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TP53 loss-of-function is commonly found in aggressive prostate cancer. However, a highly-efficient therapy for this tumor subtype is still lacking. In this study, we investigated the relationship between TP53 mutation status and autophagy in prostate cancer and assessed the efficacy of autophagy inhibitors on TP53-deficient tumors.We first evaluated the expression patterns of p53 and autophagy-related proteins, namely LC3B, ULK1 and BECLIN1, as well as their relationship in treatment-naïve and castration-resistant prostate cancer specimens through immunohistochemistry. Subsequently, we generated a Trp53-deleted genetically-engineered mouse model, established prostate tumor organoid lines from the mice and assessed the efficacy of autophagy inhibitors in overcoming Enzalutamide resistance in the tumor organoid model. We also investigated the impact of TP53 re-expression in modulating responses to autophagy inhibitors using LNCaP cell line, which harbored a TP53 missense mutation. Lastly, we attempted to identify potential autophagy-related genes that were crucial for TP53-deficient tumor maintenance.TP53 loss-of-function was associated with increased levels of autophagy-related proteins in aggressive prostate cancers and Trp53-deleted genetically-engineered mouse-derived tumors. Moreover, the generated androgen receptor-independent tumor organoids were highly vulnerable to autophagy inhibition. Upon TP53 re-expression, not only did the surviving LNCaP cells demonstrate resistance, but they also showed growth advantage in response to autophagy inhibition. Lastly, PEX14, an important peroxisomal regulator was differentially upregulated in aggressive tumors with TP53 loss-of-function mutations, thus implying the importance of peroxisome turnover in this tumor subtype.Our results support the potential use of autophagy inhibitors in prostate cancers that contain TP53 loss-of-function mutations.

Index of Supplementary Data

Supplementary Table 3: Transcriptomic analyses of the human patient samples A. TCGA Interactome B. SU2C Interactome C. Protein activity TCGA D. Protein Activity SU2C

Prioritizing treatments for individual patients with cancer remains challenging, and performing coclinical studies using patient-derived models in real time is often unfeasible. To circumvent these challenges, we introduce OncoLoop, a precision medicine framework that predicts drug sensitivity in human tumors and their preexisting high-fidelity (cognate) model(s) by leveraging drug perturbation profiles. As a proof of concept, we applied OncoLoop to prostate cancer using genetically engineered mouse models (GEMM) that recapitulate a broad spectrum of disease states, including castration-resistant, metastatic, and neuroendocrine prostate cancer. Interrogation of human prostate cancer cohorts by Master Regulator (MR) conservation analysis revealed that most patients with advanced prostate cancer were represented by at least one cognate GEMM-derived tumor (GEMM-DT). Drugs predicted to invert MR activity in patients and their cognate GEMM-DTs were successfully validated in allograft, syngeneic, and patient-derived xenograft (PDX) models of tumors and metastasis. Furthermore, OncoLoop-predicted drugs enhanced the efficacy of clinically relevant drugs, namely, the PD-1 inhibitor nivolumab and the AR inhibitor enzalutamide.Significance:OncoLoop is a transcriptomic-based experimental and computational framework that can support rapid-turnaround coclinical studies to identify and validate drugs for individual patients, which can then be readily adapted to clinical practice. This framework should be applicable in many cancer contexts for which appropriate models and drug perturbation data are available.This article is highlighted in the In This Issue feature, p. 247

Detailed Materials and Methods

Figure S1: Genomic alterations in prostate cancer represented in the GEMMs (related to Fig. 2). Figure S2: Additional phenotypic analyses of the GEMMs (related to Fig. 2). Figure S3: Phenotypic analysis of allograft and organoid models (related to Fig. 2). Figure S4: Additional transcriptomic analyses of the GEMMs (related to Fig. 3). Figure S5: Analyses of AR activity in GEMMS (related to Fig. 3). Figure S6: Regulatory sub-networks of the GEMM clusters (related to Fig. 3). Figure S7. MR-match of prostate cancer cells lines to human PCa (related to Figs. 5). Figure S8. Drug perturbation protein activity profiles from DU145 cells (related to Figs. 5, 6, 7). Figure S9. LNCaP Pharmacotyping to patients and GEMMs (related to Figs. 5). Figure S10: Additional validation of drug candidates (related to Figs 6, 7). Figure S11: Additional validation of drug candidates (related to Figs 6).

