Prostate Stem Cell Research Articles

Ligand-inducible, prostate stem cell antigen (PSCA)-directed GoCAR-T cells in advanced solid tumors: Preliminary results from a dose escalation.

283 Background: PSCA, a cell surface protein, is upregulated in many solid tumors and correlates with disease stage. BPX601 is an autologous, T-cell product engineered to contain a PSCA-CD3ζ CAR plus the small molecule rimiducid (Rim)-inducible MyD88/CD40 costimulatory domain. BPX601 is optimized for antigen-directed and independent T cell activation, proliferation and persistence, potentially enhancing efficacy in solid tumors versus traditional CARs. This first-in-human study assesses the safety, biological and clinical activity of BPX601 plus Rim in select PSCA-positive cancers. Methods: NCT02744287 is a two-part, open-label trial. Part 1 is an ongoing 3+3 cell dose escalation to identify the recommended BPX601 cell dose (Day 0) given in combination with a fixed, single Rim dose (0.4 mg/kg; Day 7). Eligibility criteria include previously treated metastatic pancreatic cancer (mPDAC) with measurable disease & positive PSCA expression. Results: Patients received only cyclophosphamide (CTX) for lymphodepletion (LD) within three days before BPX601 infusion. Nine adults have been treated across three cell dose levels (cells/kg): 1.25x106 (cells only), 1.25x106+Rim, 2.5x106+Rim. All had mPDAC with ≥ two prior therapies. Common AEs were fatigue and nausea. No DLTs, related SAEs, neurotoxicity or CRS events were reported. Rapid cell engraftment by Day 4 was observed in all patients. No evidence of LD with CTX was seen. Of six patients that received Rim: two had cell expansion 10- to 20-fold within seven days; two had cell persistence > three weeks; all had elevated serum cytokines (IP-10, TNFα) correlated with cell expansion. Best response after ≥ one scan was 4 SD ≥ eight weeks with two minor responses (not confirmed; one patient had matched CA19-9 decrease) and 2 PD. Disease control without new therapy was 16 and > 11 weeks (ongoing) in one and two patients, respectively. Conclusions: BPX601 with single-dose Rim was well-tolerated and resulted in enhanced T cell expansion and prolonged persistence in some patients despite lack of LD. Evidence of clinical benefit in this heavily pretreated mPDAC population was seen. Part 2 is planned to open soon and will include CTX/fludarabine LD to maximize engraftment as well as gastric and prostate cancers. Clinical trial information: NCT02744287.

Epigenetic demethylation of sFRPs, with emphasis on sFRP4 activation, leading to Wnt signalling suppression and histone modifications in breast, prostate, and ovary cancer stem cells.

The expression and levels of secreted frizzled-related proteins (sFRPs), important Wnt signalling antagonists, have been reported to be reduced in various cancers, and are associated with disease progression and poor prognosis. During tumour development, all sFRP (1, 2, 3, 4, and 5) genes are hypermethylated, causing transcriptional silencing. sFRPs have an ability to sensitize tumour cells to chemotherapeutic drugs, enhancing cell death. Reduced Wnt signalling is associated with loss of cancer stem cell (CSC) viability. We investigated the possible involvement of methylation-mediated silencing of the sFRP gene family in CSCs derived from breast, prostate, and ovarian tumour cell lines. Real-time RT-PCR studies indicated that loss or downregulation of sFRP (1-5) expression in tumours is associated with promoter hypermethylation. Additionally, CSCs derived from all tumour cell lines with sFRP (1-5) promotor hypermethylation expressed sFRP (1-5) mRNA after treatment with 5-Azacytidine (5-Aza), especially sFRP4, implying that DNA methylation is the predominant epigenetic mechanism for sFRP (1-5) silencing. Furthermore, post-translational modification (PTM) in total and histone proteins was observed post 5-Aza and sFRP4 treatment. Protein levels of Wnt downstream signalling components (GSK3β, active β-catenin, and phospho β-catenin) and epigenetic factors of histones (acetyl histone H3, and H3K27me3) affecting PTM were analysed. Our findings suggest that downregulation of sFRP4 expression in endocrine-related cancers can be attributed to aberrant promoter hypermethylation in conjugation with histone modification, and indicate the important role of methylation-induced gene silencing of sFRP4 in survival and proliferation of CSCs derived from these cancers.

