Long-acting Erwinia chrysanthemi, Pegcrisantaspase, induces alternate amino acid biosynthetic pathways in a preclinical model of pancreatic ductal adenocarcinoma.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease without meaningful therapeutic options beyond the first salvage therapy. Targeting PDAC metabolism through amino acid restriction has emerged as a promising new strategy, with asparaginases, enzymes that deplete plasma glutamine and asparagine, reaching clinical trials. In this study, we investigated the anti-PDAC activity of the asparaginase formulation Pegcrisantaspase (PegC) alone and in combination with standard-of-care chemotherapeutics. METHODS: Using mouse and human PDAC cell lines, we assessed the impact of PegC on cell proliferation, cell death, and cell cycle progression. We further characterized the in vitro effect of PegC on protein synthesis as well as the generation of reactive oxygen species and levels of glutathione, a major cellular antioxidant. Additional cell line studies examined the effect of the combination of PegC with standard-of-care chemotherapeutics. In vivo, the tolerability and efficacy of PegC, as well as the impact on plasma amino acid levels, was assessed using the C57BL/6-derived KPC syngeneic mouse model. RESULTS: Here we report that PegC demonstrated potent anti-proliferative activity in a panel of human and murine PDAC cell lines. This decrease in proliferation was accompanied by inhibited protein synthesis and decreased levels of glutathione. In vivo, PegC was tolerable and effectively reduced plasma levels of glutamine and asparagine, leading to a statistically significant inhibition of tumor growth in a syngeneic mouse model of PDAC. There was no observable in vitro or in vivo benefit to combining PegC with standard-of-care chemotherapeutics, including oxaliplatin, irinotecan, 5-fluorouracil, paclitaxel, and gemcitabine. Notably, PegC treatment increased tumor expression of asparagine and serine biosynthetic enzymes. CONCLUSIONS: Taken together, our results demonstrate the potential therapeutic use of PegC in PDAC and highlight the importance of identifying candidates for combination regimens that could improve cytotoxicity and/or reduce the induction of resistance pathways.

Community-engaged basic science in an NCI-designated comprehensive cancer center: antioxidants and chemotherapeutic efficacy.

PURPOSE: While community engagement has been a longstanding aspect of cancer-relevant research in social and behavioral sciences, it is far less common in basic/translational/clinical research. With the National Cancer Institute's incorporation of Community Outreach and Engagement into the Cancer Center Support Grant guidelines, successful models are desirable. We report on a pilot study supported by the University of Maryland Greenebaum Comprehensive Cancer Center (UMGCCC), that used a community-engaged, data-driven process to inform a pre-clinical study of the impact of antioxidants on the efficacy of platinum-based chemotherapeutics. METHODS: We conducted a survey of UMGCCC catchment area residents (n = 120) to identify commonly used antioxidants. We then evaluated the effect of individually combining commonly used antioxidants from the survey (vitamin C, green tea, and melatonin) with platinum agents in models of non-small cell lung cancer (A549), colon adenocarcinoma (SW620) and head and neck squamous cell carcinoma (FaDu). RESULTS: In vitro, the anti-neoplastic activity of each chemotherapy was not potentiated by any of the antioxidants. Instead, when combined at fixed ratios, most antioxidant-chemotherapy combinations were antagonistic. In vivo, addition of antioxidants did not improve chemotherapeutic efficacy and in a FaDu-tumor bearing model, cisplatin-mediated tumor growth inhibition was significantly impeded by the addition of epigallocatechin gallate, the main antioxidant in green tea. CONCLUSION: These initial findings do not support addition of antioxidant supplementation to improve platinum-based chemotherapeutic efficacy. This study's approach can serve as a model of how to bring together the two seemingly discordant areas of basic research and community engagement.

Genetic disruption of Ano5 leads to impaired osteoclastogenesis for gnathodiaphyseal dysplasia.

