Prostate Stem Cell Research Articles

Biomolecular characterization of exosomes released from cancer stem cells: Possible implications for biomarker and treatment of cancer.

Cancer recognized as one of the leading irrepressible health issues is contributing to increasing mortality-rate day-by-day. The tumor microenvironment is an important field of cancer to understand the detection, treatment and prevention of cancer. Recently, cancer stem cell (CSC) research has shown promising results aiming towards cancer diagnostics and treatment. Here, we found that prostate and breast cancer stem cells secreted vesicles of endosomal origin, called exosomes showed strong connection between autophagy and exosomes released from CSCs. Exosomes may serve as vesicles to communicate with neoplastic cells (autocrine and paracrine manner) and normal cells (paracrine and endocrine manner) and thereby suppress immune systems and regulate neoplastic growth, and metastasis. They can also be used as biomarkers for various cancers. We detected tetraspanin proteins (CD9, CD63, CD81), Alix and tumor susceptibility gene-101 (TSG101) of exosomal markers from rotenone treated CSCs. We have also detected the induction of autophagy genes, Atg7 and conversion of autophagy marker (LC3-I to LC3-II), and tetraspanin proteins (CD9, CD63, CD81) in rotenone treated CSCs by western blotting. The mRNA expression of CD9, CD63, CD81 and TSG101 analyzed by qRT-PCR showed that the rotenone induced the expression of CD9, CD63, CD81 and TSG101 in CSCs. Electron microscopy of rotenone treated CSCs showed the mitochondrial damage of CSCs as confirmed by the release of exosomes from CSCs. The constituents of exosomes may be useful to understand the mechanism of exosomes formation, release and function, and also serve as a useful biomarker and provide novel therapeutic strategies for the treatment and prevention of cancer.

Abstract 3021: Roles of EMT on stem cell properties of prostate stem and cancer cells during castration-resistant prostate cancer progression

Abstract Androgen ablation remains the mainstay of treatment for men with advanced and metastatic prostate cancer. However, despite the introduction of new generation anti-androgens, a majority of men succumb to castration resistant prostate cancer (CRPC). The molecular mechanisms governing the emergence of treatment resistance in CRPC patients are not well understood. Recent experience suggests that tumor regeneration from castration-resistant stem-like cells induce resistance to hormonal therapy. Therefore, elucidating novel targets essential for driving stem-like activity is critical to prevent and defeat CRPC. It has been shown that normal and cancer stem cells exploit normal development process of epithelial-mesenchymal transition (EMT) to survive and metastasize, and that EMT confer stem cell properties to more differentiated cancer cell progeny in breast and other cancers. However, it is unclear if EMT is linked with stem cells in normal and malignant prostate. Our lab has reported that N-cadherin, a marker of EMT, is upregulated after neoadjuvant hormone ablation and in CRPC and is sufficient to cause metastasis and CRPC. Therapeutic targeting of N-cadherin by novel N-cadherin antibody inhibited metastatic and CRPC progression. The cell population displaying N-cadherin co-expressed a number of stem cell-associated genes in CRPC models. Here, we verified EMT linked to stem cells in both normal prostate and CRPC. We found in LAPC-9 CRPC tumors, the cell population expressing N-cadherin behaved like stem cells with enhanced sphere-forming ability, which could be specifically inhibited by novel N-cadherin antibody 2A9. We isolated stem-like CD49fhi/Trop2hi cells from prostatectomy specimens and found that forced N-cadherin expression promoted sphere formation of those cells. Our evaluation of gene expression in N-cadherin-positive prostate cancer cell lines and CRPC tumors demonstrated that N-cadherin expression activated common EMT transcriptional regulators including Zeb1. We asked if Zeb1 regulated stem cell properties in normal and malignant prostate. We found that forced Zeb1 expression induced EMT with enhanced cell invasiveness in LNCaP human prostate cancer cells. However, Zeb1 overexpression inhibited cell proliferation and CRPC tumor growth of LNCaP. Zeb1 overexpression also surprisingly inhibited sphere formation of normal stem/progenitor cells from prostatectomy specimens. Our data suggest that Zeb1-regulated EMT promotes both quiescence and invasiveness in normal and malignant prostate in which the quiescent cells may survive and play a role in treatment resistance, while N-cadherin mediates stem cell proliferation and self-renewal. Our research will likely provide useful information of EMT-related biomarkers for preventing and developing efficient therapeutics to combat the treatment resistance to new generation anti-androgens. Citation Format: Shu Lin, Evelyn Kono, Joyce Yamashiro, Sean Lee, Owen Witte, Andrew Goldstein, Robert E. Reiter. Roles of EMT on stem cell properties of prostate stem and cancer cells during castration-resistant prostate cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3021. doi:10.1158/1538-7445.AM2014-3021

Silencing KRAS overexpression in arsenic-transformed prostate epithelial and stem cells partially mitigates malignant phenotype.

