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Figure S1: Additional analyses of NP and NPp53 GEM models Figure S2: Tolerance and efficacy of abiraterone in mouse prostate Figure S3: Additional analyses of tumor volumes in GEM models before and after treatment Figure S4: Analyses of abiraterone treatment of cell lines from NP and NPp53 tumors Figure S5: Examples of alternative histopathologies of NPp53 CRPC Figure S6: Additional cross-species computational analyses Figure S7: Additional analyses of expression of SOX11 and other NE genes Table S1: Description of human datasets used in this study Table S2: Summary of preclinical phenotypic analyses Table S3: Summary of histopathology of individual mouse tumors Table S4: List of master regulators Table S5: Quantitation of synaptophysin-positive cells in mouse CRPC Table S6: List of antibodies used in this study Table S7: List of primers and shRNA used in this study Detailed experimental procedures

Differentially expressed genes for mouse CRPC tumors

Prostate cancer (PCa) is the most common non-cutaneous cancer in men in Europe and is predicted to exhibit declining mortality in the European Union (EU) due to various recent improvements in treatment. The goal of this short review is to give insight into the European treatment landscape of PCa, while focusing on improvements in care.

PDF file, 3314K, Representative IHC analysis of ALDH1A1 expression in large tissues prostate sections.

PDF file, 46K, Analysis of the expression of ALDH specific isoforms in BPH and PCa surgical specimens.

PDF file, 39K, Characteristics of PCa patients.

PDF file, 1824K, Differential expression and localization of ALDH1A1 and ALDH7A1 proteins in BPH.

PDF file, 41K.

Purpose: High aldehyde dehydrogenase (ALDH) has been suggested to selectively mark cells with high tumorigenic potential in established prostate cancer cell lines. However, the existence of cells with high ALDH activity (ALDHbright) in primary prostate cancer specimens has not been shown so far. We investigated the presence, phenotype, and clinical significance of ALDHbright populations in clinical prostate cancer specimens.Experimental Design: We used ALDEFLUOR technology and fluorescence-activated cell-sorting (FACS) staining to identify and characterize ALDHbright populations in cells freshly isolated from clinical prostate cancer specimens. Expression of genes encoding ALDH-specific isoforms was evaluated by quantitative real-time PCR in normal prostate, benign prostatic hyperplasia (BPH), and prostate cancer tissues. ALDH1A1-specific expression and prognostic significance were assessed by staining two tissue microarrays that included more than 500 samples of BPH, prostatic intraepithelial neoplasia (PIN), and multistage prostate cancer.Results: ALDHbright cells were detectable in freshly excised prostate cancer specimens (n = 39) and were mainly included within the EpCAM(+) and Trop2(+) cell populations. Although several ALDH isoforms were expressed to high extents in prostate cancer, only ALDH1A1 gene expression significantly correlated with ALDH activity (P < 0.01) and was increased in cancers with high Gleason scores (P = 0.03). Most importantly, ALDH1A1 protein was expressed significantly more frequently and at higher levels in advanced-stage than in low-stage prostate cancer and BPH. Notably, ALDH1A1 positivity was associated with poor survival (P = 0.02) in hormone-naïve patients.Conclusions: Our data indicate that ALDH contributes to the identification of subsets of prostate cancer cells of potentially high clinical relevance. Clin Cancer Res; 19(19); 5361–71. ©2013 AACR.

Item does not contain fulltext Patient-derived xenografts (PDXs) are generated by engrafting human tumours into mice. Serially transplantable PDXs are used to study tumour biology and test therapeutics, linking the laboratory to the clinic. Although few prostate cancer PDXs are available in large repositories, over 330 prostate cancer PDXs have been established, spanning broad clinical stages, genotypes and phenotypes. Nevertheless, more PDXs are needed to reflect patient diversity, and to study new treatments and emerging mechanisms of resistance. We can maximize the use of PDXs by exchanging models and datasets, and by depositing PDXs into biorepositories, but we must address the impediments to accessing PDXs, such as institutional, ethical and legal agreements. Through collaboration, researchers will gain greater access to PDXs representing diverse features of prostate cancer. 01 juni 2023