OBJECTIVES: Gnathodiaphyseal dysplasia (GDD; OMIM#166260) is a rare skeletal genetic disorder characterized by sclerosis of tubular bones and cemento-osseous lesions in mandibles. TMEM16E/ANO5 gene mutations have been identified in patients with GDD. Here, Ano5 knockout (Ano5-/- ) mice with enhanced osteoblastogenesis were used to investigate whether Ano5 disruption affects osteoclastogenesis. SUBJECTS AND METHODS: The maturation of osteoclasts, formation of F-actin ring and bone resorption were detected by immunohistochemistry, TRAP, phalloidin staining and Coming Osteo assays. The expression of osteoclast-related factors was measured by qRT-PCR. Early signaling pathways were verified by western blot. RESULTS: Ano5-/- mice exhibited inhibitory formation of multinucleated osteoclasts with a reduction of TRAP activity. The expression of Nfatc1, c-Fos, Trap, Ctsk, Mmp9, Rank and Dc-stamp was significantly decreased in bone tissues and bone marrow-derived macrophages (BMMs) of Ano5-/- mice. Ano5-/- osteoclasts manifested disrupted actin ring and less mineral resorption. RANKL-induced early signaling pathways were suppressed in Ano5-/- osteoclasts and Ano5 knockdown RAW264.7 cells. Moreover, the inhibitory effects of NF-κB signalling pathway on osteoclastogenesis were partially attenuated with NF-κB signalling activator. CONCLUSIONS: Ano5 deficiency impairs osteoclastogenesis, which leads to enhanced osteogenic phenotypes mediated by bone homeostasis dysregulation.

Erwinia asparaginase (crisantaspase) increases plasma levels of serine and glycine.

The impact of asparaginases on plasma asparagine and glutamine is well established. However, the effect of asparaginases, particularly those derived from Erwinia chrysanthemi (also called crisantaspase), on circulating levels of other amino acids is unknown. We examined comprehensive plasma amino acid panel measurements in healthy immunodeficient/immunocompetent mice as well as in preclinical mouse models of acute myeloid leukemia (AML) and pancreatic ductal adenocarcinoma (PDAC) using long-acting crisantaspase, and in an AML clinical study (NCT02283190) using short-acting crisantaspase. In addition to the expected decrease of plasma glutamine and asparagine, we observed a significant increase in plasma serine and glycine post-crisantaspase. In PDAC tumors, crisantaspase treatment significantly increased expression of serine biosynthesis enzymes. We then systematically reviewed clinical studies using asparaginase products to determine the extent of plasma amino acid reporting and found that only plasma levels of glutamine/glutamate and asparagine/aspartate were reported, without measuring other amino acid changes post-asparaginase. To the best of our knowledge, we are the first to report comprehensive plasma amino acid changes in mice and humans treated with asparaginase. As dysregulated serine metabolism has been implicated in tumor development, our findings offer insights into how leukemia/cancer cells may potentially overcome glutamine/asparagine restriction, which can be used to design future synergistic therapeutic approaches.

Optical Fiber Vibration Signal Identification Method Based on Improved YOLOv4.

In the traditional peripheral-security-early-warning system, the endpoint detection and pattern recognition of the signals generated by the distributed optical fiber vibration sensors is completed step-by-step and in an orderly manner. The method by which these two processes may be placed end-to-end in a network model and processed simultaneously to improve work efficiency has increasingly become the focus of research. In this paper, the target detection algorithm combines the endpoint-detection and pattern-recognition processes of the vibration signal, which can not only quickly locate the start and end vibration positions of the signal but also accurately identify a certain type of signal. You Only Look Once v4 (YOLOv4) is one of the most advanced target detection algorithms, achieving the optimal balance of speed and accuracy. To reduce the complexity of the YOLOv4 model and solve the dataset's unbalanced sample classification problem, we use a deep separable convolution (DSC) network and a focal loss function to improve the YOLOv4 model. In this paper, the five kinds of signals collected in real-time are visualized as two different datasets in oscillograph and time-frequency diagrams as detection objects. According to the experimental results, we obtained 98.50% and 93.48% mean Average Precision (mAP) and 84.8 and 69.9 frames per second (FPS), respectively, which are improved compared to YOLOv4. Comparing the improved algorithm with other optical fiber vibration signal recognition algorithms, the mAP and FPS values were improved, and the detection speed was about 20 times faster than that of other algorithms. The improved algorithm in this paper can quickly and accurately identify the vibration signal of external intrusion, reduce the false-alarm rate of the early-warning system, and improve the real-time detection rate of the system while ensuring high recognition accuracy.