Cancer stem cells in breast and prostate: Fact or fiction?

Since the introduction of the cancer stem cell (CSC) hypothesis, accumulating evidence shows that most cancers present stem-like niches. However, therapies aimed at targeting this niche have not been as successful as expected. New evidence regarding CSCs hierarchy, similarities with normal tissue stem cells and cell plasticity might be key in understanding their role in cancer biology and how to efficiently eliminate them. In this Chapter, we discuss what is known in breast and prostate CSCs from their initial discoveries to the current therapeutic efforts in the field. Future challenges towards better CSC identification and isolation strategies will be key to shed light into how CSCs could accurately be targeted in combination to traditional therapies to ultimately prolong patient survival.

TGF-BETA IN THE NATURAL HISTORY OF PROSTATE CANCER.

SUMMARYAll transforming growth factors beta (TGFß) are cytokines that regulate several cellular functions such as cell growth, differentiation and motility. They may also have a role in immunosuppression. Their role is important for normal prostate development. TGFß is active in the regulation of balance between epithelial cell proliferation and apoptosis through stromal epithelia via the androgen receptor action. TGFß protects and maintains prostate stem cells, an important population necessary for prostate tissue regeneration. However, TGFß is shown to have a contrasting role in prostate tumor genesis. In the early stages of tumor development, TGFß acts as a tumor suppressor, whereas in the later stages, TGFß becomes a tumor promoter by inducing proliferation, invasion and metastasis. In this review, we outline complex interactions that TGFß-mediated signaling has on prostate tumor genesis, focusing on the role of these interactions during the course of prostate cancer and, in particular, during disease progression.

KLF4, A Gene Regulating Prostate Stem Cell Homeostasis, Is a Barrier to Malignant Progression and Predictor of Good Prognosis in Prostate Cancer

SUMMARYThere is a considerable need to identify those individuals with prostate cancer who have indolent disease. We propose that genes that control adult stem cell homeostasis in organs with slow turnover, such as the prostate, control cancer fate. One such gene, KLF4, overexpressed in murine prostate stem cells, regulates their homeostasis, blocks malignant transformation, and controls the self-renewal of tumor-initiating cells. KLF4 loss induces the molecular features of aggressive cancer and converts PIN lesions to invasive sarcomatoid carcinomas; its re-expression in vivo reverses this process. Bioinformatic analysis links these changes to human cancer. KLF4 and its downstream targets make up a gene signature that identifies indolent tumors and predicts recurrence-free survival. This approach may improve prognosis and identify therapeutic targets for advanced cancer.

68O - Ligand-inducible, prostate stem cell antigen (PSCA)-directed GoCAR-T™ cells in advanced solid tumors: Preliminary results from a dose escalation study

68O - Ligand-inducible, prostate stem cell antigen (PSCA)-directed GoCAR-T™ cells in advanced solid tumors: Preliminary results from a dose escalation study

Variants in the PSCA gene associated with risk of cancer and nonneoplastic diseases: systematic research synopsis, meta-analysis and epidemiological evidence.

