Tumor Evolution and Drug Response in Patient-Derived Organoid Models of Bladder Cancer.

Bladder cancer is the fifth most prevalent cancer in the U.S., yet is understudied, and few laboratory models exist that reflect the biology of the human disease. Here, we describe a biobank of patient-derived organoid lines that recapitulates the histopathological and molecular diversity of human bladder cancer. Organoid lines can be established efficiently from patient biopsies acquired before and after disease recurrence and are interconvertible with orthotopic xenografts. Notably, organoid lines often retain parental tumor heterogeneity and exhibit a spectrum of genomic changes that are consistent with tumor evolution in culture. Analyses of drug response using bladder tumor organoids show partial correlations with mutational profiles, as well as changes associated with treatment resistance, and specific responses can be validated using xenografts in vivo. Our studies indicate that patient-derived bladder tumor organoids represent a faithful model system for studying tumor evolution and treatment response in the context of precision cancer medicine.

Crosstalking between androgen and PI3K/AKT signaling pathways in prostate cancer cells.

Both androgen action and PI3K medicated signaling pathways have been implicated in prostate tumorigenesis. Our androgen receptor (AR) conditional transgenic mice developed murine prostatic intraepithelial neoplasia (mPIN) and prostatic adenocarcinoma lesions recapitulating human prostate cancer development and progression. Role of transgenic AR contributing to malignancy was demonstrated by high degree of transgenic AR expression in atypical and tumor cells in mPIN as well as prostatic adenocarcinoma lesions of the transgenic mice, but not in adjacent normal tissue. Interestingly, reduced PI3K/Akt activation also appeared in these mouse atypical and tumor cells, suggesting an interaction between androgen and PI3K/AKT pathways. In this study, we further investigated this interaction. We showed that the androgen depletion or knockdown of AR expression results in elevated levels of active phosphorylated AKT in prostate cancer cells. Castration of conditional Pten knock-out mice showed increased Akt, phosphorylated Akt, and pS6 expression in the mouse prostate. Using a series of newly generated Ar reporter and Pten knock-out compound mice, we showed that Pten loss directly represses endogenous Ar expression in prostatic epithelial cells. Moreover, Pten loss and PI3K/Akt activation reduced Ar-mediated transcription in purified Pten-null cells. This study provides novel evidence demonstrating interplay between androgen and PI3K pathways, as well as introduces unique and relevant mouse models for further studies of PI3K and AR pathways in the context of prostate tumorigenesis.

Wnt/ß-Catenin-Responsive Cells in Prostatic Development and Regeneration.

The precise role of Wnt/ß-catenin signaling during prostatic development and tumorigenesis is unclear. Axin2 is a direct transcriptional target of ß-catenin. Recent studies have shown that Axin2-expressing cells have stem/progenitor cell properties in a variety of mouse tissues. Here, we genetically labeled Axin2-expressing cells at various time points and tracked their cellular behavior at different developmental and mature stages. We found that prostatic Axin2-expressing cells mainly express luminal epithelial cell markers and are able to expand luminal cell lineages during prostatic development and maturation. They can also survive androgen withdrawal and regenerate prostatic luminal epithelial cells following androgen replacement. Deletion of ß-catenin or expression of stabilized ß-catenin in these Axin2-expressing cells results in abnormal development or oncogenic transformation, respectively. Our study uncovers a critical role of Wnt/ß-catenin-responsive cells in prostatic development and regeneration, and that dysregulation of Wnt/ß-catenin signaling in these cells contributes to prostatic developmental defects and tumorigenesis.

Identification of a novel role of ZMIZ2 protein in regulating the activity of the Wnt/ß-catenin signaling pathway.

