Human Prostate Tissue Research Articles

Purinergic smooth muscle contractions in the human prostate: estimation of relevance and characterization of different agonists

Non-adrenergic prostate smooth muscle contractions may account for the limited effectiveness of α1-adrenoceptor antagonists, which are the first-line option for medical treatment of voiding symptoms suggestive of benign prostatic hyperplasia. In non-human prostates, purinergic agonists induce contractions reaching similar magnitudes as α1-adrenergic contractions. However, evidence for the human prostate is highly limited, and pointed to much weaker purinergic contractions. Here, we examined contractions of different purinergic agonists in human prostate tissues. Tissues were obtained from radical prostatectomy. Contractions were studied in an organ bath, and expression of purinergic receptors was studied by RT-PCR. Electric field stimulation (EFS)–induced contractions amounted to 104% of KCl-induced contractions (95% CI: 84–124%). From all tested agonists, only ATP induced concentration-dependent contractions, reaching an average maximum of 18% (12–24%) of KCl. Maximum tensions following application of other agonists averaged to 7.1% of KCl for α,β-methylene-ATP (1.8–12.4%), 3.9% for β,γ-methylene-ATP (2.0–5.4%), 3.1% for 2-methylthio-ATP (− 0.1–6.3%), and 5.1% for ATPγS (1.0–9.2%). Responses were not affected by the P2X antagonist NF023 or the P2Y antagonist PPADS. mRNA expression of P2X1-4 correlated with expression of a marker for catecholaminergic nerves, although neither ATP, NF023, nor PPADS changed EFS-induced contractions. Correlation between expression of receptors and the smooth muscle marker calponin was not observed. Our findings point to a low relevance of purinergic contractions in the human prostate, compared to other contractile stimuli in the human prostate and compared to purinergic contractions in non-human prostates. Purinergic contractions in the human prostate are not sensitive to NF023 or PPADS.

Changes in the expression and functional activities of Myosin II isoforms in human hyperplastic prostate

Benign prostatic hyperplasia (BPH) is a common disease among aging males with the etiology remaining unclear. We recently found myosin II was abundantly expressed in rat and cultured human prostate cells with permissive roles in the dynamic and static components. This study aimed to explore the expression and functional activities of myosin II isoforms including smooth muscle myosin II (SMM II) and non-muscle myosin (NMM II) in the hyperplastic prostate. Human prostate cell lines and tissues from normal human and BPH patients were used. H&E, Masson's trichrome, immunohistochemical staining, in vitro organ bath, RT-PCR and Western-blotting were performed. We further created cell models with NMM II isoforms silenced and proliferation, cycle, and apoptosis of prostate cells were determined by CCK-8 assay and flow cytometry. Hyperplastic prostate SM expressed more SM1 and LC17b isoforms compared to their alternatively spliced counterparts, favoring a slower more tonic-type contraction and greater force generation. For BPH group, blebbistatin (BLEB, a selective myosin II inhibitor), exhibited a stronger effect on relaxing phenylephrine (PE) pre-contracted prostate strips and inhibiting PE induced contraction. Additionally, NMMHC-A and NMMHC-B were upregulated in hyperplastic prostate with no change in NMMHC-C. Knockdown of NMMHC-A or NMMHC-B inhibited prostate cell proliferation and induced apoptosis, with no changes in cell cycle. Our novel data demonstrates that expression and functional activities of myosin II isoforms are altered in human hyperplastic prostate, suggesting a new pathological mechanism for BPH. Thus, the myosin II system may provide potential new therapeutic targets for BPH/lower urinary tract symptoms (LUTS).

Steroidogenesis in Peripheral and Transition Zones of Human Prostate Cancer Tissue.

