Neuron-derived neurotensin promotes pancreatic cancer invasiveness and gemcitabine resistance via the NTSR1/Akt pathway.

Perineural invasion and neurogenesis are frequently observed in pancreatic ductal adenocarcinoma (PDAC) and link to poor outcome. However, how neural factors affect PDAC prognosis and the underlying mechanism as well as counteracting therapeutic are still unclear. In silico systematic analysis was performed with PROGgene to identify potential neural factor and its receptor in pancreatic cancer. In vitro assays including migration, invasion, 3D recruitment, and gemcitabine resistance were performed to study the effect of neuron-derived neurotensin (NTS) on pancreatic cancer behavior. Orthotopic animal study was used to validate the in vitro findings. Gene set enrichment analysis (GSEA) was performed to confirm the results from in silico to in vivo. Expression of NTS and its receptor 1 (NTSR1) predicted poor prognosis in PDAC. NTS synthetic peptide or neuron-derived condition medium promoted pancreatic cancer invasiveness and recruitment in 2D and 3D assays. NTS-induced effects depended on NTSR1 and PI3K activation. GDC-0941, a clinically approved PI3K inhibitor, counteracted NTS-induced effects in vitro. Inhibition of NTSR1 in pancreatic cancer cells resulted in decreased tumor dissemination and diminished PI3K activation in vivo. NTS boosted gemcitabine resistance via NTSR1 in pancreatic cancer. Our results suggest that neural cell-secreted NTS plays an important role in promoting PDAC.

RNF43 Inactivation Enhances the B-RAF/MEK Signaling and Creates a Combinatory Therapeutic Target in Cancer Cells.

RING finger 43 (RNF43), a RING-type E3 ubiquitin ligase, is a key regulator of WNT signaling and is mutated in 6-10% of pancreatic tumors. However, RNF43-mediated effects remain unclear, as only a few in vivo substrates of RNF43 are identified. Here, it is found that RNF43-mutated pancreatic cancer cells exhibit elevated B-RAF/MEK activity and are highly sensitive to MEK inhibitors. The depletion of RNF43 in normal pancreatic ductal cells also enhances MEK activation, suggesting that it is a physiologically regulated process. It is confirmed that RNF43 ubiquitinates B-RAF at K499 to promote proteasome-dependent degradation, resulting in reduced MEK activity and proliferative ability in cancer cells. In addition, phosphorylation of B-RAF at T491 suppresses B-RAF ubiquitination by decreasing the interaction between RNF43 and B-RAF. Mutations at K499 in B-RAF are identified in various cancer types. MEK and WNT inhibitors synergistically suppress the growth of RNF43-mutated pancreatic cancer cells in vitro and in vivo. Collectively, the research reveals a novel mechanism by which RNF43 inhibits B-RAF/MEK signaling to suppress tumor growth and provide a new strategy for the treatment of RNF43-inactivated pancreatic cancer.

Pancreatic cancer cell-derived semaphorin 3A promotes neuron recruitment to accelerate tumor growth and dissemination.

Perineural invasion and neurogenesis are frequently observed in pancreatic ductal adenocarcinoma (PDAC), and they are associated with a poor prognosis. Axon guidance factor semaphorin 3A (SEMA3A) is upregulated in PDAC. However, it remains unclear whether cancer-derived SEMA3A influences nerve innervation and pancreatic tumorigenesis. In silico analyses were performed using PROGgene and NetworkAnalyst to clarify the importance of SEMA3A and its receptors, plexin A1 (PLXNA1) and neuropilin 2 (NRP2), in pancreatic cancer. In vitro assays, including migration, neurite outgrowth, and 3D recruitment, were performed to study the effects of SEMA3A on neuronal behaviors. Additionally, an orthotopic animal study using C57BL/6 mice was performed to validate the in vitro findings. Expression of SEMA3A and its receptors predicted worse prognosis for PDAC. Cancer-derived SEMA3A promoted neural migration, neurite outgrowth, and neural recruitment. Furthermore, SEMA3A-induced effects depended on PLXNA1, NRP2, and MAPK activation. Trametinib, an approved MAPK kinase (MEK) inhibitor, counteracted SEMA3A-enhanced neuronal activity in vitro. Inhibition of SEMA3A by shRNA in pancreatic cancer cells resulted in decreased neural recruitment, tumor growth, and dissemination in vivo. Our results suggested that cancer-secreted SEMA3A plays an important role in promoting neo-neurogenesis and progression of PDAC.

