Among β-adrenergic receptors, β3-AR is the last identified member of this family, and it is involved in cancer cell survival and induction of stromal reactivity in the tumor microenvironment. β3-AR is well known as a strong activator of uncoupling protein 1 (UCP1) in brown fat tissue. Interestingly, β3-AR is strongly expressed in early embryo development and in many cancer tissues. Induction of uncoupling protein 2 (UCP2) has been related to cancer metabolic switch, leading to accelerated glycolysis and reduced mitochondrial activity. In this study, for the first time, we demonstrate that β3-AR is able to promote this metabolic shift in both cancer and embryonic stem cells, inducing specific glycolytic cytoplasmic enzymes and a sort of mitochondrial dormancy through the induction of UCP2. The β3-AR/UCP2 axis induces a strong reduction of mitochondrial activity by reducing ATP synthesis and mitochondrial reactive oxygen species (mtROS) content. These effects are reverted by SR59230A, the specific β3-AR antagonist, causing an increase in mtROS. The increased level of mtROS is neutralized by a strong antioxidant activity in embryonic stem cells, but not in cancer stem cells, where it causes a dramatic reduction in tumor cell viability. These results lead to the possibility of a selective antitumor therapeutic use of SR59230A. Notably, we demonstrate the presence of β3-AR within the mitochondrial membrane in both cell lines, leading to the control of mitochondrial dormancy.The early phases of embryonic development and cancer share similar strategies to improve their survival in an inhospitable environment: both proliferate in a hypoxic and catecholamine-rich context, increasing aerobic glycolysis. Recent studies show that  Both fetal development and tumor growth, despite, respectively, physiological and pathological events with feedback far away from each other, are triggered by heterogeneous interrelated pathways. Embryo and cancer, among hypoxic active stromal tissues and high stem cell potential , share aβ-adrenergic receptors (β-ARs) has been proposed as a potential therapeutic approach to cancer and, in particular, melanoma [β2-AR [β3-AR was found overexpressed in tumors, particularly in melanoma cell lines and tumor microenvironments [β3-AR expression correlates with cancer progression, angiogenesis, and tumor stromal cell reactivity [β3-AR has also been revealed in the pregnant myometrium [Several studies suggest that stress-related catecholamine release accelerates cancer progression, so the targeting of melanoma , 14. At a [β2-AR , but, moronments –18. Curractivity –18. Inteometrium  and duriometrium , 21, conβ3-AR is well known to control thermogenesis by activating uncoupling protein 1 (UCP1). At low temperature, a signal is transmitted to brown adipocytes by catecholamine release and β3-AR activation, resulting in increased UCP1 expression and activity. The UCPs are members of the mitochondrial anion carrier family. While UCP1 is predominantly expressed in brown adipocyte tissue [e tissue , UCP2 ise tissue , 24. Inte tissue –28.β3-AR/UCP2 axis on the regulation of the Warburg metabolism in cancer stem cells (CSC) and in mouse embryonic stem cells (ESC).In this study, we investigated the role of the Melanoma cells (A375) and mouse ESC (CGR8) were purchased from the European Collection of Cell Cultures .−3 M sodium pyruvate (Gibco), 10−7 M beta-mercaptoethanol (Sigma), 2 × 10−3 M glutamine (Sigma) 1% penicillin-streptomycin (Gibco), 10% fetal bovine serum (Gibco), and 1000 U/ml recombinant mouse leukemia inhibitory factor  in a humidified 5% CO2 atmosphere at 37°C [μl of a differentiation medium, whose composition was similar to that used for cell propagation except for fetal bovine serum  and without recombinant mouse leukemia inhibitory factor. After 4 days in suspension, EBs were plated on gelatin-coated plates. Starting from day 8 of differentiation, the number of beating EBs was counted by phase-contrast microscopy.A375 cells were cultured in high-glucose DMEM containing 10% FBS, while CGR8 were cultured in a propagation medium containing a BHK21 medium (Gibco), 1% nonessential amino acids (Gibco), 10 at 37°C . Differe at 37°C . Brieflyβ3-adrenergic agonist, and after 30 minutes with the β3-antagonist SR59230A and with genipin, UCP2's antagonist.A375 cells were grown to 70% confluence in complete medium DMEM high-glucose for 48 h. Then the medium was removed, and after washing in PBS solution, cells were serum-starved overnight with a starvation medium, without FCS, in order to promote the cells' entry into a quiescent G0 phase, thereby better evaluating cells' responsiveness to exogenous treatments. After 24 h, cells were treated with a single dose of BRL, a 2 on a low-attachment 100 mm plate in a Dulbecco's modified Eagle's medium/F12 supplemented with B27 and N2 media, 5 g/ml insulin, 20 ng/ml fibroblast GF-2, and 20 ng/ml epidermal GF. A375 cells were grown under these conditions for 15–20 days and formed nonadherent P0 spheres termed prostaspheres. For the evaluation of self-renewal, a single prostasphere was dissociated in single cells with Accutase, and a dilution of one cell per well into 96-well low-attachment plates was performed in order to isolate individual P1 prostaspheres. Single-cell cloning was confirmed by microscopic analysis, and single clones were counted.A375 cells were incubated for 72 h with a conditioned medium (CM) and then detached using Accutase (Sigma). Single A375 cells were plated at 150 cells/cmβ-actin (sc-1615), HKII (ab-76959), MCT-4 (376140), GLUT1 (ab-209449), SOX2 (sc-16320), total OXPHOS (ab-110143), and β3-AR (h-160).The buffer used was RIPA lysis buffer . Twenty micrograms of total proteins was loaded on SDS-PAGE, separated, and transferred onto nitrocellulose. The immunoblots were incubated in 3% bovine serum albumin, 10 mmol/l Tris-HCl (pH 7.5), 1 mmol/l EDTA, and 0.1% Tween 20 for 1 h at room temperature, probed first with specific antibodies and then with appropriate secondary antibodies. Bound antibodies were detected using the Novex ECL, HRP Chemiluminescent Substrate Reagent Kit (Life Technologies). Filters were autoradiographed, and images were acquired through the BioSpectrum Imaging System . The following antibodies were used: UCP2 (sc-6525), The lactate extrusion assay was performed using the Lactate Colorimetric Assay Kit II, following the manufacturer's instructions (BioVision®).The glucose uptake assay was performed using the Glucose Uptake Cell-Based Assay Kit (item n. 600470), following the manufacturer's instructions .ATP production assay was performed using the ATPlite Luminescence Assay System, following the manufacturer's instructions (PerkinElmer®).All experiments were collected after 48 h, and the luminescence and fluorescence were measured with a Flex spectrophotometer.μl of MitoSOX and were incubated for 15 minutes, then washed with PBS one time; the supernatant was aspirated; and the cells were incubated with Accutase. Finally, the detached cells were suspended in a final volume of 300 μl and were analyzed using MACSQuant FACS (Miltenyi Biotec).The cells were stained with 1 Membrane potential was performed using the MitoProbe™ DiIC1(5) Assay Kit for Flow Cytometry M34151, following the manufacturer's instructions (Thermo Fisher®).The ferric reducing antioxidant power (FRAP) assay, which is a measure of the antioxidant effects of nonenzymatic defences in plasma, was performed according to the method by Benzie and Strain .μg/ml fluorescent phalloidin conjugate solution in PBS, phalloidin-TRITC, for 1 h at room temperature with anti-β3-AR antibody, MitoProbe, and DAPI, then analyzed with the related laser. After several washes with PBS, the coverslips were mounted with glycerol plastine and then observed under a confocal fluorescence microscope (Leica).After washing with PBS, the cells were fixed with a 3.7% formaldehyde solution in PBS for 20 min at 4°C. Then, after extensive washes in PBS, the cells were permeabilized with 0.1% Triton X-100 in PBS and then stained with a 50 To isolate the mitochondria from A375 stem cells and ESC, the Mitochondria Isolation Kit (Miltenyi Biotec®) was used. At the end of the kit procedure, after the centrifugation at 13,000xg for 2 minutes at 4°C, we aspirated the supernatant and resuspended the mitochondria pellet in lysis buffer for Western blot analysis and in PBS for FACS analysis.Viability of tumor cells, in all conditions, was evaluated using an MTT  assay. It is a test of cell viability based on the reduction of the MTT dye by mitochondrial succinate dehydrogenase enzyme, which is active only in living cells. The enzyme cuts the tetrazolium ring of MTT (yellow substance) with the formation of a blue salt that is exocitated and precipitates.The cells were maintained in MTT for 1 h, then the formation of needle-like crystals near the membrane was visible . Then, the cell membrane was lysed and the dye solubilized developing a color whose intensity was proportional to cell viability. The absorbance was evaluated at 570 nm using a spectrophotometer.To assess the bioinformatic analysis, we chose four widely used methods: Hum-mPLoc 3.0, FUEL-mLoc, WegoLoc, and COMPARTMENTS. All the selected predictors are available as Web servers that are intended for eukaryotic proteins or are specific for human proteins. These predictors take advantage from the usage of the Gene Ontology (GO) terms, representing gene product properties. GO-based methods use the correlation between the annotations  for a protein and its subcellular location. Methods such as FUEL-mLoc, WegoLoc, and Hum-mPLoc 3.0 adopt different databases. WegoLoc and COMPARTMENTS extract the GO terms from the UniProtKB database. Hum-mPLoc 3.0 uses the SWISS-PROT portal, and FUEL-mLoc adopts two newly created compact databases, namely, ProSeq and ProSeq-GO, that allow consuming much less memory and make predictions faster.β3-AR protein.By unifying these information with evidence on protein localization from curated knowledge, high-throughput experiments, text mining, and sequence-based prediction methods, these tools infer on the subcellular localization of our In vitro data are presented as means ± standarddeviation (SD) from at least three experiments. Results were normalized versus control expression levels.Statistical analysis was performed using GraphPad Prism software  by one-way analysis variance (ANOVA), followed by a Bonferroni post hoc analysis.β3-AR affected CSC and ESC metabolism. We enriched the melanoma stem cell compartment by growing melanospheres in vitro. Interestingly, melanospheres revealed higher levels of β3-AR compared with A375 parental populations (Since embryos and cancer are enriched in nondifferentiated cells (embryonic and cancer stem cells), we investigated whether ulations . Usuallyulations . Elevateulations .