Mouse Prostate Epithelial Cells Research Articles

Aberrant activated Notch1 promotes prostate enlargement driven by androgen signaling via disrupting mitochondrial function in mouse

The prostate is a vital accessory gonad in the mammalian male reproductive system. With the ever-increasing proportion of the population over 60 years of age worldwide, the incidence of prostate diseases, such as benign prostatic hyperplasia (BPH) and prostate cancer (PCa), is on the rise and is gradually becoming a significant medical problem globally. The notch signaling pathway is essential in regulating prostate early development. However, the potential regulatory mechanism of Notch signaling in prostatic enlargement and hyperplasia remains unclear. In this study, we proved that overactivation of Notch1 signaling in mouse prostatic epithelial cells (OEx) led to prostatic enlargement via enhancing proliferation and inhibiting apoptosis of prostatic epithelial cells. Further study showed that N1ICD/RBPJ directly up-regulated the androgen receptor (AR) and enhanced prostatic sensitivity to androgens. Hyper-proliferation was not found in orchidectomized OEx mice without androgen supply but was observed after Dihydrotestosterone (DHT) supplementation. Our data showed that the number of mitochondrion in prostatic epithelial cells of OEx mice was increased, but the mitochondrial function was impaired, and the essential activity of the mitochondrial respiratory electron transport chain was significantly weakened. Disordered mitochondrial number and metabolic function further resulted in excessive accumulation of reactive oxygen species (ROS). Importantly, anti-oxidant N-Acetyl-L-Cysteine (NAC) therapy could alleviate prostatic hyperplasia caused by the over-activation of Notch1 signaling. Furthermore, we observed the incremental Notch signaling activity in progenitor-like club cells in the scRNA-seq data set of human BPH patients. Moreover, the increased number of TROP2+ progenitors and Club cells was also confirmed in our OEx mice. In conclusion, our study revealed that over-activated Notch1 signaling induces prostatic enlargement by increasing androgen receptor sensitivity, disrupting cellular mitochondrial metabolism, increasing ROS, and a higher number of progenitor cells, all of which can be effectively rescued by NAC treatment.

Estrogen receptor expression is modulated in human and mouse prostate epithelial cells during cancer progression

Substantial data posit estrogen receptors (ERs) as promising targets for prostate cancer (PCa) therapeutics. However, the trials on assessing the chemo-preventive or therapeutic potential of ER targeting drugs or selective estrogen receptor modulators (SERMs) have not yet established their clinical benefits. This could be ascribed to a possible modulation in the ER expression during PCa progression. Further it is warranted to test various ER targeting drugs in appropriate preclinical models that simulate human ER expression pattern during PCa progression. The study was undertaken to revisit the existing data on the epithelial ER expression pattern in human cancerous prostates and experimentally determine whether these patterns are replicated in TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice, a model for human PCa. Estradiol (E2) binding to the plasma membrane of the epithelial cells and its modulation during the PCa progression in TRAMP were also investigated.A reassessment of the existing data revealed a trend towards downregulation in the epithelial expression of wild-type ESR1 transcripts in high-grade PCa, compared to non-cancerous prostate in humans. Next, epithelial cell-enriched populations from TRAMP prostates (TP) displaying low-grade prostatic intraepithelial neoplasia (LGPIN), high-grade PIN (HGPIN), HGPIN with well-differentiated carcinoma (PIN + WDC), WDC (equivalent to grade 2/3 human PCa), and poorly-differentiated carcinoma (PDC-equivalent to grade 4/5 human PCa) revealed significantly higher Esr1 and Esr2 levels in HGPIN and significantly reduced levels in WDC, compared to respective age-matched control prostates. These patterns for the nuclear ERs were similar to the trend shown by E2 binding to the plasma membrane of the epithelial cells during PCa progression in TRAMP. E2 binding to epithelial cells (EpCAM+), though significantly higher in TPs displaying LGPIN, decreased significantly as the disease progressed to WDC. The study highlights a reduction in the epithelial ESR level with the PCa progression and this pattern was evident in both humans and TRAMP. These observations may have major implications in refining PCa therapeutics targeting ER.

FACS-Free isolation and purification protocol of mouse prostate epithelial cells for organoid primary culture

The prostate is a gland that contributes to men's fertility. It is highly responsive to androgens and is often the site of carcinogenesis, as prostate cancer is the most frequent cancer in men in over a hundred countries. To study the normal prostate, few in vitro models exist, and most of them do not express the androgen receptor (AR). To overcome this issue, prostate epithelial cells can be grown in primary culture ex vivo in 2- and 3-dimensional culture (organoids). However, methods to purify these cells often require flow cytometry, thus necessitating specialized instruments and expertise. Herein, we present a detailed protocol for the harvest, purification, and primary culture of mouse prostate epithelial cells to grow prostate organoids ex vivo. This protocol does not require flow cytometry approaches, facilitating its implementation in most research laboratories, and organoids grown with this protocol are highly responsive to androgens. In summary, we present a new simple method that can be used to grow prostate organoids that recapitulate the androgen response of this gland in vivo.