Patient‐derived organoids (PDOs) represent promising preclinical models in various tumor types. In the context of prostate cancer (PCa), however, their establishment has been hampered by poor success rates, which impedes their broad use for translational research applications. Along with the necessity to improve culture conditions, there is a need to identify factors influencing outcomes and to determine how to assess success versus failure in organoid generation. In the present study, we report our unbiased efforts to generate PDOs from a cohort of 81 PCa specimens with diverse pathological and clinical features. We comprehensively analyzed histological features of each enrolled sample (Gleason score, tumor content, proliferation index) and correlated them with organoid growth patterns. We identified improved culture conditions favoring the generation of PCa organoids, yet no specific intrinsic tumor feature was broadly associated with sustained organoid growth. In addition, we performed phenotypic and molecular characterization of tumor–organoid pairs using immunohistochemistry, immunofluorescence, fluorescence in situ hybridization, and targeted sequencing. Morphological and immunohistochemical profiles of whole organoids altogether provided a fast readout to identify the most promising ones. Notably, primary samples were associated with an initial take‐rate of 83% (n = 60/72) in culture, with maintenance of cancer cells displaying common PCa alterations, such as PTEN loss and ERG overexpression. These cancer organoids were, however, progressively overgrown by organoids with a benign‐like phenotype. Finally, out of nine metastasis samples, we generated a novel organoid model derived from a hormone‐naïve lung metastasis, which displays alterations in the PI3K/Akt and Wnt/β‐catenin pathways and responds to androgen deprivation. Taken together, our comprehensive study explores determinants of outcome and highlights the opportunities and challenges associated with the establishment of stable tumor organoid lines derived from PCa patients. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Introduction: Muscular Invasive Bladder Cancer (MIBC) is a highly heterogeneous disease. A consensus classification, based on gene expression profiling, has identified six MIBC subgroups, ranging from basal/squamous (Ba/Sq) to luminal papillary (LumP). This classification is a static representation of the disease and does not account for cellular plasticity, which may induce phenotype switch and cancer progression. Here, we aimed at identifying factors involved in cellular plasticity using bladder cancer patient-derived organoids (PDO) models. Methods: Bladder cancer RNA sequencing data from bulk and single cell experiments were downloaded from public repositories. Samples from both datasets were classified using the consensus molecular classification. Pathway activation scores and cell-cell interaction scores were calculated for each subtype. Differential gene expression analysis was performed with the R packages edgeR and Seurat. MIBC samples were collected from bladder cancer patients undergoing surgical treatment. The samples were mechanically and enzymatically digested to generate a cell suspension which was seeded in Matrigel and immersed in growth-factor supplemented medium. Phenotypical characterization of long-term PDO lines was performed through immunofluorescence (IF). The PDOs were classified as Luminal, Basal or Mixed, based on the number of cells expressing the luminal marker CK8 or the basal marker CK5. Three selected organoids lines were cultured in alginate, immersed in a serum-free chemically-defined medium. Results: Long-term PDO lines were successfully generated from 6 out of 13 MIBC samples. Organoids from the same line showed a consistent expression of basal and luminal markers. One line was classified as Basal, 2 lines as Mixed and 3 lines as Luminal. Exploring bulk RNAseq and scRNAseq datasets, we identified several genes whose expression was significantly altered in Ba/Sq and LumP MIBC subtypes. In particular, SHH and related BMP genes were significantly less expressed in Ba/Sq samples than in LumP. In addition, we observed an overexpression of BMP3 in LumP samples and of TGFβ2 in Ba/Sq samples. As preliminary test, we formulated a chemically-defined medium containing inhibitors of the proteins encoded by the identified genes (TGFβ inhibitor and Noggin); using this medium, we were able to maintain organoids from a Luminal, Basal and Mixed subtype in culture for up to two weeks. Interestingly, after two weeks of culture, IF analysis highlighted the presence of cells expressing luminal markers in the basal organoid line, suggesting culture-induced change of phenotype. Conclusions: Bladder cancer PDOs represent promising models to study MIBC plasticity. The integration of RNA sequencing data and the successful culture of PDOs in chemically-defined culture conditions may allow to identify candidate factors impacting the phenotype of bladder cancer cells. Citation Format: Michele Garioni, Luca Roma, Renaud Mével, Tatjana Vlajnic, Heike Pueschel, Dagmar Iber, Hans-Helge Seifert, Cyrill A. Rentsch, Lukas Bubendorf, Clémentine Le Magnen. Bladder cancer patient-derived organoids to decipher cellular plasticity and cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3073.

