Tumor Evolution and Drug Response in Patient-Derived Organoid Models of Bladder Cancer.

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.

TP53 loss-of-function causes vulnerability to autophagy inhibition in aggressive prostate cancer.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSION: Our results support the potential use of autophagy inhibitors in prostate cancers that contain TP53 loss-of-function mutations.

Prostate organoid technology - the new POT of gold in prostate stem cell and cancer research.

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.

Nkx3.1 controls the DNA repair response in the mouse prostate.

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.

Exploring prostate cancer in the post-genomic era.

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.

OncoLoop: A Network-Based Precision Cancer Medicine Framework.

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.

Modeling prostate cancer: What does it take to build an ideal tumor model?

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.

Novel Mouse Models of Bladder Cancer Identify a Prognostic Signature Associated with Risk of Disease Progression.

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.

Single-cell analysis supports a luminal-neuroendocrine transdifferentiation in human prostate cancer.

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.

The role of Id-1 in chemosensitivity and epirubicin-induced apoptosis in bladder cancer cells.

Recurrence and progression are the major problems in the treatment of bladder cancer. Increased expression of Id-1, a basic helix-loop-helix transcription factor, has recently been shown in several types of advanced cancer. Some studies have provided evidence to suggest that Id-1 can be considered a potential therapeutic target. The objective of this study was to investigate the role of Id-1 in the chemosensitivity of bladder cancer cells, and the effect of Id-1 on chemotherapeutic drug-induced apoptosis in bladder cancer cells. We compared the different sensitivity to epirubicin in RT112 and MGH-U1 cell lines with different Id-1 expression. Then, we transfected different vectors into RT112 and MGH-U1 respectively, and generated the stable Id-1 up-regulation and down-regulation transfectants. The results of cell viability assay showed up-regulation of Id-1 in RT112 leading to increased sensitivity in response to epirubicin, and down-regulation of Id-1 increased cellular sensitivity to epirubicin. Furthermore, the analysis of apoptosis related protein revealed that up-regulation of Id-1 suppressed epirubicin-induced apoptosis and down-regulation of Id-1 leading to increased epirubicin-induced apoptosis. Wound closure assay showed up-regulation of Id-1 leading to improved migration abilities of bladder cancer cells under chemotherapy. Our results suggest that up-regulation of Id-1 in bladder cancer cells lead to increased cell viability in response to epirubicin by its improved anti-apoptotic role, and down-regulation of Id-1 increases cellular sensitivity to epirubicin by increased anticancer drug-induced apoptosis.

An Organoid Assay for Long-Term Maintenance and Propagation of Mouse Prostate Luminal Epithelial Progenitors and Cancer Cells.

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.

TWIST modulates prostate cancer cell-mediated bone cell activity and is upregulated by osteogenic induction.

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.

Evidence of a novel docetaxel sensitizer, garlic-derived S-allylmercaptocysteine, as a treatment option for hormone refractory prostate cancer.

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.

Decreased adhesiveness, resistance to anoikis and suppression of GRP94 are integral to the survival of circulating tumor cells in prostate cancer.

The presence of circulating tumor cells (CTC) is common in prostate cancer patients, however until recently their clinical significance was unknown. The CTC stage is essential for the formation of distant metastases, and their continuing presence after radical prostatectomy has been shown to predict recurrent or latent disease. Despite their mechanistic and prognostic importance, due both to their scarcity and difficulties in their isolation, little is known about the characteristics that enable their production and survival. The aim of this study was to investigate the molecular mechanisms underlying the survival of CTC cells. A novel CTC cell line from the bloodstream of an orthotopic mouse model of castration-resistant prostate cancer was established and compared with the primary tumor using attachment assays, detachment culture, Western blot, flow cytometry and 2D gel electrophoresis. Decreased adhesiveness and expression of adhesion molecules E-cadherin, beta4-integrin and gamma-catenin, together with resistance to detachment and drug-induced apoptosis and upregulation of Bcl-2 were integral to the development of CTC and their survival. Using proteomic studies, we observed that the GRP94 glycoprotein was suppressed in CTC. GRP94 was also shown to be suppressed in a tissue microarray study of 79 prostate cancer patients, indicating its possible role in prostate cancer progression. Overall, this study suggests molecular alterations accounting for the release and survival of CTC, which may be used as drug targets for either anti-metastatic therapy or the suppression of latent disease. We also indicate the novel involvement of GRP94 suppression in prostate cancer metastasis.

Garlic-derived S-allylmercaptocysteine is a novel in vivo antimetastatic agent for androgen-independent prostate cancer.

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.

Differential requirements of androgen receptor in luminal progenitors during prostate regeneration and tumor initiation.

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.

Significance of TWIST expression and its association with E-cadherin in bladder cancer.

Recently, TWIST, a basic helix-loop-helix transcription factor, has been reported to play a key role in the metastatic progression of several types of human cancer. The aim of this study was to investigate the significance of TWIST expression in bladder cancer using tissue microassays generated from 226 bladder tissue specimens. Using immunohistochemical staining, we studied TWIST expression levels in nonmalignant bladder tissues (n = 37), primary bladder cancer tissues (n = 164), and 25 cases of matched lymph node metastatic lesions. The association between TWIST expression levels and tumor staging and grading, as well as metastatic potential, was analyzed by statistical analysis. Our results showed that TWIST protein expression was significantly higher in bladder cancer specimens compared with nonmalignant tissues (P < .001), indicating its positive role in the development of bladder cancer. In addition, increased TWIST expression levels were associated with advanced-stage and high-grade tumors, suggesting its involvement in the progression of this cancer. Furthermore, TWIST expression was much higher in the metastatic lesion compared with its primary site (P < .05). More importantly, the increased TWIST expression in bladder cancer specimens was correlated with decreased membranous expression of E-cadherin, a cell adhesion molecule that plays a key role in the metastatic progression of human cancer. Our results demonstrate TWIST as a novel positive factor in the development and progression of bladder cancer and suggest a marker for advanced bladder cancer.

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.

Significance of Id-1 up-regulation and its association with EGFR in bladder cancer cell invasion.

Epidermal growth factor receptor (EGFR) is suggested to be one of the positive factors in the invasive progression of bladder cancer. Id-1 (inhibitor of differentiation or DNA binding), a helix-loop-helix (HLH) transcription factor, was recently identified as a key factor in the EGFR signalling pathway. The aim of this study was to investigate the role of Id-1 in bladder cancer progression and its relation-ship with EGFR. Using clinical specimens from different stages of bladder cancer, immunohistochemical staining was performed to determine if Id-1 expression was positively associated with tumour staging and EGFR expression. The direct role of Id-1 in cancer cell invasion was also investigated through ectopically expressing the Id-1 gene in a RT112 bladder cancer cell line by wound closure and collagen invasion assays. To explore the therapeutic potential of targeting the Id-1 gene in the treatment of invasive bladder cancer, we studied if inactivation of the Id-1 gene through small RNA interference could lead to the suppression of invasion in a MGHU1 bladder cancer cell line. Our results showed that the up-regulation of Id-1 was associated with increased EGFR expression, clinical staging and the invasion ability of bladder cancer cells. Inactivation of Id-1 may be a potential therapeutic target to inhibit the invasion by bladder cancer cells.