Pattern recognition receptor-initiated innate immune responses in mouse prostatic epithelial cells‡.

Three major pathogenic states of the prostate, including benign prostatic hyperplasia, prostate cancer, and prostatitis, are related to the local inflammation. However, the mechanisms underlying the initiation of prostate inflammation remain largely unknown. Given that the innate immune responses of the tissue-specific cells to microbial infection or autoantigens contribute to local inflammation, this study focused on pattern recognition receptor (PRR)-initiated innate immune responses in mouse prostatic epithelial cells (PECs). Primary mouse PECs abundantly expressed Toll-like receptor 3 (TLR3), TLR4, TLR5, melanoma differentiation-associated protein 5 (MDA5), and IFN-inducible protein 16 (p204 in mouse). These PRRs can be activated by their respective ligands: lipopolysaccharide (LPS) and flagellin of Gram-negative bacteria for TLR4 and TLR5, polyinosinic-polycytidylic acid (poly(I:C)) for TLR3 and MDA5, and herpes simplex virus DNA analog (HSV60) for p204. LPS and flagellin predominantly induced the expression of inflammatory cytokines, including tumor necrosis factor alpha (TNFA), interleukin 6 (IL6), chemokines monocyte chemoattractant protein-1 (MCP1), and C-X-C motif chemokine 10 (CXCL10). Poly(I:C) and HSV60 predominantly induced the expression of type 1 interferons (IFNA and IFNB) and antiviral proteins: Mx GTPase 1, 2',5'-oligoadenylate synthetase 1, and IFN-stimulated gene 15. The replication of mumps virus in PECs was inhibited by type 1 IFN signaling. These findings provide insights into the mechanisms underlying innate immune response in the prostate.

The Sca-1+ and Sca-1- mouse prostatic luminal cell lineages are independently sustained.

The phenotypic and functional heterogeneity of the mouse prostate epithelial cell lineages remains incompletely characterized. We show that the Sca-1+ luminal cells at the mouse proximal prostate express Sox2. These cells are replicative quiescent, castration resistant, and do not possess secretory function. We use the Probasin-CreERT2 and Sox2-CreERT2 models in concert with a fluorescent reporter line to label the Sca-1- and Sca-1+ luminal cells, respectively. By a lineage tracing approach, we show that the two luminal cell populations are independently sustained. Sox2 is dispensable for the maintenance of the Sca-1+ luminal cells but is essential for their facultative bipotent differentiation capacity. The Sca-1+ luminal cells share molecular features with the human TACSTD2+ luminal cells. This study corroborates the heterogeneity of the mouse prostate luminal cell lineage and shows that the adult mouse prostate luminal cell lineage is maintained by distinct cellular entities rather than a single progenitor population.

Slug-expressing mouse prostate epithelial cells have increased stem cell potential.

Deciphering the properties of adult stem cells is crucial for understanding of their role in healthy tissue and in cancer progression as well. Both stem cells and cancer stem cells have shown association with epithelial-to-mesenchymal transition (EMT) in various tissue types. Aiming to investigate the epithelial and mesenchymal phenotypic traits in adult mouse prostate, we sorted subpopulations of basal prostate stem cells (mPSCs) and assessed the expression levels of EMT regulators and markers with custom-designed gene expression array. The population of mPSCs defined by a Lin-/Sca-1+CD49fhi/Trop-2+ (LSC Trop-2+) surface phenotype was enriched in mesenchymal markers, especially EMT master regulator Slug, encoded by the Snai2 gene. To further dissect the role of Slug in mPSCs, we used transgenic Snai2tm1.1Wbg reporter mouse strain. Using this model, we confirmed the presence of mesenchymal traits and increase of organoid forming capacity in Slug+ population of mPSCs. The Slug+-derived organoids comprised all prostate epithelial cell types - basal, luminal, and neuroendocrine. Collectively, these data uncover the important role of Slug expression in the physiology of mouse prostate stem cells.