β3-AR antagonism with SR59230A decreased the ability of A375 cells to form melanospheres under BRL37344 (selective β3-AR agonist) stimulations, as demonstrated by the reduction of P1 populations and reduction of stem cell markers CD133 and SOX2 , as confirmed by the increased expression of HKII and MCT-4, lactate export, and glucose uptake. These effects were dramatically reduced by SR59230A in both CSC and ESC  had no effect on the reduction of the Warburg metabolism in CSC. Thus, these results indicate that CSC and ESC share similar metabolic pathways (Warburg effect) and that this shift is mediated by β3-AR. The extrusion of lactate, promoted by β3-AR, enhanced the similarities between cancer and embryonic environment. During embryo development, the dramatic production and extracellular transport of lactate reduce the pH in the microenvironment, promoting the disaggregation of uterine tissues and facilitating the trophoblast invasion [Selective  Figures . To veri Figures . Suppleminvasion . The lacβ3-AR's role has been well clarified in white and brown adipocytes. Selective, pharmacological activation of β3-AR has been shown to have strong effects on adipose tissue morphology and metabolism. The sympathetic nervous system, through β3-AR stimulations, is the main trigger of UCP1 induction and activation in brown fat mitochondria, leading to uncoupling of respiration and adaptive thermogenesis. UCP1, localized on the inner mitochondrial membrane, uncouples the activity of the respiratory chain from ATP synthesis, thereby releasing energy as heat [β3-AR agonist, leads to a marked increase in thermogenesis in brown adipose tissue (BAT) and an acute decrease in food consumption [β3-AR-induced Warburg effect, we used SR 59230A (specific β3-AR antagonist) and genipin (specific UCP2 inhibitor) under BRL37344 stimulations, in both CSC and ESC. UCP2 Western blotting expression analysis and lactate export assay at different concentrations of genipin were performed to evaluate which of them could have a similar effect to SR59230A treatment  in the culture media recovered cancer cell death mediated by SR59230A and genipin in CSC (Figures β3-AR dramatically increased A375 cell death compared to embryonic stem cells, demonstrating a strong selectivity for cancer cells. SR59230A, by inhibiting the β3-AR/UCP2 axis, strongly increases mtROS levels mainly in CSC, due to their inability to maintain antioxidant response compared to ESC. These results confirm data already present in literature, where β3-ARs and UCP2 are indicated to have a strong antioxidant role [β3-ARs and UCP2 in both cancer and embryonic stem cells is not surprising. Both types of cells need a strong ability to proliferate and strong protection from reactive oxygen species and present a highly β3-AR expression.The addition of the ectopic antioxidant  Figures . These dant role . The expThis result is in line with previous studies demonstrating a potent anticancer agent of the specific UCP2 inhibitor genipin . Our datβ3-AR/UCP2 axis promotes mitochondrial dormancy by inhibiting ATP production and mtROS content and leading both cell lines to increase aerobic glycolysis. Moreover, β3-AR antagonism promotes mitochondrial reactivation by inhibiting UCP2 activity and by increasing mtROS content.Recently, it has been shown that CSC have a markedly glycolytic profile with a decreased or dormant mitochondrial function, although the CSC phenotype can change between different cancer subtypes , 40. OurThere is accumulating evidence supporting a direct link between mitochondria, oxidative stress, and cell death. With the increasing number of newly discovered protein sequences in the postgenomic era, computational methods are required to address large-scale proteomics data and to establish new lines of experimental inquiry. Therefore, predicting subcellular location using computational tools has become a valuable approach for experimental validation.At present, several public subcellular localization predictors are available. In our study, we chose four widely used methods: Hum-mPLoc 3.0, FUEL-mLoc, WegoLoc, and COMPARTMENTS. By unifying this information with evidence on protein localization from curated knowledge, high-throughput experiments, text mining, and sequence-based prediction methods, these tools infer on the subcellular localization of a protein.Z-score computed by COMPARTMENTS, which designates the mitochondrion as the second best hit (Supplementary In order to support our previous observation, we performed a bioinformatic analysis by using the aforementioned Web tools. The results shown in Supplementary β3-AR is expressed on mitochondria of both ESC (Supplementary Surprisingly, we found that β3-AR/UCP2-mediated Warburg metabolism and enhanced the different ability of the two cell lines to respond to SR59230A that induced cell death only in CSC by inhibiting antioxidant response (β3-AR in the mitochondria, suggesting a possible cooperation with β3-AR located in the plasmatic membrane. We suggest that β3-AR could work as sensor of redox state of cells directly influencing mitochondria bioenergetic functions. The presence of β3-AR in isolated mitochondria could be explained by its protective role against the induction of apoptosis, in both cancer and embryonic stem cells. Here, we clearly demonstrated a functional role of β3-AR blockade in isolated mitochondria by its ability to decrease ATP production and to increase mtROS levels.In summary, this study highlighted the parallelism between embryonic and cancer stem cells in the regulation of the response . An impoβ3-AR in mitochondria, but the results of this study have shown a new and different possible function of this receptor in a new compartment.Further experiments need to be performed to better clarify and elucidate the role of Finally, we report that SR59230A is extremely selective in reducing the viability of CSC by blocking the Warburg metabolism and by inducing high mtROS levels. These results suggest a potential role of SR59230A as a strong and selective agent that could be used in cancer therapy.


Supplemental Digital Content is available in the text The co-existence of allergic rhinitis (AR) and obstructive sleep apnea (OSA) is a common phenomenon in clinical practice. AR has long been considered a risk factor for OSA. However, the relationship is not completely clear. Therefore, we conducted a meta-analysis to evaluate the prevalence of AR in sleep-disordered breathing (SDB) /OSA and their relationship.P < .05 and I2 > 50% indicating evidence of heterogeneity.A comprehensive literature search was performed in PubMed/Medline, Google Scholar, Wiley Online Library, EMBASE, and Web of Science. Data were analyzed and pooled to estimate effect size (ES) /odds ratio (OR) with 95% confidence intervals (95%CI). Heterogeneity was quantified and evaluated by chi-squared-based Q-test and I2 test, with P < .0001) times higher than that of non-SDB pediatric patients. There were no significant differences between OSA adults with or without AR in BMI (Body Mass Index), neck circumference, apnea hypopnea index (AHI) and epworth sleep scale score (ESS).44 studies contained 6086 participants were included in this meta-analysis. For adults, the prevalence of AR was 22.8  % in SDB and 35.2  % in OSA. In children with SDB and OSA, the prevalence of AR was 40.8  %, and 45.2  % respectively. The odds ratios of prevalence of the SDB pediatric patients with AR was 2.12 (95%CI, 1.75, 2.57; The prevalence of AR in OSA/SDB is considerably high and children with SDB suffering from a higher incidence of AR than non-SDB. OSA adults accompanied with AR do not have any influences on sleep parameters. It disrupts normal ventilation and patterns during sleep. Moreover, these breathing disorders may increase the risks of complications of cardiovascular, neurocognitive, and metabolic morbidities. Allergic diseases, allergic rhinitis (AR), asthma, and eczema are common among individuals. AR is a very common disease that affects 10% to 40% of the global population. With the increasing exposure to allergens and pollutants, the prevalence of AR has increased over the past few decades. Since it usually leads to nasal obstruction and increased upper airway resistance, AR has long been recognized as a risk factor of OSA in previous studies.–10 Some articles have reviewed the association between AR and SDB in children. However, no meta-analysis was obtained from databases. Therefore, in view of this scenario, we conducted a meta-analysis to explore and summarize the prevalence and association between OSA and AR in order to gain a deeper insight of these 2 diseases.Obstructive sleep apnea (OSA) is the most common sleep-disordered breathing (SDB). The prevalence of OSA in healthy children and adults was as high as 1% to 5% and 3.5% to 20.4%, respectively, and it was even higher for SDB.2We performed this meta-analysis in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement.2.1In accordance with the PRISMA guidelines, we identified relevant research articles through a systematic review of scientific databases . The MeSH and keywords used in different logical combinations and phrases were: allergic rhinitis, allergic rhino conjunctivitis, hay fever, nasal allergy, OSA, apnea, hypopnea, Epworth Sleepiness Scale (ESS), apnea-hypopnea index (AHI), SDB, sleep-associated breathing disorder, sleep-related disordered breathing, snoring. The search encompassed original research papers published by July 1, 2017 in online journals in English language.2.2Inclusion criterion was clinical or epidemiological studies which examined the relationship between AR and sleep disorders and reported the prevalence of AR in OSA or SDB patients. Studies were excluded from the meta-analysis if reported only the sleep quality measures other than OSA or SDB, or provided qualitative information only. Other studies that were excluded include review article, conference abstract, article not published in English, animal study, case report, article with no abstract/full text available.2.3,12 and SDB  patients, body mass index (BMI), neck circumference, AHI and ESS score, and other relevant information were obtained from the selected research articles of the respective studies and organized on data sheets. To ensure the quality of the meta-analysis, all eligible publications were reviewed by 2 researchers independently according to the standardized approach and later cross checked the work of each other. The final selection of a study for inclusion in the meta-analysis was reached in consensus.The following data were extracted from each eligible study: participants’ demographic and clinical characteristics, the prevalence of AR in OSA (diagnosed with sleep studies according to guidelines),2.4Q-test and I2 test, with P < .05 and I2 > 50% indicating evidence of heterogeneity.Random effects meta-analyses were performed with STATA 12.0 (Stata Inc. Texas) to achieve overall effect sizes of the prevalence of AR in OSA and SDB patients and to achieve a summary estimate of the odds ratio of the prevalence of AR between OSA/SDB and non-OSA/SDB patients observed in the individual studies. The significance of differences in BMI, neck circumference, AHI, and ESS between OSA/SDB patients with and without AR were carried out by STATA 12.0 with under random effects model. Heterogeneity was quantified and evaluated by the chi-squared-based 3,13–56 (6086 patients) which fulfilled the eligibility criteria  times higher in SDB patients than in non-SDB patients , neck circumference , AHI , or ESS .Data were acquired from 44 studies ria Fig.  