Inorganic arsenic is a human carcinogen that likely targets the prostate. Chronic arsenic exposure malignantly transforms the RWPE-1 human prostate epithelial line to chronic arsenic exposed-prostate epithelial (CAsE-PE) cells, and a derivative normal prostate stem cell (SC) line, WPE-stem to arsenic-cancer SCs (As-CSCs). The KRAS oncogene is highly overexpressed in CAsE-PE cells and activation precedes transformation, inferring mechanistic significance. As-CSCs also highly overexpress KRAS. Thus, we hypothesize KRAS activation is key in causing and maintaining an arsenic-induced malignant phenotype, and hence, KRAS knockdown (KD) may reverse this malignant phenotype. RNA interference using shRNAmirs to obtain KRAS KD was used in CAsE-PE and As-CSC cells. Cells analyzed 2 weeks post transduction showed KRAS protein decreased to 5% of control after KD, confirming stable KD. KRAS KD decreased phosphorylated ERK, indicating inhibition of RAS/ERK signaling, a proliferation/survival pathway activated with arsenic transformation. Secreted metalloproteinase (MMP) activity was increased by arsenic-induced malignant transformation, but KRAS KD from 4 weeks on decreased secreted MMP-9 activity by 50% in As-CSCs. Colony formation, a characteristic of cancer cells, was decreased in both KRAS KD transformants. KRAS KD also decreased the invasive capacity of both cell types. KRAS KD decreased proliferation in As-CSCs, consistent with loss of rapid tumor growth. Genes predicted to impact cell proliferation (eg, Cyclin D1, p16, and p21) changed accordingly in both KD cell types. Thus, KRAS silencing impacts aspects of arsenic-induced malignant phenotype, inducing loss of many typical cancer characteristics particularly in As-CSCs.

Combination of bladder cancer-specific oncolytic adenovirus gene therapy with cisplatin on bladder cancer in vitro.

Bladder cancer-specific oncolytic adenovirus Ad/PSCAE/UPII/E1A, carrying E1A gene regulated by human Uroplakin II (UPII) promoter and prostate stem cell antigen enhancer (PSCAE), could kill bladder tumor cells preferentially. The aim of this study was to examine the effects of Ad/PSCAE/UPII/E1A combined with cisplatin on human bladder cancer cells and to identify the underlying mechanisms. The combined effects of Ad/PSCAE/UPII/E1A and cisplatin on EJ, 5637, and BIU-87 bladder cancer cells were evaluated by MTT cell proliferation assay. Cell apoptosis was detected by flow cytometry with fluorescein isothiocyanate-conjugated annexin V (annexin V-FITC) and propidium iodide staining. The activation of the caspase pathway and the expression of Bcl-2 family proteins were determined by western blot assay. Ad/PSCAE/UPII/E1A adenovirus vector could infect bladder cancer cell lines selectively and induce growth inhibition effectively. Of note, the combination treatment of cisplatin and Ad/PSCAE/UPII/E1A could inhibit the proliferation of bladder cancer cells significantly compared with the "alone" treatment. Furthermore, Ad/PSCAE/UPII/E1A plus cisplatin combined treatment resulted in enhanced apoptosis in bladder cancer cells. The enhanced antitumor effects in vitro elicited by Ad/PSCAE/UPII/E1A plus cisplatin were closely related to the increased Fas expression and cleavage of caspase-8 and Bid and decrease in the ratio of anti- to pro-apoptotic proteins followed by activation of caspase-9 and caspase-3, which may contribute to the activation of extrinsic and intrinsic apoptotic pathways. Our results indicate that the combination of Ad/PSCAE/UPII/E1A with cisplatin exerts a synergistic antitumor effect on human bladder cancer cells and is a potential combined treatment strategy for bladder cancer.

Increased Notch signalling inhibits anoikis and stimulates proliferation of prostate luminal epithelial cells.

The prostate epithelial lineage hierarchy remains inadequately defined. Recent lineage-tracing studies have implied the existence of prostate luminal epithelial progenitors with extensive regenerative capacity. However, this capacity has not been demonstrated in prostate stem cell activity assays, probably due to the strong susceptibility of luminal progenitors to anoikis. Here we show that constitutive expression of Notch1 intracellular domain impairs secretory function of mouse prostate luminal cells, suppresses anoikis of luminal epithelial cells by augmenting NF-κB activity independent of Hes-1, stimulates luminal cell proliferation by potentiating PI3K-AKT signaling, and rescues the capacities of the putative prostate luminal progenitors for unipotent differentiation in vivo and short-term self-renewal in vitro. Epithelial cell-autonomous AR signaling is dispensable for the Notch-mediated effects. As Notch activity is increased in prostate cancers and anoikis resistance is a hallmark for metastatic cancer cells, this study suggests a pro-metastatic function of Notch signaling during prostate cancer progression.