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

Abstract 4405: Acetylation of KLF5 controls the cell fate of basal progenitors during postnatal development and regeneration of mouse prostates

Abstract Adult stem cells are responsible for lineage specification in epithelial homeostasis, and thus are essential for both postnatal development and tissue regeneration. Genetic changes in adult stem cells have been revealed to promote cancer development. Dissecting the underlying regulators is critical for understanding the intrinsic mechanisms that regulate adult stem cell function, yet whether PTM of a single molecule determines cell fate of adult stem cells during organ development has not been well established, particularly for glandular organs. Using the androgen-dependent mouse prostate as an experimental system, an ideal system for understanding epithelial hierarchy, we examined the Krüppel-like factor 5 (Klf5) transcription factor and its acetylation for their roles in postnatal development and regeneration. The absence of KLF5 eliminated basal progenitor activities in both human and mouse prostate epithelial cells, as indicated by a compromised sphere and organoid formatting capabilities, downregulation of basal cell markers, and decreased basal cells. Klf5 was also necessary for basal progenitor cells to maintain their luminal differentiation capacity, as loss of Klf5 also decreased basal progenitor-derived luminal cells and their proliferation in mouse prostates. Interestingly, the progenitor activity of Klf5 was restricted to deacetylated Klf5 (deAc-Klf5), as indicated by in vitro and in vivo assays. On the other hand, acetylated Klf5 (Ac-Klf5) was essential for proper formation and maintenance of luminal structures in mouse prostates, as deacetylation of Klf5 interrupted the formation of normal luminal structures, almost eliminated the castration resistance in basal progenitor-derived luminal cells, and attenuated the luminal regeneration after a castration-regeneration cycle, even though deAc-Klf5 promoted basal-to-luminal differentiation and the proliferation of luminal cells during postnatal development. Mechanistically, deacetylation of Klf5 increased Notch signaling activity. These findings indicate that Klf5 and its acetylation control the cell fate of prostatic basal progenitors, which have a distinct contribution to postnatal development and regeneration of the prostate. Considering that androgen deprivation therapy (ADT) is currently a major therapeutic strategy in the treatment of prostate cancer, our findings also provide a potential mechanism for prostate cancer progression, i.e., interruption of Klf5 acetylation could sensitize castration-resistant prostate cancer to ADT or slow the recurrence of prostate cancer after ADT. Citation Format: Baotong Zhang, Jin-Tang Dong. Acetylation of KLF5 controls the cell fate of basal progenitors during postnatal development and regeneration of mouse prostates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4405.

Inhibition of the CXCL12/CXCR4 axis prevents periurethral collagen accumulation and lower urinary tract dysfunction in vivo.

Several studies show that prostatic fibrosis is associated with male lower urinary tract dysfunction (LUTD). Development of fibrosis is typically attributed to signaling through the transforming growth factor β (TGF-β) pathway, but our laboratory has demonstrated that in vitro treatment of human prostatic fibroblasts with the C-X-C motif chemokine ligand 12 (CXCL12) chemokine stimulates myofibroblast phenoconversion and that CXCL12 has the capacity to activate profibrotic pathways in these cells in a TGF-β-independent manner. We have previously reported that feeding mice high-fat diet (HFD) results in obesity, type II diabetes, increased prostatic fibrosis, and urinary voiding dysfunction. The purpose of this study was to test the hypothesis that in vivo blockade of the CXCL12/CXCR4 axis would inhibit the development of fibrosis-mediated LUTD in HFD-fed mice. Two-month-old male senescence-accelerated mouse prone-6 mice were fed either a HFD or low-fat diet (LFD) for 8 months. Half of each dietary group were given constant access to normal water or water that contained the C-X-C chemokine receptor type 4 (CXCR4; CXCL12 receptor) antagonist CXCR4AIII. At the conclusion of the study, mice were weighed, subjected to oral glucose tolerance testing and cystometry, and lower urinary tract tissues collected and assessed for collagen content. HFD-fed mice became significantly obese, insulin resistant, and hyperglycemic, consistent with acquisition of metabolic syndrome, compared with LFD-fed mice. Anesthetized cystometry demonstrated that HFD-fed mice experienced significantly longer intercontractile intervals and greater functional bladder capacity than LFD-fed mice. Immunohistochemistry demonstrated high levels of CXCR4 and CXCR7 staining in mouse prostate epithelial and stromal cells. Picrosirius red staining indicated significantly greater periurethral collagen deposition in the prostates of HFD than LFD-fed mice. Treatment with the CXCR4 antagonist CXCR4AIII did not affect acquisition of metabolic syndrome but did reduce both urinary voiding dysfunction and periurethral prostate collagen accumulation. This is the first study to report that obesity-induced lower urinary tract fibrosis and voiding dysfunction can be repressed by antagonizing the activity of the CXCR4 chemokine receptor in vivo. These data suggest that targeting the CXCL12/CXCR4 signaling pathway may be a clinical option for the prevention or treatment of human male LUTD.

3D Co-culture System of Mouse Prostatic Wild-type Fibroblasts withHuman Prostate Cancer Epithelial Cells.

Heterogeneous prostatic carcinoma-associated fibroblasts (CAF) contribute to tumor progression. This was established using transgenic mouse models. Paracrine interactions between fibroblasts and epithelial cells were further interrogated using isolated 2D cell culture systems, but 3D culture systems currently being developed can better mimic reciprocal interactions potentially found in the native tissue. To understand paracrine and juxtacrine signaling among fibroblasts and epithelia, 3D co-cultures with species differences allows for further subsequent analysis of the cultures. The use of mouse and human cells, for example, in one system allows for species-specific FACS or quantitative PCR analysis. This protocol describes the use of a 3D Co-culture System of Mouse Prostatic Wild-type Fibroblasts with Human Prostate Cancer Epithelial Cells.