LINC00472 suppresses oral squamous cell carcinoma growth by targeting miR-455-3p/ELF3 axis.

LINC00472 is reported to play a role in suppressing tumors in cancers such as lung cancer and hepatocellular carcinoma, among others. We made investigations into the effects of LINC00472 in oral squamous cell carcinoma (OSCC) progression to explore the underlying molecular mechanisms. By qRT-PCR, we assessed the LINC00472 expression in OSCC tissues and cells and performed functional analysis to investigate how LINC00472/miR-455-3p/ELF3 impacts OSCC cell proliferation, apoptosis, and cell cycle. The role that LINC00472 plays in OSCC tumor growth was examined by establishing a xenograft model. Down-regulation of LINC00472 occurred in tissues and cells of an OSCC tumor. LINC00472 overexpression caused OSCC cell proliferation to be inhibited, cell apoptosis to be promoted, and cell cycle arrest to be induced. As a competing endogenous RNA (ceRNA), LINC00472 can block miR-455-3p function and further promote ELF3 expression. The overexpression of miR-455-3p or ELF3 knockdown was shown to be capable of reversing the anti-tumor effects of LINC00472 in OSCC. In vivo experiments confirmed the tumor-suppressing role of LINC00472 in the progression of OSCC. In short, we found that the novel LINC00472 inhibits OSCC growth via the miR-455-3p/ELF3 axis. LINC00472 and its targeted miR-455-3p/ELF3 axis may represent valuable targets for treating OSCC.

Characterization of Various Subunit Combinations of ADP-Glucose Pyrophosphorylase in Duckweed (Landoltia punctata).

Background: Landoltia punctata can be used as renewable and sustainable biofuel feedstock because it can quickly accumulate high starch levels. ADP-glucose pyrophosphorylase (AGPase) catalyzes the first committed step during starch biosynthesis in higher plants. The heterotetrameric structure of plant AGPases comprises pairs of large subunits (LSs) and small subunits (SSs). Although several studies have reported on the high starch accumulation capacity of duckweed, no study has explored the underlying molecular accumulation mechanisms and their linkage with AGPase. Therefore, this study focused on characterizing the roles of different L. punctate AGPases. Methodology. Expression patterns of LpAGPs were determined through comparative transcriptome analyses, followed by coexpressing their coding sequences in Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, and Nicotiana tabacum. Results: Comparative transcriptome analyses showed that there are five AGPase subunits encoding cDNAs in L. punctata (LpAGPS1, LpAGPS2, LpAGPL1, LpAGPL2, and LpAGPL3). Nutrient starvation (distilled water treatment) significantly upregulated the expression of LpAGPS1, LpAGPL2, and LpAGPL3. Coexpression of LpAGPSs and LpAGPLs in Escherichia coli generated six heterotetramers, but only four (LpAGPS1/LpAGPL3, LpAGPS2/LpAGPL1, LpAGPS2/LpAGPL2, and LpAGPS2/LpAGPL3) exhibited AGPase activities and displayed a brownish coloration upon exposure to iodine staining. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays validated the interactions between LpAGPS1/LpAGPL2, LpAGPS1/LpAGPL3, LpAGPS2/LpAGPL1, LpAGPS2/LpAGPL2, and LpAGPS2/LpAGPL3. All the five LpAGPs were fusion-expressed with hGFP in Arabidopsis protoplasts, and their green fluorescence signals were uniformly localized in the chloroplast, indicating that they are plastid proteins. Conclusions: This study uncovered the cDNA sequences, structures, subunit interactions, expression patterns, and subcellular localization of AGPase. Collectively, these findings provide new insights into the molecular mechanism of fast starch accumulation in L. punctata.