Redirection of CD4+ and CD8+ T lymphocytes via a novel antibody-based modular targeting system triggers efficient killing of PSCA+ prostate tumor cells.

There is still a need for new therapeutic options against prostate cancer. Conventional single-chain bispecific antibodies (bsAbs), that directly cross-link T cells and tumor cells, hold great potential for efficient tumor treatment. However, rapid development of novel bsAbs is hampered by laborious optimization to improve their efficacy and reduce potential side effects. To accelerate the development of a novel antibody tool for the redirection of T cells to different tumor-associated antigens, we recently introduced a modular targeting system. We here describe a novel modular system for treatment of prostate cancer by retargeting of T cells to the prostate stem cell antigen (PSCA). Functionality of the novel PSCA-specific modular system was investigated in vitro by T cell activation and chromium release assays as well as in immunodeficient mice. Similar to a conventional bsAb CD3-PSCA, the novel PSCA-specific modular system induces activation of both CD4+ and CD8+ T cells leading to secretion of pro-inflammatory cytokines and highly efficient target-specific tumor cell lysis. The novel TM was ready-to-use from the time point of construction and functional at low E:T ratios and picomolar concentrations without further optimization. In addition, the PSCA-specific modular system delays outgrowth of s.c. tumors in mice comparable to bsAb CD3-PSCA. We have developed a novel PSCA-specific modular system which triggers an efficient T cell-mediated killing of PSCA+ tumor cells in vitro and in vivo. The new Ab-based targeting strategy can functionally replace conventional bsAbs and allows a flexible redirection of T cells to different tumor-associated antigens.

Simultaneous targeting of prostate stem cell antigen and prostate-specific membrane antigen improves the killing of prostate cancer cells using a novel modular T cell-retargeting system.

Recently, we described a novel modular platform technology in which T cell-recruitment and tumor-targeting domains of conventional bispecific antibodies are split to independent components, a universal effector module (EM) and replaceable monospecific/monovalent target modules (TMs) that form highly efficient T cell-retargeting complexes. Theoretically, our unique strategy should allow us to simultaneously retarget T cells to different tumor antigens by combining the EM with two or more different monovalent/monospecific TMs or even with bivalent/bispecific TMs, thereby overcoming limitations of a monospecific treatment such as the selection of target-negative tumor escape variants. In order to advance our recently introduced prostate stem cell antigen (PSCA)-specific modular system for a dual-targeting of prostate cancer cells, two additional TMs were constructed: a monovalent/monospecific TM directed against the prostate-specific membrane antigen (PSMA) and a bivalent/bispecific TM (bsTM) with specificity for PSMA and PSCA. The functionality of the novel dual-targeting strategies was analyzed by performing T cell activation and chromium release assays. Similar to the PSCA-specific modular system, the novel PSMA-specific modular system mediates an efficient target-dependent and -specific tumor cell lysis at low E:T ratios and picomolar Ab concentrations. Moreover, by combination of the EM with either the bispecific TM directed to PSMA and PSCA or both monospecifc TMs directed to either PSCA or PSMA, dual-specific targeting complexes were formed which allowed us to kill potential escape variants expressing only one or the other target antigen. Overall, the novel modular system represents a promising tool for multiple tumor targeting.

Improved modeling of in vivo kinetics of slowly diffusing radiotracers for tumor imaging.