ZMIZ2, also named ZIMP7, is a protein inhibitor of activated STAT (PIAS)-like protein and a transcriptional coactivator. In this study, we investigated the interaction between ZMIZ2 and ß-catenin, a key regulator of the Wnt signaling pathway. We demonstrated that the expression of exogenous ZMIZ2 augments TCF (T cell factor) and ß-catenin-mediated transcription. In contrast, shRNA knockdown of ZMIZ2 expression specifically represses the enhancement of TCF/ß-catenin-mediated transcription by ZMIZ2. Using Wnt3a-conditioned medium, we demonstrated that ZMIZ2 can enhance Wnt ligand-induced TCF/ß-catenin-mediated transcription. We also showed a promotional role of ZMIZ2 in enhancing ß-catenin downstream target gene expression in human cells and in Zmiz2 null (Zmiz2(-/-)) mouse embryonic fibroblasts (MEFs). The regulatory role of Zmiz2 in Wnt-induced TCF/ß-catenin-mediated transcription can be restored in Zmiz2(-/-) MEFs that were infected with adenoviral expression vectors for Zmiz2. Moreover, enhancement of Zmiz2 on TCF/ß-catenin-mediated transcription was further demonstrated in Zmiz2 knockout and Axin2 reporter compound mice. Furthermore, the protein-protein interaction between ZMIZ2 and ß-catenin was identified by co-immunoprecipitation and in vitro protein pulldown assays. We also observed recruitment of endogenous ZMIZ2 onto the promoter region of the Axin 2 gene, a ß-catenin downstream target promoter, in a Wnt ligand-inducible manner. Finally, a promotional role of ZMIZ2 on cell growth was demonstrated in human cell lines and Zmiz2 knockout MEFs. Our findings demonstrate a novel interaction between ZMIZ2 and ß-catenin and elucidate a novel mechanism for PIAS-like proteins in regulating Wnt signaling pathways.

Deletion of leucine zipper tumor suppressor 2 (Lzts2) increases susceptibility to tumor development.

Using an Lzts2 knock-out mouse model, we characterized the biological role of Lzts2 in tumorigenesis. Both heterozygous and homozygous deletion of the Lzts2-targeted allele in mice shows an increased incidence in spontaneous tumor development, although Lzts2 homozygous knock-out mice show significantly higher incidences than heterozygous mice. Treatment of Lzts2-deficient mice with a carcinogen, N-butyl-N-(4-hydroxybutyl) nitrosamine, increases the susceptibility to N-butyl-N-(4-hydroxybutyl) nitrosamine-induced bladder carcinoma development. Examination of human prostate cancer tissue specimens shows a reduction of LZTS2 protein expression in prostate cancer cells. Further analyses of mouse embryonic fibroblasts isolated from Lzts2 knock-out embryos show that loss of Lzts2 enhances cell growth. These data provide the first line of evidence demonstrating that deletion of Lzts2 increases susceptibility to spontaneous and carcinogen-induced tumor development.

Human microRNA-27a* targets Prf1 and GzmB expression to regulate NK-cell cytotoxicity.

Perforin (Prf1) and granzyme B (GzmB) are essential effector molecules for natural killer (NK)-cell cytotoxicity, but how Prf1 and GzmB expression is regulated during arming of NK cells is poorly defined. We show that human microRNA (miR)-27a* is a negative regulator of NK-cell cytotoxicity by silencing Prf1 and GzmB expression. Human miR-27a* specifically bound to the 3' untranslated regions of Prf1 and GzmB, down-regulating expression in both resting and activated NK cells, and it functioned as a fine-tuner for homeostasis of the net amount of the effector proteins. Consistent with miR-27a* having an inhibitory role, knockdown of miR-27a* in NK cells dramatically increased cytotoxicity in vitro and decreased tumor growth in a human tumor xenograft model. Thus, NK-cell cytotoxicity is regulated, in part, by microRNA, and modulating endogenous miR-27a* levels in NK cells represents a potential immunotherapeutic strategy.

VDUP1 exacerbates bacteremic shock in mice infected with Pseudomonas aeruginosa.