A. In chria Fig.  B. The o4–59 Rhinorrhea, nasal blockage, or congestion always led to stuffy nose, which were the most complained symptoms of AR patients. The nasal obstruction may gradually increase, resulting in daytime fatigue, sleepiness, and performance decrements. In addition, symptoms brought by AR such as apnea and snoring were also considered to be risk factors for sleep-disordered breathing events and contributed to the development of OSA. In addition, some studies had demonstrated that several chemical mediators and inflammatory cytokines play interaction roles between AR and OSA, including histamine, cysteinyl leukotrienes (cysLTs), interleukin-1β(IL-1β), and interleukin-1 (IL-4) and so on.,62 Given the impact of AR to OSA, it seemed logical to investigate their relationship which may help understand overlapped subjects.Nasal obstruction had long been considered as one of the leading risk facts for the upper airway obstruction during the sleep. Further, some clinical studies had found that patients with nasal congestion caused by AR were more susceptible to disturbed sleep. Adenotonsillar hypertrophy (AH) was a common comorbidity of pediatric AR, it was reported that 92.6% of AR children also suffered AH . Furthermore, AH is the main cause of OSAS for children aged 3 to 6, yet adults patients were often caused by obesity. This meta-analysis showed no significant difference in the prevalence of AR in OSA and non-OSA patients (children and adults). This would lead to widely underdiagnosis of OSA in group of patients in clinical practice.,66 Although weight and neck circumference had been shown to be good predictors for OSA in epidemiologic studies, our study suggested that there was not enough evidence that OSA adults patients coexist AR were related to individuals’ BMI, neck circumference, ESS, and AHI. Additionally, Francesco and Alvarez reported that AR is not an aggravating factor regarding the severity of AHI in children. The relationship between the OSA and AR is remaining a long-standing controversy. In traditional view, AR was considered as a potential risk factor for OSA. However, Kramer et al revealed that AR did not influence sleeping parameters of OSA. They found no statistically significant difference in sleeping behavior or polysomnography (PSG) parameters between AR and non-AR patients. Recent studies had also revealed AR only had effect on symptoms, but did not affect PSG results for OSA patients nor belong to risk factors for OSA.,17 Similarly, a demographic study conducted in OSA children reported that allergic rhinitis did not contribute to sleep disordered breathing in Australian. Intranasal corticosteroids (INCS) were generally considered as the most effective agent in relieving nasal symptoms of allergic rhinitis. Therefore, it was believed that the effectiveness of INCS in relieving nasal congestion may have a positive effect on SDB. A randomized, controlled trial of OSA children aged 6 to 18 years old demonstrated that intranasal mometasone furoate effectively improved obstructive apnea hypopnea index and oxygen desaturation index. Beyond that, Lavigne et al found that INCS not only reduced upper airway inflammation but also improved OSA morbidity in patients with concomitant AR. A meta-analysis showed that patients receiving INCS had a better effect on decreasing the AHI; however, with limited evidences. In general, continuous positive airway pressure (CPAP) was the preferred therapy for OSA, whereas, INCS did not alleviate nasal symptoms during CPAP treatment in OSA patients. Hence, further research should be performed on INCS in these 2 entities.In this meta-analysis, we reported that the prevalence of children diagnosed with AR is 2.12 times higher in SDB patients than that of non-SDB patients. However, we did not find this tendency in adults, due to immaturity of immune system in children and the studies’ bias. AR was one of the most common chronic diseases in children. Children's immune system developed around the age of 2-year old. During this time, they were more likely to acquire Type I hypersensitivity which reflecting more T helper 2 lymphocyte (Th2) and consequently immunoglobulin E (IgE) driven response to allergen exposure.In conclusion, patients with AR were more likely to become habitual snorers, and had increasing risk of SDB and the SDB children suffered from a higher incidence of AR. Physicians may need a comprehensive understanding of the overlapping disorders before making a reasonable therapeutic strategy.5This meta-analysis revealed that the prevalence of AR in adult SDB/OSA patients was 23%, and 35%, and in children SDB/OSA patients 41%, and 45%, respectively. The odds of having AR were 2.12 times higher in SDB than that of non-SDB children patients (significantly). However, there was no significant difference between OSA patients and those who suffered AR and OSA in neck circumference simultaneously BMI, AHI, or ESS. Whether AR is a risk event of OSA need further consideration, meanwhile, patients with SDB should be cautiously focused especially accompanied with nasal symptoms.Conceptualization: Yuan Cao, Qiao Li.Data curation: Yuan Cao, Liyu Zhang.Formal analysis: Yuan Cao, Ying Yang.Funding acquisition: Liyu Zhang, Sancheng Cao.Writing – original draft: Shuang Wu.Writing – review & editing: Qiao Li.Qiao Li: 0000-0002-2575-7005.QIAO LI orcid: 0000-0002-2575-7005.


E,1'E)--)bis(methanylylidene))bis-1,4-phenylene))bis(methanylyl-idene))-bis(azanylylidene))dianiline (OIC1MS) and 7,7'---bis(azanylylidene))bis(methanylylidene))bis-1,4-phenylene))bis- (methanylylidene))bis(azanylylidene))bis(9H-fluoren-2-amine) (OIC2MS) were prepared by means of conventional and mechanochemical synthesis and characterized by FT-IR, 1H- and 13C-NMR techniques. The optical properties of the compounds were studied in solution by using UV-visible spectroscopy, and the optical effects were analyzed as a function of solvent. The results show that OIC2MS exhibits interesting photochromic properties. Furthermore, the structural and electronic properties of the compounds were analyzed by TD-DFT. It was found that the mechanosynthesis is an efficient method for the synthesis of both tetraimines.Phenyleneimine oligomers 4,4'-(((1 Experimental. % C = 79.85, % H = 7.99, % N = 6.44. FT-IR/ATR ν (cm−1): 3465, 3329, 3007, 2926, 2854, 1615, 1589, 1491; Raman [cm−1]: 1556, 1584; 1H-NMR (CDCl3) ppm: 9.03 , 8.0–6.50 , 4.16–3.54 , , , 1.97–1.17 , 1.63–1.44 , 0.87 ; 13C-NMR (CDCl3) ppm: 152.15–151.52 (C=N),144.12 (Ar), 142.51 (Ar), 127.21 (Ar), 121.59 (Ar), 121.05 (Ar), 115.68 (Ar), 114.53 (Ar), 109.16 (Ar), 68.13 (CH2α-O), 30.77 (-CH2-), 28.24 (-CH2-), 25.13 (-CH2-), 21.65 (-CH2-), 13.10 (–CH3); MS (m/z): [M-726], [M-923], [M-1100].In a 100 mL bottom flask, 2,5-bis(octyloxy)terephtaldehyde  and 1,4-phenylendiamine  or 2,5-diaminofluorene  were dissolved in toluene (20 mL). The reaction mixture was kept under an argon atmosphere with magnetic stirring, and heated at reflux for 24 h. The crude product was purified by precipitation using hexane.4,4'---)bis(methanylylidene))bis-1,4-phenylene))bis(methanylylidene))bis(azanylylidene))dianiline (OIC1): Yield 87%; orange powder; Td 152 °C; Elemental Analysis: C66H92N6O4; Calculated % C = 76.70, % H = 8.97, % N = 8.13. Observed. % C = 76.68, % H = 8.94, % N = 8.0. FT-IR/ATR ν (cm−1): 3316, 3204, 3028, 2926, 2855; 1610, 1583, 1509; Raman (cm−1): 1555, 1578; 1H-NMR (CDCl3) ppm: 8.9 , 7.82–6.50 , 4.20 , 3.71 , 1.92–1.20 , 0.89 ; 13C-NMR (CDCl3) ppm: 152.15–151.52 (C=N), 144.12 (Ar), 142.51 (Ar), 127.21 (Ar), 121.59 (Ar), 121.05 (Ar), 115.68 (Ar), 114.53 (Ar), 109.16 (Ar), 68.13 (CH2α-O), 30.77 (-CH2-), 28.24 (-CH2-), 25.13 (-CH2-), 21.65 (-CH2-), 13.10 (–CH3); MS (m/z): 1033 [M+].7,7'---bis(azanylylidene))bis(methanylylidene))bis-1,4-phenylene))bis(methanylylidene))bis(azanylylidene))bis(9H-fluoren-2-amine) (OIC2): Yield 81%; orange powder; Td 242 °C; FT-IR/ATR ν (cm−1): 3465, 3329, 3007, 2926, 2854, 1615, 1589, 1491; Raman [cm−1]: 1556, 1584; 1H-NMR (CDCl3) ppm: 9.03 , 8.0–6.50 , 4.16–3.54 , , , 1.97–1.17 , 1.63–1.44 , 0.87 ; 13C-NMR (CDCl3) ppm: 152.15–151.52 (C=N), 144.12 (Ar), 142.51 (Ar), 127.21 (Ar), 121.59 (Ar), 121.05 (Ar), 115.68 (Ar), 114.53 (Ar), 109.16 (Ar), 68.13 (CH2α-O), 30.77 (-CH2-), 28.24 (-CH2-), 25.13 (-CH2-), 21.65 (-CH2-), 13.10 (–CH3); MS (m/z): [M-726], [M-923], [M-1100].OIC2MS exhibits photochromic properties compared to OIC1MS. This is probably caused by reversible transformations between isomers that absorb at different wavelengths. These results were confirmed with DFT theoretical simulations.In the present work, mechanosynthesis is shown to be an efficient method for the synthesis of the phenyleneimine pentamers as it requires less time than the conventional method, and no solvent or inert atmosphere is used for the synthesis. The product


Balloon post-dilatation (BPD) is often needed for optimizing transcatheterheart valve (THV) implantation, since paravalvular leak (PVL) aftertranscatheter aortic valve implantation is associated with poor outcome andmortality. Quantitative assessment of PVL severity before and after BPD ismandatory to properly assess PVL, thus improving implantation results andoutcomes.To investigate a quantitative angiographic assessment of aortic regurgitation(AR) by videodensitometry before and after BPD.Videodensitometric-AR assessments (VD-AR) before and after BPD were analysedin 61 cases.VD-AR decreased significantly from 24.0[18.0-30.5]% to 12.0[5.5-19.0]% .The relative delta of VD-AR after BPD ranged from -100% (improvement) to+40% (deterioration) and its median value was -46.2%. The frequency ofimprovement, no change, and deterioration were 70% (n = 43), 25% (n = 15)and 5% (n = 3), respectively. Significant AR (VD-AR > 17%) was observedin 47 patients (77%) before and in 19 patients (31%) after BPD.VD-AR after THV implantation provides a quantitative assessment of post-TAVIregurgitation and can help in the decision-making process on performing BPDand in determining its efficacy. We have previously reported the in vitroand in vivo validation of quantitative angiographic assessment ofaortic regurgitation (AR) by videodensitometry technique after implantation of THVwith an excellent reproducibility and accuracy.12 This technique provides an accurate assessment of theseverity of PVL and it has been shown that a Videodensitometric-AR (VD-AR) > 17%correlates with increased mortality and impaired reverse cardiac remodelling asdetermined by echocardiography after TAVI.13,14 This prognostic cut-off value (VD-AR > 17%) could have thepotential to guide operators in deciding the need for BPD. However, the change ofVD-AR from before to after BPD has