Introduction of a Cys360Tyr Mutation in ANO5 Creates a Mouse Model for Gnathodiaphyseal Dysplasia.

Gnathodiaphyseal dysplasia (GDD) is a rare autosomal dominant genetic disease characterized by the osteosclerosis of tubular bones and the formation of cemento-osseous lesions in mandibles. Although genetic mutations for GDD have been identified in the ANO5/TMEM16E gene, the cellular and molecular mechanisms behind the pathogenesis of GDD remain unclear. Here, we generated the first knock-in mouse model for GDD with the expression of human mutation p.Cys360Tyr in ANO5. Homozygous Ano5 knock-in mice (Ano5KI/KI ) replicated GDD-like skeletal features, including massive jawbones, bowing tibia, bone fragility, sclerosis, and cortical thickening of the femoral and tibial diaphysis. Serum alkaline phosphatase (ALP) levels were elevated in Ano5KI/KI mice as in GDD patients with p.Cys360Tyr mutation. Calvaria-derived Ano5KI/KI osteoblast cultures showed increased osteoblastogenesis, including hypermineralized bone matrix and enhanced bone formation-related factors expression. Interestingly, Ano5KI/KI bone marrow-derived macrophage cultures showed decreased osteoclastogenesis, and Ano5KI/KI osteoclasts exhibited disrupted actin ring formation, which may be associated with some signaling pathways. In conclusion, this new mouse model may facilitate elucidation of the pathogenesis of GDD and shed more light on its treatment. © 2021 American Society for Bone and Mineral Research (ASBMR).

DUOX2, a New Biomarker for Disseminated Gastric Cancer's Response to Low Dose Radiation in Mice.

Treatment options are rather limited for gastrointestinal cancer patients whose disease has disseminated into the intra-abdominal cavity. Here, we designed pre-clinical studies to evaluate the potential application of chemopotentiation by Low Dose Fractionated Radiation Therapy (LDFRT) for disseminated gastric cancer and evaluate the role of a likely biomarker, Dual Oxidase 2 (DUOX2). Nude mice were injected orthotopically with human gastric cancer cells expressing endogenous or reduced levels of DUOX2 and randomly assigned to four treatment groups: 1; vehicle alone, 2; modified regimen of docetaxel, cisplatin and 5'-fluorouracil (mDCF) for three consecutive days, 3; Low Dose- Whole Abdomen Radiation Therapy (LD-WART) (5 fractions of 0.15 Gy in three days), 4; mDCF and LD-WART. The combined regimen increased the odds of preventing cancer dissemination (mDCF + LD-WART OR = 4.16; 80% CI = 1.0, 17.29) in the DUOX2 positive tumors, while tumors expressing lower DUOX2 levels were more responsive to mDCF alone with no added benefit from LD-WART. The molecular mechanisms underlying DUOX2 effects in response to the combined regimen include NF-κB upregulation. These data are particularly important since our study indicates that about 33% of human stomach adenocarcinoma do not express DUOX2. DUOX2 thus seems a likely biomarker for potential clinical application of chemopotentiation by LD-WART.

An Extract of Taro (Colocasia esculenta) Mediates Potent Inhibitory Actions on Metastatic and Cancer Stem Cells by Tumor Cell-Autonomous and Immune-Dependent Mechanisms.