Introduction: An important clinical need remains to improve survival of men with advanced metastatic prostate cancer (PCa). A better understanding of the mechanisms underlying PCa progression and treatment resistance is a prerequisite to address this challenge, which is currently limited by the scarcity of experimental models. Here, we aimed at establishing and characterizing new patient-derived organoids xenograft (PDOX) models of advanced PCa. Materials & methods: We used two patient-derived organoid lines (PDOs), previously generated in our laboratory. P20-11 PDOs were derived from a lung metastasis obtained from a patient with hormone-naïve PCa. P20-23 PDOs were derived from a transurethral prostate resection obtained from a patient with metastatic castration-resistant PCa, previously treated with goserelin, docetaxel, and enzalutamide. PDOX were generated by subcutaneous injection of PDOs in NOD scid gamma (NSG) male mice. Organoids were derived from the PDOX tumors (PDOX-O). Matched patients’ tumor, PDOs, PDOX and PDOX-O samples were characterized using immunohistochemistry (IHC), immunofluorescence (IF), and whole exome sequencing (WES). Organoids were treated with different concentrations of drugs and cell viability was measured using CellTiter-Glo 3D, after a 5-day long treatment. Results: P20-11 PDOs developed tumors in 2 out of 6 mice, 10 months after injection. IHC analysis highlighted a loss of PTEN expression, overexpression of ERG and P53, as well as strong AR and NKX3.1 expression in the tumor, PDOs, PDOX and PDOX-O samples. WES analysis uncovered mutations in CTNNB1, PTEN and TP53 in all samples, and approximately 87% of shared non-synonymous mutations between the PDOX and the original patients’ tumor. Similar to the original PDOs, P20-11 PDOX-O displayed androgen sensitivity in vitro. P20-23 PDOs formed tumors in 3 out of 3 mice 8 months following injection. IHC and IF analyses highlighted a strong expression of CK8, PSMA, AR and NKX3.1, as well as a loss of PTEN expression in the tumor, PDOs, PDOX and PDOX-O samples. WES identified a pathogenic mutation in the PCa-associated gene ZMYM3 in all samples, associated with loss of protein expression. The activating AR point mutation L702H, previously linked to AR signaling inhibitors resistance, was detected in the tumor and in 2 out of 3 PDOX and their derived organoids. Overall, an average of 80% of non-synonymous mutations were shared between the patients’ tumor and PDOX. Finally, P20-23 PDOX-O did not respond to docetaxel or enzalutamide but exhibited sensitivity to the PI3K/AKT inhibitor ipatasertib. Conclusion: We have successfully generated two novel PDOX models, which highly resemble the original patients’ tumor and can be further cultured as organoids. These models are representative of relevant clinical and molecular subtypes of advanced PCa, providing further opportunities for translational studies. Citation Format: Raphaëlle Servant, Zoi Diamantopoulou, Michele Garioni, Luca Roma, Tatjana Vlajnic, Arnoud J. Templeton, Heike Pueschel, Salvatore Piscuoglio, Nicola Aceto, Helge Seifert, Cyrill A. Rentsch, Bubendorf Lukas, Clémentine Le Magnen. Establishment and characterization of two novel patient-derived organoid xenograft models of advanced prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3085.

BACKGROUND Mutations of AT-rich interactive domain 1 (ARID1A) have been associated with a worse outcome after intravesical treatment with bacille Calmette-Guerin in patients with non-muscle-invasive bladder cancer (NMIBC). Loss of ARID1A protein expression in urine cytology may serve as an indication of an ARID1A mutation. Therefore, the authors examined the expression of ARID1A in urine cytology and histological specimens of bladder cancer for correlation with ARID1A mutational status. METHODS The authors constructed a tissue microarray containing samples from 164 tissue samples from 150 patients with NMIBC and 100 tissue samples from 81 patients with muscle-invasive bladder cancer. A second cohort consisted of archived cytological specimens and matched tissue sections from 62 patients with high-grade NMIBC. The authors established immunohistochemistry and immunocytochemistry (ICC) protocols, respectively, for the analysis of ARID1A protein expression in histological and cytological specimens. Confirmatory next-generation sequencing (NGS) was performed on tumor specimens using a targeted NGS panel containing all exonic regions of ARID1A. RESULTS The prevalence of ARID1A loss of expression on the tissue microarray was 3.6% in NMIBC (6 of 164 tissue samples) and 10% in muscle-invasive bladder cancer (10 of 100 tissue samples) (P = .059). Loss of ARID1A expression in cytology was concordantly immunohistochemistry negative in 6 of 8 matched tissue specimens. NGS confirmed an ARID1A mutation on all 6 histology samples with loss of ARID1A expression. When NGS demonstrated an absence of ARID1A mutation, histology was concordantly positive (16 of 16 cases). CONCLUSIONS The authors have suggest ARID1A ICC as a promising surrogate marker for ARID1A mutational status in patients with urothelial carcinoma. Pitfalls in ICC scoring include benign umbrella cells that often are negative for ARID1A. Further prospective studies are needed to determine the clinical relevance of ARID1A ICC in urinary cytology.