Variants in the prostate stem cell antigen (PSCA) gene have been linked with risk of multiple cancers and other diseases. But results have been inconclusive and no systematic research synopsis has been available. We did a comprehensive meta-analysis to investigate associations between variants in this gene and risk of nine cancers and four nonneoplastic diseases based on data from 55 publications including 81 961 cases and 442 932 controls. We graded levels of cumulative epidemiological evidence of a significant association using the Venice criteria and false-positive report probability tests. We performed functional annotation for these variants using data from the Encyclopedia of DNA Elements Project and other public databases. We found that six variants were nominally significantly associated with an increased or reduced risk of three cancers and three nonneoplastic diseases (P < 0.05). Cumulative evidence of an association was graded as strong for rs2294008 [odds ratio (OR) = 1.32, P = 5.1 × 10-33], rs2976392 (OR = 1.29, P = 1.8 × 10-8), rs9297976 (OR = 0.75, P = 1.4 × 10-7), rs2976391 (OR = 1.38, P = 6.1 × 10-5) and rs138377917 (OR = 0.53, P = 0.008) with gastric cancer, rs2294008 with bladder cancer (OR = 1.15, P = 8.0 × 10-19), gastritis (OR = 1.35, P = 1.2 × 10-5), duodenal ulcer (OR = 0.68, P = 2.4 × 10-57) and gastric ulcer (OR = 0.88, P = 1.7 × 10-7). Data from the Encyclopedia of DNA Elements Project and other databases showed that these variants and other variants correlated with them might fall in putative functional regions. In conclusion, this study provides summary evidence that variants in the PSCA gene are associated with risk of gastric and bladder cancer, gastritis, as well as duodenal and gastric ulcer and highlights the significant role of this gene in the pathogenesis of these diseases.

Evaluation of Bisphenol A (BPA) Exposures on Prostate Stem Cell Homeostasis and Prostate Cancer Risk in the NCTR-Sprague-Dawley Rat: An NIEHS/FDA CLARITY-BPA Consortium Study.

Background:Previous work determined that early life exposure to low-dose Bisphenol A (BPA) increased rat prostate cancer risk with aging. Herein, we report on prostate-specific results from CLARITY-BPA (Consortium Linking Academic and Regulatory Insights on BPA Toxicity), which aims to resolve uncertainties regarding BPA toxicity.Objectives:We sought to a) reassess whether a range of BPA exposures drives prostate pathology and/or alters prostatic susceptibility to hormonal carcinogenesis, and b) test whether chronic low-dose BPA targets prostate epithelial stem and progenitor cells.Methods:Sprague-Dawley rats were gavaged daily with vehicle, ethinyl estradiol (EE) or BPA/kg-BW during development or chronically, and prostate pathology was assessed at one year. One developmentally exposed cohort was given testosterone plus estradiol () implants at day 90 to promote carcinogenesis with aging. Epithelial stem and progenitor cells were isolated by prostasphere (PS) culture from dorsolateral prostates (DLP) of rats continuously exposed for six months to BPA/kg-BW. Gene expression was analyzed by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR).Results:Exposure to BPA alone at any dose did not drive prostate pathology. However, rats treated with EE, 2.5, 250, or BPA/kg-BW plus showed greater severity of lateral prostate intraepithelial neoplasia (PIN), and DLP ductal adenocarcinoma multiplicity was markedly elevated in tumor-bearing rats exposed to -BW. DLP stem cells, assessed by PS number, doubled with chronic EE and exposures. PS size, reflecting progenitor cell proliferation, was greater at 25 and BPA doses, which also shifted lineage commitment toward basal progenitors while reducing luminal progenitor cells.Conclusions:Together, these results confirm and extend previous evidence using a rat model and human prostate epithelial cells that low-dose BPA augments prostate cancer susceptibility and alters adult prostate stem cell homeostasis. Therefore, we propose that BPA exposures may contribute to the increased carcinogenic risk in humans that occurs with aging. https://doi.org/10.1289/EHP3953

Prostate Stem Cells and Cancer Stem Cells.

Stem/progenitor cells play central roles in processes of organogenesis and tissue maintenance, whereas cancer stem cells (CSCs) are thought to drive tumor malignancy. Here, we review recent progress in the identification and analysis of normal prostate stem/progenitor cells as well as putative CSCs in both genetically engineered mouse models as well as in human tissue. We also discuss studies that have investigated the cell type of origin for prostate cancer. In addition, we provide a critical assessment of methodologies used in stem cell analyses and outline directions for future research.

Identification of Two Types of Stem Cells in Methylene Blue-stained Sections of Untreated and Diethylstilbestrol-treated Human Prostate Cancer and Their Characterization by Immunogold Localization of CD133.