not been investigated. The aim of this study isto assess a quantitative aortographic approach of PVL by videodensitometry beforeand after BPD.The Valve Academic Research Consortium-2 (VARC-2) consensus document recommends toperform quantitative and semi-quantitative hemodynamic assessments of PVL severityand other definitions for valve failure than mild PVL only.15The study protocol was approved by the ethics committee at each of theparticipating centers and all patients provided informed written consent. Threehundred ninety-nine patients were enrolled in the Brazilian TAVI registry inthat period. VD-AR was performed and found to be analysable in 228patients.16 In thispopulation, 102 patients underwent BPD, and in 17 cases, no angiography wasavailable before BPD. Out of 85 cases with available aortograms before and afterBPD, VD-AR was analysable at both time points in 61 cases  or within thedistal third of the prosthetic valve stent (in case of a self-expanding device).The decision on the total contrast volume and speed of injection, catheter size,and the projection were left to the discretion of the operators. Visualassessment of AR was performed by experienced observers based on Sellers’grade.12-14,16,18 After drawing the contours of the aortic root(i.e. reference region) and the subaortic one third of theleft ventricle (i.e. region of interest [ROI]), the contrast time-density curveswere generated for both regions over at least three cardiac cycles aftercontrast injection. The areas under these curves (AUC) are automaticallycalculated and represent the time-density integral. VD-AR is automaticallycalculated as the ratio of the AUC of the ROI to that of the reference region by experienced observers using a dedicated software . The details of thistechnique have been described elsewhere.e region. TheoretMultislice computed tomography (MSCT) was performed following the localradiological protocol. Cover index was calculated as “ /  × 100”. Thepost-dilatation balloon size / annulus diameter ratio was calculated as“ / × 100”.19If they were not normally distributed, median and inter-quartile range [IQR]were used. Mann-Whitney test was used to compare continuous variables betweenindependent samples. Wilcoxon signed ranks test was performed to compare theserial changes between before and after BPD. All analyses were performed withSPSS 23 . A two-tailed p < 0.05 defined the statisticalsignificance.When continuous variables were normally distributed, we summarized data as mean± standard deviation.Baseline characteristics and echocardiographic data of this population (n = 61) areshown in The change of VD-AR from before- to after- BPD is shown in The THV cover index and the balloon size used in post-dilatation were bothavailable in 38 out of 61 patients – 25 (66%) among those with significantlyimprovement of PVL after BPD, 11 (29%) with no change in VD-AR, and 2 (5%) withdeterioration of AR. THV cover index was 11.5 and ranged from 0.0% to22.8% in patients with a significant improvement of AR, and 13.8,ranging -29.0% to 19.3% in those with no change or a significant deteriorationof AR. Post-dilatation balloon size / annulus diameter ratio was 0.0and ranged from -25.0% to 14.3% in patients with a significant improvement ofAR, and 0.0, ranging from -33.3% to 16.4% in patients with no changeor with a significant deterioration of AR.Before BPD, AR was visually classified as Sellers’ III in 36 patients (59%), andas Sellers’ II in 25 patients (41%). After post-dilatation, there were 3 (5%)cases with Seller’ III, 19 (31%) cases with Sellers’ II, 34 (56%) cases withSellers’ I and 5 (8%) cases with Sellers’ 0. Out of 36 patients with Sellers’III before BPD, 34 patients had their Sellers’ grade reduced  in three patients .Before BPD, VD-AR > 17%, a value that has a prognostic significance inlong-term follow-up, was observed in 47 patients (77%). Fourteen cases (23%) hada VD-AR ≤ 17%, eleven  were evaluated as Sellers’ II beforeBPD and 3  as Sellers’ III. After BPD, VD-AR > 17% was observed in19 patients  – 3 patients (16%) in Seller’sIII, 10 patients (53%) in Sellers’ II, and 6 patients in Sellers’ I (32%) . In addiPredilatation was performed in 18 patients and had no impact on the reduction ofAR assessed by VD-AR. VD-AR was 25.5% (19.5%-36.0%) with predilatation and 23.0%(16.0%-29.0%) without predilatation (p = 0.159) before PBD, and 16.5%(9.5%-22.8%) with predilatation and 11.0% (5.0%-17.0%) without predilatation (p= 0.106) after PBD. Normalized delta VD-AR was -44.5 (-60.1 – -13.0) withpredilatation and -50.0(-75.0 – -17.9) without predilatation (p = 0.569).11,20This is the first study to report the value of VD-AR in assessing periproceduralchanges in AR. In clinical practice, echocardiogram and aortography are the standardtools to define the device success. As mentioned in the Valve Academic ResearchConsortium-2 (VARC-2) consensus document, quantitative and semi-quantitativehemodynamic assessment are recommended to assess AR severity by echocardiogram andmoderate-to-severe AR is defined as valve failure.8 reportedserial changes using semi-quantitative grading based on echocardiogram and showed areduction of at least 1 degree of AR in 71% of patients. To make a decision whetherBPD is needed or not, echocardiogram is an important tool to evaluate the severityof AR. However, we must consider that with the increasing minimalist TAVI approach,the usage of TEE as a guidance of TAVI is becoming unfeasible. Moreover, lowinter-observer agreement for the PVL 4-class grading (kappa 0.481) and the 7-classgrading (kappa 0.517) has been reported,21 making a more reliable technique necessary.18 These facts support the value ofaortography with VD assessment as the most practical and objective screening tool todetermine the severity of PVL during the procedure. The technique has a median timeof execution of 3 minutes.Nombela-Franco et al.13,14 This value (VD-AR > 17%) could be decisive in helping theoperator to make a decision as whether BPD should be performed during the procedure.When BPD was performed, we showed that before BPD, 77% of patients had a VD-AR >17%, and the other patients (VD-AR ≤ 17%) (23%) would not require BPD. Thisfinding is important, since BPD is associated with higher rate of cerebrovascularevents compared to the patients without BPD.8,22 Avoiding unnecessary BPD would possibly reduce the risk ofcerebrovascular events as well as procedural costs. Moreover, most cases of VD-AR≤ 17% before BPD were found in Sellers’ II, suggesting that the visualassessment of the Sellers’ classification could lead to unnecessary PBD.We have previously shown that a VD-AR > 17% correlates with increased mortalityand with impaired cardiac reverse remodelling as determined by echocardiographyafter TAVI with excellent reproducibility.14,16After BPD, VD-AR > 17% was still seen in 31% of patients. Based on currentavailable data, for patients with residual AR (VD-AR > 17%), additional measuresshould be taken. We found higher mortality in patients with VD-AR > 17% comparedto patients with VD-AR ≤ 17% during the follow-up (34% vs. 19%). Although thedifference in mortality was not significant (log rank p = 0.273) in this smallpopulation with BPD, a tendency for high mortality was previously reported inpatients with VD-AR > 17% in a large population.8 and could be due to prosthetic overexpansion withsecondary leaflet maladaptation and transvalvular regurgitation.23VD-AR deteriorated numerically in 11 patients, and this deterioration was significantin 3. This comes in agreement with previous studies which also reported ARdeterioration in a small proportion of patients after BPD,8,24 The mechanisms of regurgitationafter implantation of THV are multifactorial as, for example, calcification of thenative aortic annulus and left ventricular outflow tract (LVOT) and cover index arewell known predicting factors of regurgitation after implantation of aTHV.19,26-34Serial changes of VD-AR showed predominantly improvement of AR. A reduction of theregurgitation by BPD was reported in 68%-91% in the literature.34,35 The mechanisms of AKI followingTAVI are multifactorial, and the role of the contrast medium volume iscontroversial.36 However,there is some evidence suggesting that a larger contrast volume is related to anincreased risk of AKI after TAVI.34,37Taking into account the important role of aortography in the minimalist TAVI era,repeated aortograms cannot be avoided. However, the possibility of reducing contrastmedium is reported using a diastolic phase-synchronized injection of only 8 ml ofcontrast medium in an in-vitro setting.12 This technique could enable the reduction of thetotal amount of contrast medium during the procedure.To make a decision whether BPD is needed or not and to judge its efficiency, repeatedinjections of large doses of contrast medium would be needed. Contrast medium volumeused in this population was 150[131-209] ml/procedure. In the setting of TAVI,peri-procedural acute kidney injury (AKI) develops in 12% to 57% of cases andportends a significant increase in early and late mortality.38 However, the effect of theguidewire on AR during TAVI is variable according to the weight of the wire.Most operators decide whether to perform BPD with or without a guidewire in LVby using echocardiography and aortography. Indeed, in the present study, VD-ARbefore BPD was analysed either with (n = 49) or without (n = 12) the guidewirebeing left in the left ventricle.After implantation of the THV, the guidewire is frequently left in the leftventricle and may produce artificial transvalvular regurgitation.39 reported how to plan anoverlap free projection for VD-AR assessment.39 A dedicated acquisition protocol would achievea high feasibility of assessment. We tried to overcome this limitation bychoosing the cases that did had an adequate acquisition of images, lowering oursample size. However, a prospective clinical study is needed to confirm thishypothetical assumption. So far, CAAS-A-valve software is available as anoffline system. Currently, attempts are being made to allow onlineassessment.40 In thenear future, online system will probably foster the VD-AR as guidance forTAVI.One limitation of our study is the absence of data on aortic regurgitation index,thus lacking the possibility of comparing this to our method. Limitations ofVD-AR assessment are its feasibility. The current report is a retrospectivestudy so that the acquisition of aortography was not dedicated for VD-ARassessment. In order to perform videodensitometric assessment appropriately, theacquisition of aortography should be done without overlapping ROI with contrastfilled ascending/descending aorta. Recently, Teng et al.In this registry, no echocardiographic parameters recorded were reported afterTHV deployment but before BPD. The information of calcification of the nativeaortic valve, annulus and LVOT from computed tomography were not available.VD-AR after THV implantation enables the operator to assess quantitativelyregurgitation, to rationalise BPD and to assess its efficacy.