Abstract B097: NRF2 antioxidant signaling mediates the ability of PIM protein kinases to induce resistance of prostate cancer to PI3K inhibitor therapy

Abstract Aberrant activation of the PI3K pathway leading to increases in AKT activity is seen in many prostate cancer patients refractory to androgen blockade therapy. A number of small-molecule inhibitors of PI3K, including BKM120 (Novartis), have entered clinical trials. As with PI3K-AKT activation, highly expressed Pim kinase has been shown to drive the growth of prostate cancer. Recent studies have demonstrated that Pim kinase plays a critical role in promoting resistance of breast cancer to PI3K inhibition. Similarly, when the Pim-1 protein kinase is overexpressed, prostate cancer cell lines become resistant to PI3K inhibitor. In both tissue culture and animal models, the combination of a Pim inhibitor, PIM447, and BKM120 acted synergistically in blocking the survival and growth of prostate cancer cells. We therefore investigated the biochemical mechanism by which Pim-1 mediates cell resistance to the PI3K inhibitor. Previously, we have demonstrated that knockout of Pim kinases led to accumulation of reactive oxygen species (ROS) accompanied by a lower level of expression of ROS scavengers. To explore whether Pim-1 overexpression reverses this phenotype in prostate epithelial cancer, using lentiviral transduction, we expressed Pim-1 in mouse prostate epithelial cancer cells derived from spontaneous prostate adenocarcinomas that arose in a mouse model in which Tp53 and Rb1 are deleted in prostate epithelium. Pim-1 overexpression promoted anchorage-independent growth and formation of mouse prostate epithelial tumors in vivo. Gene expression arrays demonstrated that Pim-1 induced an Nrf2 antioxidant response element gene signature, including increases in Heme oxygenase-1 (Hmox1), NAD(P)H quinone oxidoreductase 1 (Nqo1), and glutathione S-transferase kappa1 (Gstk1) genes. Similarly, overexpression of Pim-1 in human prostate cancer LNCaP cells results in increased expression of Nrf2 and Hmox1 protein. As expected, depletion of Nrf2 with shRNA prevented Pim-1-mediated increases in Hmox1 expression, indicating that Nrf2 activation is required for the Pim-1 induction of ROS scavengers. In line with this result, in Pim-1 overexpressing cell lines knockdown of either Hmox1 or Nrf2 restored sensitivity to BKM120 treatment. A vital role of ROS scavengers in mediating the resistance to BKM120 was further confirmed by the treatment of cells and xenografted tumors with buthionine sulfoximine (inhibitor of glutathione synthesis), which resensitized the Pim-1 overexpressing cells to BKM120 via ROS induction. Our data further demonstrated that Pim-1 overexpression decreases the ubiquitination of Nrf2 protein, thus preventing its degradation, and increasing its half-life. Conversely, inhibition of Pim kinase promoted Nrf2 ubiquitination and proteasomal degradation. Taken together, our results show that the increased expression of Pim kinases in prostate cancer blocks therapeutic efficacy of PI3K inhibitor by inducing Nrf2 and antioxidant scavengers that inhibit the induction of ROS. Citation Format: Jin H. Song, Libia A. Leuvano, Neha Singh, Andrew S. Kraft. NRF2 antioxidant signaling mediates the ability of PIM protein kinases to induce resistance of prostate cancer to PI3K inhibitor therapy [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B097.

Abstract A010: ETS-related gene mediated androgen receptor aggregation and endoplasmic reticulum stress in prostate cancer development