Protein arginine methyltransferase 3: A crucial regulator in metabolic reprogramming and gene expression in cancers.

Post-translational modification (PTM) of proteins increases proteome diversity, which is critical for maintaining cellular homeostasis. The importance of protein methylation in the regulation of diverse biological processes has been highlighted in the past decades. Methylation of the arginine residue on proteins is catalyzed by members of the protein arginine methyltransferase (PRMT) family. PRMTs play indispensable roles in various pathways that regulate cancer development, progression, and drug response. In this review, we discuss the role of PRMT3, a member of the PRMT family, in controlling oncogenic processes. Additionally, the effects of PRMT3 on the methylation of regulatory proteins involved in transcription, post-transcriptional control, ribosomal maturation, translation, biological synthesis, and metabolic signaling are summarized. Moreover, recent progresses in the development of PRMT3 inhibitors are introduced. Overall, this review highlights the importance of PRMT3 in tumorigenesis and discusses the underlying mechanisms by which PRMT3 modulates cellular metabolism and gene expression. These results also provide a molecular basis for therapeutic modalities by targeting PRMT3.

Role of high ubiquitin-conjugating enzyme E2 expression as a prognostic factor in nasopharyngeal carcinoma.

The incidence of nasopharyngeal carcinoma (NPC) in Southeast Asia and Taiwan is high due to epidemiological factors. Cisplatin-based chemoradiotherapy is an important treatment strategy with excellent outcomes for patients with NPC. However, the outcomes for patients who are refractory to cisplatin-based therapy are poor. Methods for risk stratification of patients with NPC undergoing cisplatin-based chemoradiotherapy require to be investigated. A previous study indicated that ubiquitin-conjugating enzyme E2 B (UBE2B) was able to regulate alkylating drug sensitivity in NPC cells. In the present study, the clinical significance of UBE2B expression in patients with NPC was analyzed. Analysis of the two available NPC datasets containing the UBE2B expression profile (GSE12452 and GSE68799) was performed to evaluate the UBE2B expression levels in NPC tissues compared with nasopharyngeal mucosal epithelial tissues. Furthermore, immunohistochemical staining was performed using anti-UBE2B antibodies on samples from 124 patients with NPC who underwent cisplatin-based chemoradiotherapy. Disease-specific survival (DSS), distant metastatic-free survival (DMeFS) and local recurrence-free survival (LRFS) of patients with high and low UBE2B expression was analyzed. Furthermore, the associations between UBE2B expression and the biological behavior of NPC cells were investigated in vitro. Using public NPC datasets and in vitro studies, it was identified that UBE2B expression levels were increased in NPC tumor tissues compared with those in mucosal epithelial tissues. The cell proliferation ability was decreased in UBE2B-deficient NPC cells as compared with that in UBE2B-proficient cells. Immunohistochemical analysis of 124 NPC tissues from patients who underwent cisplatin-based chemoradiotherapy indicated that high UBE2B expression levels were associated with poor DSS, DMeFS and LRFS. Multivariate regression analysis of factors influencing survival also confirmed that high UBE2B expression levels were a statistically significant independent risk factor for poor clinical outcomes in terms of DSS [hazard ratio (HR), 1.955; 95% CI 1.164-3.282], DMeFS (HR, 2.141; 95% CI 1.206-3.801) and LRFS (HR, 2.557; 95 CI 1.313-4.981). In vitro analysis indicated that O6-methylguanine-DNA methyltransferase attenuated cisplatin sensitivity induced by knockdown of UBE2B in NPC cells. In conclusion, the present study demonstrated that high UBE2B expression is associated with poor clinical outcomes for patients with NPC treated with cisplatin-based chemoradiotherapy.

Semaphorin 6C Suppresses Proliferation of Pancreatic Cancer Cells via Inhibition of the AKT/GSK3/ß-Catenin/Cyclin D1 Pathway.