Large-molecule tracers, such as labeled antibodies, have shown success in immuno-PET for imaging of specific cell surface biomarkers. However, previous work has shown that localization of such tracers shows high levels of heterogeneity in target tissues, due to both the slow diffusion and the high affinity of these compounds. In this work, we investigate the effects of subvoxel spatial heterogeneity on measured time-activity curves in PET imaging and the effects of ignoring diffusion limitation on parameter estimates from kinetic modeling. Partial differential equations (PDE) were built to model a radially symmetric reaction-diffusion equation describing the activity of immuno-PET tracers. The effects of slower diffusion on measured time-activity curves and parameter estimates were measured in silico, and a modified Levenberg-Marquardt algorithm with Bayesian priors was developed to accurately estimate parameters from diffusion-limited data. This algorithm was applied to immuno-PET data of mice implanted with prostate stem cell antigen-overexpressing tumors and injected with (124)I-labeled A11 anti-prostate stem cell antigen minibody. Slow diffusion of tracers in linear binding models resulted in heterogeneous localization in silico but no measurable differences in time-activity curves. For more realistic saturable binding models, measured time-activity curves were strongly dependent on diffusion rates of the tracers. Fitting diffusion-limited data with regular compartmental models led to parameter estimate bias in an excess of 1,000% of true values, while the new model and fitting protocol could accurately measure kinetics in silico. In vivo imaging data were also fit well by the new PDE model, with estimates of the dissociation constant (Kd) and receptor density close to in vitro measurements and with order of magnitude differences from a regular compartmental model ignoring tracer diffusion limitation. Heterogeneous localization of large, high-affinity compounds can lead to large differences in measured time-activity curves in immuno-PET imaging, and ignoring diffusion limitations can lead to large errors in kinetic parameter estimates. Modeling of these systems with PDE models with Bayesian priors is necessary for quantitative in vivo measurements of kinetics of slow-diffusion tracers.

HGF and the regulation of tight junctions in human prostate cancer cells.

Hepatocyte growth factor (HGF) may impact the metastasis of prostate cancer via its action on prostate stem cells or their progeny. Tight junctions (TJs) are crucial to the process of metastasis and have been previously shown to be regulated by HGF. The present study aimed to evaluate the effect of HGF on the function of TJs in human prostate epithelial, prostate stem cell-like and prostate cancer cell lines. Four human prostate cancer cell lines (PC-3, DU-145, PZHPV-7, CaHPV-10), normal adult prostate parental epithelial cells (RWPE-1) and a stem cell-like derivative of RWPE-1 (WPE-STEM) were used to assess HGF-induced changes in TJs. A significant difference was noted in the behaviour between the WPE-STEM, RWPE-1 and the cancer cell lines which was HGF concentration-dependent. However, in the WPE-STEM cells, the effect was biphasic, with the cells seemingly resistant to HGF-modulated TJ disruption. Closer examination revealed that HGF affected the redistribution of ZO-1, ZO-2 and ZO-3 away from the TJs of confluent cells with concurrent loss of claudin-1 and claudin-5, and western blot analysis revealed a loss in TJ protein expression of ZO-1 and ZO-2. We demonstrated for the first time that HGF regulates TJ function in human prostate cells. Moreover, this regulation was dependent on the tumourigenicity of the cells, with the most aggressive cells most susceptible and the stem cell-like cells least susceptible. These data offer an intriguing glimpse of how TJs affect the behaviour of prostate cancer cells and how HGF modulates the expression and function of the molecules maintaining TJ structure and function.

Su1940 Influence of Prostate Stem Cell Antigen Gene Polymorphisms on Risk of Gastric Cancer and Duodenal Ulcer in Japanese Patients

BACKGROUND AND AIM : IDO plays a role in self-limiting Th1 response and promoting regulatory T cells and Th2 cytokine profile, mainly represented by interleukin (IL)-4. Interleukin (IL)-17, a Th17 cells-derived cytokine up-regulated in H.pylori-infected human gastric mucosa, is involved in the host response against the bacterium and, possibly, in its clearance/ persistence. Aim of this study was to investigate the role of IDO in the host response against H.pylori. MATERIAL AND METHODS : Five antral biopsies were taken from 26 patients (13 M, median age 41 yrs, range 25-70) who underwent endoscopy for dyspeptic symptoms: 1 for urease quick test, 2 for histology (modified Sydney System score) and 2 for protein extraction. A C13-urea breath test was also performed. IDO expression was determined by Western blotting in total proteins extracted from freshly obtained gastric biopsies. In a subgroup of patients, further antral biopsies were taken, immediately placed in an organ culture chamber, and treated with and without IDO inhibitor 1-methyl-L-Trp (Sigma, St. Louis, MO). Expression of interferon (IFN)-γ mRNA and levels of T-bet, a Th1-related transcription factor, IL-17 and IL4 were determined in gastric biopsy cultures, by realtime PCR and Western blotting, respectively. The ratio of IDO, T-bet, IL-17 and IL-4 with beta-actin was calculated by densitometry. The expression of IFNγ gene was normalized to that of RPS9 housekeeping gene and showed as fold changes using the 2(-Delta Delta C(T)) (Livak) method. All values were expressed as means ± SD arbitrary units (a.u.). RESULTS : IDO expression was found enhanced in gastric biopsies from H.pylori-infected (n=11) compared to uninfected (n=15) patients (1.24±0.21 a.u. vs 0.92±0.47 a.u., P=0.033, respectively). In H.pylori-infected patients, IDO expression did not parallel gastritis score (r=-0.29, P=0.41). In gastric biopsy cultures, IDO inhibition significantly (P<0.05) increased T-bet (0.25±0.17 a.u. vs 0.51±0.26 a.u.,) and IL-17 (0.70±0.45 a.u. vs 1.00±0.58 a.u.,) while decreasing IL-4 (0.35±0.34 a.u. vs 0.24±0.23 a.u.) levels. IFN-γmRNA expression was significantly (P=0.014) higher after IDO inhibition (1±0 a.u. vs 3.1±2 a.u.). CONCLUSIONS : This study demonstrates, for the first time, that an enhanced expression of IDO takes place in H. pylori-infected human gastric mucosa. Higher IDO expression downregulates Th1 response and IL-17 levels and upregulates IL-4 production. This molecular mechanism lowers gastric inflammation, possibly contributing to the persistence of H. pylori. Targeting IDO pathway may be a new strategy for modulating H. pylori-induced mucosal immune response.