Abstract Introduction: AR-mediated activation of ETS Related Gene (ERG) represents one of the most common and validated prostate cancer driver genes. Recently, we have shown novel morphologic phenotypes of endoplasmic reticulum (ER) stress in prostate glands of ARR2PB-ERG transgenic mouse. Since AR regulates ERG expression through TMPRSS2 promoter in human prostate cancer, we continue to investigate the post-translational interactions between ERG and AR leading to ER stress and subsequently to cell survival mechanisms. Understanding these mechanisms will potentially have major therapeutic implications. Methods: Light and electron microscopy were used to examine the morphologic and subcellular differences. AR aggregations, Co-IP and Proximal Ligation Assay for protein-protein interactions were studied in LNCaP, HEK293 cells. N-terminal and C-deletions of AR were utilized to identify specific AR domain interactions with ERG. Luminal cell surface markers on the isolated mouse prostate glands and spontaneously immortalized mouse prostate epithelial cells from ERG transgenic mouse (MoE1) were analyzed by FACS analysis. Results: Coexpression of ERG and AR showed significant aggregation of AR in filter assays. Co-IP experiments and PLA assays revealed that significant interactions occur through N-terminal domain of AR with ERG. Epithelial cells of ERG-Tg mouse prostates showed ~70% increase in CD49f (low) and Sca-1 (med) population with increased sphere formation capability and resistance to radiation-induced cell death. Both epithelial cells grown into spheres and established MoE1 cells displayed increased CD49f (low) and significant increase in the EpCAM negative population. Conclusions: Overall, our experiments demonstrate the mechanistic link that the physical interactions between ERG and AR lead to ER stress in prostate epithelium through AR misfolding/aggregation. Our observations of ERG-induced AR aggregation as one of the initial events that lead to ER stress and to cell survival indicate a critical function for ERG in the etiology of prostate cancer initiation and progression. Citation Format: Taduru L. Sreenath, Shiela S. Macalindong, Natallia Mikhalkevich, Shashwat Sharad, Ahmed Mohamed, Denise Young, Borbiev Talaibek, Charles Xavier, Rishita Gupta, Muhammad Jamal, Kevin Babcock, Shyh-Han Tan Tan, Marja T. Nevalainen, Albert Dobi, Gyorgy Petrovics, Isabell A. Sesterhenn, Inger L. Rosner, Charles J. Bieberich, Peter Nelson, Valeri Vasioukhin, Shiv Srivastava. ETS-related gene mediated androgen receptor aggregation and endoplasmic reticulum stress in prostate cancer development [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A010.

Abstract 3111: Expansion of mouse prostate epithelial stem cells in serum-free ProstaCult Organoid Growth Medium

Abstract Growing prostate epithelial cells as organoids in a three-dimensional (3D) cell culture environment represents a more physiological model system than conventional 2D adherent cell culture systems for studying many different aspects of prostate epithelial cell biology. Two types of prostate epithelial organoids have been previously described: large hollow cystic organoids, and smaller solid organoids (Karthaus et al, Cell 2014). Both types of organoids are composed of androgen receptor (AR)-expressing luminal cells and keratin (K) 5-expressing basal cells, but only vary in the proportion of each cell type, with the cystic organoids enriched for luminal cells and solid organoids enriched for basal cells. We are currently developing ProstaCult™ Organoid Growth Medium, a serum-free medium for the long-term propagation of both types of organoids from mouse prostate tissue. To initiate the cultures, mouse prostates are enzymatically dissociated sequentially in collagenase type II, trypsin and dispase to generate a single cell suspension, and 5x103 the liberated cells are then embedded in Corning® Matrigel® and cultured in ProstaCult™ Organoid Growth Medium. Approximately 10±2% (mean±sem; n=6) of the seeded cells will proliferate and generate approximately equivalent numbers of cystic and solid organoids, which can be dissociated and passaged every 7 days as a single cell suspension. Typical total epithelial cell expansion observed during media development ranges from 21-240-fold per passage for a minimum of 4 passages. Experiments are ongoing to determine the long-term passageability of organoids maintained in ProstaCult™ Organoid Growth Medium. Organoids that are generated at multiple passages are composed of a polarized epithelium with AR+K18+ luminal cells and K5+ basal cells. These results demonstrate that ProstaCult™ Organoid Growth Medium efficiently generates and expands prostate epithelial organoids. Citation Format: John Stingl, David Rowbotham, Terry E. Thomas, Allen C. Eaves, Sharon A. Louis. Expansion of mouse prostate epithelial stem cells in serum-free ProstaCult Organoid Growth Medium [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3111.

Fluorescence of Picrosirius Red Multiplexed With Immunohistochemistry for the Quantitative Assessment of Collagen in Tissue Sections.

The low cost and simplicity of picrosirius red (PSR) staining have driven its popularity for collagen detection in tissue sections. We extended the versatility of this method by using fluorescent imaging to detect the PSR signal and applying automated quantification tools. We also developed the first PSR protocol that is fully compatible with multiplex immunostaining, making it possible to test whether collagen structure differs across immunohistochemically labeled regions of the tissue landscape. We compared our imaging method with two gold standards in collagen imaging, linear polarized light microscopy and second harmonic generation imaging, and found that it is at least as sensitive and robust to changes in sample orientation. As proof of principle, we used a genetic approach to overexpress beta catenin in a patchy subset of mouse prostate epithelial cells distinguished only by immunolabeling. We showed that collagen fiber length is significantly greater near beta catenin overexpressing cells than near control cells. Our fluorescent PSR imaging method is sensitive, reproducible, and offers a new way to guide region of interest selection for quantifying collagen in tissue sections.