The taro plant, Colocasia esculenta, contains bioactive proteins with potential as cancer therapeutics. Several groups have reported anti-cancer activity in vitro and in vivo of taro-derived extracts (TEs). We reported that TE inhibits metastasis in a syngeneic murine model of Triple-Negative Breast Cancer (TNBC). PURPOSE: We sought to confirm our earlier studies in additional models and to identify novel mechanisms by which efficacy is achieved. METHODS: We employed a panel of murine and human breast and ovarian cancer cell lines to determine the effect of TE on tumor cell viability, migration, and the ability to support cancer stem cells. Two syngeneic models of TNBC were employed to confirm our earlier report that TE potently inhibits metastasis. Cancer stem cell assays were employed to determine the ability of TE to inhibit tumorsphere-forming ability and to inhibit aldehyde dehydrogenase activity. To determine if host immunity contributes to the mechanism of metastasis inhibition, efficacy was assessed in immune-compromised mice. RESULTS: We demonstrate that viability of some, but not all cell lines is inhibited by TE. Likewise, tumor cell migration is inhibited by TE. Using 2 immune competent, syngeneic models of TNBC, we confirm our earlier findings that tumor metastasis is potently inhibited by TE. We also demonstrate, for the first time, that TE directly inhibits breast cancer stem cells. Administration of TE to mice elicits expansion of several spleen cell populations but it was not known if host immune cells contribute to the mechanism by which TE inhibits tumor cell dissemination. In novel findings, we now show that the ability of TE to inhibit metastasis relies on immune T-cell-dependent, but not B cell or Natural Killer (NK)-cell-dependent mechanisms. Thus, both tumor cell-autonomous and host immune factors contribute to the mechanisms underlying TE efficacy. Our long-term goal is to evaluate TE efficacy in clinical trials. Most of our past studies as well as many of the results reported in this report were carried out using an isolation protocol described earlier (TE). In preparation for a near future clinical trial, we have now developed a strategy to isolate an enriched taro fraction, TE-method 2, (TE-M2) as well as a more purified subfraction (TE-M2F1) which can be scaled up under Good Manufacturing Practice (GMP) conditions for evaluation in human subjects. We demonstrate that TE-M2 and TE-M2F1 retain the anti-metastatic properties of TE. CONCLUSIONS: These studies provide further support for the continued examination of biologically active components of Colocasia esculenta as potential new therapeutic entities and identify a method to isolate sufficient quantities under GMP conditions to conduct early phase clinical studies.

Exogenous 1',4'-trans-Diol-ABA Induces Stress Tolerance by Affecting the Level of Gene Expression in Tobacco (Nicotiana tabacum L.).

1',4'-trans-diol-ABA is a key precursor of the biosynthesis of abscisic acid (ABA) biosynthesis in fungi. We successfully obtained the pure compound from a mutant of Botrytis cinerea and explored its function and possible mechanism on plants by spraying 2 mg/L 1',4'-trans-diol-ABA on tobacco leaves. Our results showed that this compound enhanced the drought tolerance of tobacco seedlings. A comparative transcriptome analysis showed that a large number of genes responded to the compound, exhibiting 1523 genes that were differentially expressed at 12 h, which increased to 1993 at 24 h and 3074 at 48 h, respectively. The enrichment analysis demonstrated that the differentially expressed genes (DEGs) were primarily enriched in pathways related to hormones and resistance. The DEGs of transcription factors were generally up-regulated and included the bHLH, bZIP, ERF, MYB, NAC, WRKY and HSF families. Moreover, the levels of expression of PYL/PYR, PP2C, SnRK2, and ABF at the ABA signaling pathway responded positively to exogenous 1',4'-trans-diol-ABA. Among them, seven ABF transcripts that were detected were significantly up-regulated. In addition, the genes involved in salicylic acid, ethylene and jasmonic acid pathways, reactive oxygen species scavenging system, and other resistance related genes were primarily induced by 1',4'-trans-diol-ABA. These findings indicated that treatment with 1',4'-trans-diol-ABA could improve tolerance to plant abiotic stress and potential biotic resistance by regulating gene expression, similar to the effects of exogenous ABA.

Venetoclax and pegcrisantaspase for complex karyotype acute myeloid leukemia.