The experimental study of ^131I labeling prostate stem cell antigen monoclonal antibody in the treat-ment of nude mice bearing orthotopic implantation of prostate carcinomar

Objective To study the distribution, radioimmunoimaging and anti-tumor effect of ^131I labeling prostate stem cell antigen monoclonal antibody （^131I-PSCA-McAb） in nude mice bearing orthotopic implantation of prostate carcinoma. Methods Thirty-six male nude mice were randomly divided into 2 groups： LNCap cell group （androgen dependent prostate cancer） and PC3 cell group （androgen independent prostate cancer）. Each group were then subdivided into 3 subgroups： experimental group （were intrave noused 1311-PSCA-McAb 200 μg）, monoclonal control group （were intravenoused PSCA-McAb 200 μg）, iodic control group （ were intravenoused 1311 200 μg）. Continuous images of nude mice bearing LNCap cell and PC3 cell were carried out respectively at 1, 4, 8, 12 and 24 h after drugs injection. Transplantation tumor images were then observed and the radioactivity ratio of tumor/non-tumor （T/NT） was calculated. 48 h later, nude mice were sacrificed and the apoptosis of tumor tissues were measured by TdT-mediated dUTP nick end labeling （TUNEL）. Results The models of nude mice bearing orthotopic implantation of prostate carcinoma were successfully established. The peak uptake of ^131I_PSCA.McAb in orthotopic tumor of prostate carcinoma was at 8h after injection with a T/NT ratio of 4.37 ± 0. 76 （LNCap cell group） and 4. 21±0. 71 （PC3 cell group） respectively. The number of TUNEL positive cells in experimental group was larger than that of the two other control groups. Conclusion ^131 I-PSCA-McAb could effectively target PSCA and inhibit the growth of tumor, therefore was a promising radioimmunotherapy radiophamaceutical for prostate carcinoma. Key words: Models of orthotopic implantation of prostate cancer; ^131I labeling prostate stem cellantigen monoclonal antibody; Cell apoptosis; Radioimmunotherapy

Prostate stem cell antigen antibody-conjugated multiwalled carbon nanotubes for targeted ultrasound imaging and drug delivery

Multiwalled carbon nanotubes (MWCNTs) are cut short and grafted with polyethylenimine (PEI) for further covalent conjugation to fluorescein isothiocyanate (FITC) and prostate stem cell antigen (PSCA) monoclonal antibody (mAb). The in vitro and in vivo toxicity data reveal that the as-prepared CNT-PEI(FITC)-mAb has good biocompatibility. Combined flow cytometry and confocal luminescence imaging experiments confirm that the CNT-PEI(FITC)-mAb can specifically target the cancer cells which overexpress PSCA. The results of in vitro and in vivo ultrasound (US) imaging indicate that CNT-PEI(FITC)-mAb has great potential to be used as a targeted US contrast agent. The in vivo anti-cancer efficacy testing using PC-3 tumor-bearing mice as animal models demonstrates that CNT-PEI(FITC)-mAb can targetedly deliver drug to the tumors and suppress tumor growth. Findings from this study suggest that the CNT-PEI(FITC)-mAb could be used as a multifunctional platform for simultaneous US imaging and drug delivery applications.

