Membrane metalloendopeptidase suppresses prostate carcinogenesis by attenuating effects of gastrin-releasing peptide on stem/progenitor cells.

Aberrant neuroendocrine signaling is frequent yet poorly understood feature of prostate cancers. Membrane metalloendopeptidase (MME) is responsible for the catalytic inactivation of neuropeptide substrates, and is downregulated in nearly 50% of prostate cancers. However its role in prostate carcinogenesis, including formation of castration-resistant prostate carcinomas, remains uncertain. Here we report that MME cooperates with PTEN in suppression of carcinogenesis by controlling activities of prostate stem/progenitor cells. Lack of MME and PTEN results in development of adenocarcinomas characterized by propensity for vascular invasion and formation of proliferative neuroendocrine clusters after castration. Effects of MME on prostate stem/progenitor cells depend on its catalytic activity and can be recapitulated by addition of the MME substrate, gastrin-releasing peptide (GRP). Knockdown or inhibition of GRP receptor (GRPR) abrogate effects of MME deficiency and delay growth of human prostate cancer xenografts by reducing the number of cancer-propagating cells. In sum, our study provides a definitive proof of tumor-suppressive role of MME, links GRP/GRPR signaling to the control of prostate stem/progenitor cells, and shows how dysregulation of such signaling may promote formation of castration-resistant prostate carcinomas. It also identifies GRPR as a valuable target for therapies aimed at eradication of cancer-propagating cells in prostate cancers with MME downregulation.

WNT antagonists exhibit unique combinatorial antitumor activity with taxanes by potentiating mitotic cell death.

The WNT pathway mediates intercellular signaling that regulates cell fate in both normal development and cancer. It is widely appreciated that the WNT pathway is frequently dysregulated in human cancers through a variety of genetic and epigenetic mechanisms. Targets in the WNT pathway are being extensively pursued for the development of new anticancer therapies, and we have advanced two WNT antagonists for clinical development: vantictumab (anti-FZD) and ipafricept (FZD8-Fc). We examined the antitumor efficacy of these WNT antagonists in combination with various chemotherapies in a large set of patient-derived xenograft models. In responsive models, WNT blockade led to profound synergy with taxanes such as paclitaxel, and the combination activity with taxanes was consistently more effective than with other classes of chemotherapy. Taxane monotherapy increased the frequency of cells with active WNT signaling. This selection of WNT-active chemotherapy-resistant tumorigenic cells was prevented by WNT-antagonizing biologics and required sequential dosing of the WNT antagonist followed by the taxane. The WNT antagonists potentiated paclitaxel-mediated mitotic blockade and promoted widespread mitotic cell death. By blocking WNT/ß-catenin signaling before mitotic blockade by paclitaxel, we found that this treatment effectively sensitizes cancer stem cells to taxanes. This combination strategy and treatment regimen has been incorporated into ongoing clinical testing for vantictumab and ipafricept.

One-Year Mortality after Traumatic Brain Injury in Liver Cirrhosis Patients--A Ten-Year Population-Based Study.

This study investigated the 1-year mortality of patients who underwent brain surgery following traumatic brain injury (TBI) who also had alcoholic and/or nonalcoholic liver cirrhosis (LC) using a nationwide database in Taiwan. A longitudinal cohort study matched by propensity score with age, gender, length of ICU stay, HTN, DM, MI, stroke, HF, renal diseases, and year of TBI diagnosis in TBI patients with alcoholic and/or nonalcoholic LC and TBI patients without LC was conducted using the National Health Insurance Research Database in Taiwan between January 1997 and December 2007. The main outcome studied was 1-year mortality. In total, 7296 subjects (2432 TBI patients with LC and 4864 TBI patients without LC) were enrolled in this study. The main findings were (1) TBI patients with LC had a higher 1-year mortality (52.18% vs 30.61%) and a 1.75-fold increased risk of mortality (95% CI 1.61-1.90) compared with non-LC TBI patients, (2) renal diseases and HF are risk factors, but hypertension could be a protective factor in cirrhotic TBI patients, and (3) TBI patients with non-alcoholic LC and the coexistence of alcoholic and nonalcoholic LC had higher 1-year mortality compared with TBI patients with alcoholic cirrhosis. This study showed that patients with LC who have undergone brain surgery might have higher risk of 1-year mortality than those without LC. In addition, nonalcoholic and the coexistence of alcoholic and nonalcoholic LC show higher 1-year mortality risk than alcoholic in TBI patients with LC, especially in those with comorbidities of hypertension, diabetes mellitus, and stroke.

