and is required for their development. In addition, ETV1 is universally highly expressed in GISTs and is required for growth of imatinib-sensitive and resistant GIST cell lines. Transcriptome profiling and global analyses of ETV1-binding sites suggest that ETV1 is a master regulator of an ICC-GIST-specific transcription network mainly through enhancer binding. The ETV1 transcriptional program is further regulated by activated KIT, which prolongs ETV1 protein stability and cooperates with ETV1 to promote tumorigenesis. We propose that GIST arises from ICCs with high levels of endogenous ETV1 expression that, when coupled with an activating KIT mutation, drives an oncogenic ETS transcriptional program. This differs from other ETS-dependent tumours such as prostate cancer, melanoma and Ewing sarcoma where genomic translocation or amplification drives aberrant ETS expression. It also represents a novel mechanism of oncogenic transcription factor activation.

Cabozantinib resolves bone scans in tumor-naïve mice harboring skeletal injuries.

The receptor tyrosine kinase inhibitor cabozantinib (XL184, BMS-907351 Cometriq) has displayed impressive clinical activity against several indications, culminating in its recent approval for medullary thyroid cancer. Among malignancies with tropism for the bone (prostate, breast), one striking feature of early clinical reports about this drug has been the rapid and complete resolution of bone scans, a phenomenon almost never observed even among therapies already shown to confer survival benefit. In castration-resistant prostate cancer, not all conventional response indicators change as dramatically posttreatment, raising the possibility that cabozantinib may impair the ability of bone-seeking radionuclides to integrate within the remodeling bone. To test this hypothesis, we surgically induced bone remodeling via physical insult in non-tumor-bearing mice and performed 18F-sodium fluoride (18F-NaF) positron emission tomographic (PET) and technetium 99m-methylene diphosphonate (99mTc-MDP) single-photon emission computed tomographic (SPECT) scans pre- and posttreatment with cabozantinib and related inhibitors. A consistent reduction in the accumulation of either radiotracer at the site of bone remodeling was observed in animals treated with cabozantinib. Given that cabozantinib is known to inhibit several receptor tyrosine kinases, we drugged animals with various permutations of more selective inhibitors to attempt to refine the molecular basis of bone scan resolution. Neither the vascular endothelial growth factor receptor (VEGFR) inhibitor axitinib, the MET inhibitor crizotinib, nor the combination was capable of inhibiting 18F-NaF accumulation at known bioactive doses. In summary, although the mechanism by which cabozantinib suppresses radionuclide incorporation into foci undergoing bone remodeling remains unknown, that this phenomenon occurs in tumor-naïve models indicates that caution should be exercised in interpreting the clinical significance of this event.

Noninvasive measurement of androgen receptor signaling with a positron-emitting radiopharmaceutical that targets prostate-specific membrane antigen.

Despite encouraging clinical results with next generation drugs (MDV3100 and abiraterone) that inhibit androgen receptor (AR) signaling in patients with castration-resistant prostate cancer (CRPC), responses are variable and short-lived. There is an urgent need to understand the basis of resistance to optimize their future use. We reasoned that a radiopharmaceutical that measures intratumoral changes in AR signaling could substantially improve our understanding of AR pathway directed therapies. Expanding on previous observations, we first show that prostate-specific membrane antigen (PSMA) is repressed by androgen treatment in multiple models of AR-positive prostate cancer in an AR-dependent manner. Conversely, antiandrogens up-regulate PSMA expression. These expression changes, including increased PSMA expression in response to treatment with the antiandrogen MDV3100, can be quantitatively measured in vivo in human prostate cancer xenograft models through PET imaging with a fully humanized, radiolabeled antibody to PSMA, (64)Cu-J591. Collectively, these results establish that relative changes in PSMA expression levels can be quantitatively measured using a human-ready imaging reagent and could serve as a biomarker of AR signaling to noninvasively evaluate AR activity in patients with CRPC.

Timing of treatment shapes the path to androgen receptor signaling inhibitor resistance in prostate cancer.