Video of mouse procedures for post-surgical steps

Video of mouse surgical procedures for transurethral delivery of Adenovirus to bladder

Video of mouse procedures for intravesical delivery of 4-hydroxy tamoxifen into bladder

Detailed procedures to accompany the videos

To study the progression of bladder cancer from non–muscle-invasive to muscle-invasive disease, we have developed a novel toolkit that uses complementary approaches to achieve gene recombination in specific cell populations in the bladder urothelium in vivo, thereby allowing us to generate a new series of genetically engineered mouse models (GEMM) of bladder cancer. One method is based on the delivery of adenoviruses that express Cre recombinase in selected cell types in the urothelium, and a second uses transgenic drivers in which activation of inducible Cre alleles can be limited to the bladder urothelium by intravesicular delivery of tamoxifen. Using both approaches, targeted deletion of the Pten and p53 tumor suppressor genes specifically in basal urothelial cells gave rise to muscle-invasive bladder tumors. Furthermore, preinvasive lesions arising in basal cells displayed upregulation of molecular pathways related to bladder tumorigenesis, including proinflammatory pathways. Cross-species analyses comparing a mouse gene signature of early bladder cancer with a human signature of bladder cancer progression identified a conserved 28-gene signature of early bladder cancer that is associated with poor prognosis for human bladder cancer and that outperforms comparable gene signatures. These findings demonstrate the relevance of these GEMMs for studying the biology of human bladder cancer and introduce a prognostic gene signature that may help to stratify patients at risk for progression to potentially lethal muscle-invasive disease.Significance:Analyses of bladder cancer progression in a new series of genetically engineered mouse models has identified a gene signature of poor prognosis in human bladder cancer.

Video of mouse surgical procedures for delivery of Adenovirus into the bladder lumen.

This file contains an index of all supplementary materials, and the supplementary figures, legends, and tables.

Purpose: Previously, FTY720 was found to possess potent anticancer effects on various types of cancer. In the present study, we aimed to first verify the role of Runx2 in prostate cancer progression and metastasis, and, subsequently, assessed if FTY720 could modulate Runx2 expression, thus interfering downstream events regulated by this protein.Experimental Design: First, the association between Runx2 and prostate cancer progression was assessed using localized prostate cancer specimens and mechanistic investigation of Runx2-induced cancer aggressiveness was then carried out. Subsequently, the effect of FTY720 on Runx2 expression and transcriptional activity was investigated using PC-3 cells, which highly expressed Runx2 protein. Last, the involvement of Runx2 in FTY720-induced anticancer effects was evaluated by modulating Runx2 expression in various prostate cancer cell lines.Results: Runx2 nuclear expression was found to be up-regulated in prostate cancer and its expression could be used as a predictor of metastasis in prostate cancer. Further mechanistic studies indicated that Runx2 accelerated prostate cancer aggressiveness through promotion of cadherin switching, invasion toward collagen I, and Akt activation. Subsequently, we found that FTY720 treatment down-regulated Runx2 expression and its transcriptional activity, as well as inhibited its regulated downstream events. More importantly, silencing Runx2 in PC-3 enhanced FTY720-induced anticancer effects as well as cell viability inhibition, whereas overexpressing Runx2 in 22Rv1 that expressed very low endogenous Runx2 protein conferred resistance in the same events.Conclusion: This study provided a novel mechanism for the anticancer effect of FTY720 on advanced prostate cancer, thus highlighting the therapeutic potential of this drug in treating this disease.

Supplementary Data from Suppression of Androgen-Independent Prostate Cancer Cell Aggressiveness by FTY720: Validating Runx2 as a Potential Antimetastatic Drug Screening Platform

Supplementary of differential gene expression and pathway analyses

Supplementary Figure S1 from Up-Regulation of TWIST in Prostate Cancer and Its Implication as a Therapeutic Target

Androgen-independent metastatic prostate cancer is the main obstacle in the treatment of this cancer. Unlike a majority of solid cancers, prostate cancer usually shows poor response to chemotherapeutic drugs. In this study, we have shown a potential novel target, TWIST, a highly conserved bHLH transcription factor, in the treatment of prostate cancer. Using malignant and nonmalignant prostate tissues, we found that TWIST expression was highly expressed in the majority (90%) of prostate cancer tissues but only in a small percentage (6.7%) of benign prostate hyperplasia. In addition, the TWIST expression levels were positively correlated with Gleason grading and metastasis, indicating its role in the development and progression of prostate cancer. Furthermore, down-regulation of TWIST through small interfering RNA in androgen-independent prostate cancer cell lines, DU145 and PC3, resulted in increased sensitivity to the anticancer drug taxol-induced cell death which was associated with decreased Bcl/Bax ratio, leading to activation of the apoptosis pathway. More importantly, inactivation of TWIST suppressed migration and invasion abilities of androgen-independent prostate cancer cells, which was correlated with induction of E-cadherin expression as well as morphologic and molecular changes associated with mesenchymal to epithelial transition. These results were further confirmed on the androgen-dependent LNCaP cells ectopically expressing the TWIST protein. Our results have identified TWIST as a critical regulator of prostate cancer cell growth and suggest a potential therapeutic approach to inhibit the growth and metastasis of androgen-independent prostate cancer through inactivation of the TWIST gene.