A Novel Ex Vivo Isolation and Expansion Procedure for Chimeric Antigen Receptor Engrafted Human T Cells

Genetically engineered T lymphocytes are a promising option for cancer therapy. Prior to adoptive transfer they have to be expanded in vitro to reach therapeutically sufficient numbers. So far, no universal method exists for selective in vitro expansion of engineered T lymphocytes. In order to overcome this problem and for proof of concept we incorporated a novel unique peptide sequence of ten amino acids as epitope (E-Tag) into the binding domains of two novel chimeric antigen receptors (ECARs) directed against either prostate stem cell antigen (PSCA) for the treatment of prostate cancer (PCa) or CD33 for the treatment of acute myeloide leukemia (AML). The epitope tag then was utilized for expanding ECAR engrafted T cells by triggering the modified T cells via a monoclonal antibody directed against the E-Tag (Emab). Moreover, the E-Tag served as an efficient selection epitope for immunomagnetic isolation of modified T cells to high purity. ECAR engrafted T cells were fully functional and mediated profound anti-tumor effects in the respective models of PCa or AML both in vitro and in vivo. The method can be integrated straightforward into clinical protocols to improve therapeutic efficiency of tumor treatment with CAR modified T lymphocytes.

Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN) and PH Domain and Leucine-rich Repeat Phosphatase Cross-talk (PHLPP) in Cancer Cells and in Transforming Growth Factor β-Activated Stem Cells

Akt kinase controls cell survival, proliferation, and invasive growth and is a critical factor for cancer development. Here we describe a cross-talk between phosphatases that may preserve levels of activated/phosphorylated Akt and confer aggressive growth of cancer cells. In prostatic cancer cells, but not in non-transformed cells or in prostate stem cells, we found that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) overexpression down-regulated PH domain and leucine-rich repeat phosphatase (PHLPP) and that PHLPP overexpression down-regulated PTEN. We also show that silencing PTEN by siRNA increased the levels of PHLPPs. This cross-talk facilitated invasive migration and was mediated by epigenetic alterations, including activation of miR-190, miR-214, polycomb group of proteins, as well as DNA methylation. A role for the purinergic receptor P2X4, previously associated with wound healing, was indicated. We also show that TGF-β1 induced cross-talk concomitant with epithelial-mesenchymal transition in stem cells. The cross-talk emerged as an integrated part of epithelial-mesenchymal transition. We conclude that cross-talk between PTEN and PHLPPs is silenced in normal prostate cells but activated in TGF-β1 transformed prostate stem and cancer cells and facilitates invasive growth.

Regulation of PSCA expression by m‐Numb in the stomach (904.2)

Prostate stem cell antigen (PSCA) is a GPI‐anchored cell surface protein expressed in the normal epithelium of several tissues including the isthmus region of the gastric mucosa. Recently, a genome‐wide association study revealed several SNPs in the PSCA‐gene to be associated with gastric cancer and duodenal ulcer risk. However, the underlying regulatory mechanisms of PSCA are still unclear.Methods: Msi1‐KO mice with lowered expression of m‐Numb in the stomach and wild‐type ICR mice were used for in vivo studies. In vitro studies were carried out with the mouse gastric epithelial cell line MGE507. Overexpression and suppression of m‐Numb in MGE507 cells was achieved by transfection with lentiviral particles carrying an m‐Numb expression vector and shRNA expression vector, respectively. Gene expression was analysed with qRT‐PCR, and western blotting was used to measure protein levels.Results: In Msi1‐KO mice, a significantly reduced expression‐ and protein level of PSCA was found when comparing the gastric mucosa to wild‐type ICR mice. Expectedly, the protein level of m‐Numb was also verified as lower in the KO‐line. Silencing m‐Numb expression in MGE507 cells resulted in a reduction in protein level of PSCA, and overexpression of m‐Numb led to an increased PSCA‐level.Conclusion: PSCA gene expression and protein level was regulated by m‐Numb in the mouse stomach and in mouse gastric epithelial cells.

Inhibitive effects of 131I labeling prostate stem cell antigen monoclonal antibody on prostate carcinoma cell line LNCap and PC3