There is optimism that cancer drug resistance can be addressed through appropriate combination therapy, but success requires understanding the growing complexity of resistance mechanisms, including the evolution and population dynamics of drug-sensitive and drug-resistant clones over time. Using DNA barcoding to trace individual prostate tumor cells in vivo , we find that the evolutionary path to acquired resistance to androgen receptor signaling inhibition (ARSI) is dependent on the timing of treatment. In established tumors, resistance occurs through polyclonal adaptation of drug-sensitive clones, despite the presence of rare subclones with known, pre-existing ARSI resistance. Conversely, in an experimental setting designed to mimic minimal residual disease, resistance occurs through outgrowth of pre-existing resistant clones and not by adaptation. Despite these different evolutionary paths, the underlying mechanisms responsible for resistance are shared across the two evolutionary paths. Furthermore, mixing experiments reveal that the evolutionary path to adaptive resistance requires cooperativity between subclones. Thus, despite the presence of pre-existing ARSI-resistant subclones, acquired resistance in established tumors occurs primarily through cooperative, polyclonal adaptation of drug-sensitive cells. This tumor ecosystem model of resistance has new implications for developing effective combination therapy.

Constitutively active androgen receptor splice variants expressed in castration-resistant prostate cancer require full-length androgen receptor.

Androgen receptor (AR) splice variants lacking the ligand binding domain (ARVs), originally isolated from prostate cancer cell lines derived from a single patient, are detected in normal and malignant human prostate tissue, with the highest levels observed in late stage, castration-resistant prostate cancer. The most studied variant (called AR-V7 or AR3) activates AR reporter genes in the absence of ligand and therefore, could play a role in castration resistance. To explore the range of potential ARVs, we screened additional human and murine prostate cancer models using conventional and next generation sequencing technologies and detected several structurally diverse AR isoforms. Some, like AR-V7/AR3, display gain of function, whereas others have dominant interfering activity. We also find that ARV expression increases acutely in response to androgen withdrawal, is suppressed by testosterone, and in some models, is coupled to full-length AR (AR-FL) mRNA production. As expected, constitutively active, ligand-independent ARVs such as AR-V7/AR3 are sufficient to confer anchorage-independent (in vitro) and castration-resistant (in vivo) growth. Surprisingly, this growth is blocked by ligand binding domain-targeted antiandrogens, such as MDV3100, or by selective siRNA silencing of AR-FL, indicating that the growth-promoting effects of ARVs are mediated through AR-FL. These data indicate that the increase in ARV expression in castrate-resistant prostate cancer is an acute response to castration rather than clonal expansion of castration or antiandrogen-resistant cells expressing gain of function ARVs, and furthermore, they provide a strategy to overcome ARV function in the clinic.

HER2/neu kinase-dependent modulation of androgen receptor function through effects on DNA binding and stability.

Given the role of the EGFR/HER2 family of tyrosine kinases in breast cancer, we dissected the molecular basis of EGFR/HER2 kinase signaling in prostate cancer. Using the small molecule dual EGFR/HER2 inhibitor PKI-166, we show that the biologic effects of EGFR/HER-2 pathway inhibition are caused by reduced AR transcriptional activity. Additional genetic and pharmacologic experiments show that this modulation of AR function is mediated by the HER2/ERBB3 pathway, not by EGFR. This HER2/ERBB3 signal stabilizes AR protein levels and optimizes binding of AR to promoter/enhancer regions of androgen-regulated genes. Surprisingly, the downstream signaling pathway responsible for these effects appears to involve kinases other than Akt. These data suggest that the HER2/ERBB3 pathway is a critical target in hormone-refractory prostate cancer.

Myc-driven murine prostate cancer shares molecular features with human prostate tumors.