Selective cerebral hypothermia induced via hypothermic retrograde jugular vein saline flush in a porcine model.

OBJECTIVES: Multiple methods of selective brain cooling have been used to prevent cerebral ischemia secondary to trauma and pathological or iatrogenic cerebral blood flow restriction. In this study, we tested the efficacy of hypothermic retrograde jugular vein flush (HRJVF) in eliciting selective brain hypothermia in a porcine model. METHODS: Twelve swine were divided into two groups: retrograde jugular vein infusion (RJVI) with cold saline (4°C RJVI, n  =  6) and with room temperature saline (24°C RJVI, n  =  6). For 90 minutes, the following parameters were measured: brain parenchymal temperature, rectal temperature, intracranial pressure (ICP), mean arterial pressure, and heart rate (HR). RESULTS: Swine receiving 4°C RJVI experienced a drop in mean brain parenchymal temperature of 1·1 ± 0·1°C, compared to 0·1 ± 0·1°C in swine receiving 24°C RJVI. At 90 minutes, mean brain parenchymal temperature in the 4°C RJVI treatment group was 35·5 ± 0·2°C, as compared to 37·1 ± 0·2°C in the 24°C RJVI treatment group (P < 0·001). In the 4°C RJVI group, the brain-systemic temperature gradient peaked 10 minutes after initiation of cooling and remained significantly different when comparing the two experimental groups (P < 0·001) throughout the duration of the 90 minutes experiment. Of note, ICP, mean arterial pressure, and HR remained constant without any significant changes or differences between treatment groups. DISCUSSION: These results suggest that HRJVF is an effective method for selective brain hypothermia in a large animal model. Clinical application may prove effective in delaying neural ischemia.

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 18 months 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 15 months of age. Consistently, combined deficiency of p53 and miR-34 leads to acceleration 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.

Loss of survivin in the prostate epithelium impedes carcinogenesis in a mouse model of prostate adenocarcinoma.

The inhibitor of apoptosis protein survivin is expressed in most cancers. Using the conditional PTEN deletion mouse model, we previously reported that survivin levels increase with prostate tumor growth. Here we evaluated the functional role of survivin in prostate tumor growth. First, we demonstrated that mice lacking the survivin gene in prostate epithelium were fertile and had normal prostate growth and development. We then serially, from about 10-56 weeks of age, evaluated histopathologic changes in the prostate of mice with PTEN deletion combined with survivin mono- or bi-allelic gene deletion. While within this time period most of the animals with wild-type or monoallelic survivin deletion developed adenocarcinomas, the most severe lesions in the biallelic survivin deleted mice were high-grade prostatic intra-epithelial neoplasia with distinct histopathology. Many atypical cells contained large hypertrophic cytoplasm and desmoplastic reaction in the prostatic intra-epithelial neoplasia lesions of this group was minimal until the late ages. A reduced proliferation index as well as apoptotic and senescent cells were detected in the lesions of mice with compound PTEN/survivin deficiency throughout the time points examined. Survivin deletion was also associated with reduced tumor expression of another inhibitor of apoptosis member, the X-linked inhibitor of apoptosis. Our findings suggest that survivin participates in the progression of prostatic intraepithelial neoplasia to adenocarcinoma, and that survivin interference at the prostatic intraepithelial neoplasia stages may be a potential therapeutic strategy to halt or delay further progression.

