Publications by authors named "Omar E Franco"

54 Publications

Loss of ephrin B2 receptor (EPHB2) sets lipid rheostat by regulating proteins DGAT1 and ATGL inducing lipid droplet storage in prostate cancer cells.

Lab Invest 2021 Apr 6. Epub 2021 Apr 6.

Department of Surgery, Urology Division, Department of Cancer Biology, NorthShore University HealthSystem, Affiliate of the University of Chicago Pritzker School of Medicine, Evanston, IL, USA.

Lipid droplet (LD) accumulation in cancer results from aberrant metabolic reprograming due to increased lipid uptake, diminished lipolysis and/or de novo lipid synthesis. Initially implicated in storage and lipid trafficking in adipocytes, LDs are more recently recognized to fuel key functions associated with carcinogenesis and progression of several cancers, including prostate cancer (PCa). However, the mechanisms controlling LD accumulation in cancer are largely unknown. EPHB2, a tyrosine kinase (TKR) ephrin receptor has been proposed to have tumor suppressor functions in PCa, although the mechanisms responsible for these effects are unclear. Given that dysregulation in TRK signaling can result in glutaminolysis we postulated that EPHB2 might have potential effects on lipid metabolism. Knockdown strategies for EPHB2 were performed in prostate cancer cells to analyze the impact on the net lipid balance, proliferation, triacylglycerol-regulating proteins, effect on LD biogenesis, and intracellular localization of LDs. We found that EPHB2 protein expression in a panel of human-derived prostate cancer cell lines was inversely associated with in vivo cell aggressiveness. EPHB2 silencing increased the proliferation of prostate cancer cells and concurrently induced de novo LD accumulation in both cytoplasmic and nuclear compartments as well as a "shift" on LD size distribution in newly formed lipid-rich organelles. Lipid challenge using oleic acid exacerbated the effects on the LD phenotype. Loss of EPHB2 directly regulated key proteins involved in maintaining lipid homeostasis including, increasing lipogenic DGAT1, DGAT2 and PLIN2 and decreasing lipolytic ATGL and PEDF. A DGAT1-specific inhibitor abrogated LD accumulation and proliferative effects induced by EPHB2 loss. In conclusion, we highlight a new anti-tumor function of EPHB2 in lipid metabolism through regulation of DGAT1 and ATGL in prostate cancer. Blockade of DGAT1 in EPHB2-deficient tumors appears to be effective in restoring the lipid balance and reducing tumor growth.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41374-021-00583-9DOI Listing
April 2021

The role of the androgen receptor in prostate development and benign prostatic hyperplasia: A review.

Asian J Urol 2020 Jul 19;7(3):191-202. Epub 2019 Oct 19.

Department of Surgery, NorthShore University HealthSystem, Evanston, IL, USA.

Benign prostatic hyperplasia (BPH) is a benign enlargement of the prostate in which incidence increases linearly with age, beginning at about 50 years old. BPH is a significant source of morbidity in aging men by causing lower urinary tract symptoms and acute urinary retention. Unfortunately, the etiology of BPH incidence and progression is not clear. This review highlights the role of the androgen receptor (AR) in prostate development and the evidence for its involvement in BPH. The AR is essential for normal prostate development, and individuals with defective AR signaling, such as after castration, do not experience prostate enlargement with age. Furthermore, decreasing dihydrotestosterone availability through therapeutic targeting with 5α-reductase inhibitors diminishes AR activity and results in reduced prostate size and symptoms in some BPH patients. While there is some evidence that AR expression is elevated in certain cellular compartments, how exactly AR is involved in BPH progression has yet to be elucidated. It is possible that AR signaling within stromal cells alters intercellular signaling and a "reawakening" of the embryonic mesenchyme, loss of epithelial AR leads to changes in paracrine signaling interactions, and/or chronic inflammation aids in stromal or epithelial proliferation evident in BPH. Unfortunately, a subset of patients fails to respond to current medical approaches, forcing surgical treatment even though age or associated co-morbidities make surgery less attractive. Fundamentally, new therapeutic approaches to treat BPH are not currently forthcoming, so a more complete molecular understanding of BPH etiology is necessary to identify new treatment options.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajur.2019.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7385520PMC
July 2020

Stromal reactivity differentially drives tumour cell evolution and prostate cancer progression.

Nat Ecol Evol 2020 06 11;4(6):870-884. Epub 2020 May 11.

Department of Integrated Mathematical Oncology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA.

Prostate cancer (PCa) progression is a complex eco-evolutionary process driven by the feedback between evolving tumour cell phenotypes and microenvironmentally driven selection. To better understand this relationship, we used a multiscale mathematical model that integrates data from biology and pathology on the microenvironmental regulation of PCa cell behaviour. Our data indicate that the interactions between tumour cells and their environment shape the evolutionary dynamics of PCa cells and explain overall tumour aggressiveness. A key environmental determinant of this aggressiveness is the stromal ecology, which can be either inhibitory, highly reactive (supportive) or non-reactive (neutral). Our results show that stromal ecology correlates directly with tumour growth but inversely modulates tumour evolution. This suggests that aggressive, environmentally independent PCa may be a result of poor stromal ecology, supporting the concept that purely tumour epithelium-centric metrics of aggressiveness may be incomplete and that incorporating markers of stromal ecology would improve prognosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41559-020-1157-yDOI Listing
June 2020

Propagation of human prostate tissue from induced pluripotent stem cells.

Stem Cells Transl Med 2020 07 14;9(7):734-745. Epub 2020 Mar 14.

Translational and Clinical Research Institute, Newcastle University Centre for Cancer, Newcastle University, Newcastle upon Tyne, UK.

Primary culture of human prostate organoids and patient-derived xenografts is inefficient and has limited access to clinical tissues. This hampers their use for translational study to identify new treatments. To overcome this, we established a complementary approach where rapidly proliferating and easily handled induced pluripotent stem cells enabled the generation of human prostate tissue in vivo and in vitro. By using a coculture technique with inductive urogenital sinus mesenchyme, we comprehensively recapitulated in situ 3D prostate histology, and overcame limitations in the primary culture of human prostate stem, luminal and neuroendocrine cells, as well as the stromal microenvironment. This model now unlocks new opportunities to undertake translational studies of benign and malignant prostate disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/sctm.19-0286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308643PMC
July 2020

Heterogeneity of human prostate carcinoma-associated fibroblasts implicates a role for subpopulations in myeloid cell recruitment.

Prostate 2020 02 25;80(2):173-185. Epub 2019 Nov 25.

Department of Surgery, NorthShore University HealthSystem, Evanston, Illinois.