Expression of androgen receptor (AR) splice variant 7 (AR-V7) has been identified as the mechanism associated with the development of castration-resistant prostate cancer (CRPC). However, a potential link between AR-V7 expression and resistance to taxanes, such as docetaxel or cabazitaxel, has not been unequivocally demonstrated. To address this, we used LNCaP95-DR cells, which express AR-V7 and exhibit resistance to enzalutamide and docetaxel. Interestingly, LNCaP95-DR cells showed cross-resistance to cabazitaxel. Furthermore, these cells had increased levels of P-glycoprotein (P-gp) and their sensitivity to both docetaxel and cabazitaxel was restored through treatment with tariquidar, a P-gp antagonist. Results generated demonstrated that P-gp mediated cross-resistance between docetaxel and cabazitaxel. Although the LNCaP95-DR cells had increased expression of AR-V7 and its target genes , the knockdown of AR-V7 did not restore sensitivity to docetaxel or cabazitaxel. However, despite resistance to docetaxel and carbazitaxel, EPI-002, an antagonist of the AR amino-terminal domain (NTD), had an inhibitory effect on the proliferation of LNCaP95-DR cells, which was similar to that achieved with the parental LNCaP95 cells. On the other hand, enzalutamide had no effect on the proliferation of either cell line. In conclusion, our results suggested that EPI-002 may be an option for the treatment of AR-V7-driven CRPC, which is resistant to taxanes. The primary effective treatment for most recurring prostate cancer (PC) is androgen deprivation therapy (ADT). Although initially effective, the malignancy will eventually form castration-resistant prostate cancer (CRPC) . Current2. The cell line DU145 was cultured in DMEM supplemented with 10% FBS, PC-3 in RPMI 1640 with 10% FBS, LNCaP in phenol red-free RPMI 1640 with 10% FBS, and LNCaP95 in phenol red-free RPMI 1640 with 10% charcoal stripped serum (CSS). A docetaxel-resistant cell line variant, LNCaP95-DR, was developed over a period of 6 months by exposure to gradually increased concentrations of docetaxel . A time-matched parental cell line, LNCaP95-C, was developed in a medium containing vehicle (DMSO) at the corresponding concentration. Finally, LNCaP95-DR cells were maintained in medium containing 15 nM docetaxel.Prostate cancer cell lines, DU145, PC3, and LNCaP were purchased from the American Type Culture Collection (ATCC). LNCaP95 was a generous gift from Dr. Jun O. Luo . Cells were authenticated by short tandem repeat analysis  and then tested by DDC Medical  in April 2018, to ensure that the cells were mycoplasma-free. Cells were maintained as monolayer cultures at 37 °C and 5% COThe effect of drugs on cell proliferation was assessed using the Premix WST-1 Cell Proliferation Assay System (Takara Bio) according to the manufacturer’s protocol. Cell viability was normalized to the viability of vehicle-treated control cells (DMSO). BrdU ELISA was performed to evaluate the inhibitory effect of EPI-002 on the proliferation of cells. LNCaP95-P, LNCaP95-C, and LNCaP95-DR cells were treated with vehicle (DMSO) or EPI-002 for 48 h, and BrdU incorporation was measured using the BrdU ELISA kit .Western blots were performed as previously described in Reference . The priTotal RNA was isolated using the RNAqueous Total RNA Isolation Kit  and it was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit . Real-time RT-qPCR was performed in triplicate for each biological sample. Transcript levels for each gene were normalized to levels of the GAPDH transcript. Primers were purchased from Applied Biosystems: AR (Hs00171172_m1), KLK3 (Hs02576345_m1), FKBP5 (Hs01561006_m1), UBE2C (Hs00964100_g1), CDC20 (Hs00426680_mH), GAPDH (Hs00266705_g1), AR-V7 .PSA (6.1kb)-luciferase reporter plasmid encodes nucleotides −6000/+12 relative to the transcription start site of the human PSA/KLK3 gene and it includes the PSA promoter, with AREII (−395 to 376) and AREI (−170 to −156), and enhancer regions with AREIII (−4148 to −4134), as described in References ,28. LNCaTM RNAiMAX  according to the manufacturer’s instructions. AR-V7 siRNAs (Silencer® Select siRNAs) were obtained from Life TechnologiesTM . The sense sequence of siRNA for AR-V7 was 5′-GUAGUUGUGAGUAUCAUGATT-3′.AR-V7 expression was transiently knocked down in LNCaP95-DR cells using Lipofectaminep < 0.05 was considered to indicate a statistically significant difference.Statistical analyses were performed using GraphPad Prism 7 . All experiments were performed in triplicates for each biological sample. Data for cell proliferation assays, real-time RT-qPCR, and luciferase assays were depicted as mean ± SD from 3 to 4 independent experiments. IC50 values and 95% confidence intervals (CI) were calculated using the nonlinear regression analysis of percentage inhibition. The comparison of LogIC50 was calculated using the extra sum-of-squares F-test. One-way ANOVA followed by a Sidak’s post hoc test was used to assess the difference between the data of real-time RT-qPCR and luciferase assay. To evaluate the inhibitory effect of docetaxel and cabazitaxel on prostate cancer cell lines, the MTT assay was performed A,B. LNCaConsistent with a known mechanism of acquired resistance to taxanes, P-gp was overexpressed in LNCaP95-DR cells as measured by the Western blot analysis A. To tesTo elucidate other potential contributing factors involved in the mechanism of taxane resistance and provide clues for possible intervention, we compared the levels of expression of several key genes in LNCaP95-DR cells using Western blot analysis and real-time RT-qPCR. LNCaP95-DR cells had higher levels of glucocorticoid receptor (GR), UBE2C, and phosphorylated S6 (pS6), but lower levels of BRN-2 proteins as compared to levels in LNCaP95-C C.Real-time RT-qPCR revealed that the transcript levels of FL-AR and its target gene KLK3 in LNCaP95-DR cells did not differ from those in LNCaP-C cells, whereas the transcript levels of AR-V7 and its target genes, UBE2C and CDC20, were all increased in LNCaP95-DR cells as compared to levels in LNCaP-C cells . InteresBRN2 is a transcription factor that is proposed to play a role in enzalutamide-induced neuroendocrine transdifferentiation . LNCaP95Glucocorticoid receptor (GR), is proposed to play a role in CRPC as an alternative steroid receptor for AR . AlthougAlterations in expression of components of the PI3K/Akt/mTOR occur in 42% of primary prostate tumors and 100% of metastatic tumors . Thus, tGiven the expression of AR-V7, and its target genes UBE2C and CDC20 being increased in LNCaP95-DR, we examined whether AR-V7 may contribute to the acquisition of resistance to taxanes and whether the targeting of AR-V7 might be a good intervention strategy. To test these we used two approaches, knockdown of AR-V7 and an inhibitor of the AR-Vs transcriptional activities. When AR-V7 expression was transiently knocked down in LNCaP95-DR cells using small interfering RNA (siRNA) A, prolifAR-NTD targeting drugs are a potential treatment strategy for CRPC represented by LNCaP95-DR cells which have acquired resistance to enzalutamide, docetaxel, and cabazitaxel. This is because AR-NTD is essential for the transcriptional activities exerted by both the FL-AR and AR-Vs. Thus, antagonists of AR-NTD, such as EPI-002, could have a therapeutic effect on LNCaP95 driven by AR-V7. Importantly, EPI-002 had an inhibitory effect on the proliferation of LNCaP95-DR cells that was similar to the effect measured with the parental LNCaP95 cells D. TogethAR-V7 is a major splice variant expressed in human prostate cancer that is associated with the development and progression of CRPC ,40. AR-VResistance to taxanes can be associated with the overexpression of P-gp ,17, whicCabazitaxel is a next-generation semisynthetic taxane chemotherapeutic agent that is effective in patients with docetaxel-resistant CRPC . In the Expression of AR-V7 is clinically important and has been proposed for the assessment of which patients should receive inhibitors of the androgen receptor or taxanes . In thisSome recent reports suggest that taxanes can inhibit AR signaling in prostate cancer cells ,52,53. HAR-NTD is essential for the transcriptional activities of both FL-AR and AR-Vs. Therefore, AR-NTD-targeting therapy has benefits over the drugs targeting the AR-LBD. EPI-002 targets the NTD of the AR and can block the signaling induced by the FL-AR and AR-Vs . In thisIn summary, we have demonstrated that docetaxel-resistant LNCaP95 cells are cross-resistant to cabazitaxel. We showed that resistance to docetaxel and cabazitaxel depended on the increased expression of P-gp and the inhibition of P-gp with tariquidar restored to docetaxel and cabazitaxel. Furthermore, expression of AR-V7-regulated genes was increased in LNCaP95-DR cells, although AR-V7 did not contribute to taxane resistance. Finally, EPI-002, an antagonist of AR-NTD, inhibited proliferation of LNCaP95-DR. In conclusion, the present study described a potential option for the treatment of docetaxel-resistant, AR-V7-driven CRPC.