Abstract 4504: Androgen receptor aggregation is a key regulator of endoplasmic reticulum stress and cell survival in ETS-related gene driven prostate carcinogenesis

Abstract Introduction and Objectives: Deregulated androgen receptor (AR) signaling due to either mutations or altered expression of the AR and its cofactors (activators or suppressors) has been identified as critical in prostate cancer development and progression. AR regulated oncogenic activation of Ets Related Gene (ERG) represents one of the most common and validated prostate cancer driver gene. In our recent studies using prostate specific ERG transgenic mouse prostate glands, we observed novel morphological phenotypes of endoplasmic reticulum (ER) stress. Since AR was the critical regulator of ERG expression through TMPRSS2 promoter in human prostate cancer, the present study was aimed towards understanding the post-translational interactions between ERG and AR in ER stress and subsequent cell survival mechanisms in mouse and cell culture models. Understanding such mechanistic insights will potentially have major therapeutic implications. Methods: Histological phenotype in the mouse prostate glands were examined by light and electron microscopy. Cell culture models of LNCaP, HEK293 and COS7 cells were utilized to examine the AR aggregations, Co-IP and Proximal Ligation Assay in the presence and absence of ERG. Various domain deletions of AR were utilized to identify specific AR domain interactions with ERG and its contribution in AR aggregation. Luminal cell surface markers on the isolated mouse prostate glands and spontaneously immortalized mouse prostate epithelial cells from ERG transgenic mouse (MoE1) were analyzed by FACS analysis. Results: Co-expression of ERG and AR in LNCaP and COS-7 cells showed significant aggregation of AR in filter assays. Co-IP experiments and PLA assays in VCaP, LNCaP and HEK 293 cell revealed that ERG physically interacts with AR. Epithelial cells of ERG-Tg mouse prostates showed ~70% increase in CD49f (low) and Sca-1 (med) population with increased sphere formation capability and resistance to radiation induced cell death. Both epithelial cells grown into spheres and established MoE1 cells displayed increased CD49f (low) and significant increase in the EpCAM negative population. Conclusions: Overall, our experiments demonstrate the mechanistic link that the physical interactions between ERG and AR initiate the ER stress in prostate epithelium through AR misfolding/aggregation. Our observation of ERG induced AR aggregation is one of the initial events that lead to ER stress to cell survival indicate a critical function for ERG in the etiology of prostate cancer initiation and progression. Funding: This research in part was supported by the National Cancer Institute R01CA162383 (S. S.) and HU0001-10-2-0002 funds. Citation Format: Taduru L. Sreenath, Natallia Mikhalkevich, Shashwat Sharad, Rishita Gupta, Oluwatosin Diaro, Kevin Babcock, Charles Xavier, Ahmed Mohamed, Muhammad Jamal, Shyh-Han Tan, Albert Dobi, Gyorgy Petrovics, Isabell A. Sesterhenn, David G. McLeod, Inger L. Rosner, Shiv Srivastava. Androgen receptor aggregation is a key regulator of endoplasmic reticulum stress and cell survival in ETS-related gene driven prostate carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4504. doi:10.1158/1538-7445.AM2017-4504

MP87-19 ETS RELATED GENE (ERG) DRIVEN ANDROGEN RECEPTOR AGGREGATION IS A KEY REGULATOR OF ENDOPLASMIC STRESS AND CELL SURVIVAL DURING PROSTATE CARCINOGENESIS