Background: Carcinoma-associated fibroblasts (CAF) are a heterogeneous group of cells within the tumor microenvironment (TME) that can promote tumorigenesis in the prostate. By understanding the mechanism(s) by which CAF contributes to tumor growth, new therapeutic targets for the management of this disease may be identified. These studies determined whether unique sub-populations of human prostate CAF can be identified and functionally characterized.

Methods: Single-cell RNA-seq of primary human prostate CAF followed by unsupervised clustering was utilized to generate cell clusters based on differentially expressed (DE) gene profiles. Potential communication between CAF and immune cells was analyzed using in vivo tissue recombination by combining CAF or normal prostate fibroblasts (NPF) with non-tumorigenic, initiated prostate epithelial BPH-1 cells. Resultant grafts were assessed for inflammatory cell recruitment.

Results: Clustering of 3321 CAF allows for visualization of six subpopulations, demonstrating heterogeneity within CAF. Sub-renal capsule recombination assays show that the presence of CAF significantly increases myeloid cell recruitment to resultant tumors. This is supported by significantly increased expression of chemotactic chemokines CCL2 and CXCL12 in large clusters compared to other subpopulations. Bayesian analysis topologies also support differential communication signals between chemokine-related genes of individual clusters. Migration of THP-1 monocyte cells in vitro is stimulated in the presence of CAF conditioned medium (CM) compared with NPF CM. Further in vitro analyses suggest that CAF-derived chemokine CCL2 may be responsible for CAF-stimulated migration of THP-1 cells, since neutralization of this chemokine abrogates migration capacity.

Conclusions: CAF clustering based on DE gene expression supports the concept that clusters have unique functions within the TME, including a role in immune/inflammatory cell recruitment. These data suggest that CCL2 produced by CAF may be involved in the recruitment of inflammatory cells, but may also directly regulate the growth of the tumor. Further studies aimed at characterizing the subpopulation(s) of CAF which promote immune cell recruitment to the TME and/or stimulate prostate cancer growth and progression will be pursued.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pros.23929DOI Listing
February 2020

Injection molded open microfluidic well plate inserts for user-friendly coculture and microscopy.

Lab Chip 2020 01 12;20(1):107-119. Epub 2019 Nov 12.

Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195, USA. and Department of Urology, University of Washington School of Medicine, Seattle, WA 98195, USA.

Open microfluidic cell culture systems are powerful tools for interrogating biological mechanisms. We have previously presented a microscale cell culture system, based on spontaneous capillary flow of biocompatible hydrogels, that is integrated into a standard cell culture well plate, with flexible cell compartment geometries and easy pipet access. Here, we present two new injection molded open microfluidic devices that also easily insert into standard cell culture well plates and standard culture workflows, allowing seamless adoption by biomedical researchers. These platforms allow culture and study of soluble factor communication among multiple cell types, and the microscale dimensions are well-suited for rare primary cells. Unique advances include optimized evaporation control within the well, manufacture with reproducible and cost-effective rapid injection molding, and compatibility with sample preparation workflows for high resolution microscopy (following well-established coverslip mounting procedures). In this work, we present several use cases that highlight the usability and widespread utility of our platform including culture of limited primary testis cells from surgical patients, microscopy readouts including immunocytochemistry and single molecule fluorescence in situ hybridization (smFISH), and coculture to study interactions between adipocytes and prostate cancer cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c9lc00706gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6917835PMC
January 2020

Lipid droplet velocity is a microenvironmental sensor of aggressive tumors regulated by V-ATPase and PEDF.

Lab Invest 2019 12 13;99(12):1822-1834. Epub 2019 Aug 13.

Department of Surgery, NorthShore University Research Institute, Affiliate of University of Chicago Pritzker School of Medicine, Evanston, IL, 60201, USA.

Lipid droplets (LDs) utilize microtubules (MTs) to participate in intracellular trafficking of cargo proteins. Cancer cells accumulate LDs and acidify their tumor microenvironment (TME) by increasing the proton pump V-ATPase. However, it is not known whether these two metabolic changes are mechanistically related or influence LD movement. We postulated that LD density and velocity are progressively increased with tumor aggressiveness and are dependent on V-ATPase and the lipolysis regulator pigment epithelium-derived factor (PEDF). LD density was assessed in human prostate cancer (PCa) specimens across Gleason scores (GS) 6-8. LD distribution and velocity were analyzed in low and highly aggressive tumors using live-cell imaging and in cells exposed to low pH and/or treated with V-ATPase inhibitors. The MT network was disrupted and analyzed by α-tubulin staining. LD density positively correlated with advancing GS in human tumors. Acidification promoted peripheral localization and clustering of LDs. Highly aggressive prostate, breast, and pancreatic cell lines had significantly higher maximum LD velocity (LDVmax) than less aggressive and benign cells. LDVmax was MT-dependent and suppressed by blocking V-ATPase directly or indirectly with PEDF. Upon lowering pH, LDs moved to the cell periphery and carried metalloproteinases. These results suggest that acidification of the TME can alter intracellular LD movement and augment velocity in cancer. Restoration of PEDF or blockade of V-ATPase can normalize LD distribution and decrease velocity. This study identifies V-ATPase and PEDF as new modulators of LD trafficking in the cancer microenvironment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41374-019-0296-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289525PMC
December 2019

Hyperglycemia and T Cell infiltration are associated with stromal and epithelial prostatic hyperplasia in the nonobese diabetic mouse.

Prostate 2019 06 18;79(9):980-993. Epub 2019 Apr 18.

Department of Surgery, NorthShore University HealthSystem, Evanston, Illinois.

Background: Prostatic inflammation and various proinflammatory systemic comorbidities, such as diabetes and obesity are associated with human benign prostatic hyperplasia (BPH). There is a paucity of in vivo models reflecting specific aspects of BPH pathogenesis. Our aim was to investigate the nonobese diabetic (NOD) mouse as a potential model for subsequent intervention studies.

Materials And Methods: We used the NOD mouse, a model of autoimmune inflammation leading to type 1 diabetes to examine the effects of systemic inflammation and diabetes on the prostate. We assessed changes in prostatic histology, infiltrating leukocytes, and gene expression associated with aging and diabetic status.

Results: Both stromal expansion and epithelial hyperplasia were observed in the prostates. Regardless of diabetic status, the degree of prostatic hyperplasia varied. Local inflammation was associated with a more severe prostatic phenotype in both diabetic and nondiabetic mice. Testicular atrophy was noted in diabetic mice, but prostate glands showed persistent focal cell proliferation. In addition, a prostatic intraepithelial neoplasia (PIN)-like phenotype was seen in several diabetic animals with an associated increase in c-Myc and MMP-2 expression. To examine changes in gene and cytokine expression we performed microarray and cytokine array analysis comparing the prostates of diabetic and nondiabetic animals. Microarray analysis revealed several differentially expressed genes including CCL3, CCL12, and TNFS10. Cytokine array analysis revealed increased expression of cytokines and proteases such as LDLR, IL28 A/B, and MMP-2 in diabetic mice.