The peripheral zone (PZ) and transition zone (TZ) represent about 70% of the human prostate gland with each zone having differential ability to develop prostate cancer. Androgens and their receptor are the primary driving cause of prostate cancer growth and eventually castration-resistant prostate cancer (CRPC). De novo steroidogenesis has been identified as a key mechanism that develops during CRPC. Currently, there is very limited information available on human prostate tissue steroidogenesis. The purpose of the present study was to investigate steroid metabolism in human prostate cancer tissues with comparison between PZ and TZ. Human prostate cancer tumors were procured from the patients who underwent radical prostatectomy without any neoadjuvant therapy. Human prostate homogenates were used to quantify steroid levels intrinsically present in the tissues as well as formed after incubation with 2 µg/mL of 17-hydroxypregnenolone (17-OH-pregnenolone) or progesterone. A Waters Acquity ultraperformance liquid chromatography coupled to a Quattro Premier XE tandem quadrupole mass spectrometer using a C18 column was used to measure thirteen steroids from the classical and backdoor steroidogenesis pathways. The intrinsic prostate tissue steroid levels were similar between PZ and TZ with dehydroepiandrosterone (DHEA), dihydrotestosterone (DHT), pregnenolone and 17-OH-pregnenolone levels higher than the other steroids measured. Interestingly, 5-pregnan-3,20-dione, 5-pregnan-3-ol-20-one, and 5-pregnan-17-ol-3,20-dione formation was significantly higher in both the zones of prostate tissues, whereas, androstenedione, testosterone, DHT, and progesterone levels were significantly lower after 60 min incubation compared to the 0 min control incubations. The incubations with progesterone had a similar outcome with 5-pregnan-3,20-dione and 5-pregnan-3-ol-20-one levels were elevated and the levels of DHT were lower in both PZ and TZ tissues. The net changes in steroid formation after the incubation were more observable with 17-OH-pregnenolone than with progesterone. In our knowledge, this is the first report of comprehensive analyses of intrinsic prostate tissue steroids and precursor-driven steroid metabolism using a sensitive liquid chromatography-mass spectrometry assay. In summary, the PZ and TZ of human prostate exhibited similar steroidogenic ability with distinction in the manner each zone utilizes the steroid precursors to divert the activity towards backdoor pathway through a complex matrix of steroidogenic mechanisms.

Hyperpolarized 15N-labeled, deuterated tris(2-pyridylmethyl)amine as an MRI sensor of freely available Zn2+

Dynamic nuclear polarization (DNP) coupled with 15N magnetic resonance imaging (MRI) provides an opportunity to image quantitative levels of biologically important metal ions such as Zn2+, Mg2+ or Ca2+ using appropriately designed 15N enriched probes. For example, a Zn-specific probe could prove particularly valuable for imaging the tissue distribution of freely available Zn2+ ions, an important known metal ion biomarker in the pancreas, in prostate cancer, and in several neurodegenerative diseases. In the present study, we prepare the cell-permeable, 15N-enriched, d6-deuterated version of the well-known Zn2+ chelator, tris(2-pyridylmethyl)amine (TPA) and demonstrate that the polarized ligand had favorable T1 and linewidth characteristics for 15N MRI. Examples of how polarized TPA can be used to quantify freely available Zn2+ in homogenized human prostate tissue and intact cells are presented.

Ciliary neurotrophic factor (CNTF) and its receptor (CNTFRα) signal through MAPK/ERK pathway in human prostate tissues: a morphological and biomolecular study.

Ciliary neurotrophic factor (CNTF) is a member of interleukin-6 type cytokine family. The CNTF receptor complex is a heterodimer including gp130 and CNTF receptor α (CNTFRɑ) proteins triggering the activation of multiple intracellular signaling pathways including AKT/PI3K, MAPK/ERK and Jak/STAT pathways. At present no data are available on the localization of CNTF and CNTFRɑ in prostate as well as on the role of CNTF in this organ. In this study we have analyzed the localization of CNTF and CNTFRɑ by immunohistochemistry and we have used PWR-1E cell line as a model for normal glandular cell to investigate the role of this cytokine. Our results show that CNTF and CNTFRɑ are expressed in the staminal compart of the prostate and that CNTF selectively inhibits ERK pathway. In conclusion, we suggest that CNTF could be considered as key molecule to maintain the epithelium homeostasis via pERK downregulation by an autocrine mechanism. Further CNTF studies in prostate cancer could be useful to verify the potential role of this cytokine in carcinogenesis.

Abstract 4698: Increased CHST15 expression follows declines in Arylsulfatase B (ARSB) and DKK3 and disinhibition of non-canonical WNT signaling