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology III1 Apr 2017MP87-19 ETS RELATED GENE (ERG) DRIVEN ANDROGEN RECEPTOR AGGREGATION IS A KEY REGULATOR OF ENDOPLASMIC STRESS AND CELL SURVIVAL DURING PROSTATE CARCINOGENESIS Taduru Sreenath, Natallia Mikhalkevich, Shashwat Sharad, Rishita Gupta, Oluwatosin Diaro, Kevin Babcock, Charles Xavier, Ahmed Mohamed, Muhammad Jamal, Shyh-Han Tan, Albert Dobi, Gyorgy Petrovics, Isabell Sesterhenn, David McLeod, Inger Rosner, and Shiv Srivastava Taduru SreenathTaduru Sreenath More articles by this author , Natallia MikhalkevichNatallia Mikhalkevich More articles by this author , Shashwat SharadShashwat Sharad More articles by this author , Rishita GuptaRishita Gupta More articles by this author , Oluwatosin DiaroOluwatosin Diaro More articles by this author , Kevin BabcockKevin Babcock More articles by this author , Charles XavierCharles Xavier More articles by this author , Ahmed MohamedAhmed Mohamed More articles by this author , Muhammad JamalMuhammad Jamal More articles by this author , Shyh-Han TanShyh-Han Tan More articles by this author , Albert DobiAlbert Dobi More articles by this author , Gyorgy PetrovicsGyorgy Petrovics More articles by this author , Isabell SesterhennIsabell Sesterhenn More articles by this author , David McLeodDavid McLeod More articles by this author , Inger RosnerInger Rosner More articles by this author , and Shiv SrivastavaShiv Srivastava More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2723AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Deregulated androgen receptor (AR) signaling due to either mutations or altered expression of the AR and its cofactors (activators or suppressors) has been identified as critical in prostate cancer development and progression. AR regulated oncogenic activation of Ets Related Gene (ERG) represents one of the most common and validated prostate cancer driver gene. In our recent studies using prostate specific ERG transgenic mouse prostate glands, we a observed novel morphological phenotypes of endoplasmic reticulum (ER) stress. Since AR was the critical regulator of ERG expression through TMPRSS2 promoter in human prostate cancer, the present study was aimed towards understanding the post-translational interactions between ERG and AR in ER stress and subsequent cell survival mechanisms in mouse and cell culture models. Understanding such mechanistic insights will potentially have major therapeutic implications. METHODS Histological phenotype in the mouse prostate glands were examined by light and electron microscopy. Cell culture models of LNCaP, HEK293 and COS7 cells were utilized to examine the AR aggregations, Co-IP and Proximal Ligation Assay in the presence and absence of ERG. Various domain deletions of AR were utilized to identify specific AR domain interactions with ERG and its contribution in AR aggregation. Luminal cell surface markers on the isolated mouse prostate glands and spontaneously immortalized mouse prostate epithelial cells from ERG transgenic mouse (MoE1) were analyzed by FACS analysis. RESULTS Co-expression of ERG and AR in LNCaP and COS-7 cells showed significant aggregation of AR in filter assays. Co-IP experiments and PLA assays in VCaP, LNCaP and HEK 293 cell revealed that ERG physically interacts with AR. Epithelial cells of ERG-Tg mouse prostates showed ~70% increase in CD49f (low) and Sca-1 (med) population with increased sphere formation capability and resistance to radiation induced cell death. Both epithelial cells grown into spheres and established MoE1 cells displayed increased CD49f (low) and significant increase in the EpCAM negative population. CONCLUSIONS Overall, our experiments demonstrate the mechanistic link that the physical interactions between ERG and AR initiate the ER stress in prostate epithelium through AR misfolding/aggregation. Our observation of ERG induced AR aggregation is one of the initial events that lead to ER stress to cell survival indicate a critical function for ERG in the etiology of prostate cancer initiation and progression. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1173 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Taduru Sreenath More articles by this author Natallia Mikhalkevich More articles by this author Shashwat Sharad More articles by this author Rishita Gupta More articles by this author Oluwatosin Diaro More articles by this author Kevin Babcock More articles by this author Charles Xavier More articles by this author Ahmed Mohamed More articles by this author Muhammad Jamal More articles by this author Shyh-Han Tan More articles by this author Albert Dobi More articles by this author Gyorgy Petrovics More articles by this author Isabell Sesterhenn More articles by this author David McLeod More articles by this author Inger Rosner More articles by this author Shiv Srivastava More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Copper as a target for prostate cancer therapeutics: copper-ionophore pharmacology and altering systemic copper distribution.

Copper-ionophores that elevate intracellular bioavailable copper display significant therapeutic utility against prostate cancer cells in vitro and in TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) mice. However, the pharmacological basis for their anticancer activity remains unclear, despite impending clinical trails. Herein we show that intracellular copper levels in prostate cancer, evaluated in vitro and across disease progression in TRAMP mice, were not correlative with copper-ionophore activity and mirrored the normal levels observed in patient prostatectomy tissues (Gleason Score 7 & 9). TRAMP adenocarcinoma cells harbored markedly elevated oxidative stress and diminished glutathione (GSH)-mediated antioxidant capacity, which together conferred selective sensitivity to prooxidant ionophoric copper. Copper-ionophore treatments [CuII(gtsm), disulfiram & clioquinol] generated toxic levels of reactive oxygen species (ROS) in TRAMP adenocarcinoma cells, but not in normal mouse prostate epithelial cells (PrECs). Our results provide a basis for the pharmacological activity of copper-ionophores and suggest they are amendable for treatment of patients with prostate cancer. Additionally, recent in vitro and mouse xenograft studies have suggested an increased copper requirement by prostate cancer cells. We demonstrated that prostate adenocarcinoma development in TRAMP mice requires a functional supply of copper and is significantly impeded by altered systemic copper distribution. The presence of a mutant copper-transporting Atp7b protein (tx mutation: A4066G/Met1356Val) in TRAMP mice changed copper-integration into serum and caused a remarkable reduction in prostate cancer burden (64% reduction) and disease severity (grade), abrogating adenocarcinoma development. Implications for current clinical trials are discussed.