Conclusion: Overall, NOD mice provide a model to examine the effects of hyperglycemia and chronic inflammation on the prostate, demonstrating relevance to some of the mechanisms present underlying BPH and potentially the initiation of prostate cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pros.23809DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591734PMC
June 2019

DGAT1 Inhibitor Suppresses Prostate Tumor Growth and Migration by Regulating Intracellular Lipids and Non-Centrosomal MTOC Protein GM130.

Sci Rep 2019 02 28;9(1):3035. Epub 2019 Feb 28.

Department of Surgery, NorthShore University Research Institute, Affiliate of University of Chicago Pritzker School of Medicine, Evanston, IL, 60201, United States.

Acyl-CoA:diacylglycerol acyltransferase I (DGAT1) is a key enzyme in lipogenesis which is increased in metabolically active cells to meet nutrient requirements. DGAT1 has been recognized as an anti-obesity target; however, its role in the tumor microenvironment remains unclear. We postulated that, in prostate cancer (PCa) cells, augmented lipogenesis and growth are due to increased DGAT1 expression leading to microtubule-organizing center (MTOC) amplification. Thus, therapeutic targeting of DGAT1 potentially has tumor suppressive activity. We tested whether blocking DGAT1 in PCa cells altered MTOC and lipid signaling. Western blot and immunofluorescence were performed for MTOC and triglyceride mediators. Treatment with a DGAT1 inhibitor was evaluated. We found a stepwise increase in DGAT1 protein levels when comparing normal prostate epithelial cells to PCa cells, LNCaP and PC-3. Lipid droplets, MTOCs, and microtubule-regulating proteins were reduced in tumor cells treated with a DGAT1 inhibitor. Depletion of the non-centrosomal MTOC protein GM130 reduced PCa cell proliferation and migration. Inhibition of DGAT1 reduced tumor growth both in vitro and in vivo, and a negative feedback loop was discovered between DGAT1, PEDF, and GM130. These data identify DGAT1 as a promising new target for suppressing PCa growth by regulating GM130, MTOC number and disrupting microtubule integrity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-39537-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395665PMC
February 2019

Tyrosine kinase inhibitor therapy prescribed for non-urologic diseases can modify PSA titers in urology patients.

Prostate 2019 02 28;79(3):259-264. Epub 2018 Oct 28.

Department of Surgery, NorthShore University HealthSystem, Affiliate of University of Chicago Pritzker School of Medicine, Evanston, Illinois.

Background: The tyrosine kinase inhibitors (TKI), imatinib and nilotinib, are used to treat chronic myelogenous leukemia (CML). In three CML patients being monitored for urologic diseases, we observed that switching of TKI therapy affected prostate-specific antigen (PSA) titers. Urologists and other medical professionals need to be aware of the potential side-effects of drugs that patients may be receiving for other indications to modify this important prostate diseases indicator. TKIs may affect PSA titers independent of prostate growth or volume.

Materials And Methods: We followed PSA levels in urology patients who were also undergoing TKI treatment for CML. We determined the effects of nilotinib and imatinib on proliferation, AR and PSA expression in the LNCaP and 22Rv1 prostate cancer (PCa) cell lines using real-time PCR and Western blotting.

Results: Clinically, nilotinib and dasatinib reversibly reduced PSA titers compared to imatinib. At high doses nilotinib and imatinib both demonstrated antiproliferative effects in the PCa cells. At low doses expression of AR and PSA was decreased by both drugs, at mRNA and protein levels. Nilotinib exerted greater effects at lower doses than imatinib.

Conclusions: Nilotinib down-regulates serum PSA in patients being treated for non-urological indications, potentially masking a clinical useful marker, we cannot exclude a similar but smaller effect of imatinib. Nilotinib and imatinib both decreased AR and PSA expression in PCa cell lines with the nilotinib effect evident at lower doses. Urologists must appreciate the effects of drugs provided for other diseases on PSA titers and be aware that sudden changes may not reflect underlying prostatic disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pros.23730DOI Listing
February 2019

Elevation of Stromal-Derived Mediators of Inflammation Promote Prostate Cancer Progression in African-American Men.

Cancer Res 2018 11 4;78(21):6134-6145. Epub 2018 Sep 4.

Department of Surgery, NorthShore University HealthSystem Research Institute, Evanston, Illinois.

Progress in prostate cancer racial disparity research has been hampered by a lack of appropriate research tools and better understanding of the tumor biology. Recent gene expression studies suggest that the tumor microenvironment (TME) may contribute to racially disparate clinical outcomes in prostate cancer. Analysis of the prostate TME has shown increased reactive stroma associated with chronic inflammatory infiltrates in African-American (AA) compared with European-American (EA) patients with prostate cancer. To better understand stromal drivers of changes in TME, we isolated prostate fibroblasts (PrF) from AA (PrF) and EA (PrF) prostate cancer tissues and studied their functional characteristics. PrF showed increased growth response to androgens FGF2 and platelet-derived growth factor. Compared with PrF, conditioned media from PrF significantly enhanced the proliferation and motility of prostate cancer cell lines. Expression of markers associated with myofibroblast activation (αSMA, vimentin, and tenascin-C) was elevated in PrF tumorigenicity of an AA patient-derived prostatic epithelial cell line E006AA was significantly increased in the presence of PrF compared with PrF, and RNA-seq data and cytokine array analysis identified a panel of potential proinflammatory paracrine mediators (BDNF, CHI3L1, DPPIV, FGF7, IL18BP, IL6, and VEGF) to be enriched in PrF E006AA cell lines showed increased responsiveness to BDNF ligand compared with EA-derived LNCaP and C4-2B cells. Addition of a TrkB-specific antagonist significantly reduced the protumorigenic effects induced by PrF compared with PrF These findings suggest that fibroblasts in the TME of AA patients may contribute to the health disparity observed in the incidence and progression of prostate cancer tumors. These findings suggest that stromal cells in the tumor microenvironment of African-American men promote progression of prostate cancer by increasing levels of a specific set of pro-inflammatory molecules compared with European-American men. http://cancerres.aacrjournals.org/content/canres/78/21/6134/F1.large.jpg .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-17-3810DOI Listing
November 2018

PEDF regulates plasticity of a novel lipid-MTOC axis in prostate cancer-associated fibroblasts.

J Cell Sci 2018 07 11;131(13). Epub 2018 Jul 11.