Complex karyotype acute myeloid leukemia (CK-AML) has a dismal outcome with current treatments, underscoring the need for new therapies. Here, we report synergistic anti-leukemic activity of the BCL-2 inhibitor venetoclax (Ven) and the asparaginase formulation Pegylated Crisantaspase (PegC) in CK-AML in vitro and in vivo. Ven-PegC combination inhibited growth of multiple AML cell lines and patient-derived primary CK-AML cells in vitro. In vivo, Ven-PegC showed potent reduction of leukemia burden and improved survival, compared with each agent alone, in a primary patient-derived CK-AML xenograft. Superiority of Ven-PegC, compared to single drugs, and, importantly, the clinically utilized Ven-azacitidine combination, was also demonstrated in vivo in CK-AML. We hypothesized that PegC-mediated plasma glutamine depletion inhibits 4EBP1 phosphorylation, decreases the expression of proteins such as MCL-1, whose translation is cap dependent, synergizing with the BCL-2 inhibitor Ven. Ven-PegC treatment decreased cellular MCL-1 protein levels in vitro by enhancing eIF4E-4EBP1 interaction on the cap-binding complex via glutamine depletion. In vivo, Ven-PegC treatment completely depleted plasma glutamine and asparagine and inhibited mRNA translation and cellular protein synthesis. Since this novel mechanistically-rationalized regimen combines two drugs already in use in acute leukemia treatment, we plan a clinical trial of the Ven-PegC combination in relapsed/refractory CK-AML.

The Chemokine Receptor CXCR3 Isoform B Drives Breast Cancer Stem Cells.

We are seeking to identify molecular targets that are relevant to breast cancer cells with stem-like properties. There is growing evidence that cancer stem cells (CSCs) are supported by inflammatory mediators expressed in the tumor microenvironment. The chemokine receptor CXCR3 binds the interferon-γ-inducible, ELR-negative CXC chemokines CXCL9, CXCL10, and CXCL11 and malignant cells have co-opted this receptor to promote tumor cell migration and invasion. There are 2 major isoforms of CXCR3: CXCR3A and CXCR3B. The latter is generated from alternative splicing and results in a protein with a longer N-terminal domain. CXCR3 isoform A is generally considered to play a major role in tumor metastasis. When the entire tumor cell population is examined, CXCR3 isoform B is usually detected at much lower levels than CXCR3A and for this, and other reasons, was not considered to drive tumor progression. We have shown that CXCR3B is significantly upregulated in the subpopulation of breast CSCs in comparison with the bulk tumor cell population in 3 independent breast cancer cell lines (MDA-MB-231, SUM159, and T47D). Modulation of CXCR3B levels by knock in strategies increases CSC populations identified by aldehyde dehydrogenase activity or CD44+CD24- phenotype as well as tumorsphere-forming capacity. The reverse is seen when CXCR3B is gene-silenced. CXCL11 and CXCL10 directly induce CSC. We also report that novel CXCR3 allosteric modulators BD064 and BD103 prevent the induction of CSCs. BD103 inhibited experimental metastasis. This protective effect is associated with the reversal of CXCR3 ligand-mediated activation of STAT3, ERK1/2, CREB, and NOTCH1 pathways. We propose that CXCR3B, expressed on CSC, should be explored further as a novel therapeutic target.

Nicotine dependence in community-dwelling Chinese patients with schizophrenia.

BACKGROUND: Smoking is a serious public health problem. Patients with schizophrenia usually have a higher prevalence of smoking than the general population, but the level of nicotine dependence is seldom studied, especially for patients living in the communities. AIMS: This study aimed to examine the level of nicotine dependence in Chinese community-dwelling patients with schizophrenia and explored its associated sociodemographic and clinical factors. METHODS: A total of 621 patients with schizophrenia treated in the primary care centres of Guangzhou were consecutively recruited. The level of nicotine dependence was assessed with the Chinese version of the Fagerström Test for Nicotine Dependence (FTND). RESULTS: 148 patients with schizophrenia were current smokers, and the mean (SD) score of FTND was 5.06 (2.55) for all the current smokers. The prevalence of nicotine addiction was 48.0% (95% CI: 40.0%-56.0%) in patients with current smoking. The patients with schizophrenia had a significantly higher level of nicotine dependence than the Chinese general population. Multiple linear regression analysis revealed that male gender, being unemployed, having a family history of psychiatric disorders, having major medical conditions, first illness episode and less severe positive symptoms were significantly associated with a higher level of nicotine dependence. CONCLUSION: Community-dwelling patients with schizophrenia in China, especially male patients, had a higher level of nicotine dependence than the general population.