Detection and organ-specific ablation of neuroendocrine cells by synaptophysin locus-based BAC cassette in transgenic mice.

The role of cells of the diffuse neuroendocrine system in development and maintenance of individual organs and tissues remains poorly understood. Here we identify a regulatory region sufficient for accurate in vivo expression of synaptophysin (SYP), a common marker of neuroendocrine differentiation, and report generation of Tg(Syp-EGFP(loxP)-DTA)147(Ayn) (SypELDTA) mice suitable for flexible organ-specific ablation of neuroendocrine cells. These mice express EGFP and diphtheria toxin fragment A (DTA) in SYP positive cells before and after Cre-loxP mediated recombination, respectively. As a proof of principle, we have crossed SypELDTA mice with EIIA-Cre and PB-Cre4 mice. EIIA-Cre mice express Cre recombinase in a broad range of tissues, while PB-Cre4 mice specifically express Cre recombinase in the prostate epithelium. Double transgenic EIIA-Cre; SypELDTA embryos exhibited massive cell death in SYP positive cells. At the same time, PB-Cre4; SypELDTA mice showed a substantial decrease in the number of neuroendocrine cells and associated prostate hypotrophy. As no increase in cell death and/or Cre-loxP mediated recombination was observed in non-neuroendocrine epithelium cells, these results suggest that neuroendocrine cells play an important role in prostate development. High cell type specificity of Syp locus-based cassette and versatility of generated mouse model should assure applicability of these resources to studies of neuroendocrine cell functions in various tissues and organs.

Ovarian surface epithelium at the junction area contains a cancer-prone stem cell niche.

Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer deaths among women in the United States, but its pathogenesis is poorly understood. Some epithelial cancers are known to occur in transitional zones between two types of epithelium, whereas others have been shown to originate in epithelial tissue stem cells. The stem cell niche of the ovarian surface epithelium (OSE), which is ruptured and regenerates during ovulation, has not yet been defined unequivocally. Here we identify the hilum region of the mouse ovary, the transitional (or junction) area between the OSE, mesothelium and tubal (oviductal) epithelium, as a previously unrecognized stem cell niche of the OSE. We find that cells of the hilum OSE are cycling slowly and express stem and/or progenitor cell markers ALDH1, LGR5, LEF1, CD133 and CK6B. These cells display long-term stem cell properties ex vivo and in vivo, as shown by our serial sphere generation and long-term lineage-tracing assays. Importantly, the hilum cells show increased transformation potential after inactivation of tumour suppressor genes Trp53 and Rb1, whose pathways are altered frequently in the most aggressive and common type of human EOC, high-grade serous adenocarcinoma. Our study supports experimentally the idea that susceptibility of transitional zones to malignant transformation may be explained by the presence of stem cell niches in those areas. Identification of a stem cell niche for the OSE may have important implications for understanding EOC pathogenesis.

Molecular characterization of a phenylalanine ammonia-lyase gene (BoPAL1) from Bambusa oldhamii.

Phenylalanine ammonia-lyase is the first enzyme of general phenylpropanoid pathway. A PAL gene, designated as BoPAL1, was cloned from a Bambusa oldhamii cDNA library. The open reading frame of BoPAL1 was 2,139 bp in size and predicted to encode a 712-amino acid polypeptide. BoPAL1 was the first intronless PAL gene found in angiosperm plant. Several putative cis-acting elements such as P box, GT-1motif, and SOLIPs involved in light responsiveness were found in the 5'-flanking sequence of BoPAL1 which was obtained by TAIL-PCR method. Recombinant BoPAL1 protein expressed in Pichia pastoris was active. The optimum temperature and pH for BoPAL1 activity was 50°C and 9.0, respectively. The molecular mass of recombinant BoPAL1 was estimated as 323 kDa using gel filtration chromatography and the molecular mass of full-length BoPAL was about 80 kDa, indicating that BoPAL1 presents as a homotetramer. The Km and kcat values of BoPAL1 for L-Phe were 1.01 mM and 10.11 s(-1), respectively. The recombinant protein had similar biochemical properties with PALs reported in other plants.