The basal compartment of the prostatic acinus harbors stem and basal cells, whereas the luminal compartment contains cuboidal and columnar cells. Mutation in the genes of stem cells is required for benign (normal) prostate to develop into prostatic adenocarcinoma. Stem/basal cells survive androgen deprivation therapy in humans and castrated mice to repopulate glandular cells by proliferation when stimulated by androgen. We hypothesized that using different embedding and staining methods, it would be possible to identify two types of stem cells in human prostate by localization of CD133. Prostate biopsy or prostatectomy pieces from 13 untreated and eight diethylstilbestrol-treated men with prostate cancer were sectioned, stained by methylene blue and CD133 was localized by immunogold technique. Methylene blue stained basic proteins in dark basal cells, but not in light cells. Light basal cells expressed androgen receptors and dark cells estrogen receptors. Light and dark cells expressed CD133, indicating them to be stem cells. Light stem cells produced the lineage of columnar/cuboidal cells. Estrogen-dependent dark cells produced a lineage of columnar/cuboidal cells, that also expressed estrogen receptors. Our analysis indicates that stem/basal cells are privileged cells in the basal compartment. Stem cells are not under the regulation of steroid hormones, whereas their lineage of cuboidal/columnar cells are. The lineage of androgen-dependent cells are columnar/cuboidal cells and the lineage of estrogen-dependent cells are also columnar/cuboidal cells. Epon-embedding and methylene blue staining showed two types of CD133-positive stem cells in prostate. Paraffin sections did not show two types of stem cells in prostate and bone marrow leukemia cells. Our study indicates the continuity of embryonic stem cells into adult prostate as organ-specific stem cells. To our knowledge, this is the first study to identify two types of stem cells in human prostate.

The application of prostate specific membrane antigen in CART‑cell therapy for treatment of prostate carcinoma (Review).

Adoptive cell transfer (ACT) is an emerging immunotherapy technique that restricts tumor growth and invasion in cancer patients. Among the different types of ACT, chimeric antigen receptor (CAR)T‑cell therapy is considered to be the most advanced and a potentially powerful technique for the treatment of cancer in clinical trials. The primary aim of CART‑cell therapy is to destroy cancer cells and therefore, it serves an important role in tumor immunotherapy. CART‑cell therapy has been demonstrated to mainly treat blood cancer by targeting cluster of differentiation (CD)‑19, CD20, CD22, CD33 and CD123. However, the use of CART‑cell therapy for treating solid tumors is currently under extensive investigation. With respect to prostate cancer, prostatic acid phosphatase, prostate‑specific antigen, prostate‑specific membrane antigen (PSMA), prostate stem cell antigen, T‑cell receptorγ alternate reading frame protein, transient receptor potential‑p8 and six‑transmembrane epithelial antigen of the prostate 1 are among the identified target antigens for prostate tumors. However, mesothelin, fibroblast activation protein, epidermal growth factor receptor, carcinoembryonic antigen, disialoganglioside‑2 and human epidermal growth factor 2 are among the main targets of CART‑cell therapy in the case of other types of solid tumors. The main challenges in CART‑cell therapy are the selection of the target antigens and the modulation of the ideal tumor microenvironment for T‑cells to fight against the cancer. The present review focuses on the 1st, 2nd, 3rd and 4th generations of anti‑PSMA CARs and their application for combating prostate carcinoma.

Quantum dot-based fluorescent probes for targeted imaging of the EJ human bladder urothelial cancer cell line.

QDs are a type of inorganic nanoparticle with unique optical properties. As a fluorescent label, QDs are widely used in biomedical fields. In the present study, fluorescent probes of quantum dots (QDs) conjugated with a prostate stem cell antigen (PSCA) monoclonal antibody (QD-PSCA) were prepared to study the targeted imaging of QD-PSCA probes in EJ human bladder urothelial cancer cells and analyze the feasibility of QD-based non-invasive tumor-targeted imaging in vivo. QDs with an emission wavelength of 605 nm (QD605) were conjugated with PSCA to prepare QD605-PSCA fluorescent probes by chemical covalent coupling. The optical properties of the probes coupled and uncoupled with PSCA were measured and assessed using an ultraviolet spectrophotometer and a fluorescence spectrophotometer. Direct immune-fluorescent labeling was utilized to detect and analyze imaging of the probes for EJ cells. The results revealed that QD605-PSCA probes retained the fluorescent properties of QD605 and the immunogenicity of the PSCA protein. The probes were able to specifically recognize the PSCA protein expressed in bladder cancer cells, while fluorescence was stable and had a long duration. The present study suggests that QD-PSCA fluorescent probes may be useful for specific targeted labeling and imaging in bladder urothelial cancer cells. Furthermore, the probes possess good optical stability and may be useful for research into non-invasive targeted imaging, early diagnosis and targeted in vivo tumor therapy.