Vitamin-D3 upregulated protein-1 (VDUP1) is a stress response protein. Pseudomonas aeruginosa (P. aeruginosa) infection is a leading cause of death. Mice infected with live P. aeruginosa exhibit significantly decreased VDUP1 expression. However, the function of VDUP1 during P. aeruginosa-induced mouse bacteremic shock is unknown. To address the function of VDUP1 in P. aeruginosa-infected mice, we constructed a bacteremic shock model wherein both wild-type and VDUP1-deficient mice were infected intra-peritoneally with live P. aeruginosa. We found that VDUP1-deficient mice were more resistant to P. aeruginosa-induced bacteremic shock than wild-type mice, as shown by the increased survival, accelerated bacterial clearance and suppression of cytokine overproduction of the VDUP1-deficient mice. VDUP1 promoted the recruitment of neutrophils into the peritoneal cavities of infected mice. VDUP1 impeded the phagocytosis of non-opsonized P. aeruginosa via phosphatidylinositide 3-kinase (PI3K) pathway in macrophages. P. aeruginosa infection induced the generation of reactive oxygen species (ROS), and the increased production of ROS by the peritoneal cells of VDUP1-deficient mice was advantageous in clearing the bacteria. Overall, VDUP1 aggravates bacteremic shock; thus, VDUP1 can be considered a target molecule for the inhibition of P. aeruginosa-induced bacteremic shock.

Suppressor of cytokine signaling 2 regulates IL-15-primed human NK cell function via control of phosphorylated Pyk2.

NK cells are capable of killing virus-infected or tumor cells and producing IFN-gamma. Resting NK cells, however, have only minimal cytolytic activity and secrete a low level of IFN-gamma. The cytokine IL-15 can promote the expression of effector functions by resting NK cells. In this study, we demonstrate that suppressor of cytokine signaling 2 (SOCS2) has a novel role in IL-15-primed human NK cell function. SOCS2 expression was upregulated in NK cells following stimulation with IL-15. During IL-15-mediated NK cell priming, SOCS2 interacted with phosphorylated proline-rich tyrosine kinase 2 (Pyk2) at tyrosine 402 (p-Pyk2(Tyr402)) and induced the proteasome-mediated degradation of p-Pyk2(Tyr402) via ubiquitination. Knockdown of SOCS2 resulted in the accumulation of p-Pyk2(Tyr402) and blocked NK cell effector functions. In addition, NK cell cytolytic activity and IFN-gamma production were inhibited by overexpression of the wild-type of Pyk2 but not by the overexpression of tyrosine 402 mutant of Pyk2. These results suggest that SOCS2 regulates human NK cell effector functions via control of phosphorylated Pyk2 depending on IL-15 existence.

Pseudomonas aeruginosa eliminates natural killer cells via phagocytosis-induced apoptosis.

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes the relapse of illness in immunocompromised patients, leading to prolonged hospitalization, increased medical expense, and death. In this report, we show that PA invades natural killer (NK) cells and induces phagocytosis-induced cell death (PICD) of lymphocytes. In vivo tumor metastasis was augmented by PA infection, with a significant reduction in NK cell number. Adoptive transfer of NK cells mitigated PA-induced metastasis. Internalization of PA into NK cells was observed by transmission electron microscopy. In addition, PA invaded NK cells via phosphoinositide 3-kinase (PI3K) activation, and the phagocytic event led to caspase 9-dependent apoptosis of NK cells. PA-mediated NK cell apoptosis was dependent on activation of mitogen-activated protein (MAP) kinase and the generation of reactive oxygen species (ROS). These data suggest that the phagocytosis of PA by NK cells is a critical event that affects the relapse of diseases in immunocompromised patients, such as those with cancer, and provides important insights into the interactions between PA and NK cells.

RasGRP1 is required for human NK cell function.