P < 0.001), T classification (P = 0.002), and pN stage (P = 0.032). Kaplan–Meier curves revealed that low C6orf141 expression was significantly associated with shorter disease-specific survival (DSS) in patients with OSCC (log-rank P = 0.007). Multivariate analysis indicated that low C6orf141 expression was an independent prognostic biomarker for DSS . Additionally, ectopic C6orf141 expression could significantly suppress oral cancer cell proliferation, colony formation, and migratory and invasive abilities. Xenograft tumor growth assay revealed that C6orf141 could significantly suppress oral tumor growth in vivo. Our results suggest that C6orf141 plays a novel tumor-suppressive role in oral cancer cell growth and motility. Furthermore, C6orf141 dysfunction could be a potential prognostic biomarker for OSCC and provide new therapeutic strategies in the future.C6orf141 (Chromosome 6 open reading frame 141) is a novel gene, and its role in oral cancer progression remains unclear. C6orf141 expression in oral squamous cell carcinoma (OSCC) and adjacent normal tissues from 428 patients was examined through immunohistochemistry (IHC). Our results revealed that C6orf141 expression was significantly reduced in OSCC compared with adjacent normal tissues. Low C6orf141 expression was significantly associated with a poor American Joint Committee on Cancer pathological stage ( It is the fourth cause of cancer death in male individuals and the top cancer in young male adults (25–44 years old) in Taiwan4. More than 90% of oral cancers are classified as oral squamous cell carcinoma (OSCC), typically observed on the tongue, buccal mucosa, and lips5. Habitual substance use, betel quid chewing, tobacco smoking, and alcohol drinking were reported to be associated with increased incidence and mortality rates of oral cancer6. Despite considerable progress in cancer treatment and management, the mortality rate associated with patients with OSCC remains unchanged. In Taiwan, the 5-year overall survival rate of patients with OSCC has remained at approximately 50% for several decades4. Metastasis and local recurrence are major problems due to therapy failure. However, most patients with OSCC were reported to be diagnosed at an advanced stage, leading to a low 5-year survival rate3. Therefore, developing a favorable biomarker for diagnosis or diagnosis using human genome information and molecular technologies may help to improve the survival rate of patients with OSCC.Oral cancer is the sixth most common malignancy worldwide7. Gene dysfunctions are clinically attractive as candidate prognostic markers and therapeutic targets. Accumulating studies have identified several biomarkers for predicting OSCC progression, including moesin, Caspase 3, fibronectin 1, CD44, and SOX21-AS111. In our previous study, we identified several deregulated genes in oral cancer through a next-generation sequencing approach10. Among these dysfunctional genes, chromosome 6 open reading frame 141 (C6orf141) located at chromosome 6p12.3 was identified as being involved in deregulating OSCC. However, the role of C6orf141 in human cancer is unknown.Carcinogenesis is a complex multistep process in which genetic events within signal transduction pathways governing normal cellular physiology are quantitatively altered12. In addition, an analysis of The Cancer Genome Atlas (TCGA) database revealed that low expression levels of C6orf141 were associated with a poor survival rate in patients with breast cancer, endometrial cancer, and head and neck cancer12. However, the clinical effect of C6orf141 expression on OSCC remains largely unknown, and details of its role has not yet been fully elucidated. In this study, we examined the expression levels of C6orf141 in OSCC and adjacent normal tissues by using immunohistochemistry (IHC). Furthermore, we assessed the association between C6orf141 expression and clinical pathological features. We also assessed that effect of C6orf141 expression on cell growth and the invasion of OSCC cell lines.According to the Human Protein Atlas (HPA) database, C6orf141 was expressed in some human cancers, including cancers of the brain, colon, duodenum, endometrium, esophagus, gall bladder, skin, stomach, and testis12. The UCSC Genome Browser also reveals that C6orf141 can generate several noncoding RNA transcripts through alternative splicing  than corresponding adjacent normal tissues . Furthermore, the expression levels of C6orf141 expression was also significantly attenuated in adjacent normal tissues compared with normal uvula tissues , large tumor size , and advance N stage  in patients with OSCC, whereas a higher C6orf141 expression level was weakly correlated with older patients (P = 0.062). Additionally, C6orf141 expression in tumor tissues was significantly different among the BMSCC and TSCC (Table P < 0.001). Thus, a stratification analysis was performed according to the 2 different subsites. Our results reveal a significantly lower C6orf141 expression level along with advanced pathological stage  and large tumor size  in BMSCC, but not in TSCC , but the model indicated no significant with DFS  . The analysis also showed a borderline significant association of low C6orf141 expression with poor DSS in patients with TSCC  but no significant difference in DFS . Further multivariate analysis revealed that low C6orf141 expression was associated with poor DSS in patients with BMSCC , but the results indicated no correlation between DSS and TSCC  analysis. Based on this cutoff value, the patients were separated into two groups, which represented higher and lower C6orf141 expression in oral cancer. Our results showed that low C6orf14 expression was associated with an unfavorable DSS rate in patients with OSCC , the expression levels of C6orf141 could be silenced in breast cancer cell lines with ZNF217 (Zinc finger protein 217) overexpression13. ZNF217 was reported to play a crucial role in promoting breast cancer cell metastasis to bones14. Freier at al reported that 6.2% of patients with head and neck squamous cell carcinoma exhibited ZNF217 gene amplification15. These results suggest that C6orf141 downregulation might partially be a result of ZNF217 amplification in OSCC. In addition, through analyzing the GEO database, we observed that ectopic miR-34a and miR-335 expression could silence C6orf141 expression17. Previous studies have revealed that the expression levels of miR-335 were upregulated in the cancer-associated fibroblasts of patients with head and neck cancer. Furthermore, miR-335 expression could induce cancer cell motility through the suppression of phosphatase and tensin homolog signaling19. Perhaps, miR-335 suppressed cancer cell motility through inhibiting C6orf141 expression in OSCC. These potential mechanisms resulting in C6orf141 depletion in OSCC require further analysis in future works, including microRNA, and transcription factors.OSCC is an aggressive malignancy and results in cancer deaths for male individuals in Taiwan20. Together with some genes involved in tumor proliferation and inflammation, C6orf141 has been identified as significantly influenced by cetuximab21. Moreover, inhibition of OSCC development by C6orf141 may be achieved through the down-regulation of BRG1. C6orf141 was one of the downregulated genes in BRG1 knockdown cell lines. Previous studies have shown that BRG1 loss results in widespread changes in chromatin organization at regions including transcriptional start sites of cancer-associated genes and increased tumorigenic potential23.Little information for C6orf141 on the roles in tumor-related function has been identified. There are some possible mechanisms to elucidate why C6orf141 underexpression occurs in OSCC. Cetuximab  is a chimeric immunoglobulin (Ig) G1 monoclonal antibody directed against the epidermal growth factor receptor (EGFR). In combination with curative-intent radiotherapy, cetuximab has been reported to increase median survival in locally advanced head and neck carcinoma24. In multicellular organisms, the squared Pearson correlation coefficient (R2) ranges from 0.09 to 0.46. The low correlation between protein and mRNA levels results from translation efficiency, protein degradation, and mRNA stability. With these combined results, using mRNA to evaluate the clinical effects of C6orf141 in human cancer might result in some uncertainties. In the current study, we used protein levels of C6orf141 in OSCC to evaluate its clinical effects and observed that low C6orf141 was significantly associated with the prognosis of patients with OSCC. The specificity of aiti-C6orf141 antibody was confirmed using Western blotting (data not shown). Consistent with its clinical effects, ectopic C6orf141 expression could suppress oral cancer cell growth and invasion, implying that C6orf141 plays a tumor-suppressive role in OSCC progression. Previous studies revealed that the expression levels of tumor suppressor genes frequently were silenced with a DNA hypermethylated promoter25. In a human genome, a CpG-rich region is located upstream of C6orf141, and our results indicated that C6orf141 plays a tumor-suppressive role in oral cancer. Abe et al. also reported that promoter CpG islands of C6orf141 were methylated in neuroblastomas26. In addition, Jiang et al. reported that DNA methylation of C6orf141 has an interesting causal relationship with high-density lipoprotein changes27. Taken together, these results implied that the transcriptional activity of C6orf141 may be controlled through DNA methylation. We examined the relationships between the DNA methylation status of the CpG islands of C6orf141 and its expression levels. No methylation modification at the promoter regions of C6orf141 was observed in OSCC (data not shown). We also examined the expression levels of C6orf141 in oral cancer cells after 5-Aza-dC treatment. Results revealed that the expression levels of C6orf141 could not be increased in most oral cancer cell lines, except for SAS cells, after 5-Aza-dC treatment. Taken together, the results suggested that DNA methylation is not the major factor resulting in low C6orf141 expression in oral cancer progression.Herein, we first report that C6orf141 protein was significantly reduced in OSCC tissues compared with adjacent normal tissues. An analysis of the relevant databases indicated RNA levels of C6orf141 to be inconsistent in different human cancers. The expression levels of C6orf141 were determined to be significantly reduced in thyroid cancer and testicular cancer, whereas those of C6orf141 were determined to be increased in colon cancer and lung cancer (data not shown). Furthermore, the effects of C6orf141 expression on the survival curve of patients with cancer were noted to be inconsistent. Based on TCGA database, high C6orf141 expression was noted to be significantly correlated with poor survival rates in patients with liver cancer, pancreatic cancer, kidney cancer, and gastric cancer. In contrast, high C6orf141 expression was observed to be associated with a more favorable survival curve than that of low C6orf141 expression in patients with head and neck cancer, breast cancer, ovarian cancer, and endometrial cancer. These results indicate that C6orf141 might have a distinct biological function in different cancer types. Our data also demonstrate that the expression levels of the protein-coding transcript of C6orf141 were clearly different from those of the noncoding transcripts of C6orf141 in patients with OSCC Fig. . The datIn summary, we provide a novel insight that C6orf141 expression could suppress the growth and invasive abilities of