50-Gy Stereotactic Body Radiation Therapy to the Dominant Intraprostatic Nodule: Results From a Phase 1a/b Trial

Although localized prostate cancer (PCa) is multifocal, the dominant intraprostatic nodule (DIN) is responsible for disease progression after radiation therapy. PCa expresses antigens that could be recognized by the immune system. We therefore hypothesized that stereotactic dose escalation to the DIN is safe, may increase local control, and may initiate tumor-specific immune responses. Patients with localized PCa were treated with stereotactic extreme hypofractionated doses of 36.25Gy in 5 fractions to the whole prostate while simultaneously escalating doses to the magnetic resonance image-visible DIN (45Gy, 47.5Gy, and 50Gy in 5 fractions). The phase 1a part was designed to determine the recommended phase 1b dose in a "3+3" cohort-based, dose-escalation design. The primary endpoint was dose-limiting toxicities defined as≥grade 3 gastrointestinal (GI) or genitourinary (GU) toxicity (or both) by National Cancer Institute Common Terminology Criteria for Adverse Events (version 4) up to 90days after the first radiation fraction. The secondary endpoints were prostate-specific antigen kinetics, quality of life (QoL), and blood immunologic responses. Nine patients were treated in phase 1a. No dose-limiting toxicities were observed at either level, and therefore the maximum tolerated dose was not reached. Further characterization of tolerability, efficacy, and immunologic outcomes was conducted in the subsequent 11 patients irradiated at the highest dose level (50Gy) in the phase 1b expansion cohort. Toxicity was 45% and 25% for grades 1 and 2 GU, and 20% and 5% for grades 1 and 2 GI, respectively. No grade 3 or worse toxicity was reported. The average (±standard error of the mean) of the QoL assessments at baseline and at 3-month posttreatment were 0.8 (±0.8) and 3.5 (±1.5) for the bowel (mean difference, 2.7; 95% confidence interval, 0.1-5), and 6.4 (±0.8) and 7.27 (±0.9) for the International Prostate Symptom Score (mean difference, 0.87; 95% confidence interval, 0.3-1.9), respectively. A subset of patients developed antigen-specific immune responses against prostate-specific membrane antigen (n=2), prostatic acid phosphatase (n=1), prostate stem cell antigen (n=4), and prostate-specific antigen (n=2). Irradiation of the whole prostate with 36.25Gy in 5 fractions and dose escalation to 50Gy to the DIN was tolerable and determined as the recommended phase 1b dose. This treatment has promising antitumor activity, which will be confirmed by the ongoing phase 2 part. Preliminary QoL analysis showed minimal impact in GU, GI, and sexual domains. Stereotactic irradiation induced antigen-specific immune responses in a subset of patients.

Genome-Wide Association of Genetic Variation in the PSCA Gene with Gastric Cancer Susceptibility in a Korean Population

PurposeHalf of the world’s gastric cancer cases and the highest gastric cancer mortality rates are observed in Eastern Asia. Although several genome-wide association studies (GWASs) have revealed susceptibility genes associated with gastric cancer, no GWASs have been conducted in the Korean population, which has the highest incidence of gastric cancer. Materials and MethodsWe performed genome scanning of 450 gastric cancer cases and 1,134 controls via Affymetrix Axiom Exome 319 arrays, followed by replication of 803 gastric cancer cases and 3,693 healthy controls. ResultsWe showed that the rs2976394 in the prostate stem cell antigen (PSCA) gene is a gastriccancer-susceptibility gene in a Korean population, with genome-wide significance and an odds ratio (OR) of 0.70 (95% confidence interval [CI], 0.64 to 0.77). A strong linkage disequilibrium with rs2294008 was also found, indicating an association with susceptibility. Individuals with the CC genotype of the PSCA gene showed an approximately 2-fold lower risk of gastric cancer compared to those with the TT genotype (OR, 0.47; 95% CI, 0.39 to 0.57). The effect of the PSCA gene on gastric cancer was more prominent in the female population and for diffuse type gastric cancer.ConclusionOur result confirmed that the PSCA gene may be the most important susceptibility gene for gastric cancer risk in a Korean population.