Semaphorins (SEMAs) are axon guidance factors that participate in axonal connections and nerve system development. However, the functional roles of SEMAs in tumorigenesis are still largely uncovered. By using in silico data analysis, we found that SEMA6C was downregulated in pancreatic cancer, and its reduction was correlated with worse survival rates. RNA sequencing revealed that cell cycle-related genes, especially cyclin D1, were significantly altered after blockage of SEMA6C by neutralizing antibodies or ectopic expressions of SEMA6C. Mechanistic investigation demonstrated that SEMA6C acts as a tumor suppressor in pancreatic cancer by inhibiting the AKT/GSK3 signaling axis, resulting in a decrease in cyclin D1 expression and cellular proliferation. The enhancement of cyclin D1 expression and cyclin-dependent kinase activation in SEMA6C-low cancer created a druggable target of CDK4/6 inhibitors. We also elucidated the mechanism underlying SEMA6C downregulation in pancreatic cancer and demonstrated a novel regulatory role of miR-124-3p in suppressing SEMA6C. This study provides new insights of SEMA6C-mediated anti-cancer action and suggests the treatment of SEMA6C-downregulated cancer by CDK4/6 inhibitors.

Generation of induced pluripotent stem cells FIRDIi001-A from a Taiwanese subject carrying ALDH2 pE487K mutation.

The ALDH2 mutation (ALDH2*2) is caused by an amino acid substitution ALDH2 rs671 G>A (pE487K) which reduces ALDH2 enzyme activity. When individuals with the ALDH2 mutation consume alcohol, accumulating acetaldehyde in the blood can cause reddened face, headache, nausea, and palpitations; symptoms referred to as Alcohol Flushing Reaction. We report the production of an induced pluripotent stem cell (iPSC) line, FIRDIi001-A, developed from peripheral blood mononuclear cells of a 39-year-old male subject with the ALDH2*2 mutation. The ALDH2-pE487K iPSCs will be valuable in investigating pathogenic mechanisms involved in the link between the ALDH2 polymorphism and alcohol-related diseases.

Lgr5+ pericentral hepatocytes are self-maintained in normal liver regeneration and susceptible to hepatocarcinogenesis.

Emerging evidence suggests that hepatocytes are primarily maintained by self-renewal during normal liver homeostasis, as well as in response to a wide variety of hepatic injuries. However, how hepatocytes in distinct anatomic locations within the liver lobule are replenished under homeostasis and injury-induced regeneration remains elusive. Using a newly developed bacterial artificial chromosome (BAC)-transgenic mouse model, we demonstrate that Lgr5 expression in the liver is restricted to a unique subset of hepatocytes most adjacent to the central veins. Genetic lineage tracing revealed that pericentral Lgr5+ hepatocytes have a long lifespan and mainly contribute to their own lineage maintenance during postnatal liver development and homeostasis. Remarkably, these hepatocytes also fuel the regeneration of their own lineage during the massive and rapid regeneration process following two-thirds partial hepatectomy. Moreover, Lgr5+ hepatocytes are found to be the main cellular origin of diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) and are highly susceptible to neoplastic transformation triggered by activation of Erbb pathway. Our findings establish an unexpected self-maintaining mode for a defined subset of hepatocytes during liver homeostasis and regeneration, and identify Lgr5+ pericentral hepatocytes as major cells of origin in HCC development.

Ubiquitin-conjugating enzyme E2 B regulates the ubiquitination of O6-methylguanine-DNA methyltransferase and BCNU sensitivity in human nasopharyngeal carcinoma cells.