Abstract CHST15 (carbohydrate sulfotransferase 15; chondroitin 4-sulfate-6-sulfotransferase), is the sulfotransferase enzyme which adds 6-sulfate to chondroitin 4-sulfate to make chondroitin 4,6-sulfate, known as CSE. Increased CHST15 expression has been identified in malignant pancreas, renal, colon, ovary, and prostate tissues. We now report a pathway by which expression of CHST15 is increased following decline in the enzyme ARSB (arylsulfatase B; N-acetylgalactosamine-4-sulfatase), which is required for removal of 4-sulfate groups from chondroitin 4-sulfate (C4S). Previous work has shown that decline in ARSB and the resulting increase in C4S leads to decline in activity of SHP2, a ubiquitous non-receptor tyrosine phosphatase, due to enhanced binding of SHP2 with C4S when ARSB is reduced. Decline in SHP2 leads to enhanced ERK1/2 phosphorylation and subsequent downstream effects, including hypermethylation of the DKK (Dickkopf WNT signaling pathway inhibitor)3 promoter and suppressed DKK3 expression. Since DKK3 acts to inhibit Wnt signaling, Wnt signaling is enhanced in prostate epithelium when ARSB is reduced. Experiments were performed to identify the pathway by which CHST15 expression is up-regulated when ARSB is reduced and to assess the role of Wnt. The impact of ARSB silencing on CHST15 expression was determined in normal and malignant human prostate cells, in normal and malignant prostate tissue obtained by laser-capture microdissection, in HepG2 cells, and in prostate tissue of the ARSB-null mouse. Effects of selective inhibitors of Wnt and MAPK signaling pathways on CHST15 expression were evaluated. CHST15 expression was increased in malignant prostate epithelial tissue, in ARSB-null mouse prostate tissue, and following ARSB silencing in human prostate epithelial cells. The activation of Wnt signaling which followed declines in ARSB and DKK3 was required for increased CHST15 expression. Inhibition of Wnt3A blocked the increase in CHST15 expression in prostate epithelial cells co-cultured with prostate stromal cells, whereas DKK3 antibody increased CHST15. Inhibition of Rac-1 GTPase and of phospho-p38 MAPK signaling blocked the observed increase in CHST15 expression, whereas inhibition of JNK, Sp1, or of Rho kinase had no effect. These results indicate that the increase in epithelial CHST15 occurred in the malignant prostate cells and tissue due to effects of non-canonical Wnt signaling leading to enhanced phospho-p38 MAPK. The resultant increase in CHST15 when ARSB activity is reduced may contribute to determination of epithelial vs. stromal characteristics. Citation Format: Joanne Kramer Tobacman, Sumit Bhattacharyya, Leo Feferman. Increased CHST15 expression follows declines in Arylsulfatase B (ARSB) and DKK3 and disinhibition of non-canonical WNT signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4698.

Recapitulation of prostate tissue cell type-specific transcriptomes by an in vivo primary prostate tissue xenograft model.

Studies of the normal functions and diseases of the prostate request in vivo models that maintain the tissue architecture and the multiple-cell type compartments of human origin in order to recapitulate reliably the interactions of different cell types. Cell type-specific transcriptomes are critical to reveal the roles of each cell type in the functions and diseases of the prostate. A primary prostate tissue xenograft model was developed using fresh human prostate tissue specimens transplanted onto male mice that were castrated surgically and implanted with a device to maintain circulating testosterone levels comparable to adult human males. Endothelial cells and epithelial cells were isolated from 7 fresh human prostate tissue specimens and from primary tissue xenografts established from 9 fresh human prostate tissue specimens, using antibody-conjugated magnetic beads specific to human CD31 and human EpCAM, respectively. Transcriptomes of endothelial, epithelial and stromal cell fractions were obtained using RNA-Seq. Global and function-specific gene expression profiles were compared in inter-cell type and inter-tissue type manners. Gene expression profiles in the individual cell types isolated from xenografts were similar to those of cells isolated from fresh tissue, demonstrating the value of the primary tissue xenograft model for studies of the inter-relationships between prostatic cell types and the role of such inter-relationships in organ development, disease progression, and response to drug treatments.

Abstract B094: Regulation of megalin by vitamin D as the mechanism for differential levels of intra-prostatic androgens between African American and Caucasian men

Abstract Prostate cancer (PCa) is a hormonally driven cancer and is currently the third most common cancer in the US. African American (AA) men are disproportionately at risk for both PCa and vitamin D (vitD) deficiency compared to white men. The numerous chemopreventative properties of vitD and epidemiological relationship of vitD status with PCa aggressiveness and mortality has led to the hypothesis that vitD deficiency is a biological contributor to PCa disparity in AA men. Our lab recently reported an unexpected relationship between serum and intraprostatic vitD metabolites 25-hydroxyvitamin D (25(OH)D) and 1,25-dihydroxyvitamin D in AA men. We also observed that Megalin, a multi-liganded endocytic membrane receptor encoded by the gene LRP2, was expressed in the prostate epithelium and is regulated by vitD. Extra-renal activity of Megalin has not been well studied as the widely accepted Free Hormone Hypothesis assumes passive diffusion of circulating free hormones into tissues. The presence of megalin suggests that globulin bound hormones from the circulation, including 25(OH)D bound to vitamin D binding protein (DBP) and testosterone (T) bound to sex hormone binding globulin (SHBG), are imported into the prostate in a regulated manner. Here we examine megalin as a potential mechanism to regulate globulin bound hormone import into the prostate. 25(OH)D decreased expression of LRP2 in primary prostate epithelial cells and fresh human prostate tissue slice explants. DBP-bound 25(OH)D and SHBG-bound T were imported into these prostate models and transcriptionally active. Lastly, we quantified T and its active metabolite dihydrotestosterone (DHT) in the patient cohort from our prior study. Prostatic DHT levels inversely correlated with serum 25(OH)D status. AA men had higher levels of DHT in prostate tissue compared to white men. These clinical findings support our hypothesis that vitD status regulates intraprostatic hormone levels. In summary, we report the presence of a negative feedback loop in which vitD deficiency increases hormone import into prostate epithelium via megalin. Therefore, the upregulation of megalin in the setting of vitamin D deficiency may facilitate increased import of circulating sex steroids into the prostate contributing to carcinogenesis in AA men. Citation Format: Jason Garcia, Zachary Richards, Morgan Zenner, Yancai Wang, Peter Gann, Gail Prins, Larisa Nonn. Regulation of megalin by vitamin D as the mechanism for differential levels of intra-prostatic androgens between African American and Caucasian men [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B094.