Enhancing the Potency and Specificity of Engineered T Cells for Cancer Treatment.

The adoptive transfer of chimeric antigen receptor (CAR)-modified T cells has produced tumor responses even in patients with refractory diseases. However, the paucity of antigens that are tumor selective has resulted, on occasion, in "on-target, off-tumor" toxicities. To address this issue, we developed an approach to render T cells responsive to an expression pattern present exclusively at the tumor by using a trio of novel chimeric receptors. Using pancreatic cancer as a model, we demonstrate how T cells engineered with receptors that recognize prostate stem cell antigen, TGFβ, and IL4, and whose endodomains recapitulate physiologic T-cell signaling by providing signals for activation, costimulation, and cytokine support, produce potent antitumor effects selectively at the tumor site. In addition, this strategy has the benefit of rendering our cells resistant to otherwise immunosuppressive cytokines (TGFβ and IL4) and can be readily extended to other inhibitory molecules present at the tumor site (e.g., PD-L1, IL10, and IL13).Significance: This proof-of-concept study demonstrates how sophisticated engineering approaches can be utilized to both enhance the antitumor efficacy and increase the safety profile of transgenic T cells by incorporating a combination of receptors that ensure that cells are active exclusively at the tumor site. Cancer Discov; 8(8); 972-87. ©2018 AACR.See related commentary by Achkova and Pule, p. 918This article is highlighted in the In This Issue feature, p. 899.

Abstract 3063: Stem cell lineage hierarchy by keratin profiling in normal human prostate epithelial cells and prostate cancer

Abstract Background: Both mechanical and nonmechanical functions of keratins have been implicated in stem cells and cancers. Using a sphere-based label retention assay, we recently isolated and characterized prostate stem cells and cancer stem-like cells from mixed progenitor populations (PMID 28651114), identifying keratin 13 (KRT13) as a specific prostate stem cell marker regulating self-renewal. Herein we utilize detailed keratin profiles to further clarify the human prostate epithelial lineage hierarchy and identify prostate cancer stem-like cells. Methods and Results: Primary prostate epithelial cells were 3D cultured (5 days) to form prostaspheres (PS), followed by PS-based long-term label retention and FACS to separate stem cells from progenitors. In normal prostate tissues from three healthy donors, RNA-seq revealed enrichment of KRT13, 23, 80, 78, 86 and 4 in label-retaining prostate stem cells while KRT6, 17, 14, 5, 8, 18 and P63 were enriched in nonretaining progenitors. We next used Fluidigm C1 captured single-cell RNA-seq and identified three major clusters in the label-retaining stem cell population; Cluster I represents quiescent stem cells (KRT13, 23, 80, 78, 4 enriched) and Clusters II and III contain active stem cells and bipotent progenitors, respectively (KRT16, 17, 6 enriched). GSEA analysis found stem cell and cancer-related pathways enrichment in Cluster I. Three additional clusters were identified in nonretaining progenitor cells, with Cluster IV representing unipotent basal progenitor cells (KRT5, 14, 6, 16 enriched) and Clusters V and VI early- and late-stage unipotent luminal progenitors (KRT8, 18, 10 enriched). Cancer stem-like cells were similarly isolated from three prostate cancer specimens and RNA-seq with MetaCore pathway analysis found enrichment of cytoskeleton remodeling keratin filaments. Interestingly, in addition to normal stem cell keratins (KRT13, 23, 80, 78, 4), other keratins (KRT10, 19, 6, 75, 16, 79, 3, 82) were enriched in cancer stem-like cells. Surprisingly, stem-like cells from patient-matched benign regions revealed a similar keratin profile, suggesting a cancer field effect for stem-like cell populations. Conclusion: Taken together, using gene profiling with an emphasis on keratin patterns, we have delineated the lineage hierarchy of human prostate stem cells originating from the activation of quiescent stem cells to bipotent progenitors that give rise to unipotent basal and luminal progenitor cells. We have identified common keratins enriched in stem cells from normal prostate and cancer/benign tissues as well as keratins unique in stem-like cells from prostate cancer. This clarification of the stem cell lineage hierarchy and keratin profiling of human prostate stem cells and cancer stem-like cells may provide enhanced opportunities for translational studies that target therapeutic-resistant cancer stem-like cells. (CA-172220) Citation Format: Wenyang Hu, Danping Hu, Lishi Xie, Hong Hu, Ye Li, Larisa Nonn, Toshi Shioda, Gail S. Prins. Stem cell lineage hierarchy by keratin profiling in normal human prostate epithelial cells and prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3063.