Prioritizing treatments for individual patients with cancer remains challenging, and performing coclinical studies using patient-derived models in real time is often unfeasible. To circumvent these challenges, we introduce OncoLoop, a precision medicine framework that predicts drug sensitivity in human tumors and their preexisting high-fidelity (cognate) model(s) by leveraging drug perturbation profiles. As a proof of concept, we applied OncoLoop to prostate cancer using genetically engineered mouse models (GEMM) that recapitulate a broad spectrum of disease states, including castration-resistant, metastatic, and neuroendocrine prostate cancer. Interrogation of human prostate cancer cohorts by Master Regulator (MR) conservation analysis revealed that most patients with advanced prostate cancer were represented by at least one cognate GEMM-derived tumor (GEMM-DT). Drugs predicted to invert MR activity in patients and their cognate GEMM-DTs were successfully validated in allograft, syngeneic, and patient-derived xenograft (PDX) models of tumors and metastasis. Furthermore, OncoLoop-predicted drugs enhanced the efficacy of clinically relevant drugs, namely, the PD-1 inhibitor nivolumab and the AR inhibitor enzalutamide. Significance: OncoLoop is a transcriptomic-based experimental and computational framework that can support rapid-turnaround coclinical studies to identify and validate drugs for individual patients, which can then be readily adapted to clinical practice. This framework should be applicable in many cancer contexts for which appropriate models and drug perturbation data are available. This article is highlighted in the In This Issue feature, p. 247

To study the progression of bladder cancer from non–muscle-invasive to muscle-invasive disease, we have developed a novel toolkit that uses complementary approaches to achieve gene recombination in specific cell populations in the bladder urothelium in vivo, thereby allowing us to generate a new series of genetically engineered mouse models (GEMM) of bladder cancer. One method is based on the delivery of adenoviruses that express Cre recombinase in selected cell types in the urothelium, and a second uses transgenic drivers in which activation of inducible Cre alleles can be limited to the bladder urothelium by intravesicular delivery of tamoxifen. Using both approaches, targeted deletion of the Pten and p53 tumor suppressor genes specifically in basal urothelial cells gave rise to muscle-invasive bladder tumors. Furthermore, preinvasive lesions arising in basal cells displayed upregulation of molecular pathways related to bladder tumorigenesis, including proinflammatory pathways. Cross-species analyses comparing a mouse gene signature of early bladder cancer with a human signature of bladder cancer progression identified a conserved 28-gene signature of early bladder cancer that is associated with poor prognosis for human bladder cancer and that outperforms comparable gene signatures. These findings demonstrate the relevance of these GEMMs for studying the biology of human bladder cancer and introduce a prognostic gene signature that may help to stratify patients at risk for progression to potentially lethal muscle-invasive disease. Significance: Analyses of bladder cancer progression in a new series of genetically engineered mouse models has identified a gene signature of poor prognosis in human bladder cancer.

In the Special Issue on Prostate Cancer, we have invited 25 researchers or clinicians from prostate cancer community to review the cutting-edge topics in this field. In particular, the mini-reviews have covered various basic science and clinical aspects in prostate cancer, including prostate epithelial stem cells or progenitors, androgen and androgen receptor pathways, tumor modeling, genomics, different cell-autonomous and non-cell-autonomous mechanisms as well as various clinical issues encompassing diagnosis, risk stratification and treatments.

Prostate cancer is frequently characterized as a multifocal disease with great intratumoral heterogeneity as well as a high propensity to metastasize to bone. Consequently, modeling prostate tumor has remained a challenging task for researchers in this field. In the past decades, genomic advances have led to the identification of key molecular alterations in prostate cancer. Moreover, resistance towards second-generation androgen-deprivation therapy, namely abiraterone and enzalutamide has unveiled androgen receptor-independent diseases with distinctive histopathological and clinical features. In this review, we have critically evaluated the commonly used preclinical models of prostate cancer with respect to their capability of recapitulating the key genomic alterations, histopathological features and bone metastatic potential of human prostate tumors. In addition, we have also discussed the potential use of the emerging organoid models in prostate cancer research, which possess clear advantages over the commonly used preclinical tumor models. We anticipate that no single model can faithfully recapitulate the complexity of prostate cancer, and thus, propose the use of a cost- and time-efficient integrated tumor modeling approach for future prostate cancer investigations.

Organoids are self-organized cellular clusters in three-dimensional culture, which can be derived from a single stem cell, progenitor or cell clusters of different lineages resembling in vivo tissue architecture of an organ. In the recent years, organoids technology has contributed to the revolutionary changes in stem cell and cancer fields. In this review, we have briefly overviewed the emerging landscape of prostate organoid technology (POT) in prostate research. In addition, we have also summarized the potential application of POT in the understanding of prostate stem cell and cancer biology and the discovery of novel therapeutic strategies for prostate cancer. Lastly, we have critically discussed key challenges that lie in the current state of POT and provided a future perspective on the second-generation of POT, which should better recapitulate cellular behaviors and drug responses of prostate cancer patients.