Morphological change and characteristics of myofibroblasts during the growth process of benign prostatic hyperplasia.

To clarify the morphological change and characteristics of myofibroblast during the growth process of benign prostatic hyperplasia. This study examined the characteristics of myofibroblasts during the growth process of the prostate in the stromal component-dominant benign prostatic hyperplasia rat model. Transforming growth factor-β1 and insulin-like growth factor-binding protein3 expression were evaluated by western blotting (n=6). We used double immunohistochemical staining to evaluate the number of myofibroblasts positive for α-smooth muscle actin and vimentin in benign prostatic hyperplasia tissues. Expression and histological analyses of the benign prostatic hyperplasia were also carried out in rats at 2, 3 and 8weeks after urogenital sinus implantation (n=6). To evaluate the fine morphological characteristics of myofibroblasts in human benign prostatic hyperplasia tissues, electron microscopy analysis was additionally carried out. There was a significant upregulation of the transforming growth factor-β1 and insulin-like growth factor-binding protein3 expression in benign prostatic hyperplasia (P<0.05). There was a significant increase in the number of myofibroblasts in benign prostatic hyperplasia (P<0.05) compared with normal prostate, with these abundantly located in the stromal area. The transforming growth factor-β1 and insulin-like growth factor-binding protein3 expression and number of myofibroblasts showed a time-dependent increase (P<0.05), with growth factor expressions preceding the myofibroblast increase. Electron microscopy confirmed that the myofibroblast progenitor cells, which possess abundant stress fibers, were predominantly located around fibrous areas in human benign prostatic hyperplasia. Differentiation into myofibroblasts induced by transforming growth factor-β1 and insulin-like growth factor-binding protein3 actively occurs during the growth process of benign prostatic hyperplasia. Myofibroblast progenitor cells seem to be associated with prostatic fibrosis in human benign prostatic hyperplasia.

Inhibition of neurogenic and thromboxane A2 -induced human prostate smooth muscle contraction by the integrin α2β1 inhibitor BTT-3033 and the integrin-linked kinase inhibitor Cpd22.

Prostate smooth muscle contraction is critical for etiology and treatment of lower urinary tract symptoms in benign prostatic hyperplasia (BPH). Integrins connect the cytoskeleton to membranes and cells to extracellular matrix, what is essential for force generation in smooth muscle contraction. Integrins are composed of different subunits and may cooperate with integrin-linked kinase (ILK). Here, we examined effects of inhibitors for different integrin heterodimers and ILK on contraction of human prostate tissues. Prostate tissues were obtained from radical prostatectomy. Integrins and ILK were detected by Western blot, real-time polymerase chain reaction (RT-PCR), and double fluorescence staining. Smooth muscle contractions of prostate strips were studied in an organ bath. Contractions were compared after application of solvent (controls), the ILK inhibitor Cpd22 (N-methyl-3-(1-(4-(piperazin-1-yl)phenyl)-5-(4'-(trifluoromethyl)-[1,1'-biphenyl]-4-yl)-1H-pyrazol-3-yl)propanamide), the integrin α2β1 inhibitor BTT-3033 (1-(4-fluorophenyl)-N-methyl-N-[4[[(phenylamino)carbonyl]amino]phenyl]-1H-pyrazole-4-sulfonamide), or the integrin α4β1/α9β1 inhibitor BOP (N-(benzenesulfonyl)- l-prolyl- l-O-(1-pyrrolidinylcarbonyl)tyrosine sodium salt). Western blot analyses of prostate tissues using antibodies raised against integrins α2b, α4, α9, β1, and ILK revealed bands matching the expected sizes of corresponding antigens. Expression of integrins and ILK was confirmed by RT-PCR. Individual variations of expression levels occurred independently from divergent degree of BPH, reflected by different contents of prostate-specific antigen. Double fluorescence staining of prostate sections using antibodies raised against integrins α2 and β1, or against ILK resulted in immunoreactivity colocalizing with calponin, suggesting localization in prostate smooth muscle cells. Electric field stimulation (EFS) induced frequency-dependent contractions, which were inhibited by Cpd22 (3 µM) and BTT-3033 (1 µM) (inhibition around 37% by Cpd22 and 46% by BTT-3033 at 32 Hz). The thromboxane A2 analog U46619-induced concentration-dependent contractions, which were inhibited by Cpd22 and BTT-3033 (around 67% by Cpd22 and 39% by BTT-3033 at 30 µM U46619). Endothelin-1 induced concentration-dependent contractions, which were not affected by Cpd22 or BTT-3033. Noradrenaline and the α1 -adrenergic agonists methoxamine and phenylephrine-induced concentration-dependent contractions, which were not or very slightly inhibited by Cpd22 and BTT-3033. BOP did not change EFS- or agonist-induced contraction. Integrin α2β1 and ILK inhibitors inhibit neurogenic and thromboxane A2 -induced prostate smooth muscle contraction in human BPH. A role for these targets for prostate smooth muscle contraction may appear possible.