Clues to cancer from an identity change The prostate and seminal vesicle have closely related developmental histories and both are regulated by the same androgenic hormones. A better understanding of the molecular mechanisms controlling the development of the two tissues could help solve why cancer arises frequently in the prostate but only rarely in seminal vesicles. Working with cell and mouse models, Dutta et al. show that forced expression of a single gene, the homeobox gene NKX3.1 , causes seminal vesicle epithelium to differentiate into prostate. NKX3.1 regulates the expression of a gene program associated with prostate differentiation by interacting with the G9a histone methyltransferase. Disruption of this regulatory network probably contributes to prostate cancer development. Science , this issue p. 1576

Although it is known that inflammation plays a critical role in prostate tumorigenesis, the underlying processes are not well understood. Based on analysis of genetically engineered mouse models combined with correlative analysis of expression profiling data from human prostate tumors, we demonstrate a reciprocal relationship between inflammation and the status of the NKX3.1 homeobox gene associated with prostate cancer initiation. We find that cancer initiation in aged Nkx3.1 mutant mice correlates with enrichment of specific immune populations and increased expression of immunoregulatory genes. Furthermore, expression of these immunoregulatory genes is similarly increased in human prostate tumors having low levels of NKX3.1 expression. We further show that induction of prostatitis in Nkx3.1 mutant mice accelerates prostate cancer initiation, which is coincident with aberrant cellular plasticity and differentiation. Correspondingly, human prostate tumors having low levels of NKX3.1 have de-regulated expression of genes associated with these cellular processes. We propose that loss of function of NKX3.1 accelerates inflammation-driven prostate cancer initiation potentially via aberrant cellular plasticity and impairment of cellular differentiation. This article has an associated First Person interview with the first author of the paper., Summary: Chronic inflammation collaborates with loss of function of the prostate-specific tumor-suppressor NKX3.1 to promote prostate cancer initiation, increase cellular plasticity and impair cellular differentiation.

Historically, it has been widely presumed that differentiated cells are determined during development and become irreversibly committed to their designated fates. In certain circumstances, however, differentiated cells can display plasticity by changing their identity, either by dedifferentiation to a progenitor-like state or by transdifferentiation to an alternative differentiated cell type. Such cellular plasticity can be triggered by physiological or oncogenic stress, or it can be experimentally induced through cellular reprogramming. Notably, physiological stresses that promote plasticity, such as severe tissue damage, inflammation, or senescence, also represent hallmarks of cancer. Furthermore, key drivers of cellular plasticity include major oncogenic and tumor suppressor pathways and can be exacerbated by drug treatment. Thus, plasticity may help cancer cells evade detection and treatment. We propose that cancer can be considered as a disease of excess plasticity, a notion that has important implications for intervention and treatment.

The hTERT-immortalization of human bone marrow-derived Mesenchymal Stromal Cells (hMSCs) was proposed to address availability/standardization issues for experimental or clinical studies, but raised concerns due to possible uncontrolled growth or malignant cell transformation.Here we report a method to generate a hMSCs line with controlled survival, through the implementation of a pre-established suicide system (inducible caspase 9, iCasp9) in hTERT-transduced hMSCs.Primary hMSCs were successfully immortalized (>280 PD) and further transduced with the iCasp9 device. A clone was selected and shown to maintain typical properties of primary hMSCs, including phenotype, differentiation and immunomodulation capacities. The successive transductions did not induce tumorigenic transformation, as assessed by analysis of cell cycle regulators and in vivo luciferase-based cell tracking. Cells could be efficiently induced toward apoptosis (>95%) both in vitro and in vivo.By combining the opposite concepts of ‘induced-life’ and ‘inducible-death’, we generated a hMSCs line with defined properties and allowing for temporally controlled survival. The cell line represents a relevant tool for medical discovery in regenerative medicine and a potential means to address availability, standardization and safety requirements in cell & gene therapy. The concept of a hTERT-iCasp9 combination, here explored in the context of hMSCs, could be extended to other types of progenitor/stem cells.

Purpose: High aldehyde dehydrogenase (ALDH) has been suggested to selectively mark cells with high tumorigenic potential in established prostate cancer cell lines. However, the existence of cells with high ALDH activity (ALDHbright) in primary prostate cancer specimens has not been shown so far. We investigated the presence, phenotype, and clinical significance of ALDHbright populations in clinical prostate cancer specimens. Experimental Design: We used ALDEFLUOR technology and fluorescence-activated cell-sorting (FACS) staining to identify and characterize ALDHbright populations in cells freshly isolated from clinical prostate cancer specimens. Expression of genes encoding ALDH-specific isoforms was evaluated by quantitative real-time PCR in normal prostate, benign prostatic hyperplasia (BPH), and prostate cancer tissues. ALDH1A1-specific expression and prognostic significance were assessed by staining two tissue microarrays that included more than 500 samples of BPH, prostatic intraepithelial neoplasia (PIN), and multistage prostate cancer. Results: ALDHbright cells were detectable in freshly excised prostate cancer specimens (n = 39) and were mainly included within the EpCAM(+) and Trop2(+) cell populations. Although several ALDH isoforms were expressed to high extents in prostate cancer, only ALDH1A1 gene expression significantly correlated with ALDH activity (P < 0.01) and was increased in cancers with high Gleason scores (P = 0.03). Most importantly, ALDH1A1 protein was expressed significantly more frequently and at higher levels in advanced-stage than in low-stage prostate cancer and BPH. Notably, ALDH1A1 positivity was associated with poor survival (P = 0.02) in hormone-naïve patients. Conclusions: Our data indicate that ALDH contributes to the identification of subsets of prostate cancer cells of potentially high clinical relevance. Clin Cancer Res; 19(19); 5361–71. ©2013 AACR.