Neuroendocrine prostate cancer is one of the most aggressive subtypes of prostate tumor. Although much progress has been made in understanding the development of neuroendocrine prostate cancer, the cellular architecture associated with neuroendocrine differentiation in human prostate cancer remain incompletely understood. Here, we use single-cell RNA sequencing to profile the transcriptomes of 21,292 cells from needle biopsies of 6 castration-resistant prostate cancers. Our analyses reveal that all neuroendocrine tumor cells display a luminal-like epithelial phenotype. In particular, lineage trajectory analysis suggests that focal neuroendocrine differentiation exclusively originate from luminal-like malignant cells rather than basal compartment. Further tissue microarray analysis validates the generality of the luminal phenotype of neuroendocrine cells. Moreover, we uncover neuroendocrine differentiation-associated gene signatures that may help us to further explore other intrinsic molecular mechanisms deriving neuroendocrine prostate cancer. In summary, our single-cell study provides direct evidence into the cellular states underlying neuroendocrine transdifferentiation in human prostate cancer., Using single-cell RNA sequencing, Dong, Miao, Wang et al. profile the transcriptomes of 21,292 cells from biopsies of 6 castration-resistant prostate cancers. They find that all neuroendocrine tumor cells display a luminal-like epithelial phenotype, providing insights into the cellular states underlying neuroendocrine transdifferentiation in human prostate cancer.

Background: Uterine clear cell carcinoma (UCCC) is a rare histological type of endometrial cancer with poor prognosis and high risk of tumor relapse. Although adjuvant chemotherapy (CT) and/or radiotherapy (RT) are often recommended for early stage UCCC patients, the effectiveness of these treatment strategies remains unclear. Methods: Systematic review and meta-analysis were applied to evaluate treatment-related outcomes of stage I-II UCCC patients. Search strategy was applied using electronic databases until June 1st, 2019. Inclusion criteria were retrospective, observational and prospective studies that reported outcome of UCCC patients receiving adjuvant therapy. Clinical endpoints like overall survival (OS) and progression-free survival (PFS) were evaluated. Data were extracted by two independent reviewers and a meta-analysis was performed. Results: 12 articles with a total of 3845 patients were analyzed. Overall, adjuvant CT after surgery could improve 5 year-OS significantly compared to patients without CT (odds ratio (OR): 0.75, 95% confidence interval (CI): 0.58-0.95; p=0.02). In comparison, RT could also improve OS in UCCC patients of early stage (OR: 0.61, 95% CI: 0.46-0.82; p=0.001) compared to the patients without RT, in US and Europe patients. Lastly, when comparing the patients undergoing both CT and RT with those receiving CT or RT alone, no further improvement in OS was observed (OR: 0.95, 95% CI: 0.53-1.72; P=0.88). Conclusions: Either CT or RT after surgery could improve the OS of early stage of UCCC patients. However, combinatorial CT and RT treatment did not improve the OS compared with CT or RT treatment alone.

The basal cell compartment in many epithelial tissues is generally believed to serve as an important pool of stem cells. However, basal cells are heterogenous and the stem cell subpopulation within basal cells is not well elucidated. Here we uncover that the core epithelial-to-mesenchymal transition (EMT) inducer Zeb1 is expressed in a prostate basal cell subpopulation. The Zeb1+ prostate epithelial cells are multipotent prostate basal stem cells (PBSCs) that can self-renew and generate functional prostatic glandular structures at the single-cell level. Genetic ablation studies reveal an indispensable role for Zeb1 in prostate basal cell development. Utilizing unbiased single-cell transcriptomic analysis of over 9000 mouse prostate basal cells, we confirm the existence of the Zeb1+ basal cell subset. Moreover, Zeb1+ epithelial cells can be detected in mouse and human prostate tumors. Identification of the PBSC and its transcriptome profile is crucial to advance our understanding of prostate development and tumorigenesis., Heterogeneous populations of basal cells in the prostate epithelium contain stem cells. Here the authors show that Zeb1 marks a pool of prostate epithelial stem cells that self-renew, generate prostate glandular structures with all 3 epithelial cell types and are required for prostate basal cell development.