SUN-143 Prostatic Acid Phosphatase Is Not Regulated by Androgens During Prostate Development and Tumorigenesis

INTRODUCTION: Prostatic acid phosphatase (PAP) is a soluble factor secreted by prostate luminal epithelial cells. PAP expression correlates with prostate cancer (PCa) bone metastases and poor survival. The androgenic regulation of PAP in prostate development and tumorigenesis is not fully understood. We investigated the relationship between PAP and androgens in human prostate specimens and in vivo. HYPOTHESIS AND OBJECTIVES: We hypothesized that PAP expression was independent of androgens. Our objectives were to determine the immunohistochemical expression of PAP in human fetal prostate tissue, human PCa bone metastases, and xenograft and surgical castration mouse models. METHODS: Immunohistochemical staining for PAP and three androgen-regulated proteins, the Androgen Receptor (AR), Prostate-Specific Antigen (PSA), and ETS-related gene (ERG) protein, was carried out on human fetal prostate (9.5, 11.5, 13, 16.5, 18 and 20 weeks of gestational age), archival human PCa bone metastases, and PCa mouse models. For xenograft studies, PAP-expressing PCa cell lines, LNCaP, C42B, and VCaP cells, were inoculated subcutaneously into SCID mice. A castration study with surgical or sham castration was performed after VCaP tumors were palpable. Mouse tumor growth and weight were measured biweekly, and tumor tissue isolated after mouse sacrifice. RESULTS: PAP expression was observed in the fetal prostate as early as 11.5 weeks of gestational age. Strong PAP expression was noted in all human PCa bone metastases examined, both treatment-naive and castrate-resistant (n=10). However, AR and ERG expression was absent in two of four castrate-resistant specimens. PSA was weakly expressed in human castration-resistant bone metastatic prostate specimens. In vivo, PAP expression was observed in all tumor models; however, the expression of PAP differed among androgen-sensitive models; LNCaP (low PAP), C42B (moderate PAP) and VCaP (high PAP). Castrated VCaP tumors underwent tumor stasis and were significantly smaller compared to intact mice. Strong expression of PAP was observed after castration. In contrast, AR, PSA, and ERG expression were reduced in castrated VCaP tumors compared to tumors from intact mice. Double staining of tumors for PAP and AR demonstrated a population of cells that were positive for PAP but negative for AR expression located in hypoxic areas near necrosis. CONCLUSIONS: Our findings demonstrated that PAP is expressed early in normal human fetal prostate development prior to the secretion of significant androgens or expression of AR. In mouse xenografts and human PCa bone metastases, androgens did not significantly regulate PAP expression. These data demonstrate that PAP is a marker of early progenitor cells in the normal prostate and is persistently expressed after castration. PAP may be a suitable target for the treatment of castration-resistant metastatic disease.

Regenerative potential of prostate luminal cells revealed by single-cell analysis.

Androgen deprivation is the cornerstone of prostate cancer treatment. It results in involution of the normal gland to ~90% of its original size because of the loss of luminal cells. The prostate regenerates when androgen is restored, a process postulated to involve stem cells. Using single-cell RNA sequencing, we identified a rare luminal population in the mouse prostate that expresses stemlike genes (Sca1 + and Psca +) and a large population of differentiated cells (Nkx3.1 +, Pbsn +). In organoids and in mice, both populations contribute equally to prostate regeneration, partly through androgen-driven expression of growth factors (Nrg2, Rspo3) by mesenchymal cells acting in a paracrine fashion on luminal cells. Analysis of human prostate tissue revealed similar differentiated and stemlike luminal subpopulations that likewise acquire enhanced regenerative potential after androgen ablation. We propose that prostate regeneration is driven by nearly all persisting luminal cells, not just by rare stem cells.