Department of Surgery, NorthShore University Research Institute, Affiliate of University of Chicago Pritzker School of Medicine, Evanston, IL 60201, United States

Prostate tumors make metabolic adaptations to ensure adequate energy and amplify cell cycle regulators, such as centrosomes, to sustain their proliferative capacity. It is not known whether cancer-associated fibroblasts (CAFs) undergo metabolic re-programming. We postulated that CAFs augment lipid storage and amplify centrosomal or non-centrosomal microtubule-organizing centers (MTOCs) through a pigment epithelium-derived factor (PEDF)-dependent lipid-MTOC signaling axis. Primary human normal prostate fibroblasts (NFs) and CAFs were evaluated for lipid content, triacylglycerol-regulating proteins, MTOC number and distribution. CAFs were found to store more neutral lipids than NFs. Adipose triglyceride lipase (ATGL) and PEDF were strongly expressed in NFs, whereas CAFs had minimal to undetectable levels of PEDF or ATGL protein. At baseline, CAFs demonstrated MTOC amplification when compared to 1-2 perinuclear MTOCs consistently observed in NFs. Treatment with PEDF or blockade of lipogenesis suppressed lipid content and MTOC number. In summary, our data support that CAFs have acquired a tumor-like phenotype by re-programming lipid metabolism and amplifying MTOCs. Normalization of MTOCs by restoring PEDF or by blocking lipogenesis highlights a previously unrecognized plasticity in centrosomes, which is regulated through a new lipid-MTOC axis.This article has an associated First Person interview with the first author of the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.213579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6051346PMC
July 2018

Cancer-associated fibroblasts promote directional cancer cell migration by aligning fibronectin.

J Cell Biol 2017 11 11;216(11):3799-3816. Epub 2017 Oct 11.

Department of Biological Sciences, Vanderbilt University, Nashville, TN.

Cancer-associated fibroblasts (CAFs) are major components of the carcinoma microenvironment that promote tumor progression. However, the mechanisms by which CAFs regulate cancer cell migration are poorly understood. In this study, we show that fibronectin (Fn) assembled by CAFs mediates CAF-cancer cell association and directional migration. Compared with normal fibroblasts, CAFs produce an Fn-rich extracellular matrix with anisotropic fiber orientation, which guides the cancer cells to migrate directionally. CAFs align the Fn matrix by increasing nonmuscle myosin II- and platelet-derived growth factor receptor α-mediated contractility and traction forces, which are transduced to Fn through α5β1 integrin. We further show that prostate cancer cells use αv integrin to migrate efficiently and directionally on CAF-derived matrices. We demonstrate that aligned Fn is a prominent feature of invasion sites in human prostatic and pancreatic carcinoma samples. Collectively, we present a new mechanism by which CAFs organize the Fn matrix and promote directional cancer cell migration.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201704053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5674895PMC
November 2017

Interaction of prostate carcinoma-associated fibroblasts with human epithelial cell lines in vivo.

Differentiation 2017 Jul - Aug;96:40-48. Epub 2017 Jul 20.

Department of Surgery, NorthShore University HealthSystem, Evanston, IL, USA. Electronic address:

Stromal-epithelial interactions play a crucial and poorly understood role in carcinogenesis and tumor progression. Mesenchymal-epithelial interactions have a long history of research in relation to the development of organs. Models designed to study development are often also applicable to studies of benign and malignant disease. Tumor stroma is a complex mixture of cells that includes a fibroblastic component often referred to as cancer-associated fibroblasts (CAF), desmoplasia or "reactive" stroma. Here we discuss the history of, and approaches to, understanding these interactions with particular reference to prostate cancer and to in vivo modeling using human cells and tissues. A series of studies have revealed a complex mixture of signaling molecules acting both within the stromal tissue and between the stromal and epithelial tissues. We are starting to understand the interactions of some of these pathways, however the work is still ongoing. This area of research provide a basis for new medical approaches aimed at stabilizing early stage cancers rendering them chronic rather than acute problems. Such work is especially relevant to slow growing tumors found in older patients, a class that would include many prostate cancers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.diff.2017.07.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5669818PMC
June 2018

Pathomimetic avatars reveal divergent roles of microenvironment in invasive transition of ductal carcinoma in situ.

Breast Cancer Res 2017 05 15;19(1):56. Epub 2017 May 15.

Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI, 48201, USA.

Background: The breast tumor microenvironment regulates progression of ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). However, it is unclear how interactions between breast epithelial and stromal cells can drive this progression and whether there are reliable microenvironmental biomarkers to predict transition of DCIS to IDC.

Methods: We used xenograft mouse models and a 3D pathomimetic model termed mammary architecture and microenvironment engineering (MAME) to study the interplay between human breast myoepithelial cells (MEPs) and cancer-associated fibroblasts (CAFs) on DCIS progression.

Results: Our results show that MEPs suppress tumor formation by DCIS cells in vivo even in the presence of CAFs. In the in vitro MAME model, MEPs reduce the size of 3D DCIS structures and their degradation of extracellular matrix. We further show that the tumor-suppressive effects of MEPs on DCIS are linked to inhibition of urokinase plasminogen activator (uPA)/urokinase plasminogen activator receptor (uPAR)-mediated proteolysis by plasminogen activator inhibitor 1 (PAI-1) and that they can lessen the tumor-promoting effects of CAFs by attenuating interleukin 6 (IL-6) signaling pathways.

Conclusions: Our studies using MAME are, to our knowledge, the first to demonstrate a divergent interplay between MEPs and CAFs within the DCIS tumor microenvironment. We show that the tumor-suppressive actions of MEPs are mediated by PAI-1, uPA and its receptor, uPAR, and are sustained even in the presence of the CAFs, which themselves enhance DCIS tumorigenesis via IL-6 signaling. Identifying tumor microenvironmental regulators of DCIS progression will be critical for defining a robust and predictive molecular signature for clinical use.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13058-017-0847-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5433063PMC
May 2017

Genome-wide analysis of AR binding and comparison with transcript expression in primary human fetal prostate fibroblasts and cancer associated fibroblasts.

Mol Cell Endocrinol 2018 08 5;471:1-14. Epub 2017 May 5.