Increased Myc gene copy number is observed in human prostate cancer. To define Myc's functional role, we generated transgenic mice expressing human c-Myc in the mouse prostate. All mice developed murine prostatic intraepithelial neoplasia followed by invasive adenocarcinoma. Microarray-based expression profiling identified a Myc prostate cancer expression signature, which included the putative human tumor suppressor NXK3.1. Human prostate tumor databases revealed modules of human genes that varied in concert with the Myc prostate cancer signature. This module includes the Pim-1 kinase, a gene known to cooperate with Myc in tumorigenesis, and defines a subset of human, "Myc-like" human cancers. This approach illustrates how genomic technologies can be applied to mouse cancer models to guide evaluation of human tumor databases.

Loss of CHD1 Promotes Heterogeneous Mechanisms of Resistance to AR-Targeted Therapy via Chromatin Dysregulation.

Metastatic prostate cancer is characterized by recurrent genomic copy number alterations that are presumed to contribute to resistance to hormone therapy. We identified CHD1 loss as a cause of antiandrogen resistance in an in vivo small hairpin RNA (shRNA) screen of 730 genes deleted in prostate cancer. ATAC-seq and RNA-seq analyses showed that CHD1 loss resulted in global changes in open and closed chromatin with associated transcriptomic changes. Integrative analysis of this data, together with CRISPR-based functional screening, identified four transcription factors (NR3C1, POU3F2, NR2F1, and TBX2) that contribute to antiandrogen resistance, with associated activation of non-luminal lineage programs. Thus, CHD1 loss results in chromatin dysregulation, thereby establishing a state of transcriptional plasticity that enables the emergence of antiandrogen resistance through heterogeneous mechanisms.

Tumor Microenvironment-Derived NRG1 Promotes Antiandrogen Resistance in Prostate Cancer.

Despite the development of second-generation antiandrogens, acquired resistance to hormone therapy remains a major challenge in treating advanced prostate cancer. We find that cancer-associated fibroblasts (CAFs) can promote antiandrogen resistance in mouse models and in prostate organoid cultures. We identify neuregulin 1 (NRG1) in CAF supernatant, which promotes resistance in tumor cells through activation of HER3. Pharmacological blockade of the NRG1/HER3 axis using clinical-grade blocking antibodies re-sensitizes tumors to hormone deprivation in vitro and in vivo. Furthermore, patients with castration-resistant prostate cancer with increased tumor NRG1 activity have an inferior response to second-generation antiandrogen therapy. This work reveals a paracrine mechanism of antiandrogen resistance in prostate cancer amenable to clinical testing using available targeted therapies.

GREB1 amplifies androgen receptor output in human prostate cancer and contributes to antiandrogen resistance.

Genomic amplification of the androgen receptor (AR) is an established mechanism of antiandrogen resistance in prostate cancer. Here, we show that the magnitude of AR signaling output, independent of AR genomic alteration or expression level, also contributes to antiandrogen resistance, through upregulation of the coactivator GREB1. We demonstrate 100-fold heterogeneity in AR output within human prostate cancer cell lines and show that cells with high AR output have reduced sensitivity to enzalutamide. Through transcriptomic and shRNA knockdown studies, together with analysis of clinical datasets, we identify GREB1 as a gene responsible for high AR output. We show that GREB1 is an AR target gene that amplifies AR output by enhancing AR DNA binding and promoting EP300 recruitment. GREB1 knockdown in high AR output cells restores enzalutamide sensitivity in vivo. Thus, GREB1 is a candidate driver of enzalutamide resistance through a novel feed forward mechanism.

Transgenic mouse model for rapid pharmacodynamic evaluation of antiandrogens.

Persistent androgen receptor signaling has been implicated as a critical factor in prostate cancer progression even at the hormone-refractory stage and provides strong rationale for developing novel androgen receptor antagonists. Traditional models for in vivo evaluation of antiandrogens are cumbersome because they rely on physiologic end points, such as the size of androgen-dependent tissues. Here, we describe a transgenic mouse (ARR2 Pb-Lux) that expresses luciferase specifically in the prostate in an androgen-dependent fashion. This signal is reduced by castration or by treatment with bicalutamide and can be quantified through noninvasive bioluminescent imaging. ARR2 Pb-Lux mice provide a novel method for rapid pharmacodynamic evaluation of novel pharmacologic compounds designed to inhibit androgen receptor signaling.