Cross-linking of NK activating receptors activates phospholipase-gamma and subsequently induces diacylglycerol and Ca(2+) as second messengers of signal transduction. Previous studies reported that Ras guanyl nucleotide-releasing protein (RasGRP) 1, which is activated by diacylglycerol and Ca(2+), is crucial for TCR-mediated Ras-ERK activation. We now report that RasGRP1, which can also be detected in human NK cells, plays an essential role in NK cell effector functions. To examine the role of RasGRP1 in NK cell functions, the expression of RasGRP1 was suppressed using RNA interference. Knockdown of RasGRP1 significantly blocked ITAM-dependent cytokine production as well as NK cytotoxicity. Biochemically, RasGRP1-knockdown NK cells showed markedly decreased ability to activate Ras, ERK, and JNK. Activation of the Ras-MAPK pathway was independently shown to be indispensable for NK cell effector functions via the use of specific pharmacological inhibitors. Our results reveal that RasGRP1 is required for the activation of the Ras-MAPK pathway leading to NK cell effector functions. Moreover, our data suggest that RasGRP1 might act as an important bridge between phospholipase-gamma activation and NK cell effector functions via the Ras-MAPK pathway.

Thioredoxin-interacting protein regulates hematopoietic stem cell quiescence and mobilization under stress conditions.

Hematopoietic stem cells (HSCs) are maintained in a quiescent state in bone marrow (BM) niches by intrinsic and extrinsic signals. The mechanisms regulating the quiescence and mobilization of HSCs, however, remain unclear. In this study, we report that the expression of thioredoxin-interacting protein (TXNIP) is decreased during HSC activation. In Txnip(-/-) mice, the long-term reconstituting HSC population is decreased and exhausted, and its capacity to repopulate is rapidly lost. These effects are associated with hyperactive Wnt signaling, an active cell cycle, and reduced p21 expression under conditions of stress. TXNIP deficiency reduced the CXCL12- and osteopontin-mediated interaction between HSCs and the bone marrow, and impaired homing and retention in the osteoblastic niche, resulting in mobilized HSCs. Therefore, we propose that TXNIP is essential for maintaining HSC quiescence and the interaction between HSCs and the BM niche.

The Role of Personality Traits Toward Organizational Commitments and Service Quality Commitments.

Service providers personality traits is one of important determinants to deliver proper service to customers to make them satisfied in service delivery. Despite numerous studies on personality traits and emotional labor, little empirical work has been conducted to investigate the causal effects of hotel middle managers' personality traits on their commitment to the hospitality industry. Thus, this study aims to examine the effects of hotel middle managers' personality on two dimensions of commitments: organizational commitment and service quality commitment meditated by emotional variables: emotional labor and emotional exhaustion. The sample of this study consists of 266 department managers from full-service hotels in a metropolitan city in the Southern United States. The results confirmed the significant role of hotel middle managers' personality traits, especially expressive personality, in organizational commitment and service quality commitment. Hotel operators should foster a work setting that consistently promotes congruent emotions via regular training and screening to reducing employees' emotional exhaustion, increasing organizational commitment and service quality commitment, ultimately, reducing employees' turnover intentions.

Tumor necrosis factor-alpha enhances IL-15-induced natural killer cell differentiation.

The differentiation of natural killer (NK) cells is regulated by various factors including soluble growth factors and transcription factors. Here, we have demonstrated that tumor necrosis factor-alpha (TNF-alpha) is a positive regulator of NK cell differentiation. TNF-alpha augmented the IL-15-induced expression of NK1.1 and CD122 in mature NK cells, and TNF-alpha alone also induced NK cell maturation as well as IL-15. TNF-alpha also increased IFN-gamma production in NK cells in the presence of IL-15. Meanwhile, mRNA expression of several transcription factors, including T-bet and GATA-3, was increased by the addition of TNF-alpha and IL-15. In addition, TNF-alpha increased nuclear factor-kappa B (NF-kappaB) activity in NK cells and inhibition of NF-kappaB impeded TNF-alpha-enhanced NK cell maturation. Overall, these data suggest that TNF-alpha significantly increased IL-15-driven NK cell differentiation by increasing the expression of transcription factors that play crucial roles in NK cell maturation and inducing the NF-kappaB activity.

Research on How Emotional Expressions of Emotional Labor Workers and Perception of Customer Feedbacks Affect Turnover Intentions: Emphasis on Moderating Effects of Emotional Intelligence.