Department of Surgery, Division of Urology, McGill University and the Cancer Research Program of the McGill University Health Centre Research Institute, Montreal, Quebec, H4A 3J1, Canada. Electronic address:

The androgen receptor (AR) is a transcription factor, and key regulator of prostate development and cancer, which has discrete functions in stromal versus epithelial cells. AR expressed in mesenchyme is necessary and sufficient for prostate development while loss of stromal AR is predictive of prostate cancer progression. Many studies have characterized genome-wide binding of AR in prostate tumour cells but none have used primary mesenchyme or stroma. We applied ChIPseq to identify genomic AR binding sites in primary human fetal prostate fibroblasts and patient derived cancer associated fibroblasts, as well as the WPMY1 cell line overexpressing AR. We identified AR binding sites that were specific to fetal prostate fibroblasts (7534), cancer fibroblasts (629), WPMY1-AR (2561) as well as those common among all (783). Primary fibroblasts had a distinct AR binding profile versus prostate cancer cell lines and tissue, and showed a localisation to gene promoter binding sites 1 kb upstream of the transcriptional start site, as well as non-classical AR binding sequence motifs. We used RNAseq to define transcribed genes associated with AR binding sites and derived cistromes for embryonic and cancer fibroblasts as well as a cistrome common to both. These were compared to several in vivo ChIPseq and transcript expression datasets; which identified subsets of AR targets that were expressed in vivo and regulated by androgens. This analysis enabled us to deconvolute stromal AR targets active in stroma within tumour samples. Taken together, our data suggest that the AR shows significantly different genomic binding site locations in primary prostate fibroblasts compared to that observed in tumour cells. Validation of our AR binding site data with transcript expression in vitro and in vivo suggests that the AR target genes we have identified in primary fibroblasts may contribute to clinically significant and biologically important AR-regulated changes in prostate tissue.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.mce.2017.05.006DOI Listing
August 2018

Reduced Contractility and Motility of Prostatic Cancer-Associated Fibroblasts after Inhibition of Heat Shock Protein 90.

Cancers (Basel) 2016 Aug 24;8(9). Epub 2016 Aug 24.

Division of Urology, Department of Surgery, Cancer Research Program, McGill University Health Centre Research Institute, 1001 Decarie Blvd, Montreal, QC H4A 3J1, Canada.

Background: Prostate cancer-associated fibroblasts (CAF) can stimulate malignant progression and invasion of prostatic tumour cells via several mechanisms including those active in extracellular matrix;

Methods: We isolated CAF from prostate cancer patients of Gleason Score 6-10 and confirmed their cancer-promoting activity using an in vivo tumour reconstitution assay comprised of CAF and BPH1 cells. We tested the effects of heat shock protein 90 (HSP90) inhibitors upon reconstituted tumour growth in vivo. Additionally, CAF contractility was measured in a 3D collagen contraction assay and migration was measured by scratch assay;

Results: HSP90 inhibitors dipalmitoyl-radicicol and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG) reduced tumour size and proliferation in CAF/BPH1 reconstituted tumours in vivo. We observed that the most contractile CAF were derived from patients with lower Gleason Score and of younger age compared with the least contractile CAF. HSP90 inhibitors radicicol and 17-DMAG inhibited contractility and reduced the migration of CAF in scratch assays. Intracellular levels of HSP70 and HSP90 were upregulated upon treatment with HSP90 inhibitors. Inhibition of HSP90 also led to a specific increase in transforming growth factor beta 2 (TGFβ2) levels in CAF;

Conclusions: We suggest that HSP90 inhibitors act not only upon tumour cells, but also on CAF in the tumour microenvironment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040979PMC
http://dx.doi.org/10.3390/cancers8090077DOI Listing
August 2016

Review of Prostate Anatomy and Embryology and the Etiology of Benign Prostatic Hyperplasia.

Urol Clin North Am 2016 Aug;43(3):279-88

Department of Biochemistry and Cancer Biology, Meharry Medical College, 1005 DR DB Todd JR Blvd, Nashville, TN 37208, USA; Department of Surgery, NorthShore University HealthSystem Research Institute, 1001 University Place, Evanston, IL 60201, USA. Electronic address:

Prostate development follows a common pattern between species and depends on the actions of androgens to induce and support ductal branching morphogenesis of buds emerging from the urogenital sinus. The human prostate has a compact zonal anatomy immediately surrounding the urethra and below the urinary bladder. Rodents have a lobular prostate with lobes radiating away from the urethra. The human prostate is the site of benign hyperplasia, prostate cancer, and prostatitis. The rodent prostate has little naturally occurring disease. Rodents can be used to model aspects of human benign hyperplasia, but care should be taken in data interpretation and extrapolation to the human condition.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ucl.2016.04.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4968575PMC
August 2016

Altered TGF-α/β signaling drives cooperation between breast cancer cell populations.

FASEB J 2016 10 6;30(10):3441-3452. Epub 2016 Jul 6.

Department of Surgery, NorthShore University HealthSystem, Evanston, Illinois, USA; Department of Urologic Surgery, Vanderbilt University Medical Center, Vanderbilt University, Nashville, Tennessee, USA; and Department of Cancer Biology, Vanderbilt University Medical Center, Vanderbilt University, Nashville, Tennessee, USA

The role of tumor heterogeneity in regulating disease progression is poorly understood. We hypothesized that interactions between subpopulations of cancer cells can affect the progression of tumors selecting for a more aggressive phenotype. We developed an in vivo assay based on the immortalized nontumorigenic breast cell line MCF10A and its Ras-transformed derivatives AT1 (mildly tumorigenic) and CA1d (highly tumorigenic). CA1d cells outcompeted MCF10A, forming invasive tumors. AT1 grafts were approximately 1% the size of CA1d tumors when initiated using identical cell numbers. In contrast, CA1d/AT1 mixed tumors were larger than tumors composed of AT1 alone (100-fold) or CA1d (3-fold), suggesting cooperation in tumor growth. One of the mechanisms whereby CA1d and AT1 were found to cooperate was by modulation of TGF-α and TGF-β signaling. Both of these molecules were sufficient to induce changes in AT1 proliferative potential in vitro. Reisolation of AT1 tumor-derived (AT1) cells from these mixed tumors revealed that AT1 cells grew in vivo, forming tumors as large as tumorigenic CA1d cells. Cooperation between subpopulations of cancer epithelium is an understudied mechanism of tumor growth and invasion that may have implications on tumor resistance to current therapies.-Franco, O. E., Tyson, D. R., Konvinse, K. C., Udyavar, A. R., Estrada, L., Quaranta, V., Crawford, S. E., Hayward, S. W. Altered TGF-α/β signaling drives cooperation between breast cancer cell populations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.201500187RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5024699PMC
October 2016

NF-κB and androgen receptor variant 7 induce expression of SRD5A isoforms and confer 5ARI resistance.

Prostate 2016 08 16;76(11):1004-18. Epub 2016 May 16.

Department of Urologic Surgery, Vanderbilt University Medical Center, Nashville, Tennessee.

Background: Benign prostatic hyperplasia (BPH) is treated with 5α-reductase inhibitors (5ARI). These drugs inhibit the conversion of testosterone to dihydrotestosterone resulting in apoptosis and prostate shrinkage. Most patients initially respond to 5ARIs; however, failure is common especially in inflamed prostates, and often results in surgery. This communication examines a link between activation of NF-κB and increased expression of SRD5A2 as a potential mechanism by which patients fail 5ARI therapy.