The basal cell compartment in many epithelial tissues such as the prostate, bladder, and mammary gland are generally believed to serve as an important pool of stem cells. However, basal cells are heterogenous and the stem cell subpopulation within basal cells is not well elucidated. Here we uncover that the core epithelial-to-mesenchymal transition (EMT) inducer Zeb is exclusively expressed in a prostate basal cell subpopulation based on both immunocytochemical and cell lineage tracing analysis. The Zeb1+prostate epithelial cells are multipotent prostate basal stem cells (PBSCs) that can self-renew and generate functional prostatic glandular structures with all three epithelial cell types at the single-cell level. Genetic ablation studies reveal an indispensable role for Zeb1 in prostate basal cell development. Utilizing unbiased single cell transcriptomic analysis of over 9000 mouse prostate basal cells, we find that Zeb1+basal cell subset shares gene expression signatures with both epithelial and mesenchymal cells and stands out uniquely among all the basal cell clusters. Moreover, Zeb1+epithelial cells can be detected in mouse and clinical samples of prostate tumors. Identification of the PBSC and its transcriptome profile is crucial to advance our understanding of prostate development and tumorigenesis.

Historically, prostate luminal epithelial progenitors and cancer cells have been difficult to culture, thus hampering the generation of representative models for the study of prostate homeostasis, epithelial lineage hierarchy relationship and cancer drug efficacy assessment. Here, we describe a newly developed culture methodology that can efficiently grow prostate luminal epithelial progenitors and cancer cells as organoids. Notably, the organoid assay favors prostate luminal cell growth, thus minimizing basal cell dominance upon the establishment and continuous propagation of prostate epithelial cells. Importantly, organoids cultured under this condition have demonstrated preservation of androgen responsiveness and intact androgen receptor signaling, providing a representative system to study castration resistance and androgen receptor independence.

Historically, prostate luminal epithelial progenitors and cancer cells have been difficult to culture, thus hampering the generation of representative models for the study of prostate homeostasis, epithelial lineage hierarchy relationship and cancer drug efficacy assessment. Here, we describe a newly developed culture methodology that can efficiently grow prostate luminal epithelial progenitors and cancer cells as organoids. Notably, the organoid assay favors prostate luminal cell growth, thus minimizing basal cell dominance upon the establishment and continuous propagation of prostate epithelial cells. Importantly, organoids cultured under this condition have demonstrated preservation of androgen responsiveness and intact androgen receptor signaling, providing a representative system to study castration resistance and androgen receptor independence.

BACKGROUND The human prostate tumor suppressor NKX3.1 mediates the DNA repair response and interacts with the androgen receptor to assure faithful completion of transcription thereby protecting against TMPRSS2-ERG gene fusion. To determine directly the effect of Nkx3.1 in vivo we studied the DNA repair response in prostates of mice with targeted deletion of Nkx3.1. METHODS Using both drug-induced DNA damage and γ-irradiation, we assayed expression of γ-histone 2AX at time points up to 24 hr after induction of DNA damage. RESULTS We demonstrated that expression of Nkx3.1 influenced both the timing and magnitude of the DNA damage response in the prostate. CONCLUSIONS Nkx3.1 affects the DNA damage response in the murine prostate and is haploinsufficient for this phenotype. Prostate © 2015 The Authors. The Prostate published by Wiley Periodicals, Inc.

Master regulatory genes of tissue specification play key roles in stem/progenitor cells and are often important in cancer. In the prostate, androgen receptor (AR) is a master regulator essential for development and tumorigenesis, but its specific functions in prostate stem/progenitor cells have not been elucidated. We have investigated AR function in CARNs (CAstration-Resistant Nkx3.1-expressing cells), a luminal stem/progenitor cell that functions in prostate regeneration. Using genetically--engineered mouse models and novel prostate epithelial cell lines, we find that progenitor properties of CARNs are largely unaffected by AR deletion, apart from decreased proliferation in vivo. Furthermore, AR loss suppresses tumor formation after deletion of the Pten tumor suppressor in CARNs; however, combined Pten deletion and activation of oncogenic Kras in AR-deleted CARNs result in tumors with focal neuroendocrine differentiation. Our findings show that AR modulates specific progenitor properties of CARNs, including their ability to serve as a cell of origin for prostate cancer., eLife digest Most prostate tumors rely on male hormones – called androgens – to survive. Aggressive prostate cancer is often treated with drugs that block androgens, which usually cause the prostate tumors to shrink. One class of the drugs works by binding to and inactivating the androgen receptor protein on prostate cancer cells. However, aggressive prostate tumors can often become resistant to these anti-androgen therapies. It is not clear where the resistant cancer cells come from. In 2009, researchers showed that the normal prostate contains some cells that appear to be independent of androgens. A subset of these cells – also known as CARNs – can act as stem or progenitor cells that can repair the prostate after injury. These normal androgen-independent cells can also be the cells from which prostate tumors arise. Here, Chua et al. – including one of the researchers from the 2009 study – investigated how these CARN cells behave when the androgen receptor is deleted. When the androgen receptor was genetically removed in CARN cells of otherwise healthy mice, the behavior of CARN cells was unaffected. When the androgen receptor was deleted together with a protein that normally suppresses the formation of tumors, it protected the mice from prostate cancer. However, Chua et al. also observed that deleting the androgen receptor could not prevent the tumor from growing when two cancer-causing mutations were present. These tumors were similar to human prostate tumors that are resistant to anti-androgen therapy. Since CARN cells may also exist in humans, this new way of making prostate cancers in mice may be used to study how these resistances arise in patients. A better understanding of how prostate tumors develop might lead to new treatments in which the androgen receptor is blocked in combination with other new protein targets.