Objective To investigate the inhibitive effects of 131Ⅰ labeling prostate stem cell antigen monoclonal antibody (131 Ⅰ-PSCA-McAb) on prostate carcinoma cell line LNCap and PC3 in vitro.Methods Prostate carcinoma LNCap cells and PC3 cells were cultured in vitro,and each cell line was divided into 4 groups:experimental group (adding 131Ⅰ-PSCA-McAb 2 g/L),monoclonal control group (adding PSCA-McAb 2 g/L),iodic control group (adding 131I 2 g/L),blank control group (adding saline solution 2 g/L).Cell inhibition of proliferation in each group was tested by methyl thiazol tetrazolium (MTF) assay; tumor apoptosis in each group was measured by flow cytometry; and invasiveness of cells was detected by Transwell invasion test.Results The inhibitory rates of LNCap experimental group and PC3 exerimental group reached (83.67 ± 2.52)％ and (80.33 ± 4.51)％,which obviously higher than monoclonal control groups [(73.33 ± 3.51)％ and (66.67 ± 5.03)％,P＜ 0.05] and iodic control groups [(53.33 ±4.51) ％ and (56.33 ± 2.52) ％,P ＜ 0.05].The apoptosis rates of LNCap experimental group and PC3 exerimental group reached (50.24 ±5.52)％ and (54.71 ±5.84)％ respectively,which obviously higher than monoclonal control groups [(42.23 ± 4.87)％ and (45.61 ± 5.60)％,P ＜0.05],iodic control groups [(26.75 ± 3.43) ％ and (22.26 ± 3.71) ％,P ＜ 0.05] and blank control groups [(14.18 ± 3.97) ％ and (15.14 ± 3.28) ％,P ＜ 0.05].The number of cells invading the membrane in LNCap experimental group and PC3 exerimental group was 33.34 ± 5.82 and 34.91 ± 6.10 respectively,which obviously less than monoclonal control groups (89.15 ± 7.96 and 92.04 ± 8.44,P ＜0.05),iodic control groups (97.83 ± 9.21 and 88.30 ± 8.12,P＜ 0.05) and blank control groups (119.04 ± 10.57 and 115.56 ± 11.08,P ＜ 0.05).Conclusion 131 Ⅰ-PSCA-mAb could effectively inhibit the growth and invasion ability of prostate carcinoma cell line LNCap and PC3,promote the apoptosis,and has the potential to become a new targeted therapy drug for the treatment of prostate carcinoma.131 Ⅰ-PSCA-McAb could effectively target PSCA and inhibit the growth of tumor,therefore was a promising radioimmunotherapy radiophamaceutical for prostate cancer. Key words: 131Ⅰ labeling prostate stem cell antigen monoclonal antibody; Cell proliferation; Cell apoptosis; Cell invasion; Radioimmunotherapy

MiR-34 Cooperates with p53 in Suppression of Prostate Cancer by Joint Regulation of Stem Cell Compartment

The miR-34 family was originally found to be a direct target of p53 and is a group of putative tumor suppressors. Surprisingly, mice lacking all mir-34 genes show no increase in cancer formation by 18months of age, hence placing the physiological relevance of previous studies in doubt. Here, we report that mice with prostate epithelium-specific inactivation of mir-34 and p53 show expansion of the prostate stem cell compartment and develop early invasive adenocarcinomas and high-grade prostatic intraepithelial neoplasia, whereas no such lesions are observed after inactivation of either the mir-34 or p53 genes alone by 15months of age. Consistently, combined deficiency of p53 and miR-34 leads toacceleration of MET-dependent growth, self-renewal, and motility of prostate stem/progenitor cells. Our study provides direct genetic evidence that mir-34 genes are bona fide tumor suppressors and identifies joint control of MET expression by p53 and miR-34 as a key component of prostate stem cell compartment regulation, aberrations in which may lead to cancer.

Kinetics of Tumor Destruction by Chimeric Antigen Receptor-modified T Cells

The use of chimeric antigen receptor (CAR)-modified T cells as a therapy for hematologic malignancies and solid tumors is becoming more widespread. However, the infusion of a T-cell product targeting a single tumor-associated antigen may lead to target antigen modulation under this selective pressure, with subsequent tumor immune escape. With the purpose of preventing this phenomenon, we have studied the impact of simultaneously targeting two distinct antigens present on tumor cells: namely mucin 1 and prostate stem cell antigen, both of which are expressed in a variety of solid tumors, including pancreatic and prostate cancer. When used individually, CAR T cells directed against either tumor antigen were able to kill target-expressing cancer cells, but tumor heterogeneity led to immune escape. As a combination therapy, we demonstrate superior antitumor effects using both CARs simultaneously, but this was nevertheless insufficient to achieve a complete response. To understand the mechanism of escape, we studied the kinetics of T-cell killing and found that the magnitude of tumor destruction depended not only on the presence of target antigens but also on the intensity of expression-a feature that could be altered by administering epigenetic modulators that upregulated target expression and enhanced CAR T-cell potency.

Environmental Estrogen Exposure During Fetal Life: A Time Bomb for Prostate Cancer