BACKGROUND: Cancer initiation and progression might be driven by small populations of cells endowed with stem cell-like properties. Here we comparatively addressed the expression of genes encoding putative stemness regulators including c-Myc, Klf4, Nanog, Oct4A and Sox2 genes in benign prostatic hyperplasia (BPH) and prostate cancer (PCA). METHODS: Fifty-eight PCA and thirty-nine BPH tissues samples were used for gene expression analysis, as evaluated by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of specific Klf4 isoforms was tested by conventional PCR. Klf4 specific antibodies were used for protein detection in a tissue microarray including 404 prostate samples. RESULTS: Nanog, Oct4A and Sox2 genes were comparably expressed in BPH and PCA samples, whereas c-Myc and Klf4 genes were expressed to significantly higher extents in PCA than in BPH specimens. Immunohistochemical studies revealed that Klf4 protein is detectable in a large majority of epithelial prostatic cells, irrespective of malignant transformation. However, in PCA, a predominantly cytoplasmic location was observed, consistent with the expression of a differentially spliced Klf4alpha isoform. CONCLUSION: Klf4 is highly expressed at gene and protein level in BPH and PCA tissues but a cytoplasmic location of the specific gene product is predominantly detectable in malignant cells. Klf4 location might be of critical relevance to steer its functions during oncogenesis.

Current treatments for castration-resistant prostate cancer (CRPC) that target androgen receptor (AR) signaling improve patient survival, yet ultimately fail. Here, we provide novel insights into treatment response for the antiandrogen abiraterone by analyses of a genetically engineered mouse (GEM) model with combined inactivation of Trp53 and Pten, which are frequently comutated in human CRPC. These NPp53 mice fail to respond to abiraterone and display accelerated progression to tumors resembling treatment-related CRPC with neuroendocrine differentiation (CRPC-NE) in humans. Cross-species computational analyses identify master regulators of adverse response that are conserved with human CRPC-NE, including the neural differentiation factor SOX11, which promotes neuroendocrine differentiation in cells derived from NPp53 tumors. Furthermore, abiraterone-treated NPp53 prostate tumors contain regions of focal and/or overt neuroendocrine differentiation, distinguished by their proliferative potential. Notably, lineage tracing in vivo provides definitive and quantitative evidence that focal and overt neuroendocrine regions arise by transdifferentiation of luminal adenocarcinoma cells. These findings underscore principal roles for TP53 and PTEN inactivation in abiraterone resistance and progression from adenocarcinoma to CRPC-NE by transdifferentiation. Significance: Understanding adverse treatment response and identifying patients likely to fail treatment represent fundamental clinical challenges. By integrating analyses of GEM models and human clinical data, we provide direct genetic evidence for transdifferentiation as a mechanism of drug resistance as well as for stratifying patients for treatment with antiandrogens. Cancer Discov; 7(7); 736–49. ©2017 AACR. See related commentary by Sinha and Nelson, p. 673. This article is highlighted in the In This Issue feature, p. 653

MAGE-A10 is a highly immunogenic member of the MAGE-A family of cancer/testis tumor-associated antigens (C/T TAAs). Studies performed with broadly reactive antibodies have helped to initially characterize this TAA. However, no specific reagents have been developed so far, thus preventing a thorough analysis of its expression in healthy and tumoral tissues. We have produced MAGE-A10 gene product in soluble recombinant form, and we have used it to generate specific monoclonal antibodies (mAbs). One of these reagents, recognizing an epitope located at the COOH terminus of the MAGE-A10 gene product, was used to stain a multitumor tissue microarray comprising more than 2,500 paraffin-embedded specimens including healthy tissues, benign tumors and malignancies of different histological origin. MAGE-A10 protein was identified as an intranuclear protein of an apparent molecular weight of 70 kDa, expressed in normal spermatogonia and spermatocytes but in no other healthy tissue. Most importantly, this C/T TAA appears to be expressed in high (>50%) percentages of cancer cells from a number of malignancies, including lung, skin and urothelial tumors. Unexpectedly, high expression of MAGE-A10 TAA at the protein level was also detectable in gynecological malignancies and stomach and gall bladder cancers. The characterization of MAGE-A10-specific reagents might set the stage for the development of targeted active immunotherapy by clarifying potential indications and by allowing the selection of patients eligible for treatment and the monitoring of its effectiveness.