Methods: Tissue was collected from "Surgical" patients, treated specifically for lower urinary tract symptoms secondary to advanced BPH; and, cancer free transition zone from "Incidental" patients treated for low grade, localized peripheral zone prostate cancer. Clinical, molecular and histopathological profiles were analyzed. Human prostatic stromal and epithelial cell lines were genetically modified to regulate NF-κB activity, androgen receptor (AR) full length (AR-FL), and AR variant 7 (AR-V7) expression.

Results: SRD5A2 is upregulated in advanced BPH. SRD5A2 was significantly associated with prostate volume determined by Transrectal Ultrasound (TRUS), and with more severe lower urinary tract symptoms (LUTS) determined by American Urological Association Symptom Score (AUASS). Synthesis of androgens was seen in cells in which NF-κB was activated. AR-FL and AR-V7 expression increased SRD5A2 expression while forced activation of NF-κB increased all three SRD5A isoforms. Knockdown of SRD5A2 in the epithelial cells resulted in significant reduction in proliferation, AR target gene expression, and response to testosterone (T). In tissue recombinants, canonical NF-κB activation in prostatic epithelium elevated all three SRD5A isoforms and resulted in in vivo growth under castrated conditions.

Conclusion: Increased BPH severity in patients correlates with SRD5A2 expression. We demonstrate that NF-κB and AR-V7 upregulate SRD5A expression providing a mechanism to explain failure of 5ARI therapy in BPH patients. Prostate 76:1004-1018, 2016. © 2016 Wiley Periodicals, Inc.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pros.23195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4912960PMC
August 2016

Cells Comprising the Prostate Cancer Microenvironment Lack Recurrent Clonal Somatic Genomic Aberrations.

Mol Cancer Res 2016 Apr 11;14(4):374-84. Epub 2016 Jan 11.

Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, Washington. Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington. Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, Washington. Department of Pathology, University of Washington, Seattle, Washington. Department of Urology, University of Washington, Seattle, Washington. Department of Medicine, University of Washington, Seattle, Washington.

Unlabelled: Prostate cancer-associated stroma (CAS) plays an active role in malignant transformation, tumor progression, and metastasis. Molecular analyses of CAS have demonstrated significant changes in gene expression; however, conflicting evidence exists on whether genomic alterations in benign cells comprising the tumor microenvironment (TME) underlie gene expression changes and oncogenic phenotypes. This study evaluates the nuclear and mitochondrial DNA integrity of prostate carcinoma cells, CAS, matched benign epithelium and benign epithelium-associated stroma by whole-genome copy-number analyses, targeted sequencing of TP53, and FISH. Array comparative genomic hybridization (aCGH) of CAS revealed a copy-neutral diploid genome with only rare and small somatic copy-number aberrations (SCNA). In contrast, several expected recurrent SCNAs were evident in the adjacent prostate carcinoma cells, including gains at 3q, 7p, and 8q, and losses at 8p and 10q. No somatic TP53 mutations were observed in CAS. Mitochondrial DNA (mtDNA) extracted from carcinoma cells and stroma identified 23 somatic mtDNA mutations in neoplastic epithelial cells, but only one mutation in stroma. Finally, genomic analyses identified no SCNAs, LOH, or copy-neutral LOH in cultured cancer-associated fibroblasts, which are known to promote prostate cancer progression in vivo

Implications: The gene expression changes observed in prostate cancer-adjacent stroma and the attendant contribution of the stroma to the development and progression of prostate cancer are not due to frequent or recurrent genomic alterations in the TME.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1541-7786.MCR-15-0330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5582956PMC
April 2016

NF-κB and androgen receptor variant expression correlate with human BPH progression.

Prostate 2016 Apr 28;76(5):491-511. Epub 2015 Dec 28.

Department of Urologic Surgery, Vanderbilt University Medical Center, Nashville, Tennessee.

Background: Benign prostatic hyperplasia (BPH) is a common, chronic progressive disease. Inflammation is associated with prostatic enlargement and resistance to 5α-reductase inhibitor (5ARI) therapy. Activation of the nuclear factor-kappa B (NF-κB) pathway is linked to both inflammation and ligand-independent prostate cancer progression.

Methods: NF-κB activation and androgen receptor variant (AR-V) expression were quantified in transition zone tissue samples from patients with a wide range of AUASS from incidental BPH in patients treated for low grade, localized peripheral zone prostate cancer to advanced disease requiring surgical intervention. To further investigate these pathways, human prostatic stromal and epithelial cell lines were transduced with constitutively active or kinase dead forms of IKK2 to regulate canonical NF-κB activity. The effects on AR full length (AR-FL) and androgen-independent AR-V expression as well as cellular growth and differentiation were assessed.

Results: Canonical NF-κB signaling was found to be upregulated in late versus early stage BPH, and to be strongly associated with non-insulin dependent diabetes mellitus. Elevated expression of AR-variant 7 (AR-V7), but not other AR variants, was found in advanced BPH samples. Expression of AR-V7 significantly correlated with the patient AUASS and TRUS volume. Forced activation of canonical NF-κB in human prostatic epithelial and stromal cells resulted in elevated expression of both AR-FL and AR-V7, with concomitant ligand-independent activation of AR reporters. Activation of NF-κB and over expression of AR-V7 in human prostatic epithelial cells maintained cell viability in the face of 5ARI treatment.

Conclusion: Activation of NF-κB and AR-V7 in the prostate is associated with increased disease severity. AR-V7 expression is inducible in human prostate cells by forced activation of NF-κB resulting in resistance to 5ARI treatment, suggesting a potential mechanism by which patients may become resistant to 5ARI therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pros.23140DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4763342PMC
April 2016

Isolation and analysis of discreet human prostate cellular populations.

Differentiation 2016 Apr-Jun;91(4-5):139-51. Epub 2015 Nov 3.

Department of Surgery, NorthShore University Health System, Research Institute, Evanston, IL, USA. Electronic address:

The use of lineage tracing in transgenic mouse models has revealed an abundance of subcellular phenotypes responsible for maintaining prostate homeostasis. The ability to use fresh human tissues to examine the hypotheses generated by these mouse experiments has been greatly enhanced by technical advances in tissue processing, flow cytometry and cell culture. We describe in detail the optimization of protocols for each of these areas to facilitate research on solving human prostate diseases through the analysis of human tissue.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.diff.2015.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4854811PMC
September 2017

Il-6 signaling between ductal carcinoma in situ cells and carcinoma-associated fibroblasts mediates tumor cell growth and migration.

BMC Cancer 2015 Aug 13;15:584. Epub 2015 Aug 13.

Department of Pharmacology, Wayne State University, 540 East Canfield, Detroit, MI, 48201, USA.