In this issue, Prins et al (1) elegantly describe how exposure to bisphenol A (BPA) during development could modify prostate cell fate in adulthood. This group, involved in research to understand prostate cancer (PCa) development for many years, first determined the effect of BPA on human prostate epithelial stem-like cells obtained from young men. This allowed them to determine that these cells are highly and equally sensitive to estrogens and BPA and are able to amplify. Estradiol and BPA maintain the stem-like state within the normal prostate epithelial population as demonstrated by a higher accumulation of genes such as TBX3 and NANOG. Conversely, epithelial differentiation genes such as NKX3.1 and CK18 are at a low level. In addition to this work on cell culture, Prins et al (1) demonstrated estradiol and BPA effects during prostate development using an ingenious mouse model by xenografting chimeric human-rat prostate tissues in Nude mice (2) and found that low-dose BPA exposure increases estrogen-driven carcinogenesis of human prostate epithelium. As a result of this experimental tour de force, this team has described the missing link between estrogen-like endocrine disrupters and PCa. The Holy Grail has been found: environmental estrogen exposure will modify the fate of prostate stem cells, making them more sensitive to estrogen during adulthood and more prone to develop PCa. Development is a tightly controlled process that involves thousands of genes and hundreds of signaling molecules (hormones and growth factors) and cell interactions (junctions and synapses) that will allow correct cell migration. Like a Lego model, all of the interlocking “bricks” must be assembled correctly to produce a normal individual. Such precise mechanisms are involved that it is easy to understand that any defect in a signaling molecule or, on the contrary, an abnormal activation of a usually silent pathway can produce cell defects. Estrogen-like endocrine disruptors have been considered for many years as putatively responsible for various health defects by interfering with the synthesis, metabolism, binding, or cellular responses of natural estrogens (3). These molecules can be found in various plastic products, flame retardants, pesticides, and many other products that are needed for daily use. Among them, BPA is considered as the paradigm of these environmental estrogen-like molecules. Even though BPA was banned from being used in baby bottles by the European Union in 2011 (4) and by the United States in 2012, over 3 million tons per year of this estrogenic monomer have been used to manufacture polycarbonate plastic products, in resins lining metal cans, in dental sealants, and in blends with other types of plastic products, thus suggesting that adults can be overexposed to it. The ester bond linking BPA molecules in polycarbonate and resins undergoes hydrolysis, resulting in the release of free BPA into food, beverages, and the environment, and numerous monitoring studies now show almost ubiquitous human exposure to biologically active levels of this chemical. BPA exerts estrogenic effects through the classical nuclear estrogen receptors, and BPA acts as a selective estrogen receptor modulator (5). However, BPA also initiates rapid responses via estrogen receptors presumably associated with the plasma membrane. Similar to estradiol, BPA causes changes in some cell functions at the tiniest con-

Quantitative ImmunoPET of Prostate Cancer Xenografts with 89Zr- and 124I-Labeled Anti-PSCA A11 Minibody

Prostate stem cell antigen (PSCA) is expressed on the cell surface in 83%-100% of local prostate cancers and 87%-100% of prostate cancer bone metastases. In this study, we sought to develop immunoPET agents using (124)I- and (89)Zr-labeled anti-PSCA A11 minibodies (scFv-CH3 dimer, 80 kDa) and evaluate their use for quantitative immunoPET imaging of prostate cancer. A11 anti-PSCA minibody was alternatively labeled with (124)I- or (89)Zr-desferrioxamine and injected into mice bearing either matched 22Rv1 and 22Rv1×PSCA or LAPC-9 xenografts. Small-animal PET data were obtained and quantitated with and without recovery coefficient-based partial-volume correction, and the results were compared with ex vivo biodistribution. Rapid and specific localization to PSCA-positive tumors and high-contrast imaging were observed with both (124)I- and (89)Zr-labeled A11 anti-PSCA minibody. However, the differences in tumor uptake and background uptake of the radiotracers resulted in different levels of imaging contrast. The nonresidualizing (124)I-labeled minibody had lower tumor uptake (3.62 ± 1.18 percentage injected dose per gram [%ID/g] 22Rv1×PSCA, 3.63 ± 0.59 %ID/g LAPC-9) than the residualizing (89)Zr-labeled minibody (7.87 ± 0.52 %ID/g 22Rv1×PSCA, 9.33 ± 0.87 %ID/g LAPC-9, P < 0.0001 for each), but the (124)I-labeled minibody achieved higher imaging contrast because of lower nonspecific uptake and better tumor-to-soft-tissue ratios (22Rv1×PSCA:22Rv1 positive-to-negative tumor, 13.31 ± 5.59 (124)I-A11 and 4.87 ± 0.52 (89)Zr-A11, P = 0.02). Partial-volume correction was found to greatly improve the correspondence between small-animal PET and ex vivo quantification of tumor uptake for immunoPET imaging with both radionuclides. Both (124)I- and (89)Zr-labeled A11 anti-PSCA minibody showed high-contrast imaging of PSCA expression in vivo. However, the (124)I-labeled A11 minibody was found to be the superior imaging agent because of lower nonspecific uptake and higher tumor-to-soft-tissue contrast. Partial-volume correction was found to be essential for robust quantification of immunoPET imaging with both (124)I- and (89)Zr-labeled A11 minibody.