Many newly diagnosed prostate cancers present as low Gleason score tumors that require no treatment intervention. Distinguishing the many indolent tumors from the minority of lethal ones remains a major clinical challenge. We now show that low Gleason score prostate tumors can be distinguished as indolent and aggressive subgroups on the basis of their expression of genes associated with aging and senescence. Using gene set enrichment analysis, we identified a 19-gene signature enriched in indolent prostate tumors. We then further classified this signature with a decision tree learning model to identify three genes—FGFR1, PMP22, and CDKN1A—that together accurately predicted outcome of low Gleason score tumors. Validation of this three-gene panel on independent cohorts confirmed its independent prognostic value as well as its ability to improve prognosis with currently used clinical nomograms. Furthermore, protein expression of this three-gene panel in biopsy samples distinguished Gleason 6 patients who failed surveillance over a 10-year period. We propose that this signature may be incorporated into prognostic assays for monitoring patients on active surveillance to facilitate appropriate courses of treatment.

Maintenance of cell differentiation is tightly coordinated by specific gene expression programs, which are often deregulated during tumorigenesis. While the homeodomain-containing transcription factor, NKX3.1, plays essential roles in both prostate differentiation and prostate cancer, the mechanisms governing these events are still unclear. Here, we aim to elucidate detailed mechanisms governing Nkx3.1-driven prostate epithelial differentiation and explain how deregulation of such can lead to prostate tumorigenesis. Using an Nkx3.1 mutant mouse model, we find that loss-of-function of Nkx3.1 in the prostate results in the down-regulation of genes associated with prostate differentiation as well as the up-regulation of genes that are not normally expressed in prostate. Interestingly, these mice also develop pre-malignant lesions resembling prostatic intraepithelial neoplasia (PIN). Conversely, gain of function of Nkx3.1 in a fully differentiated non-prostate epithelium is sufficient to induce prostate differentiation in renal grafts in vivo. Using a DNA binding mutant, we show that these activities are mediated via NKX3.1 homeodomain in vitro and in vivo. Exploiting mass spectrometry to identify NKX3.1-interacting proteins, we find that NKX3.1 interacts with the histone methyltransferase G9a and that this interaction is required for induction of prostate differentiation in vitro and in vivo. Finally, we show that these NKX3.1-driven activities are mediated, at least in part, by regulation of target genes such as the male-specific factor UTY (KDM6c). We propose that the NKX3.1-G9a-UTY network is essential for proper prostate differentiation whereas disruption of these activities predisposes to cancer. Citation Format: Clementine Le Magnen, Aditya Dutta, Antonina Mitrofanova, Andrea Califano, Cory Abate-Shen. Identification of an NKX3.1-G9a-UTY regulatory network that controls proper prostate differentiation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2888.

Background Chronic inflammation has been suggested to favour prostate cancer (PCA) development. Interleukins (IL) represent essential inflammation mediators. IL-2, IL-7, IL-15 and IL-21, sharing a common receptor γ chain (c-γ), control T lymphocyte homeostasis and proliferation and play major roles in regulating cancer-immune system interactions. We evaluated local IL-2, IL-7, IL-15 and IL-21 gene expression in prostate tissues from patients with early stage PCA or benign prostatic hyperplasia (BPH). As control, we used IL-6 gene, encoding an IL involved in PCA progression. IL-6, IL-7 and IL-15 titres were also measured in patients' sera. Methods Eighty patients with BPH and 79 with early (1 to 2c) stage PCA were enrolled. Gene expression in prostate tissues was analyzed by quantitative real-time PCR (qRT-PCR). Serum IL concentrations and acute phase protein titres were evaluated by ELISA. Mann-Whitney, Wilcoxon and χ2 tests were used to compare IL gene expression and serum titers in the two groups of patients. Receiver operating characteristic (ROC) curves were constructed to evaluate the possibility to distinguish sera from different groups of patients based on IL titers. Results IL-2 and IL-21 gene expression was comparably detectable, with low frequency and at low extents, in PCA and BPH tissues. In contrast, IL-6, IL-7 and IL-15 genes were expressed more frequently (p < 0.0001, p = 0.0047 and p = 0.0085, respectively) and to significantly higher extents (p = 0.0051, p = 0.0310 and p = 0.0205, respectively) in early stage PCA than in BPH tissues. Corresponding proteins could be detected to significantly higher amounts in sera from patients with localized PCA, than in those from patients with BPH (p = 0.0153, p = 0.0174 and p = 0.0064, respectively). Analysis of ROC curves indicates that IL-7 (p = 0.0039), but not IL-6 (p = 0.2938) or IL-15 (p = 0.1804) titres were able to distinguish sera from patients with malignancy from those from patients with benign disease. Serum titres of C reactive (CRP), high mobility group B1 (HMGB1) and serum amyloid A (SAA) acute phase proteins were similar in both groups of patients. Conclusions Expression IL-7 and IL-15 genes in prostate tissues and corresponding serum titres are significantly increased in patients with early stage PCA as compared with patients with BPH.