Stromal Gli signaling regulates the activity and differentiation of prostate stem and progenitor cells

Interactions between cells in the stroma and epithelium facilitate prostate stem cell activity and tissue regeneration capacity. Numerous molecular signal transduction pathways, including the induction of sonic hedgehog (Shh) to activate the Gli transcription factors, are known to mediate the cross-talk of these two cellular compartments. However, the details of how these signaling pathways regulate prostate stem and progenitor cell activity remain elusive. Here we demonstrate that, although cell-autonomous epithelial Shh-Gli signaling is essential to determine the expression levels of basal cell markers and the renewal potential of epithelial stem and progenitor cells, stromal Gli signaling regulates prostate stem and progenitor cell activity by increasing the number and size of prostate spheroids in vitro Blockade of stromal Gli signaling also inhibited prostate tissue regeneration in vivo The inhibition of stromal Gli signaling suppressed the differentiation of basal and progenitor cells to luminal cells and limited prostate tubule secretory capability. Additionally, stromal cells were able to compensate for the deficiency of epithelial Shh signaling in prostate tissue regeneration. Mechanistically, suppression of Gli signaling increased the signaling factor transforming growth factor β (TGFβ) in stromal cells. Elevation of exogenous TGFβ1 levels inhibited prostate spheroid formation, suggesting that a stromal Gli-TGFβ signaling axis regulates the activity of epithelial progenitor cells. Our study illustrates that Gli signaling regulates epithelial stem cell activity and renewal potential in both epithelial and stromal compartments.

Decline in arylsulfatase B expression increases EGFR expression by inhibiting the protein-tyrosine phosphatase SHP2 and activating JNK in prostate cells

Epidermal growth factor receptor (EGFR) has a crucial role in cell differentiation and proliferation and cancer, and its expression appears to be up-regulated when arylsulfatase B (ARSB or GalNAc-4-sulfatase) is reduced. ARSB removes 4-sulfate groups from the nonreducing end of dermatan sulfate and chondroitin 4-sulfate (C4S), and its decreased expression has previously been reported to inhibit the activity of the ubiquitous protein-tyrosine phosphatase, nonreceptor type 11 (SHP2 or PTPN11). However, the mechanism by which decline in ARSB leads to decline in SHP2 activity is unclear. Here, we show that SHP2 binds preferentially C4S, rather than chondroitin 6-sulfate, and confirm that SHP2 activity declines when ARSB is silenced. The reduction in ARSB activity, and the resultant increase in C4S, increased the expression of EGFR (Her1/ErbB1) in human prostate stem and epithelial cells. The increased expression of EGFR occurred after 1) the decline in SHP2 activity, 2) enhanced c-Jun N-terminal kinase (JNK) activity, 3) increased nuclear DNA binding by c-Jun and c-Fos, and 4) EGFR promoter activation. In response to exogenous EGF, there was increased bromodeoxyuridine incorporation, consistent with enhanced cell proliferation. These findings indicated that ARSB and chondroitin 4-sulfation affect the activation of an important dual phosphorylation threonine-tyrosine kinase and the mRNA expression of a critical tyrosine kinase receptor in prostate cells. Restoration of ARSB activity with the associated reduction in C4S may provide a new therapeutic approach for managing malignancies in which EGFR-mediated tyrosine kinase signaling pathways are active.