Quality of life in Chinese patients with schizophrenia treated in primary care.

In China, maintenance treatment for clinically stable patients with schizophrenia is usually provided by primary care physicians. This study examined the quality of life (QOL) in patients with schizophrenia treated in primary care and explored the demographic and clinical characteristics associated with QOL. Altogether, 612 patients with schizophrenia treated in 22 randomly selected primary care services in China formed the study sample. QOL, psychotic and depressive symptoms, extra-pyramidal symptoms and insight were assessed using standardized instruments. Data analyses were conducted with the one sample t-test and multiple linear regression analyses. Compared with the normative data for the Chinese general population, significantly lower scores in physical and mental QOL domains were found in the patient group. Older age, being unemployed, major medical conditions, no smoking, more severe depressive and negative symptoms, more frequent insomnia, and suicidality were independently associated with poor physical QOL. Male gender, more severe depressive and anxiety symptoms, more frequent insomnia, and suicidality were independently associated with poor mental QOL. Patients with schizophrenia treated in primary care had lower level of QOL in comparison with general population. Effective measures need to be implemented to improve their QOL.

Multiple drug resistance-associated protein (MRP4) exports prostaglandin E2 (PGE2) and contributes to metastasis in basal/triple negative breast cancer.

Cyclooxygenase-2 (COX-2) and its primary enzymatic product, prostaglandin E2 (PGE2), are associated with a poor prognosis in breast cancer. In order to elucidate the factors contributing to intratumoral PGE2 levels, we evaluated the expression of COX-2/PGE2 pathway members MRP4, the prostaglandin transporter PGT, 15-PGDH (PGE2 metabolism), the prostaglandin E receptor EP4, COX-1, and COX-2 in normal, luminal, and basal breast cancer cell lines. The pattern of protein expression varied by cell line reflecting breast cancer heterogeneity. Overall, basal cell lines expressed higher COX-2, higher MRP4, lower PGT, and lower 15-PGDH than luminal cell lines resulting in higher PGE2 in the extracellular environment. Genetic or pharmacologic suppression of MRP4 expression or activity in basal cell lines led to less extracellular PGE2. The key finding is that xenografts derived from a basal breast cancer cell line with stably suppressed MRP4 expression showed a marked decrease in spontaneous metastasis compared to cells with unaltered MRP4 expression. Growth properties of primary tumors were not altered by MRP4 manipulation. In addition to the well-established role of high COX-2 in promoting metastasis, these data identify an additional mechanism to achieve high PGE2 in the tumor microenvironment; high MRP4, low PGT, and low 15-PGDH. MRP4 should be examined further as a potential therapeutic target in basal breast cancer.

A monoclonal antibody against KCNK9 K(+) channel extracellular domain inhibits tumour growth and metastasis.

Two-pore domain potassium (K2P) channels act to maintain cell resting membrane potential--a prerequisite for many biological processes. KCNK9, a member of K2P family, is implicated in cancer, owing to its overexpression in human tumours and its ability to promote neoplastic cell survival and growth. However, KCNK9's underlying contributions to malignancy remain elusive due to the absence of specific modulators. Here we describe the development of monoclonal antibodies against the KCNK9 extracellular domain and their functional effects. We show that one antibody (Y4) with the highest affinity binding induces channel internalization. The addition of Y4 to KCNK9-expressing carcinoma cells reduces cell viability and increases cell death. Systemic administration of Y4 effectively inhibits growth of human lung cancer xenografts and murine breast cancer metastasis in mice. Evidence for Y4-mediated carcinoma cell autonomous and immune-dependent cytotoxicity is presented. Our study reveals that antibody-based KCNK9 targeting is a promising therapeutic strategy in KCNK9-expressing malignancies.

Antipsychotic polypharmacy and quality of life in patients with schizophrenia treated in primary care in China.