Background: Ductal carcinoma in situ (DCIS) is a non-obligate precursor lesion of invasive breast cancer in which approximately half the patients will progress to invasive cancer. Gaining a better understanding of DCIS progression may reduce overtreatment of patients. Expression of the pro-inflammatory cytokine interleukin-6 increases with pathological stage and grade, and is associated with poorer prognosis in breast cancer patients. Carcinoma associated fibroblasts (CAFs), which are present in the stroma of DCIS patients are known to secrete pro-inflammatory cytokines and promote tumor progression.

Methods: We hypothesized that IL-6 paracrine signaling between DCIS cells and CAFs mediates DCIS proliferation and migration. To test this hypothesis, we utilized the mammary architecture and microenvironment engineering or MAME model to study the interactions between human breast CAFs and human DCIS cells in 3D over time. We specifically inhibited autocrine and paracrine IL-6 signaling to determine its contribution to early stage tumor progression.

Results: Here, DCIS cells formed multicellular structures that exhibited increased proliferation and migration when cultured with CAFs. Treatment with an IL-6 neutralizing antibody inhibited growth and migration of the multicellular structures. Moreover, selective knockdown of IL-6 in CAFs, but not in DCIS cells, abrogated the migratory phenotype.

Conclusion: Our results suggest that paracrine IL-6 signaling between preinvasive DCIS cells and stromal CAFs represent an important factor in the initiation of DCIS progression to invasive breast carcinoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12885-015-1576-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4535667PMC
August 2015

Glucocorticoids suppress renal cell carcinoma progression by enhancing Na,K-ATPase beta-1 subunit expression.

PLoS One 2015 2;10(4):e0122442. Epub 2015 Apr 2.

Helen F. Graham Cancer Center, Christiana Care Health System, Newark, Delaware, United States of America; Department of Biological Sciences, University of Delaware, Newark, Delaware, United States of America; Therapy Architects, 2700 Silverside Road, Wilmington, Delaware, United States of America.

Glucocorticoids are commonly used as palliative or chemotherapeutic clinical agents for treatment of a variety of cancers. Although steroid treatment is beneficial, the mechanisms by which steroids improve outcome in cancer patients are not well understood. Na,K-ATPase beta-subunit isoform 1 (NaK-β1) is a cell-cell adhesion molecule, and its expression is down-regulated in cancer cells undergoing epithelial-to mesenchymal-transition (EMT), a key event associated with cancer progression to metastatic disease. In this study, we performed high-throughput screening to identify small molecules that could up-regulate NaK-β1 expression in cancer cells. Compounds related to the glucocorticoids were identified as drug candidates enhancing NaK-β1 expression. Of these compounds, triamcinolone, dexamethasone, and fluorometholone were validated to increase NaK-β1 expression at the cell surface, enhance cell-cell adhesion, attenuate motility and invasiveness and induce mesenchymal to epithelial like transition of renal cell carcinoma (RCC) cells in vitro. Treatment of NaK-β1 knockdown cells with these drug candidates confirmed that these compounds mediate their effects through up-regulating NaK-β1. Furthermore, we demonstrated that these compounds attenuate tumor growth in subcutaneous RCC xenografts and reduce local invasiveness in orthotopically-implanted tumors. Our results strongly indicate that the addition of glucocorticoids in the treatment of RCC may improve outcome for RCC patients by augmenting NaK-β1 cell-cell adhesion function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0122442PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4383530PMC
April 2016

Tumor-secreted Hsp90 subverts polycomb function to drive prostate tumor growth and invasion.

J Biol Chem 2015 Mar 10;290(13):8271-82. Epub 2015 Feb 10.

From the Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29425 and

Prostate cancer remains the second highest contributor to male cancer-related lethality. The transition of a subset of tumors from indolent to invasive disease is associated with a poor clinical outcome. Activation of the epithelial to mesenchymal transition (EMT) genetic program is a major risk factor for cancer progression. We recently reported that secreted extracellular Hsp90 (eHsp90) initiates EMT in prostate cancer cells, coincident with its enhanced expression in mesenchymal models. Our current work substantially extended these findings in defining a pathway linking eHsp90 signaling to EZH2 function, a methyltransferase of the Polycomb repressor complex. EZH2 is also implicated in EMT activation, and its up-regulation represents one of the most frequent epigenetic alterations during prostate cancer progression. We have now highlighted a novel epigenetic function for eHsp90 via its modulation of EZH2 expression and activity. Mechanistically, eHsp90 initiated sustained activation of MEK/ERK, a signal critical for facilitating EZH2 transcriptional up-regulation and recruitment to the E-cadherin promoter. We further demonstrated that an eHsp90-EZH2 pathway orchestrates an expanded repertoire of EMT-related events including Snail and Twist expression, tumor cell motility, and anoikis resistance. To evaluate the role of eHsp90 in vivo, eHsp90 secretion was stably enforced in a prostate cancer cell line resembling indolent disease. Remarkably, eHsp90 was sufficient to induce tumor growth, suppress E-cadherin, and initiate localized invasion, events that are exquisitely dependent upon EZH2 function. In summary, our findings illuminate a hitherto unknown epigenetic function for eHsp90 and support a model wherein tumor eHsp90 functions as a rheostat for EZH2 expression and activity to orchestrate mesenchymal properties and coincident aggressive behavior.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M115.637496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4375482PMC
March 2015

A novel model of urinary tract differentiation, tissue regeneration, and disease: reprogramming human prostate and bladder cells into induced pluripotent stem cells.

Eur Urol 2013 Nov 6;64(5):753-61. Epub 2013 Apr 6.

Northern Institute for Cancer Research, Newcastle University, UK.

Background: Primary culture and animal and cell-line models of prostate and bladder development have limitations in describing human biology, and novel strategies that describe the full spectrum of differentiation from foetal through to ageing tissue are required. Recent advances in biology demonstrate that direct reprogramming of somatic cells into pluripotent embryonic stem cell (ESC)-like cells is possible. These cells, termed induced pluripotent stem cells (iPSCs), could theoretically generate adult prostate and bladder tissue, providing an alternative strategy to study differentiation.

Objective: To generate human iPSCs derived from normal, ageing, human prostate (Pro-iPSC), and urinary tract (UT-iPSC) tissue and to assess their capacity for lineage-directed differentiation.

Design, Setting, And Participants: Prostate and urinary tract stroma were transduced with POU class 5 homeobox 1 (POU5F1; formerly OCT4), SRY (sex determining region Y)-box 2 (SOX2), Kruppel-like factor 4 (gut) (KLF4), and v-myc myelocytomatosis viral oncogene homolog (avian) (MYC, formerly C-MYC) genes to generate iPSCs.

Outcome Measurements And Statistical Analysis: The potential for differentiation into prostate and bladder lineages was compared with classical skin-derived iPSCs. The student t test was used.