COP1/DET1/ETS axis regulates ERK transcriptome and sensitivity to MAPK inhibitors.

Aberrant activation of MAPK signaling leads to the activation of oncogenic transcriptomes. How MAPK signaling is coupled with the transcriptional response in cancer is not fully understood. In 2 MAPK-activated tumor types, gastrointestinal stromal tumor and melanoma, we found that ETV1 and other Pea3-ETS transcription factors are critical nuclear effectors of MAPK signaling that are regulated through protein stability. Expression of stabilized Pea3-ETS factors can partially rescue the MAPK transcriptome and cell viability after MAPK inhibition. To identify the players involved in this process, we performed a pooled genome-wide RNAi screen using a fluorescence-based ETV1 protein stability sensor and identified COP1, DET1, DDB1, UBE3C, PSMD4, and COP9 signalosome members. COP1 or DET1 loss led to decoupling between MAPK signaling and the downstream transcriptional response, where MAPK inhibition failed to destabilize Pea3 factors and fully inhibit the MAPK transcriptome, thus resulting in decreased sensitivity to MAPK pathway inhibitors. We identified multiple COP1 and DET1 mutations in human tumors that were defective in the degradation of Pea3-ETS factors. Two melanoma patients had de novo DET1 mutations arising after vemurafenib treatment. These observations indicate that MAPK signaling-dependent regulation of Pea3-ETS protein stability is a key signaling node in oncogenesis and therapeutic resistance to MAPK pathway inhibition.

Context-dependent hormone-refractory progression revealed through characterization of a novel murine prostate cancer cell line.

Insights into the molecular basis of hormone-refractory prostate cancer have principally relied on human prostate cancer cell lines, all of which were derived from patients who had already failed hormonal therapy. Recent progress in developing genetically engineered mouse prostate cancer models provides an opportunity to isolate novel cell lines from animals never exposed to hormone ablation, avoiding any potential bias conferred by the selective pressure of the castrate environment. Here we report the isolation of such a cell line (Myc-CaP) from a c-myc transgenic mouse with prostate cancer. Myc-CaP cells have an amplified androgen receptor gene despite no prior exposure to androgen withdrawal and they retain androgen-dependent transgene expression as well as androgen-dependent growth in soft agar and in mice. Reexpression of c-Myc from a hormone-independent promoter rescues growth in androgen-depleted agar but not in castrated mice, showing a clear distinction between the molecular requirements for hormone-refractory growth in vitro versus in vivo. Myc-CaP cells represent a unique reagent for dissecting discreet steps in hormone-refractory prostate cancer progression and show the general utility of using genetically engineered mouse models for establishing new prostate cancer cell lines.

Deletion of 3p13-14 locus spanning FOXP1 to SHQ1 cooperates with PTEN loss in prostate oncogenesis.

A multigenic locus at 3p13-14, spanning FOXP1 to SHQ1, is commonly deleted in prostate cancer and lost broadly in a range of cancers but has unknown significance to oncogenesis or prognosis. Here, we report that FOXP1-SHQ1 deletion cooperates with PTEN loss to accelerate prostate oncogenesis and that loss of component genes correlates with prostate, breast, and head and neck cancer recurrence. We demonstrate that Foxp1-Shq1 deletion accelerates prostate tumorigenesis in mice in combination with Pten loss, consistent with the association of FOXP1-SHQ1 and PTEN loss observed in human cancers. Tumors with combined Foxp1-Shq1 and Pten deletion show increased proliferation and anaplastic dedifferentiation, as well as mTORC1 hyperactivation with reduced Akt phosphorylation. Foxp1-Shq1 deletion restores expression of AR target genes repressed in tumors with Pten loss, circumventing PI3K-mediated repression of the androgen axis. Moreover, FOXP1-SHQ1 deletion has prognostic relevance, with cancer recurrence associated with combined loss of PTEN and FOXP1-SHQ1 genes.