OBJECTIVE: In China, maintenance treatment for clinically stable patients with schizophrenia is usually provided by primary care physicians, but their prescribing patterns have not been studied. This study examined the frequency as well as demographic and clinical correlates of antipsychotic polypharmacy (APP) and its impact on quality of life (QOL) in patients with schizophrenia treated in primary care in China. METHOD: A total of 623 community-dwelling patients from 18 randomly selected primary care services were interviewed. Patients' socio-demographic and clinical characteristics, including number of hospitalizations, antipsychotic drug-induced side effects, and QOL were recorded using a standardized protocol and data collection procedure. RESULTS: The rate of APP prescription was 31% (193/623). Of the patients on APP, 89.6% received 2 antipsychotics, 10.4% received 3 or more antipsychotics. Clozapine (35.6%) was the most commonly prescribed second generation antipsychotic (SGA), while perphenazine (17.8%) was the most commonly prescribed first generation antipsychotic (FGA). Multiple logistic regression analyses revealed that patients on APP were more likely to receive SGAs and anticholinergics, had fewer hospitalizations, younger age of onset, and higher doses of antipsychotics. There were no significant differences between the two groups in any of the QOL domains. CONCLUSIONS: Approximately a third of Chinese patients with schizophrenia in primary care receive APP. Further examination of the rationale and appropriateness of APP and its alternatives is warranted.

Smoking and its associations with sociodemographic and clinical characteristics and quality of life in patients with schizophrenia treated in primary care in China.

OBJECTIVE: Maintenance treatment for clinically stable patients with schizophrenia is usually provided by Chinese primary care physicians, but no study has investigated smoking rates in this population. This study investigated the rate of smoking and its associations with sociodemographic and clinical characteristics and quality of life (QOL) in patients with schizophrenia treated in primary care in China. METHODS: This was a cross-sectional, community-based survey. A total of 621 schizophrenia patients were recruited from 22 primary care services in Guangzhou, China, in 2013. Patients' sociodemographic and clinical characteristics, smoking status, and QOL were recorded. RESULTS: The frequency of current smoking was 23.8% in the whole sample; 41.5% for men and 2.5% for women. Multiple logistic regression analysis revealed that male gender, married status, alcohol use, older age at onset, fewer major medical conditions, lower education level and more hospitalizations were independently associated with current smoking. CONCLUSION: The frequency of smoking in Chinese schizophrenia patients treated by primary care physicians is lower than most figures reported from Western and Chinese psychiatric settings.

Identification of AGO3-associated miRNAs and computational prediction of their targets in the green alga Chlamydomonas reinhardtii.

The unicellular green alga Chlamydomonas reinhardtii harbors many types of small RNAs (sRNAs) but little is known about their role(s) in the regulation of endogenous genes and cellular processes. To define functional microRNAs (miRNAs) in Chlamydomonas, we characterized sRNAs associated with an argonaute protein, AGO3, by affinity purification and deep sequencing. Using a stringent set of criteria for canonical miRNA annotation, we identified 39 precursor miRNAs, which produce 45 unique, AGO3-associated miRNA sequences including 13 previously reported miRNAs and 32 novel ones. Potential miRNA targets were identified based on the complementarity of miRNAs with candidate binding sites on transcripts and classified, depending on the extent of complementarity, as being likely to be regulated through cleavage or translational repression. The search for cleavage targets identified 74 transcripts. However, only 6 of them showed an increase in messenger RNA (mRNA) levels in a mutant strain almost devoid of sRNAs. The search for translational repression targets, which used complementarity criteria more stringent than those empirically required for a reduction in target protein levels, identified 488 transcripts. However, unlike observations in metazoans, most predicted translation repression targets did not show appreciable changes in transcript abundance in the absence of sRNAs. Additionally, of three candidate targets examined at the protein level, only one showed a moderate variation in polypeptide amount in the mutant strain. Our results emphasize the difficulty in identifying genuine miRNA targets in Chlamydomonas and suggest that miRNAs, under standard laboratory conditions, might have mainly a modulatory role in endogenous gene regulation in this alga.