Summary Bladder cancer is the fifth most prevalent cancer in the U.S., yet is understudied, and few laboratory models exist that reflect the biology of the human disease. Here, we describe a biobank of patient-derived organoid lines that recapitulates the histopathological and molecular diversity of human bladder cancer. Organoid lines can be established efficiently from patient biopsies acquired before and after disease recurrence and are interconvertible with orthotopic xenografts. Notably, organoid lines often retain parental tumor heterogeneity and exhibit a spectrum of genomic changes that are consistent with tumor evolution in culture. Analyses of drug response using bladder tumor organoids show partial correlations with mutational profiles, as well as changes associated with treatment resistance, and specific responses can be validated using xenografts in vivo . Our studies indicate that patient-derived bladder tumor organoids represent a faithful model system for studying tumor evolution and treatment response in the context of precision cancer medicine.

The intrinsic ability to display self-organizing morphogenetic properties in ex vivo culture may represent a general property of tissue stem cells. Here we show that single luminal stem/progenitor cells can generate prostate organoids in a three-dimensional culture system in the absence of stroma. Organoids generated from CARNs (castration-resistant Nkx3.1-expressing cells) or normal prostate epithelium exhibit tissue architecture containing luminal and basal cells, undergo long-term expansion in culture, and display functional androgen receptor signaling. Lineage-tracing demonstrates that luminal cells are favored for organoid formation, and generate basal cells in culture. Furthermore, tumor organoids can initiate from CARNs after oncogenic transformation, and from mouse models of prostate cancer, and can facilitate analyses of drug response. Finally, we provide evidence supporting the feasibility of organoid studies of human prostate tissue. Our studies underscore the progenitor properties of luminal cells, and identify in vitro approaches for studying prostate biology.

correlated with disease-free (p 10 had a 0 risk score, whereas a patient with a node positive, pT3/4 and an immune marker score

One of the common features in advanced prostate cancer is bone metastasis. In this study, we investigated the clinical relevance of a bone factor, MSX2, in predicting the metastatic ability of prostate adenocarcinoma. Evaluation of MSX2 expression was performed using prostate cell lines as well as patient specimens. A sharp decrease in MSX2 was found in primary prostate cancer cells, 22Rv1, when compared with the non-malignant counterparts, followed by a gradual increase in more aggressive prostate cancer cell lines. Interestingly, the MSX2 protein was upregulated and predominantly expressed in the nucleus in aggressive prostate cancer cell line, C4-2b, compared with the less aggressive 22Rv1. Consistent with the in vitro results, MSX2 nuclear expression was significantly higher in nodular hyperplasia when compared with high-grade prostatic intraepithelial neoplasia (PIN), while MSX2 nuclear expression in prostate adenocarcinoma was higher than that in high-grade PIN. Importantly, MSX2 expression was increased significantly in tumors with metastasis compared with those without metastasis. Finally, MSX2 nuclear scores were significantly increased in patients with preoperative serum PSA >20 ng/mL. No correlation between MSX2 nuclear score and Gleason score was found. Taken together, MSX2 may serve as a potential biomarker in predicting primary prostate tumors with higher metastatic capability.

TWIST, a helix-loop-helix transcription factor, is highly expressed in many types of human cancer. We have previously found that TWIST confers prostate cancer cells with an enhanced metastatic potential through promoting epithelial-mesenchymal transition (EMT) and a high TWIST expression in human prostate cancer is associated with an increased metastatic potential. The predilection of prostate cancer cells to metastasize to bone may be due to two interplaying mechanisms (i) by increasing the rate of bone remodeling and (ii) by undergoing osteomimicry. We further studied the role of TWIST in promoting prostate cancer to bone metastasis. TWIST expression in PC3, a metastatic prostate cancer cell line, was silenced by small interfering RNA and we found that conditioned medium from PC3 with lower TWIST expression had a lower activity on stimulating osteoclast differentiation and higher activity on stimulating osteoblast mineralization. In addition, we found that these effects were, at least partly, associated with TWIST-induced expression of dickkopf homolog 1 (DKK-1), a factor that promotes osteolytic metastasis. We also examined TWIST and RUNX2 expressions during osteogenic induction of an organ-confined prostate cancer cell, 22Rv1. We observed increased TWIST and RUNX2 expressions upon osteogenic induction and downregulation of TWIST through short hairpin RNA reduced the induction level of RUNX2. In summary, our results suggest that, in addition to EMT, TWIST may also promote prostate cancer to bone metastasis by modulating prostate cancer cell-mediated bone remodeling via regulating the expression of a secretory factor, DKK-1, and enhancing osteomimicry of prostate cancer cells, probably, via RUNX2.