O6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that removes the alkyl groups from the O6 position of guanine and is then degraded via ubiquitin-mediated degradation. Previous studies indicated that 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) facilitates the ubiquitination and degradation of MGMT in several types of cancer cells. However, the underlying mechanism of MGMT ubiquitination remains unclear. In this study, we demonstrated for the first time that ubiquitin-conjugating enzyme E2 B (UBE2B) is a novel regulator of MGMT ubiquitination mediated by BCNU in nasopharyngeal carcinoma (NPC) cells. The E3 ubiquitin ligase RAD18, a partner of UBE2B, is also involved in BCNU-mediated MGMT ubiquitination. Overexpression/knockdown of UBE2B enhanced/reduced BCNU-mediated MGMT ubiquitination. Surprisingly, UBE2B knockdown significantly increased BCNU cytotoxicity in NPC cells. Therefore, loss of UBE2B seems to disrupt ubiquitin-mediated degradation of alkylated MGMT. We found that UBE2B knockdown reduced MGMT activity, suggesting that loss of UBE2B leads to the accumulation of deactivated MGMT and suppresses MGMT protein turnover in BCNU-treated cells. These findings indicate that UBE2B modulates sensitivity to BCNU in NPC cells by regulating MGMT ubiquitination.

Role of adiponectin gene variants, adipokines and hydrometry-based percent body fat in metabolically healthy and abnormal obesity.

OBJECTIVE: Metabolically healthy obesity (MHO) subjects have better metabolic parameters than metabolically abnormal obesity (MAO) subjects, but the possible mechanisms underlying this remain unknown. Our study was designed to investigate the interrelationships among genes, adipokines, body fat and its distribution in MHO and MAO. METHODS: From 2007 to 2009, 103 males and 131 females aged 18-50 years were enrolled by an intention-to-treat design in a weight management clinic. Participants were divided into MHO and MAO groups. Percent body fat (PBF) was measured by a deuterium oxide dilution method. Four polymorphic variants, including PPARγ2 (Pro12Ala and C1431T) and adiponectin (T45G and G276T) genes, and three adipokines (adiponectin, leptin and resistin) were obtained. RESULTS: Of the 234 obese subjects, 130 (55.6%) were MHO. In the univariate analysis, the MAO group has significantly higher anthropometric, metabolic indices and leptin levels than the MHO group. Logistic regression analysis revealed that age, male gender, the T allele of adiponectin T45G polymorphism, leptin and PBF were positively associated with MAO. ANCOVA analysis revealed that the T allele of adiponectin T45G polymorphism was associated with higher fasting and postprandial glucose levels. We further found that TT genotype has a lower high molecular weight (HMW)/low molecular weight (LMW) adiponectin ratio than GG genotype. CONCLUSIONS: The factors associated with MAO are age, male gender, the T allele of adiponectin T45G polymorphism, leptin, and PBF. The net effects of T45G polymorphism on the MAO phenotype may be achieved by changes in the adiponectin oligomer ratio and glucose levels.

Generation of an induced pluripotent stem cell line, IBMS-iPSC-014-05, from a female autosomal dominant polycystic kidney disease patient carrying a common mutation of R803X in PKD2.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most commonly inherited forms of polycystic kidney disease, and is characterized by the growth of numerous cysts in both kidneys. Here we generated an induced pluripotent stem cell (iPSC) line from the peripheral blood mononuclear cells (PBMCs) of a 63-year-old female ADPKD patient carrying an R803X mutation in the PKD2 gene using the Sendai-virus delivery system. Downstream characterization of these iPSCs showed that they possessed normal karyotyping, were free of genomic integration, retained the disease-causing PKD2 mutation, expressed pluripotency markers and could differentiate into three germ layers.

Intensify the application of ZnO-based nanodevices in humid environment: O2/H2 plasma suppressed the spontaneous reaction of amorphous ZnO nanowires.

In this work, we have demonstrated that amorphous ZnO nanobranches (a-ZnO NBs) could spontaneously react from the crystalline ZnO NWs (c-ZnO NWs) at specific humid environment. The spontaneous reaction mechanism and result can be analyzed by humidity controlling and optical microscope (OM)/scanning electron microscope (SEM)/Kelvin probe force microscopy (KPFM)/transmission electron microscopy (TEM) system. We can make the c-ZnO NWs spontaneous reaction happen at different humid environments and suppress the a-ZnO NBs spontaneous reaction by oxygen/hydrogen plasma surface passivation. The hydrogen plasma surface treatment also can improve the UV sensing sensitivity more than twofold. This work provides the mechanism and methods of the a-ZnO NBs spontaneous growth and offers the passivation treatment for strengthening and enhancing ZnO-based nanodevice application in humid environment and UV light detection, respectively.