Equal opportunity tissue regeneration

Tissue Regeneration Tissue regeneration is thought to be driven primarily by rare stem cells with distinctive properties. Single-cell RNA sequencing allows rigorous testing of this hypothesis. Karthaus et al. examined the regeneration of normal prostate tissue in mice after androgen ablation, a common treatment for prostate cancer (see the Perspective by Kelly). Unexpectedly, they found that in addition to rare stem cells, a large population of differentiated cells was a major contributor to prostate regeneration, a result that they confirmed in a study of human prostate tissue. Investigation of the molecular mechanism by which the differentiated cells acquired regenerative potential yielded insights that could potentially lead to improved therapies for prostate cancer. Science , this issue p. [497][1]; see also p. [467][2] [1]: /lookup/doi/10.1126/science.aay0267 [2]: /lookup/doi/10.1126/science.abb7052

Estrogen regulates the proliferation and inflammatory expression of primary stromal cell in benign prostatic hyperplasia.

BackgroundTo investigate the expression of estrogen receptor (ER) in prostate tissues of benign prostatic hyperplasia (BPH) individuals, and the effects of estrogen regulating the proliferation and inflammatory expressions of primary prostate stromal cells in BPH.MethodsA total of 44 human BPH prostate tissues were collected to explore the expression of ER by immunohistochemistry (IHC). Cell proliferation, mRNA and protein expressions were analyzed in primary prostate stromal cells treated with estrogen or estrogen plus fulvestrant through cell count kit-8 (CCK-8) assay, quantitative real-time polymerase chain reaction (qPCR), IHC and western blot, respectively.ResultsFirstly, ERβ was positive, and ERα was negative in the transition zone of prostate among all the 44 individuals with BPH. Secondly, the effects could be partially inhibited by fulvestrant, of estrogen promoting the proliferation of primary prostate stromal cells cultured in dulbecco’s modified eagle medium (DMEM) supplemented with 2% fetal bovine serum (FBS). Thirdly, estrogen up-regulates the mRNA levels of C-C chemokine receptor type 3 (CCR3), CD40 ligand (CD 40L), C-X-C motif chemokine ligand 9 (CXCL9) and interleukin 10 (IL10), and down-regulates the mRNA levels of C-C chemokine receptor type 4 (CCR4) and interleukin 17C (IL17C). Then, the protein expressions of CCR3, CCR4, CD40L, IL10 and IL17C are positive, and CXCL9 is negative in the third-generation primary prostate stromal cells. Finally, the effects could be partially inhibited by fulvestrant, of estrogen up-regulating the protein levels of CD40L and IL10.ConclusionsThe expressions of ER in human BPH prostate tissues are zone-dependent. Estrogen promoting the proliferation of primary prostate stromal cells cultured in DMEM supplemented with 2% FBS. The expressions of CCR3, CCR4, CD 40L, IL17C, CXCL9 and IL10 are regulated by estrogen in primary prostate stromal cells.

Transient Sox9 Expression Facilitates Resistance to Androgen-Targeted Therapy in Prostate Cancer.

Patients with metastatic prostate cancer are increasingly presenting with treatment-resistant, androgen receptor-negative/low (AR-/Low) tumors, with or without neuroendocrine characteristics, in processes attributed to tumor cell plasticity. This plasticity has been modeled by Rb1/p53 knockdown/knockout and is accompanied by overexpression of the pluripotency factor, Sox2. Here, we explore the role of the developmental transcription factor Sox9 in the process of prostate cancer therapy response and tumor progression. Unique prostate cancer cell models that capture AR-/Low stem cell-like intermediates were analyzed for features of plasticity and the functional role of Sox9. Human prostate cancer xenografts and tissue microarrays were evaluated for temporal alterations in Sox9 expression. The role of NF-κB pathway activity in Sox9 overexpression was explored. Prostate cancer stem cell-like intermediates have reduced Rb1 and p53 protein expression and overexpress Sox2 as well as Sox9. Sox9 was required for spheroid growth, and overexpression increased invasiveness and neural features of prostate cancer cells. Sox9 was transiently upregulated in castration-induced progression of prostate cancer xenografts and was specifically overexpressed in neoadjuvant hormone therapy (NHT)-treated patient tumors. High Sox9 expression in NHT-treated patients predicts biochemical recurrence. Finally, we link Sox9 induction to NF-κB dimer activation in prostate cancer cells. Developmentally reprogrammed prostate cancer cell models recapitulate features of clinically advanced prostate tumors, including downregulated Rb1/p53 and overexpression of Sox2 with Sox9. Sox9 is a marker of a transitional state that identifies prostate cancer cells under the stress of therapeutic assault and facilitates progression to therapy resistance. Its expression may index the relative activity of the NF-κB pathway.