The recent introduction of docetaxel in the treatment of hormone refractory prostate cancer (HRPC) has made a small but significant impact on patient survival. However, its effect is limited by intolerance and resistance. The aim of our study was to investigate if the garlic-derived compound, S-allylmercaptocysteine (SAMC), was able to act as a docetaxel sensitizing agent. First, the effect of SAMC on docetaxel sensitivity was examined on 3 HRPC cell lines by colony forming assay. We found that SAMC increased the efficacy of docetaxel on colony forming inhibition by 9-50% compared to single agent treatment. Second, using the HRPC CWR22R nude mice model, we found that the combination of SAMC and docetaxel was 53% more potent than docetaxel alone (p = 0.037). In addition, there was no additive toxicity in the mice treated with the combination therapy evidenced by histological and functional analysis of liver, kidney and bone marrow. These results suggest that SAMC is able to increase the anticancer effect of docetaxel without causing additional toxic effect in vivo. Third, flow cytometry and Western blotting analysis on HRPC cell lines demonstrated that SAMC promoted docetaxel-induced G2/M phase cell cycle arrest and apoptotic induction. In addition, immunohistochemistry on CWR22R xenograft revealed a suppression of Bcl-2 expression and upregulation of E-cadherin in the SAMC and docetaxel treated animals. These results suggest that SAMC may promote docetaxel-induced cell death through promoting G2/M cell cycle arrest and apoptosis. Our study implies a potential role for SAMC in improving docetaxel based chemotherapy for the treatment of HRPC.

Aim: Development of metastasis is one of the main causes of prostatic cancer-related death. We have previously found that up-regulation of TWIST, a highly conserved basic helix–loop–helix transcription factor, in prostatic cancer cells can promote epithelial to mesenchymal transition through down-regulation of E-cadherin. The present study aimed to investigate the prognostic significance of TWIST and to correlate TWIST and E-cadherin expression in prostatic cancer specimens. Methods and results: TWIST and E-cadherin expression was studied in 115 prostatic cancer specimens, eight cases of prostatic intraepithelial neoplasia and 37 cases of benign prostatic hyperplasia by immunohistochemistry. Increased cytoplasmic expression of TWIST was associated with malignant transformation of prostatic epithelium and histological progression of prostatic cancer, while nuclear TWIST expression was significant in predicting the metastatic potential of the primary prostatic cancer. In addition, high levels of TWIST expression were also significantly associated with aberrant E-cadherin expression. Conclusions: These results suggest that TWIST may serve as a prognostic marker for high-grade prostatic cancer. In addition, up-regulation of TWIST in combination with aberrant E-cadherin expression in primary prostatic cancer specimens may predict development of distal metastatic disease.

Purpose: There is epidemiologic evidence that high garlic consumption decreases the incidence of prostate cancer, and compounds isolated from garlic have been shown to have cancer-preventive and tumor-suppressive effects. Recent in vitro studies in our laboratory have shown that garlic-derived organosulfur compound S-allylmercaptocysteine suppresses invasion and cell motility of androgen-independent prostate cancer cells via the up-regulation of cell-adhesion molecule E-cadherin. S-allylmercaptocysteine is therefore a potential antimetastatic drug with broad clinical applications that we tested in vivo for the first time in this study.Experimental Design: We used a newly established fluorescent orthotopic androgen-independent prostate cancer mouse model to assess the ability of S-allylmercaptocysteine to inhibit tumor growth and dissemination.Results: We showed that oral S-allylmercaptocysteine not only inhibited the growth of primary tumors by up to 71% (P < 0.001) but also reduced the number of lung and adrenal metastases by as much as 85.5% (P = 0.001) without causing notable toxicity. This metastatic suppression was accompanied by a 91% reduction of viable circulating tumor cells (P = 0.041), suggesting that S-allylmercaptocysteine prevents dissemination by decreasing tumor cell intravasation.Conclusions: Our results provide in vivo evidence supporting the potential use of S-allylmercaptocysteine as an E-cadherin up-regulating antimetastatic agent for the treatment of androgen-independent prostate cancer. This is the first report of the in vivo antimetastatic properties of garlic, which may also apply to other cancer types.