Cloning and expression of a phenylalanine ammonia-lyase gene (BoPAL2) from Bambusa oldhamii in Escherichia coli and Pichia pastoris.

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is the first committed enzyme of phenylpropanoid pathway. A PAL gene, designated as BoPAL2, was cloned from a Bambusa oldhamii cDNA library. The open reading frame of BoPAL2 was 2142bp in size encoding a 713-amino acid polypeptide. BoPAL2 was heterologous expressed in Escherichia coli and Pichia pastoris. The recombinant proteins were exhibited PAL and tyrosine ammonia-lyase activities. The recombinant BoPAL2 had a subunit mass of 80kDa and existed as a homotetramer. The optimum temperature and pH of BoPAL2 were 50-60 degrees C and 8.5-9.0, respectively. The K(m) and k(cat) values of BoPAL2 expressed in E. coli were 250microM and 10.12s(-1). The K(m) and k(cat) values of BoPAL2 expressed in P. pastoris were 331microM and 16.04s(-1). The recombinant proteins had similar biochemical properties and kinetic parameters with PALs reported in other plants.

Extracellular matrix proteases - cytokine regulation role in cancer and pregnancy.

The extracellular matrix proteases act in diverse physiological and pathological processes involving tumor growth, angiogenesis, and pregnancy through the cleavage of extracellular matrix (ECM) and non-matrix proteinaceous substrates. Matrix metalloproteinases (MMPs) constitute a main family among the ECM proteases. Endogenous tissue inhibitors of metalloproteinases (TIMPs), as one kind of MMPs inhibitors (MMPIs), reduce the excessive proteolytic ECM degradation by MMPs. The balance between MMPs and TIMPs plays a major role in cancer tumorigenesis, angiogenesis, as well as embryo implantation and trophoblastic invasion during pregnancy. A variety of literature concerns the correlated changes in MMPs and MMPIs during the formation of cancer and pregnancy-related complications. Importantly, MMPs and TIMPs may act as regulators of signaling pathways through the cleavage of non-matrix substrates, including cytokines, chemokines, and growth factors. In this review, we concentrate on mutual interactions between ECM proteases and cytokines during cancer development and pregnancy. The current knowledge in the field of identified ECM proteases will be contributive to the innovative therapeutic intervention in both cancer and pregnancy-related processes.

Cytokine regulation networks in the cancer microenvironment.

During carcinoma formation, cancer cells release various cytokines and growth factors into their surroundings and recruit and reprogram many other types of cells in order to establish a tumor microenvironment. Consequently, the tumor tissues almost always contain a large number of endothelial cells, fibroblasts, and infiltrating inflammatory cells that in turn produce a variety of cytokines. The cytokines produced by these cells have been posited as key factors in modulating immune response either against or in favor of tumorigenesis in the microenvironment. The interactions that take place between immune and cancer cells are complex, involving multiple cascades of cytokines, chemokines, and/or growth factors. In this review, we address the essential pro- and anti-tumorigenic roles of cytokines in the tumor microenvironment. As the interaction of cytokines, growth factors, and cancer cells forms a comprehensive network at the tumor site that is then responsible for the overall progression or rejection of the tumor, the current review links the microenvironment-derived cytokines and growth factors to a number of different kinds of human carcinogenesis models. Multifunctional cytokines, extracellular matrix mediators, and regulatory cytokines in the cancer environment are all shown to be key factors in the different cancer immune-editing systems. The characterization of cytokine networks in various types of cancer cells may yield important information for understanding the immune-related mechanisms of cancer development, and this knowledge may have subsequent application in cancer immunotherapy.