Previous studies have used various external variables and parameters as well as moderator variables such as emotional intelligence have been to understand emotional labor and its related problems. However, a comprehensive model to study such variables' correlations with each other and their overall effect on emotional labor has not yet been established. This study used a structural equation model to understand the relationship between employees' expression of emotional labor and perception of customer feedbacks. The study also looked at how the perception of customer feedback affects emotional exhaustion in order to understand how emotional exhaustion affects job satisfaction and turnover intentions. Further, in order to fully understand the effects of emotion on emotional labor at the service contact points, this study developed and tested a model of emotional labor with four factors of emotional intelligence as moderating factors. Five hundred and seventy nine emotional labor workers in service industries in the United States were collected and 577 valid survey results have been analyzed. The result shows that there exists moderating effects of emotional intelligence on how employees' Deep Acting and Surface Acting recognize customers' reactions, both positive and negative, that would affect employees' Emotional Exhaustion and Job Satisfaction, and hence, Turnover Intention. The result suggests that employees with better understanding of their own emotions, although they are more likely to recover from emotional exhaustion, experience a greater negative effect when there is a discrepancy between what they feel and how they should act.

Cell types of origin for prostate cancer.

Analyses of cell types of origin for prostate cancer should result in new insights into mechanisms of tumor initiation, and may lead to improved prognosis and selection of appropriate therapies. Here, we review studies using a range of methodologies to investigate the cell of origin for mouse and human prostate cancer. Notably, analyses using tissue recombination assays support basal epithelial cells as a cell of origin, whereas in vivo lineage-tracing studies in genetically-engineered mice implicate luminal cells. We describe how these results can be potentially reconciled by a conceptual distinction between cells of origin and cells of mutation, and outline how new experimental approaches can address the potential relationship between cell types of origin and disease outcome.

Conditional deletion of the Pten gene in the mouse prostate induces prostatic intraepithelial neoplasms at early ages but a slow progression to prostate tumors.

The PTEN tumor suppressor gene is frequently inactivated in human prostate cancer. Using Osr1 (odd skipped related 1)-Cre mice, we generated a novel conditional Pten knockout mouse strain, Pten(LoxP):Osr1-Cre. Conditional biallelic and monoallelic Pten knockout mice were viable. Deletion of Pten expression was detected in the prostate of Pten(LoxP/LoxP):Osr1-Cre mice as early as 2 weeks of age. Intriguingly, Pten(LoxP/LoxP):Osr1-Cre mice develop high-grade prostatic intraepithelial neoplasms (PINs) with high penetrance as early as one-month of age, and locally invasive prostatic tumors after 12-months of age. Pten(LoxP/+):Osr1-Cre mice show only mild oncogenic changes after 8-weeks of age. Castration of Pten(LoxP/LoxP):Osr1-Cre mice shows no significant regression of prostate tumors, although a shift of androgen receptor (AR) staining from the nuclei to cytoplasm is observed in Pten null tumor cells of castrated mice. Enhanced Akt activity is observed in Pten null tumor cells of castrated Pten(LoxP/LoxP):Osr1-Cre. This study provides a novel mouse model that can be used to investigate a primary role of Pten in initiating oncogenic transformation in the prostate and to examine other genetic and epigenetic changes that are required for tumor progression in the mouse prostate.

Oxygen tension regulates NK cells differentiation from hematopoietic stem cells in vitro.

Natural killer (NK) cells are differentiated from hematopoietic stem cells (HSCs) which are located at the lowest end of an oxygen gradient within the bone marrow (BM). In this report, we investigated whether oxygen tension could affect NK cell differentiation from hematopoietic cells in vitro. We found that hypoxia led to an inhibition of differentiation in NK cells, and increased oxygen supply alleviated this inhibition and restored NK cell differentiation under hypoxic condition. Hypoxia-treated cells demonstrated reduced mRNA expression of transcription factors (TFs) that have important roles in NK cell differentiation, such as EOMES, T-bet, GATA-3 and ETS-1. Moreover, hypoxia-pretreated cells recovered mRNA expression of TFs when the oxygen tension was changed to normoxia. Our findings suggest that oxygen tension modulates in vitro differentiation of NK cells through the regulation of TF expression.