MP06-10 INHIBITION OF HUMAN PROSTATE SMOOTH MUSCLE CONTRACTION BY THE INTEGRIN α2β1-SELECTIVE INHIBITOR BTT3033 AND THE INTEGRIN-LINKED KINASE INHIBITOR CPD22

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology (MP06)1 Apr 2020MP06-10 INHIBITION OF HUMAN PROSTATE SMOOTH MUSCLE CONTRACTION BY THE INTEGRIN α2β1-SELECTIVE INHIBITOR BTT3033 AND THE INTEGRIN-LINKED KINASE INHIBITOR CPD22 Bingsheng Li*, Xiaolong Wang, Beata Rutz, Anna Ciotkowska, Christian Gratzke, Alexander Tamalunas, Raphaela Waidelich, Christian Stief, and Martin Hennenberg Bingsheng Li*Bingsheng Li* More articles by this author , Xiaolong WangXiaolong Wang More articles by this author , Beata RutzBeata Rutz More articles by this author , Anna CiotkowskaAnna Ciotkowska More articles by this author , Christian GratzkeChristian Gratzke More articles by this author , Alexander TamalunasAlexander Tamalunas More articles by this author , Raphaela WaidelichRaphaela Waidelich More articles by this author , Christian StiefChristian Stief More articles by this author , and Martin HennenbergMartin Hennenberg More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000820.010AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Integrins connect the cytoskeleton of cells to membranes and extracellular matrix, what is essential for force generation by smooth muscle contraction. Suprisingly, their role for contraction has not been tested yet in any organ. In the prostate, smooth muscle contraction is critical for lower urinary tract symptoms in benign prostatic hyperplasia (BPH). Integrins are composed by different subunits in different organs, so that subunit-specific inhibitors may be attractive to attain prostate-specific inhibition of smooth muscle contraction. Here we examined effects of the integrin α2β1 inhibitor BTT3033 and the integrin-linked kinase (ILK) inhibitor Cpd22 on contractions of human prostate tissues. METHODS: Prostate tissues were obtained from radical prostatectomy (n=56 patients). Contraction of prostate strips was studied in an organ bath. Integrins and ILK were detected by Western blot and fluorescence staining. RESULTS: Electric field stimulation (EFS) induced frequency-dependent contractions of prostate tissues, which were inhibited by BTT3033 (1 µM) (55% inhibition at 32 Hz; p<0.004 for whole frequency-response curves; n=5) and Cpd22 (3 µM) (47% inhibition at 32 Hz, p<0.02, n=5). The α1-adrenoceptor agonists noradrenaline and methoxamine induced concentration-dependent, which were inhibited BTT3033 (1 µM) (p<0.004 for noradrenaline, n=5; p<0.03 for methoxamine, n=6), and by Cpd22 (3 µM) (p<0.008 for noradrenaline, n=6; p<0.02 for methoxamine, n=5). The thromboxane A2 analog U46619 induced concentration-dependent contractions, which were inhibited by BTT3033 (1 µM) (p<0.03, n=5), and by Cpd22 (p<0.001, n=5). Western blot analyses of prostate tissues revealed bands with sizes matching the expected sizes for integrin α2b, integrin β1 and ILK, which were observed in each included sample (n=9). Double fluorescence staining of prostate sections (n=5) resulted in immunoreactivity colocalizing with calponin, suggesting localization in prostate smooth muscle cells. CONCLUSIONS: Integrin and ILK inhibitors inhibit agonist-induced and neurogenic prostate smooth muscle contraction in BPH. As integrins show organ-specific composition, targeting the prostate-specific integrin code by subunit-specific inhibitors may offer a promising approach to attain inhibition in the prostate, without side effects in the cardiovascular system. Source of Funding: Deutsche Forschungsgemeinschaft (DFG), Chinese Scholarship Council (CSC) © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e54-e55 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Bingsheng Li* More articles by this author Xiaolong Wang More articles by this author Beata Rutz More articles by this author Anna Ciotkowska More articles by this author Christian Gratzke More articles by this author Alexander Tamalunas More articles by this author Raphaela Waidelich More articles by this author Christian Stief More articles by this author Martin Hennenberg More articles by this author Expand All Advertisement PDF downloadLoading ...

Pre-clinical evaluation of an image-guided in-situ Raman spectroscopy navigation system for targeted prostate cancer interventions.