Background Maintenance of epithelial cell identity is tightly coordinated by tissue-specific gene expression programs, which are often deregulated during tumorigenesis. The homeodomain-containing transcription factor, Nkx3.1, is a key regulator of normal prostatic development and is frequently lost at early stages of prostate cancer initiation. In this study, we aim to elucidate detailed mechanisms governing Nkx3.1-driven maintenance of prostate identity and how deregulation of such can lead to prostate tumorigenesis. Models and Methods We evaluated the consequences of Nkx3.1 loss or gain of function in vivo using genetically-engineered mouse models and cell-recombination assays. RNA sequencing was performed to generate gene expression profiles, which were analyzed using Gene Set Enrichment analysis (GSEA), and validated by quantitative real-time PCR. In parallel, protein expression was assessed by immunofluorescence and western blot. Immunoprecipitation (IP) and chromatin-immunoprecipitation (ChIP) assays were performed using RWPE1 prostate epithelial cells. Results Here, we show that loss of function of Nkx3.1 leads to the progressive down-regulation of a prostate-specific gene expression program and to aberrant expression of genes that are not typically expressed in the prostate epithelium. Conversely, gain of function of Nkx3.1 in non-prostatic epithelium leads to the acquisition of a prostate-like morphology and expression of prostate-related genes. Our findings indicate that the underlying mechanism by which Nkx3.1 promotes prostatic identity is via epigenetic regulation of gene expression. In particular, we show that Nkx3.1 interacts with the histone methyl-transferase complex G9a/Glp. Finally, we demonstrate that this interaction is necessary for maintenance of prostate identity in vivo and that Nkx3.1 and G9a cooperate to control expression of genes that coordinate prostatic epithelial integrity. Conclusions Our results suggest that Nkx3.1 promotes prostatic identity by interacting with histone modifying enzymes to coordinate the expression of prostate-specific genes and that the loss of this function results in a failure to maintain prostate identity associated with early stages of prostate tumorigenesis. Citation Format: Clémentine Le Magnen, Aditya Dutta, Cory Abate-Shen. The Nkx3.1 homeobox gene maintains prostatic identity while its loss leads to prostate cancer initiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 88. doi:10.1158/1538-7445.AM2015-88

With more than 230,000 estimated new cases in 2013, prostate cancer represents the most commonly diagnosed cancer in American men. While a subgroup of patients will develop an aggressive and lethal prostate cancer, most present with a low-risk form of the disease that requires little or no treatment. It is therefore crucial to distinguish patients whose tumors are likely to progress to aggressive disease from those whose tumors will remain indolent and for whom treatment may be more detrimental than beneficial. In our recent study, we hypothesized that low Gleason score prostate tumors can be distinguished as indolent and aggressive based on expression of genes associated with aging and senescence (1). Using gene set enrichment analyses and a decision tree learning model, we identified a 3-gene panel_FGFR1, PMP22, and CDKN1A_that is enriched in indolent prostate cancers and accurately predicts outcome of low Gleason score tumors at both the RNA and protein levels. In particular, protein expression of this 3-gene panel in biopsy samples correctly distinguished Gleason 6 patients who failed active surveillance over a 10-year period. Following from our initial study, our current efforts are focused on exploiting this 3-gene panel to develop a robust clinical assay for distinguishing indolent and aggressive tumors. We are currently using qRT-PCR, immunofluorescence, and immunohistochemistry assays to assess the most effective way to quantify the simultaneous expression of the 3-gene panel in low Gleason score tumor biopsies/tissues. This quantitative assay will ultimately be applied to a large cohort study assessing the predictive power of the 3-gene signature in patients under active surveillance. We envision that such a prognostic test will significantly improve our ability to predict the outcome of patients with low Gleason score tumors and facilitate appropriate decisions of treatment for men under active surveillance. 1. Irshad S, et al. (2013) A molecular signature predictive of indolent prostate cancer. Science translational medicine 5(202):202ra122. Citation Format: Shazia Irshad, Mukesh Bansal, Clementine Le Magnen, Risham Dillon, Mireia Castillo-Martin, Tian Zheng, Alvaro Aytes, Sven Wenske, Paolo Guarnieri, Pavel Sumazin, Mitchell Benson, Michael M. Shen, Andrea Califano, Cory Abate-Shen. A molecular signature predictive of indolent prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2873. doi:10.1158/1538-7445.AM2014-2873