Abstract 2383: EGFR expression increases following decline in activity of N-acetylgalactosamine 4-sulfatase (ARSB) and SHP2 (PTPN11) and increases in chondroitin 4-sulfate and JNK in prostate

Abstract The enzyme arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase) removes 4-sulfate groups from the non-reducing end of dermatan sulfate and chondroitin 4-sulfate (C4S) and is required for degradation of these sulfated glycosaminoglycans. Decline in ARSB and the resultant increase in C4S was previously reported to inhibit the activity of the enzyme SHP2 (PTPN11), a ubiquitous, non-receptor tyrosine phosphatase, and to lead to transcriptional events mediated by the sustained phosphorylation of phospho-ERK1,2 and phospho-p38. Previous experiments had shown that decline in ARSB was associated with increased expression of the epidermal growth factor receptor (EGFR; Her1/ErbB1) in human prostate malignant tissue and in human prostate cells, but the mechanism for this increase was unknown. The current experiments were performed to determine how decline in ARSB leads to increased expression of EGFR. Experiments were performed in human prostate stem and epithelial cells and normal and malignant human prostate tissues. Measurements included: EGFR promoter activity, chromatin immunoprecipitation assay, and binding studies of chondroitin sulfates with SHP2. Materials used included: SR11302, an inhibitor of c-fos binding with nuclear DNA; c-Jun peptide that acts as a c-Jun mimetic and inhibits c-Jun phosphorylation by JNK; JNK-selective inhibitor peptide that inhibits JNK phosphorylation; SHP2 dominant negative and constitutively active DNA constructs; PHPS1, an inhibitor of SHP2; ARSB siRNA, and galectin-3 siRNA. Increased EGFR promoter activation and EGFR expression followed decline in ARSB, decline in activity of SHP2 (PTPN11), enhanced activity of c-Jun N-terminal kinase (JNK), increased nuclear DNA binding of c-Jun and c-Fos, and EGFR promoter activation. These effects demonstrate an impact of ARSB and chondroitin 4-sulfation on the activation of a critical dual phosphorylation threonine-tyrosine kinase and on the mRNA expression of an important tyrosine kinase receptor in prostate cells. Restoration of ARSB activity may provide a new therapeutic approach to malignancies in which there is activation of EGFR-mediated tyrosine kinase signaling pathways. Citation Format: Sumit Bhattacharyya, Leo Feferman, Joanne K. Tobacman. EGFR expression increases following decline in activity of N-acetylgalactosamine 4-sulfatase (ARSB) and SHP2 (PTPN11) and increases in chondroitin 4-sulfate and JNK in prostate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2383.

2185 The effects of autoimmune inflammation on proliferation, differentiation, and androgen receptor signaling in adult prostate stem cells

OBJECTIVES/SPECIFIC AIMS: The primary goal of this project is to verify findings from a murine prostatitis model in the human setting. METHODS/STUDY POPULATION: Methods include primary cell isolation and culture, FACS, adoptive transfer, 3D cell culture, histology, immunofluorescence, xenograft, and tissue recombination. The study population includes patients undergoing HoLEP or radical prostatectomy due to hyperplasia or adjacent bladder or prostate cancer. RESULTS/ANTICIPATED RESULTS: Having verified similar sensitivities to androgen receptor (AR) inhibitors between naive murine and human basal prostate stem cells, we anticipate that autoimmune inflammation in humans affects the response of basal prostate stem cells in a manner similar to the murine setting as well. This includes increased proliferation, increased differentiation, and decreased response to AR inhibitors. DISCUSSION/SIGNIFICANCE OF IMPACT: The identification of survival mechanisms used by basal prostate stem cells in an androgen deprived environment may give insight to the process by which prostate cancer becomes androgen independent. The effect of inflammation on proliferation, survival, and AR signaling in these cells may also provide information relevant to cancer initiation and progression.