Inhibitory effect of litchi (Litchi chinensis Sonn.) flower on lipopolysaccharide-induced expression of proinflammatory mediators in RAW264.7 cells through NF-κB, ERK, and JAK2/STAT3 inactivation.

Litchi (Litchi chinensis Sonn.) flower ethanolic extract (LFEE) was found to contain five flavanoids [total amount, 102.73 ± 5.50 mg/g of dried extract (gDE)], nine phenolic acids (total amount, 60.31 ± 4.52 mg/gDE), and proanthocyanidin A2 (79.31 ± 2.95 mg/gDE). LFEE was used to evaluate the inhibitory effects on lipopolysaccharide- (LPS-) induced pro-inflammatory mediators in RAW264.7 cells. The results showed that LFEE treatment could suppress the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), the productions of nitric oxide (NO) and prostaglandin E2 (PGE2), and the secretions of pro-inflammatory cytokines [interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor α (TNF-α)] in the LPS-mediated RAW264.7 cells. The attenuation of LPS-induced inflammatory responses by LFEE was found to be closely related to the inhibition of the translocation of nuclear factor κB (NF-κB) p50/p65 subunits correlated with suppression of the activation of the inhibitor of κB kinase (IKK) α/ß and downregulation of activation of extracellular signal-regulated kinase (ERK) and Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3).

The Wilms' tumor suppressor Wt1 regulates Coronin 1B expression in the epicardium.

Coronin 1B has been shown to be critical for cell motility and various actin-dependent processes. To understand its role more extensively, the expression and transcriptional regulation of Coro1b gene during mouse development were explored. Coronin 1B is ubiquitously expressed in the whole embryo but nevertheless shows distinct expression pattern in developing heart. In addition to the localization in endocardium, Coronin 1B is specifically expressed in the endocardial cushion and epicardium where cardiac EMT processes take place as the heart develops. Promoter deletion analysis identified the positions between -1038 and -681 is important for Coro1b basal promoter activity. In addition to a correlation of Coronin 1B localization with Wt1 expression in the epicardium, we also identified putative Wt1 binding sequences within Coro1b promoter. Direct binding of Wt1 to GC-rich sequences within the Coro1b promoter is required for the regulation of Coro1b gene expression. In accordance with the motility defect found in Coronin 1B-knockdown cells, a modest decrease in expression of Coronin 1B in the remaining epicardium of Wt1(EGFPCre/EGFPCre) mutant embryos was observed. These findings seem to shed light on the role of Wt1 during cell migration and suggest that, at least in part, this involves transcriptional control of Coro1b gene expression.

The role of the characteristics of humic substances in binding with benzo[h]quinoline.

The binding constants (K(DOC)) of the mixture of benzo[h]quinoline and its protonated analog, benzo[h]quinolinium, to four types of humic substances obtained from the International Humic Substances Society were determined by the fluorescence quenching method. A simple mixing model was used to eliminate the fluorescent interference from the minor analog in the solution and to deduce K(mix), which represents the overall binding as the sum of that for the individual analogs. The characteristics of humic substances, especially their hydrophobicity and aromaticity, established by principal component analysis of structural and elemental compositions, were the main determinants of the binding affinity with both benzo[h]quinoline and benzo[h]quinolinium (K(BQ) and K (BQH+) across a range of pH values. The strongest overall affinity of benzo[h]quinoline for humic substances is observed near pH 4 and with more hydrophobic humic substances, which suggests possible choices in attempts at remediation of benzo[h]quinoline containing particles with humic substances.

pH dependence of binding benzo[h]quinoline and humic acid and effects on fluorescence quenching.