OBJECTIVE: Tumor initiation and progression might be driven by rare populations of cells endowed with stem-properties, and therefore defined as cancer stem cells (CSC). High Aldehyde dehydrogenase (ALDH) activity has been suggested to selectively identify CSC in several tumor types including breast cancer, and, possibly, prostate cancer (PCA). In this study, we investigated presence and potential CSC characteristics of cells with high ALDH activity (ALDH bright) in PCA cell lines, fresh surgical specimens, and primary cultures. MATERIALS AND METHODS: PC3, Du145, VCaP, and LNCaP PCA cell lines were evaluated. Surgical specimens, including Benign Prostate Hyperplasia (BPH) and PCA, were used directly for gene expression studies. Surgical samples were also enzymatically digested for functional analysis and the establishment of primary cultures. ALDH activity was tested using ALDEFLUOR® technology. Cells were sorted from individual cell lines by flow cytometry and evaluated for CSC properties, including spheroid formation ability, clonogenicity, stemness-related gene expression, ALDH specific isoforms expression, and tumorigenicity upon injection in NOD/SCID mice. RESULTS: PCA cell lines and primary cultures displayed heterogeneous ALDH activity and expression of specific ALDH isoforms. Despite a higher expression of Oct4A and Klf4 stemness associated genes, ALDH bright cells isolated from Du145 and PC3 did not show improved spheroid formation or clonogenic capacity, as compared to their dim counterparts. Interestingly, however, ALDH bright cells were associated with a significantly higher tumorigenic capacity in vivo as compared to ALDH low cells. Nevertheless, ALDH bright cells lost their higher tumorigenic capacity following serial “in vivo” passages. Most importantly, a well defined ALDH bright population could also be detected in cells isolated from PCA specimens (n>20). ALDH activity was consistent with a strong expression of several ALDH specific isoforms in PCA tissues. Notably, a significant increase of defined ALDH isoforms such as ALDH1A3 (p=0.001) was detectable in tissues obtained from patients with PCA as compared to BPH or normal specimens. CONCLUSIONS: ALDH bright subsets can be detected in cells isolated from fresh PCA tissue samples, PCA cell lines and primary cultures. These populations appear to be associated with increased “in vivo” tumorigenicity rather than enhanced “in vitro” stem properties in the cell lines investigated. Importantly, expression of ALDH specific isoforms appears to be increased in clinical PCA as compared to BPH and “normal” samples, thereby suggesting a putative role of ALDH in PCA tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3384. doi:1538-7445.AM2012-3384

Prostate cancer (PCA) is a leading cause of cancer death in men. Underlying immunosuppressive mechanisms in benign prostate hyperplasia (BPH) and PCA patients are not fully clarified. We analyzed homeostatic proliferation of CD8+ T cells upon stimulation with common receptor γ chain IL-2, IL-7 and IL-15 cytokines in PCA as compared to BPH patients. CD8+ T cells exhaustion was assessed by evaluating Programmed Death-1 (PD-1) receptor and its ligand PD-L1 expression in PBMC and tissue infiltrating CD8+ T cells from PCA and BPH patients. 57 BPH and 76 PCA patients were enrolled. Gene expression was quantified by Real-Time PCR. T cell proliferation was evaluated by CFSE dilution. CD132, CD122, PD-1 and PD-Ll expression were assessed by flow cytometry. IL-2, IL-7 and IL-15 gene expression were quantified in PCA (n=57) and BPH (n=32) tissues. IL-7 and IL-15 gene expression were significantly increased in PCA tissues as compared to BPH (p=0.024; p=0.031). No significant differences were observed for IL-2 gene expression. CD8+ cells from BPH (n=17) and PCA (n=21) patients showed a significantly decreased responsiveness to IL-7 and IL-15 (p=0.021; p=0.015) as compared to healthy donors (n=9). Response to IL-2 was similar. A trend towards a lower response to IL-15 in PCA as compared to BPH patients was detectable. Percentage of CD8+ T cells expressing the common γ chain (CD132) of homeostatic cytokines receptor is significantly decreased in PCA (n=13) and BPH (n=16) patients as compared to healthy donors (n=8) (p= 0.024; p Taken together these data indicate that both BPH and PCA patients display a decreased responsiveness to IL-7 and IL-15 homeostatic cytokines. Interestingly, high percentages of peripheral blood CD8+ T cells express PD-1 in BPH and PCA patients. Notably, PD-1 and its ligand are highly expressed in tissue infiltrating CD8+ T cells in BPH and PCA patients, thus raising the issue of the role of T cells exhaustion in PCA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4782.