OSCC cells. Furthermore, C6orf141 expression levels might serve as an unfavorable prognostic biomarker for patients with OSCC.In this study, the collection of clinical samples was approved by the Institutional Review Board (IRB) of Kaohsiung Veterans General Hospital (KVGH) (IRB number: VGHKS14-CT6-18). A total of 95 OSCC tissues and 31 corresponding adjacent normal tissues were collected from patients with OSCC, who provided informed consent and underwent surgical operations at the Department of Dentistry and the Department of Otorhinolaryngology of KVGH. The methods were carried out in accordance with the approved guidelines and all patients provided informed consent. The clinical and pathological information of the patients is summarized in Table Total RNA was extracted using the TRIzol reagent , in accordance with the instruction manual. Briefly, tissue samples were first homogenized in 1 mL of TRIzol reagent, and then proteins and DNA were extracted using 0.2 mL of chloroform. Finally, total RNA was precipitated with 0.6 mL of isopropanol.Tissue microarrays containing 428 paraffin-embedded oral SCC cell samples were selected; these comprised 183 samples of buccal mucosal SCC and 245 samples of tongue SCC. The clinical and pathological information of the patients is summarized in Table In this study, a Novolink max polymer detection system  was used for IHC analysis. The slides were deparaffinized in xylene and rehydrated in grade alcohol. Antigen retrieval was performed by immersion in Tris-EDTA  for 10 minutes at 125 °C in a pressure boiler. Endogenous peroxidase activity was blocked through incubating the slides for 30 minutes with protein blocks . After blocking at room temperature (RT), primary antibodies were immediately applied and slides were incubated overnight at 4 °C in a wet chamber. The primary antibody used in this study was rabbit polyclonal anti-C6orf141 , in primary antibody diluent . After being washed in phosphate-buffered saline, the slides were incubated with a horseradish peroxidase-labeled secondary antibody for 10 minutes at RT, and the sections were counterstained with hematoxylin. For the antibody control studies, the serial sections were treated with phosphate-buffered saline, normal mouse lgG, normal rabbit IgG, and normal goat IgG, instead of the primary antibodies, and were confirmed to be unstained.At the beginning of the analysis, a senior pathologist accompanied a technician to evaluate the slides until all discrepancies were resolved. Then, the technician independently reviewed all slides. Finally, 5%20% core samples at each staining intensity were randomly selected for re-evaluation by the senior pathologist. During the evaluation, both the senior pathologist and the technician were unaware of the clinical outcomes of the patients. We graded the immunoreactivity through a semiquantitative approach. Marker scores for nuclear and cytoplasmic staining were calculated based on staining intensity  and on the proportion of positively stained tumor cells in 5 high-power fields . The marker score was the sum of the staining intensity score and the percentage of positive tumor cell score. For survival analysis, C6orf141 expression levels were dichotomized as low expression and high expression with the cutoff set at the 50th percentile. The expression scores had an overall median score of 5.00 , for C6orf141.2 at 37 °C.Eight human oral cancer cell lines include three tongue SCC,  and four buccal SCC cells , a gingival SCC cell (CA9-22), and a normal epithelia cells (DOK) were used in this study. The cells were maintained in Dulbecco modified Eagle medium , supplemented with 10% fetal bovine serum  and penicillin–streptomycin  in a humidified atmosphere containing 5% COFor reverse transcription polymerase chain reaction (PCR), 2 μg of total RNA was reverse transcribed using random primers and SuperScript III Reverse Transcriptase, according to the manufacturer instructions . Gene expression was detected using an SYBR Green I assay , and C6orf141 expression was normalized to that of Glyceraldehyde 3-phosphate dehydrogenase . The individual primers were shown in Supplementary Table The full length of C6orf141 was synthesized and digested with the restriction enzyme EcoRI and SpeI. Then, the full length of C6orf141 was cloned into the pLVX-IRES-Neo vector . Stable SAS cells with C6orf141 expression were generated by transfecting oral cancer SAS cells with pLVX-C6orf141 for 48 h followed by G418 (1200 μg/mL) selection for 14 days. Subsequently, overexpression efficiency was evaluated through real-time PCR.Small interfering RNA (siRNA) oligonucleotides targeting C6orf141 (si-sense: 5′-CUAAUGAGUAGCUCGAGAAdTdT-3′ and antisense: 5′-UUCUCGTGCUACUCAUUAGdTdT-3′) and a scrambled oligonucleotide as a negative control were designed and synthesized by SIGMA . Oral cancer cells were transfected with a 10 mM  siC6orf141 or scrambled control using Lipofectamine RNAiMAX . After transfection for 24 h, knockdown efficiency was evaluated through real-time PCR.2 incubator at 37 °C for 2 weeks until colony formation. The cells were then fixed and stained with crystal violet solution   for 20 minutes at RT. Subsequently, the colony formation was determined using a microscope . For the cell proliferation assay, 1.5 × 103 SAS, TW1.5, TW2.6 and Ca9-22 cells transfected with C6orf141 expression vector, siC6orf141 or a scramble control, were seeded in a 96-well plate. Proliferation was determined at 0, 1, 2, 3, and 4 days using the CellTiter-Glo One Solution assay, according to the manufacturer’s instructions . All experiments were independently repeated 3 times.For the clonogenic assay, a gradient number of SAS cells  were seeded in 6-well plates and transfected with a C6orf141 construct or a scramble control as aforementioned. The cells were incubated in a CO5 were resuspended in DMEM  with 2% FBS supplemented with penicillin–streptomycin . The cells were then added to the upper chamber of the Transwells  without Matrigel  for the migration assay or Matrigel coating for the invasion assay. Subsequently, DMEM  supplemented with 10% FBS  and penicillin–streptomycin  was added to the lower chambers for the invasion assay. The chambers were incubated in a CO2 incubator at 37 °C for 24 or 48 hours. The remaining cells in the upper chamber were then removed using cotton swabs, and the cells under the surface of the Transwell plates were fixed with 4% formaldehyde solution for 10 minutes at RT. The cells were stained with crystal violet solution   for 20 minutes at RT, and the oral cancer cells in the 3 fields of view were counted through phase-contrast microscopy. All experiments were repeated 3 times.The oral cancer cells were analyzed using Transwell assays. Briefly, transfected cells at a density of 1.0 × 106 SAS cells with C6orf141 stable expression or control cells were suspended in PBS and implanted on the backs of nude mice. Animal were observed, and tumor volume was evaluated as V (in mm3) = largest length × 0.52 × (shortest length)2 each week for 4 weeks. At 4 weeks after implantation, the animals were sacrificed, and tumor size and weight were assessed. All research was performed in accordance with relevant guidelines/regulationsAnimal experiments were approved by the Kaohsiung Veterans Hospital Laboratory Animal Center and Use Committee. Nude mice (4 weeks old) were used in this study; 2 × 10SAS cells with C6orf141 overexpression and control cells were used as total RNA sources. The RNA samples were first examined with TapeStation 4200 to ensure that their RNA integrity number (RIN) values reached up to 7, followed by preparation with WT PLUS reagent . Then, the prepared samples were hybridized on human Clariom D microarray chips  and scanned using a GeneChip Scanner 3000 7 G . The microarray raw data passing quality control were analyzed using Partek  to identify differentially expressed genes (p < 0.05). As a result, we identified 2,306 significant genes, as illustrated in the heat map figure, among which 1,130 and 1,176 genes were upregulated and downregulated, respectively. All microarray data were submitted to the NCBI GEO and are freely available with the accession number GSE123456. We determined whether the functions of the C6orf141-regulated genes were involved by investigating the pathways. The differentially expressed genes were selected from the microarray data; subsequently, candidate genes were mapped onto Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.6 cells were collected and mixed with 70% ethanol in a fixative and incubated at −20 °C overnight. The cells were then stained with 4′,6-diamidino-2-phenylindole  and detected by the NucleoCounter NC-3000 and analyzed using NucleoView NC-3000 software (ChemoMetec).A total of 1 × 10TM ECL  and detected using the BioSpectrumTW 500 Imaging System . The detailed information has been described in our previous study28. In this study, the following primary antibodies were used: CCNA2 , CCNB1, CCND1 , CDK4 , CDKN1B , CDKN1A , and ACTB .The SAS cells with and without C6orf141 expression were harvested 24 h after transient transfection. Total protein was collected and subjected to Western blot analysis. Total protein were separated by 6%-10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose filter membranes . Finally, the proteins were visualized using WesternBrightt test, analysis of variance (ANOVA), Mann–Whitney U test, or Kruskal–Wallis one-way ANOVA was used to evaluate the correlation of C6orf141 expression with different oral tissues or clinicopathological parameters. Clinicopathological outcome is usually defined as the time from initial diagnosis or surgery to a specific event of interest. Disease-specific survival (DSS) was measured from the time of the initial resection of the primary tumor to the date of the cancer-specific death or last follow-up. Cumulative survival curves were estimated using the Kaplan–Meier method, and comparisons between the survival curves were conducted using the log-rank test. A Cox proportional hazards model was used to determine independent predictors of survival using factors that were deemed significant in the univariate analysis as covariates. A P value of <0.05 was considered statistically significant.The chi-squared test, Fisher exact test, Student Supplementay table and information of figure 9c