The binding constant (K(DOC)) between humic acid and the nitrogen-containing polycyclic aromatic compound (N-PAC), benzo[h]quinoline, was measured at varying pH levels using fluorescence quenching (FQ). Because fluorescence characteristics of benzo[h]quinoline change with pH, determination required two optimum sets of excitation and emission wavelength pairs. A simple mixing model was used to eliminate the inherent fluorescence interference between benzo[h]quinoline and its protonated form, benzo[h]quinolinium, when estimating binding constants. Hydrophobic interaction is likely to control the binding between humic acid and benzo[h]quinoline and benzo[h]quinolinium, in lower and higher pH ranges (pH <3, pH >6). In contrast, cation exchange seems to control the binding affinity of benzo[h]quinolinium in the middle range of pH. The estimates of K(DOC) were up to 70% smaller after elimination of interference. This indicates that the contribution of the minor form influences estimates of the K(DOC)-pH trend for benzo[h]quinoline, and potentially explains the large discrepancy reported in the literature between results based on using FQ and those based on equilibrium dialysis methods. Previous FQ measurements overestimate K(DOC) at some pH values and lead to an underestimation of bioavailability in an aquatic environment. The application of our models appears to be necessary when using FQ for determining the K(DOC)-pH trend for organic compounds with acid-base pair analogs.

Establishment of a chimeric reporting system for the universal detection and high-throughput screening of G protein-coupled receptors.

G proteins, further divided into four subfamilies (G(s), G(q), G(12) and G(i)) based on their Galpha subunits, are the primary components activated by G protein-coupled receptors (GPCRs). Current GPCR assays are limited to the evaluation of selective Galpha signaling and do not allow comprehensive screening for orphan GPCRs without a known coupled Galpha. Therefore, our aim was to design a chimeric reporting system that covers responses from all Galpha subfamilies simultaneously. Because G(s) activates cAMP response element (CRE)-driven genes whereas G(q) and G(12) activate serum response element (SRE)-driven genes, we therefore incorporated 2x CRE and 5x SRE (2CRE5SRE) into a promoter for driving luciferase expression. To further report G(i) signals, a 2CRE5SRE-driven chimeric G(qi), in which the C-terminus of G(q) is replaced by that of G(i), was integrated to switch the responses of G(i)-coupled GPCRs to the G(q) signaling. The novel reporter system showed a strong signal amplification when activated by neuromedin U receptor 1 (mainly activates G(q)), neuromedin U receptor 2 (mainly activates G(i)) or luteinizing hormone receptor (mainly through the G(s) and G(q) pathways). In addition, 293T cells stably carrying our reporter construct showed a similar sensitivity to the radioactive cAMP assay when revealing the constitutive signal from gain-of-function mutants of luteinizing hormone receptor. To our knowledge, this is the first reporting system capable of covering the G(s), G(q), G(12) and G(i) signals and revealing the phenomena of constitutively active GPCRs. Such a universal platform will benefit future high-throughput screening and drug designs for any GPCR.

Molecular dissection of G protein preference using Gsalpha chimeras reveals novel ligand signaling of GPCRs.

Although only 16 genes have been identified in mammals, several Galpha subunits can be simultaneously activated by G protein-coupled receptors (GPCRs) to modulate their complicated functions. Current GPCR assays are limited in the evaluation of selective Galpha activation, thus not allowing a comprehensive pathway screening. Because adenylyl cyclases are directly activated by G(s)alpha and the carboxyl termini of the various Galpha proteins determine their receptor coupling specificity, we proposed a set of chimeric G(s)alpha where the COOH-terminal five amino acids are replaced by those of other Galpha proteins and used these to dissect the potential Galpha linked to a given GPCR. Unlike G(q)alpha, G(12)alpha, and G(i)alpha outputs, compounding the signals from several Galpha members, the chimeric G(s)alpha proteins provide a superior molecular approach that reflects the previously uncharacterized pathways of GPCRs under the same cAMP platform. This is, to our knowledge, the first time allowing verification of the whole spectrum of Galpha coupling preference of adenosine A1 receptor, reported to couple to multiple G proteins and modulate many physiological processes. Furthermore, we were able to distinguish the uncharacterized pathways between the two neuromedin U receptors (NMURs), which distribute differently but are stimulated by a common agonist. In contrast to the G(q) signals mainly conducted by NMUR1, NMUR2 routed preferentially to the G(i) pathways. Dissecting the potential Galpha coupling to these GPCRs will promote an understanding of their physiological roles and benefit the pharmaceutical development of agonists/antagonists by exploiting the selective affinity toward a certain Galpha subclass.