Transrectal ultrasound (TRUS) image guidance is the standard of care for diagnostic and therapeutic interventions in prostate cancer (PCa) patients, but can lead to high false-negative rates, compromising downstream effectiveness of therapeutic choices. A promising approach to improve in-situ detection of PCa lies in using the optical properties of the tissue to discern cancer from healthy tissue. In this work, we present the first in-situ image-guided navigation system for a spatially tracked Raman spectroscopy probe integrated in a PCa workflow, capturing the optical tissue fingerprint. The probe is guided with fused TRUS/MR imaging and tested with both tissue-simulating phantoms and ex-vivo prostates. The workflow was designed to be integrated the clinical workflow for trans-perineal prostate biopsies, as well as for high-dose rate (HDR) brachytherapy. The proposed system developed in 3D Slicer includes an electromagnetically tracked Raman spectroscopy probe, along with tracked TRUS imaging automatically registered to diagnostic MRI. The proposed system is tested on both custom gelatin tissue-simulating optical phantoms and biological tissue phantoms. A random-forest classifier was then trained on optical spectrums from ex-vivo prostates following prostatectomy using our optical probe. Preliminary in-human results are presented with the Raman spectroscopy instrument to detect malignant tissue in-situ with histopathology confirmation. In 5 synthetic gelatin and biological tissue phantoms, we demonstrate the ability of the image-guided Raman system by detecting over 95% of lesions, based on biopsy samples. The included lesion volumes ranged from 0.1 to 0.61cc. We showed the compatibility of our workflow with the current HDR brachytherapy setup. In ex-vivo prostates of PCa patients, the system showed a 81% detection accuracy in high grade lesions. Pre-clinical experiments demonstrated promising results for in-situ confirmation of lesion locations in prostates using Raman spectroscopy, both in phantoms and human ex-vivo prostate tissue, which is required for integration in HDR brachytherapy procedures.

OXPHOS remodeling in high-grade prostate cancer involves mtDNA mutations and increased succinate oxidation

Rewiring of energy metabolism and adaptation of mitochondria are considered to impact on prostate cancer development and progression. Here, we report on mitochondrial respiration, DNA mutations and gene expression in paired benign/malignant human prostate tissue samples. Results reveal reduced respiratory capacities with NADH-pathway substrates glutamate and malate in malignant tissue and a significant metabolic shift towards higher succinate oxidation, particularly in high-grade tumors. The load of potentially deleterious mitochondrial-DNA mutations is higher in tumors and associated with unfavorable risk factors. High levels of potentially deleterious mutations in mitochondrial Complex I-encoding genes are associated with a 70% reduction in NADH-pathway capacity and compensation by increased succinate-pathway capacity. Structural analyses of these mutations reveal amino acid alterations leading to potentially deleterious effects on Complex I, supporting a causal relationship. A metagene signature extracted from the transcriptome of tumor samples exhibiting a severe mitochondrial phenotype enables identification of tumors with shorter survival times.

Propagation of human prostate tissue from induced pluripotent stem cells.

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.

Prostate field cancerization and exosomes: Association between CD9, early growth response 1 and fatty acid synthase.

Intracapsular and well‑defined adenocarcinomas of the prostate are often surrounded by tissue areas that harbor molecular aberrations, including those of genetic, epigenetic and biochemical nature. This is known as field cancerization, or a field effect and denotes a state of pre‑malignancy. Such alterations in histologically normal tumor‑adjacent prostatic tissues have been recognized as clinically important and are potentially exploitable as biomarkers of disease and/or targets for preventative/therapeutic intervention. The authors have previously identified and validated two protein markers of field cancerization: The expressional upregulation of the transcription factor early growth response1 (EGR‑1) and the lipogenic enzyme fatty acid synthase (FASN). However, the molecular etiology of prostate field cancerization, including EGR‑1 and FASN upregulation, remains largely unknown. It was thus hypothesized that extracellular vesicles, notably exosomes, released by tumor lesions may induce molecular alterations in the surrounding tissues, resulting in field cancerization, priming the tissue, and ultimately promoting multifocal tumorigenesis, which is often observed in prostate cancer. Towards testing this hypothesis, the current study, to the best of our knowledge, for the first time, presents correlative protein expression data, generated in disease‑free, tumor‑adjacent and cancerous human prostate tissues by quantitative immunofluorescence, between the exosomal marker CD9, and EGR‑1 and FASN. Despite the pilot character of the present study, and the static nature and heterogeneity of human tissues, the data suggest that CD9 expression itself is part of a field effect. In support of this hypothesis, the results suggest a possible contribution of exosomes to the induction of field cancerization in the prostate, particularly for EGR‑1. These findings were corroborated in established cell models of cancerous (LNCaP) and non‑cancerous (RWPE‑1) human prostate epithelial cells. The findings of this study warrant further investigation into the functional interface between exosomes and field cancerization, as a detailed understanding of this characterization may lead to the development of clinical applications related to diagnosis and/or prognosis and targeted intervention to prevent progression from pre‑malignancy to cancer.