Association between plasma hepatocyte growth factor and gefitinib resistance in patients with advanced non-small cell lung cancer.

PURPOSE: It has been suggested that hepatocyte growth factor (HGF) and insulin-like growth factor binding protein (IGFBP)-3 are associated with gefitinib resistance in non-small cell lung cancer (NSCLC). We investigated the predictive and prognostic roles of these proteins in NSCLC patients treated with gefitinib. PATIENTS AND METHODS: Of 106 patients enrolled in a randomized phase II study of gefitinib, 97 had plasma samples available for ELISA testing. Of these samples, seven and eight, respectively, had HGF and IGFBP-3 values that could not be measured. Therefore, the correlations between clinical outcomes and plasma levels of HGF and IGFBP-3 were evaluated in 90 and 89 patients, respectively. RESULTS: Plasma HGF levels were significantly higher in older patients, male patients, patients with squamous cell carcinoma, current smokers, and patients with epidermal growth factor receptor (EGFR) wild-type tumors. Low HGF levels were significantly associated with higher response rate, and longer progression-free survival (PFS) and overall survival (OS) irrespective of EGFR mutation status. In a multivariate analysis, the presence of EGFR mutations (P=0.002) and low HGF levels (P=0.031) were independently predictive of longer PFS, and an ECOG PS of 0 (P=0.001) and low HGF levels (P=0.002) were independently predictive of longer OS. No statistically significant differences were found for IGFBP-3. CONCLUSION: High HGF levels are significantly associated with resistance to gefitinib and can be used as a predictive marker for the differential outcome of gefitinib treatment in NSCLC irrespective of EGFR mutation status.

IL-15-induced IL-10 increases the cytolytic activity of human natural killer cells.

Interleukin 10 (IL-10) is a multifunctional cytokine that regulates diverse functions of immune cells. Natural killer (NK) cells express the IL-10 and IL-10 receptor, but little is known about the function of IL-10 on NK cell activation. In this study, we show the expression and role of IL-10 in human NK cells. Among the cytokines tested, IL-15 was the most potent inducer of IL-10, with a maximal peak expression at 5 h after treatment. Furthermore, IL-10 receptor was shown to be expressed in NK cells. IL-10 alone had a significant effect on NK cytotoxicity which additively increased NK cell cytotoxicity in the presence of IL-15. Neutralizing IL-10 with anti-IL-10 antibody suppressed the inductive effect of IL-10 on NK cell cytotoxicity; however, IL-10 had no effect on IFN-γ or TNF-α production or NK cell activatory receptor expression. STAT signals are implicated as a key mediator of IL-10/IL-15 cytotoxicity response. Thus, the effect of IL-10 on NK cells is particularly interesting with regard to the STAT3 signal that was enhanced by IL-10 or IL-15.

TOX regulates the differentiation of human natural killer cells from hematopoietic stem cells in vitro.

Natural killer (NK) cells act important roles in innate immunity and adaptive immunity. However, the mechanisms governing NK cell development have not been clearly elucidated. Previous studies have shown that an HMG (high-mobility group) protein, TOX, is important for regulating the differentiation program of developing T cells in mice. In this study, we examined the role of TOX in differentiation of human NK cells. Knockdown of TOX in differentiating cells decreased the NK cell population identified by expression of NK surface markers and receptors. In addition, over-expression of TOX enhanced the differentiation of NK cells which give rise to a population showing effector functions of mature NK cells. Moreover, TOX influenced expression of T-bet (T-box expressed in T cells, also as known as Tbx21) during NK cell development. Overall, these results suggest that TOX is required for IL-15-mediated NK cell differentiation and affected expression of T-bet that plays critical roles in NK differentiation and maturation.