Aims: Epidermal growth factor receptor (EGFR) is suggested as one of the positive regulators in the invasive progression of renal cell cancer (RCC). Recently, Id-1 (inhibitor of differentiation or DNA binding), a helix-loop-helix transcription factor, has been identified as one of the key factors in the EGFR signalling pathway. The aim of this study was to investigate the significance of Id-1 expression in renal cell cancer and to study its relationship with EGFR. Methods and results: Id-1 and EGFR expression was examined in tissue microarray (TMA) samples of 107 RCC and 32 normal kidney specimens by immunohistochemistry. Relative Id-1 and EGFR protein expression was quantified by estimating the staining intensity on a four-grade scale. We found that while negative to weak expression of Id-1 and EGFR was observed in non-malignant kidney tissues, most RCCs showed significant positive Id-1 and EGFR expression in tumour cells. In addition, Id-1 immunostaining intensity was positively associated with increased tumour staging, grading and EGFR expression. Conclusion: Overexpression of Id-1 is a novel marker for advanced RCC which is positively correlated with EGFR expression. Our results suggest that Id-1 may play an important role in the development of RCC and indicate that Id-1 is a potential marker of patients with a poor prognosis.

A major cause of treatment failure for prostate cancer is the development of androgen-independent metastatic disease. Id protein family, a group of basic helix-loop-helix transcription factors, has been shown to be involved in carcinogenesis and a prognostic marker in several types of human cancers. In this study, we examined the expressions of four Id proteins, Id-1, -2, -3 and -4, in 125 clinical prostate cancer specimens as well as 40 nodular hyperplasia specimens by immunohistochemistry. The expressions of Id proteins were correlated with Gleason grading and metastatic progress of the tumors. We found that Id proteins were dysregulated in prostate cancer. Id-1 and -2 expressions were elevated while Id-3 and -4 expressions were reduced in prostate cancers compared to nodular hyperplasia. Cytoplasmic staining of Id-1 (P=0.013) and nuclear staining of Id-2 (P=0.001) and Id-4 (P

Androgen-independent metastatic prostate cancer is the main obstacle in the treatment of this cancer. Unlike a majority of solid cancers, prostate cancer usually shows poor response to chemotherapeutic drugs. In this study, we have shown a potential novel target, TWIST, a highly conserved bHLH transcription factor, in the treatment of prostate cancer. Using malignant and nonmalignant prostate tissues, we found that TWIST expression was highly expressed in the majority (90%) of prostate cancer tissues but only in a small percentage (6.7%) of benign prostate hyperplasia. In addition, the TWIST expression levels were positively correlated with Gleason grading and metastasis, indicating its role in the development and progression of prostate cancer. Furthermore, down-regulation of TWIST through small interfering RNA in androgen-independent prostate cancer cell lines, DU145 and PC3, resulted in increased sensitivity to the anticancer drug taxol-induced cell death which was associated with decreased Bcl/Bax ratio, leading to activation of the apoptosis pathway. More importantly, inactivation of TWIST suppressed migration and invasion abilities of androgen-independent prostate cancer cells, which was correlated with induction of E-cadherin expression as well as morphologic and molecular changes associated with mesenchymal to epithelial transition. These results were further confirmed on the androgen-dependent LNCaP cells ectopically expressing the TWIST protein. Our results have identified TWIST as a critical regulator of prostate cancer cell growth and suggest a potential therapeutic approach to inhibit the growth and metastasis of androgen-independent prostate cancer through inactivation of the TWIST gene.

FTY720, a derivative of fungus, has demonstrated dramatic anticancer effect in several malignancies recently. Our study evaluates the therapeutic potential of FTY720 in the treatment of androgen-independent prostate cancer using a human prostate cancer xenograft in nude mice. CWR22R, an androgen-independent human prostate tumor xenograft was inoculated into castrated nude mice and the animals were administrated with either normal saline or FTY720 (10 mg/kg) through intraperitoneal (i.p.) injection for 20 days. Body weight and tumor volume were recorded every 2 days, and serum prostate specific antigen (PSA) levels were also measured before and after the treatment. The effect of FTY720 on tumor cell proliferation was examined using antibodies against PCNA and Ki-67 by immunohistochemical staining, MTT assay and colony forming assay, whereas apoptotic effect of FTY720 was evaluated by TUNEL assay and immunostaining using antibodies against cleaved caspase 3 and Bcl-2. In addition, the potential inhibitory effect of FTY720 on prostate cancer angiogenesis and metastasis was investigated by immunostaining of CD31, VEGF, E-cadherin and beta-catenin. Our results showed that FTY720 treatment led to suppression of CWR22R tumor growth without causing any detectable side effects in nude mice. The FTY720-induced tumor suppression was correlated with decreased serum PSA level as well as reduced proliferation rate, suppression of angiogenic factors, and restoration of E-cadherin and beta-catenin expression. In addition, the FTY720-treated tumors showed increased apoptosis rate demonstrated by increased TUNEL- and cleaved caspase 3-positive cells, and decreased Bcl-2 expression. Our results suggest a potential novel agent in the suppression of androgen-independent prostate cancer.