Abstract SY13-03: Signal pathways in prostate cancer stem cells

Abstract New therapeutic strategies are needed to treat prostate cancer as it progresses to the castration resistant stage following treatment with drugs to reduce androgen production or interfere with androgen receptor function. Genetic analyses of DNA alterations and RNA expression show that multiple pathways can be involved in this cancer progression. An understanding of the cellular components needed to initiate prostate cancer and sustain it during the process of metastasis may help to elucidate new pathways for therapy. We have developed techniques to transform mouse and human benign prostate epithelial basal stem cells and define them as one cell of origin which can respond to multiple stimuli to produce cancers which are capable of maturing to more differentiated cell types including luminal, squamous and neuroendocrine phenotypes. Different subtypes of cells with the properties of cancer stem cells capable of transplantation of the cancer phenotype have been defined. Current work is focusing on the activation of specific kinase driven pathways to provide new targets for therapy in metastatic prostate cancer. We previously demonstrated that castration resistant prostate cancer (CRPC) tissues exhibit increased tyrosine phosphorylation, suggesting that enhanced tyrosine kinase activity may contribute to advanced disease. To identify specific kinase activation patterns in metastatic CRPC, we utilized phospho-tyrosine peptide enrichment and quantitative mass spectrometry using tissue from CRPC patients obtained at rapid autopsy. Evaluation of these rare metastatic CRPC samples for tyrosine phosphorylation and kinases revealed activated SRC, EGFR, RET, ALK, and other targets. Importantly, these kinase activation patterns exhibited intrapatient similarity and interpatient heterogeneity implying clonal origins of these lesions. To evaluate the function of these kinases in metastatic CRPC, we are analyzing their role during specific steps of the metastatic cascade. We hope our approach will improve an understanding of the mechanistic role of kinase activation in prostate cancer and identify promising therapeutic strategies targeting non-mutated kinases for metastatic CRPC. Citation Format: Owen N. Witte. Signal pathways in prostate cancer stem cells. [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 SY13-03. doi:10.1158/1538-7445.AM2014-SY13-03

Abstract 445: Coordinate loss of CHD1 and MAP3K7 promotes aggressive prostate cancer

Abstract The genomic complexity of prostate cancer contributes to disease progression, resistance to therapy, and development of metastases. The genes CHD1 and MAP3K7 are signficantly co-deleted in 10-15% of primary tumors and combined loss of CHD1 and MAP3K7 expression is associated with poor disease-free survival. We recently validated MAP3K7 as a prostate tumor suppressor, but CHD1's role in prostate tumorigenesis has not been evaluated. In this study, we used a mouse prostate epithelial stem cell (PrP/SC) model and a human prostate cancer cell line (LNCaP) model to investigate how loss of CHD1 alone or in combination with MAP3K7 suppression affects prostate tumorigenesis. Suppression of Map3k7 increased proliferation of PrP/SCs. Chd1 suppression decreased growth of wildtype PrP/SCs but not PrP/SCs already deficient in Map3k7 expression. Similarly, suppression of CHD1 in LNCaP cells decreased proliferation when MAP3K7 expression was present, but not when MAP3K7 was suppressed. These data suggest that loss of CHD1 alone is deleterious to growth, but that MAP3K7 suppression can compensate for this growth inhibition. Chd1 and Map3k7-deficient PrP/SCs were then evaluated in vivo using tissue recombination. When Chd1-deficient PrP/SCs were recombined with fetal rat urogenital mesenchyme (rUGM) and implanted under the renal capsules of immunocompromised mice, grafts contained mostly benign glandular tissue with focal areas of mild hyperplasia/prostatic intraepithelial neoplasia (PIN). A few nuclear abnormalities were observed. Map3k7-deficient recombinants generated a mixture of benign tissue, PIN, and adenocarcinoma (consistent with our previous findings). The nuclear morphology of these grafts was unremarkable. Strikingly, dual Chd1-Map3k7 knockdown recombinants displayed massive glandular atypia with glands completely filled with AR-negative and p63-negative cells. These cells had large nuclei, prominent nucleoli, and abundant cytoplasm. Tumorigenicity of CHD1 and/or MAP3K7-deficient LNCaP cells was also assessed by subcutaneous implantation of cells into immunocompromised mice. Mice implanted with CHD1-deficient xenografts formed smaller tumors and had increased survival relative to mice injected with control LNCaP cells (p=0.0083). MAP3K7-deficient xenografts were similar in size and in survival rate compared to control LNCAP cells. Dual CHD1-MAP3K7 deficient xenografts, in contrast, showed increased tumor growth and decreased survival relative to control (p=0.0242), MAP3K7 alone (p=0.0462) and CHD1 alone (p=0.0002). Collectively, these data suggest that loss of CHD1 alone is anti-tumorigenic but that combined loss of CHD1 and MAP3K7 drives aggressive prostate cancer development. Citation Format: Lindsey Ulkus, Leah Rider, Cera Nieto, Lina Romero, Haitao Chen, Massimo Loda, Wennuan Liu, Jianfeng Xu, Scott Cramer. Coordinate loss of CHD1 and MAP3K7 promotes aggressive 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 445. doi:10.1158/1538-7445.AM2014-445