Results And Limitations: Successful reprogramming of prostate tissue into Pro-iPSCs and bladder and ureter into UT-iPSCs was demonstrated by characteristic ESC morphology, marker expression, and functional pluripotency in generating all three germ-layer lineages. In contrast to conventional skin-derived iPSCs, Pro-iPSCs showed a vastly increased ability to generate prostate epithelial-specific differentiation, as characterised by androgen receptor and prostate-specific antigen induction. Similarly, UT-iPSCs were shown to be more efficient than skin-derived iPSCs in undergoing bladder differentiation as demonstrated by expression of urothelial-specific markers: uroplakins, claudins, and cytokeratin; and stromal smooth muscle markers: α-smooth-muscle actin, calponin, and desmin. These disparities are likely to represent epigenetic differences between individual iPSC lines and highlight the importance of organ-specific iPSCs for tissue-specific studies.

Conclusions: IPSCs provide an exciting new model to characterise mechanisms regulating prostate and bladder differentiation and to develop novel approaches to disease modelling. Regeneration of bladder cells also provides an exceptional opportunity for translational tissue engineering.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.eururo.2013.03.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3819995PMC
November 2013

Deficiency in metabolic regulators PPARγ and PTEN cooperates to drive keratinizing squamous metaplasia in novel models of human tissue regeneration.

Am J Pathol 2013 Feb 6;182(2):449-59. Epub 2012 Dec 6.

Department of Urologic Surgery, Vanderbilt-Ingram Comprehensive Cancer Center, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2765, USA.

Hindgut-derived endoderm can differentiate into rectal, prostatic, and bladder phenotypes. Stromal-epithelial interactions are crucial for this development; however, the precise mechanisms by which epithelium responds to stromal cues remain unknown. We have previously reported ectopic expression of peroxisome proliferator-activated receptor-γ2 (PPARγ2) increased androgen receptor expression and promoted differentiation of mouse prostate epithelium. PPARγ is also implicated in urothelial differentiation. Herein we demonstrate that knockdown of PPARγ2 in benign human prostate epithelial cells (BHPrEs) promotes urothelial transdifferentiation. Furthermore, in vitro and in vivo heterotypic tissue regeneration models with embryonic bladder mesenchyme promoted urothelial differentiation of PPARγ2-deficient BHPrE cells, and deficiency of both PPARγ isoforms 1 and 2 arrested differentiation. Because PTEN deficiency is cooperative in urothelial pathogenesis, we engineered BHPrE cells with combined knockdown of PPARγ and PTEN and performed heterotypic recombination experiments using embryonic bladder mesenchyme. Whereas PTEN deficiency alone induced latent squamous differentiation in BHPrE cells, combined PPARγ and PTEN deficiency accelerated the development of keratinizing squamous metaplasia (KSM). We further confirmed via immunohistochemistry that gene expression changes in metaplastic recombinants reflected human urothelium undergoing KSM. In summary, these data suggest that PPARγ isoform expression provides a molecular basis for observations that adult human epithelium can be transdifferentiated on the basis of heterotypic mesenchymal induction. These data also implicate PPARγ and PTEN inactivation in the development of KSM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ajpath.2012.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3562729PMC
February 2013

Reduction of pro-tumorigenic activity of human prostate cancer-associated fibroblasts using Dlk1 or SCUBE1.

Dis Model Mech 2013 Mar 7;6(2):530-6. Epub 2012 Nov 7.

MRC Human Reproductive Sciences Unit, The Queen's Medical Research Institute, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK.

Human prostatic cancer-associated fibroblasts (CAFs) can elicit malignant changes in initiated but non-tumorigenic human prostate epithelium, demonstrating that they possess pro-tumorigenic properties. We set out to reduce the pro-tumorigenic activity of patient CAFs using the Dlk1 and SCUBE1 molecules that we had previously identified in prostate development. Our hypothesis was that mesenchymally expressed molecules might reduce CAF pro-tumorigenic activity, either directly or indirectly. We isolated primary prostatic CAFs and characterised their expression of CAF markers, expression of Notch2, Dlk1 and SCUBE1 transcripts, and confirmed their ability to stimulate BPH1 epithelial cell proliferation. Next, we expressed Dlk1 or SCUBE1 in CAFs and determined their effects upon tumorigenesis in vivo following recombination with BPH1 epithelia and xenografting in SCID mice. Tumour size was reduced by about 75% and BPH1 proliferation was reduced by about 50% after expression of Dlk1 or SCUBE1 in CAFs, and there was also a reduction in invasion of BPH1 epithelia into the host kidney. Inhibition of Notch signalling, using inhibitor XIX, led to a reduction in BPH1 cell proliferation in CAF-BPH1 co-cultures, whereas inhibition of Dlk1 in NIH3T3-conditioned media led to an increase in BPH1 growth. Our results suggest that pro-tumorigenic CAF activity can be reduced by the expression of developmental pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/dmm.010355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597035PMC
March 2013

Cathepsin D acts as an essential mediator to promote malignancy of benign prostatic epithelium.

Prostate 2013 Apr 19;73(5):476-88. Epub 2012 Sep 19.

Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2765, USA.

Background: Stromal-epithelial interactions are important in both development and prostate cancer. Stromal changes have been shown to be powerful prognostic indicators of prostate cancer progression and of patient death helping to define lethal versus indolent phenotypes. The specific molecular underpinnings of these interactions are incompletely understood. We investigated whether stromal cathepsin D (CathD) overexpression affects prostate tumorigenesis through a paracrine mechanism.

Methods: Normal prostate fibroblasts (NPF) were retrovirally transduced to overexpress cyclin D1 (CD1) and were designated NPF(CD1) . Cathepsin D expression was knocked down using shRNA in cancer associated fibroblasts (CAF) and NPF(CD1) . We analyzed these stromal cell lines using immunohistochemistry, Western blot, and tissue recombination.

Results: An examination of human prostate tissue revealed significantly increased stromal staining of CathD in malignant prostate tissue. Overexpression of CD1 in normal prostate fibroblasts (NPF(CD1) ) produced a phenotype similar to, but more moderate than, CAF in a tissue recombination model. Knockdown studies revealed that CathD is required for NPF(CD1) motility and invasive growth in vitro. BPH-1 cell proliferation was found to be induced when cultured with NPF(CD1) conditioned medium, this effect was inhibited when CathD was knocked down in NPF(CD1) cells. Overexpression of CathD in prostate stromal cells induced malignancy in adjacent epithelium, and this transformation was inhibited when stromal CathD expression was knocked down in CAF.

Conclusions: The study presented here demonstrates increased CathD expression is seen in human CAF. The upregulation of CD1 results in concomitant increases in CathD expression. Elevated CathD expression in the stroma contributes to tumor promotion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/pros.22589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3594371PMC
April 2013