Progression to a castration resistance state is the main cause of deaths in prostate cancer (PCa) patients. Androgen Receptor (AR) signaling plays the central role in progression of Castration Resistant Prostate Cancer (CRPC), therefore understanding the mechanisms of AR activation in the milieu of low androgen is critical to discover novel approach to treat CRPC.Firstly, we explore the CRPC associated lncRNAs by transcriptome microarray. The expression and clinical features of lnc-LBCS are analyzed in three independent large-scale cohorts. The functional role and mechanism of lnc-LBCS are further investigated by gain and loss of function assays in vitro.The expression of Lnc-LBCS was lower in CRPC cells lines and tissues. LBCS downregulation was correlated with higher Gleason Score, T stage and poor prognosis of PCa patients. LBCS overexpression decreases, whereas LBCS knockdown increases, the traits of castration resistance in prostate cancer cells under androgen ablated or AR blocked condition. Moreover, knockdown of LBCS was sufficient to activate AR signaling in the absence of androgen by elevating the translation of AR protein. Mechanistically, LBCS interacted directly with hnRNPK to suppress AR translation efficiency by forming complex with hnRNPK and AR mRNA.Lnc-LBCS functions as a novel AR translational regulator that suppresses castration resistance of prostate cancer by interacting with hnRNPK. This sheds a new insight into the regulation of CRPC by lncRNA mediated AR activation and LBCS-hnRNPK-AR axis provides a promising approach to the treatment of CRPC.The online version of this article (10.1186/s12943-019-1037-8) contains supplementary material, which is available to authorized users. Prostate cancer (PCa) is the most commonly diagnosed malignancy and the second leading cause of male cancer-related death in the United States . AndrogePRNCR1 and PCGEM1 are overexpressed in aggressive PCa and bind successively to the AR to enhance the AR-mediated gene activation program and induce PCa growth [Long noncoding RNAs (lncRNAs) are RNA transcripts that are longer than 200 nucleotides but have rare protein coding potential . Emergina growth . AlthougIn the current study, we discovered that lnc-LBCS is significantly downregulated in CRPC cell lines and tissues of CRPC patients. Furthermore, LBCS inhibits prostate cancer viability under castration condition by repressing AR signaling. Interestingly, LBCS act as the scaffold to recruit hnRNPK to AR mRNA and then inhibit the translation of AR protein. Our findings suggest strongly that LBCS-hnRNPK-AR axis participates in the progression of PCa and is a promising therapeutic target.2 at 37 °C. The construction of two CRPC LNCaP sublines was described in our previous article [The cell lines used in this study included the human prostate cancer cells LNCaP , and the CRPC-like cell LNCaP-Bic and LNCaP-AI. LNCaP cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 , supplemented with 10% FBS , LNCaP-AI cells were cultured in phenol red free RPMI-1640 containing 10% charcoal stripped FBS ; whereas LNCaP-Bic were cultured with 20 μM bicalutamide . All media were supplemented with 1% penicillin/streptomycin. LNCaP cells were cultured in phenol red free medium with charcoal striped FBS for at least 2 days before any experiment. Cells were grown in a humidified atmosphere of 5% COA total of 130 cases paraffin embedded PCa tissues and 32 cases of BPH tissues, termed Cohort 1, were obtained by surgery or needle biopsy with the written consent of patients who underwent surgery at Sun Yat-sen Memorial Hospital, Sun Yat-sen University. All the samples were pathologically confirmed as prostatic adenocarcinoma by two pathologists. Ethical consent was approved by Sun Yat-sen University’s Committees for Ethical Review of Research involving Human Subjects. The characteristics and clinicopathological features of the patients are listed in Table The microarray screening for differently expressed lncRNAs in CRPC was reported previously . All prihttps://cancergenome.nih.gov/. The expression of LBCS in PCa was obtained from TANRIC [http://ibl.mdanderson.org/tanric/_design/basic/query.html). The TCGA prostate adenocarcinoma cohort comprising of 374 patients was used for the analysis. The survival of TCGA PCa patients were analyzed using GEPIA [http://gepia.cancer-pku.cn/).Patients’ clinical profiles in the TCGA prostate adenocarcinoma cohort  are avaim TANRIC   according to the manufacturer’s protocol. Total RNA was reverse transcribed with a PrimerScript RT-PCR kit . Real time qPCR was conducted using a standard SYBR Green PCR kit  protocol with a CFX real-time instrument . The relative expression was calculated using the 2LBCS expression was also examined using ISH in formalin-fixed, paraffin-embedded (FFPE) samples, as previously described . The IHCWestern blotting was performed as previously described , 17. Pri3 cells/well. Then, the absorbance was measured at a wavelength of 490 nm for 5 days using a SpectraMax M5 .The methyl thiazolyl tetrazolium  colorimetric assay was used to detect cell viability. Cells were seeded in 96-well plates at a density of 2 × 10For the colony formation assay, the cells were seeded in a 96-well plate at a density of 500 cells per well. Seven days later, the clones were washed with 1× phosphate buffered saline (PBS) and stained with crystal violet for approximately 20 min. The clones were then imaged and quantified.50), data were fitted in Graph Pad Prism 5  and dose-response curve was plotted using the equation log (inhibitor) vs. response- Variable slope. This is also called a four-parameter dose-response curve: Y=Bottom + (Top-Bottom)/ (1 + 10^ ((Log IC50-X) *HillSlope)) [Cells were treated with different concentration of bicalutamide or R1881  for 120 h. The cell viability was measured using the same method as MTT assay. For calculation of half inhibition concentration (IClSlope)) .The fluorescent in situ hybridization kit was purchased from Ribo Bio  and the experiment is performed according to the manufacturer’s instruction and previously described , then vi7 cells were harvested, resuspended in 1 mL of ice cold RNase-free PBS, 1 ml of buffer C1  and 3 ml of RNase-free water, and incubated for 15 min on ice. Then cells were centrifuged for 15 min at 2500 rpm, the supernatant containing cytoplasmic constituent and the nuclear pellet were kept for RNA extraction.The cellular fraction was isolated as described previously . BrieflyRNA pulldown was conducted as previously reported . LBCS fuThe RNA isolation by RNA purification was conducted using a Magna ChIRP RNA Interactome Kit (Millipore) according to the manufacturer’s instructions and as described previously . ChIP waQuantitative data were presented as the means ± the standard deviation (SD) of three independent experiments. Differences between two groups were analyzed with the unpaired/paired Student’s t test (two-tailed tests), and one-way ANOVA followed by.p-values were provided in Additional file P value < 0.05 was considered significant.Dunnett’s multiple comparisons tests was performed when more than two groups were compared. Data of clinical analysis were shown as median with the interquartile range. The Mann-Whitney U test was used for independent samples when the population could not be assumed to be normally distributed. Pearson’s chi-square test was used to analyze the clinical variables. Spearman’s correlation analysis was performed to determine the correlation between two variables. Cumulative survival time was calculated using the Kaplan–Meier method and analyzed by the log-rank test. The best point cutoff value was used to define LBCS expression level (Low VS High) for analyzing TCGA cohort. The median H-score were used as cutoff value to define LBCS expression level (Low VS High) for analysis for cohort 2. A multivariate Cox proportional hazards model was used to estimate the adjusted hazard ratios and 95% confidence intervals, and to identify independent prognostic factors. All statistical analyses in this study were performed using SPSS 19.0 software. Actual Supplemental Materials and Methods was provided as Additional file In our previous study, we had reported the differently expressed lncRNAs in LNCaP cells and its two castration resistant sublines, which we name as LNCaP-AI and LNCaP-Bic . We idenTo investigate whether LBCS was involved in clinical PCa progression, we detected and analyzed LBCS expression in three independent cohorts of PCa specimens. Firstly, we analyzed its expression in Cohort 1 containing 130 PCa and 32 benign prostate hypertrophy (BPH) tissues from our hospital by in situ hybridization (ISH). We found that the LBCS expression was significantly lower in PCa compared with BPH tissues  and progression-free survival (PFS) Additional file 2:Table S2. The primers used in real time qPCR. (DOCX 13 kb)Additional file 3:Table S3. The probes used in this article. (DOCX 14 kb)Additional file 4:Table S4. The primers used in ChIP-real time qPCR. (DOCX 13 kb)Additional file 5:Table S5. The primers used for RNA isolation by RNA purification-real time qPCR. (DOCX 13 kb)Additional file 6:Table S6. Actual p-values of all figures. (DOCX 16 kb)Additional file 7:Supplemental Material and Method. (DOCX 17 kb)Additional file 8:Table S7. The primers used in gene clone (DOCX 14 kb)Additional file 9:Figure S1. The clinical significance of lnc-LBCS in prostate cancer. (A-B) Lnc-LBCS was detected between N0 and N1, M0 and M1 groups from cohort 1 by ISH. ISH of lnc-LBCS expression was quantified by the expression score (0–300). Patients with unavailable information was excluded for analysis. The whiskers indicate median ± interquartile in the plots. (C-D) Lnc-LBCS was detected between N0 and N1, M0 and M1 groups from cohort 2 by ISH. ISH of lnc-LBCS expression was quantified by the expression score (0–300). Patients with unavailable information was excluded for analysis. The whiskers indicate median ± interquartile in the plots. (E-F) The overall survival and disease-free survival rates of the 492 PCa patients from TCGA were analyzed by GEPIA. *p < 0.05, **p < 0.01. (JPG 1018 kb)Additional file 10:Table S8. Association between lnc-LBCS expression and clinicopathological features of prostate cancer from Cohort 2. (DOCX 15 kb)Additional file 11:Table S9. Univariate and multivariate analysis of factors associated with progression-free survival in prostate cancer Cohort 1. (DOCX 15 kb)Additional file 12:Figure S2. LBCS restores the castration sensitivity of CRPC cells. (A-B) LBCS was overexpressed in LNCaP-AI and LNCaP-Bic cells and knocked down in LNCaP cells, then cell cycles were analyzed by flow cytometry. (C-D) The caspase 3/7 activity was measured in lnc-LBCS overexpressed LNCaP-AI and LNCaP-Bic cells, and lnc-LBCS knockdown LNCaP cells treated with bicalutamide. *p < 0.05, **p < 0.01. (JPG 922 kb)Additional file 13:Figure S3. The illustration of the change of AR grey scale after treated with cycloheximide for different hours in either LBCS knockdown or control group. (TIFF 93 kb)Additional file 14:Table S10. The predicted LBCS-AR mRNA interacting sequences and oligos used for FRET. (DOCX 14 kb)


CYP19A1/aromatase (Ar) is a prognostic biomarker of gastric cancer (GCa). Ar is a critical enzyme for converting androstenedione to oestradiol in the steroidogenesis cascade. For decades, Ar has been targeted with Ar inhibitors (ARIs) in gynaecologic malignancies; however, it is unexplored in GCa. A single‐cohort tissue microarray examination was conducted to study the association between Ar expression and disease outcome in Asian patients with GCa. The results revealed that Ar was a prognostic promoter. Bioinformatics analyses conducted on a Caucasian‐based cDNA microarray databank showed Ar to be positively associated with GCa prognosis for multiple clinical modalities, including surgery, 5‐Fluorouracil (5‐FU) for adjuvant chemotherapy, or HER2 positivity. These findings imply that targeting Ar expression exhibits a potential for fulfilling unmet medical needs. Hence, Ar‐targeting compounds were tested, and the results showed that exemestane exhibited superior cancer‐suppressing efficacy to other ARIs. In addition, exemestane down‐regulated Ar expression. Ablating Ar abundance with short hairpin (sh)Ar could also suppress GCa cell growth, and adding 5‐FU could facilitate this effect. Notably, adding oestradiol could not prevent exemestane or shAr effects, implicating a nonenzymatic mechanism of Ar in cancer growth. Regarding translational research, treatment with exemestane alone exhibited tumour suppression efficacy in a dose‐dependent manner. Combining subminimal doses of 5‐FU and exemestane exerted an excellent tumour suppression effect without influencing bodyweight. This study validated the therapeutic potentials of exemestane in GCa. Combination of metronomic 5‐FU and exemestane for GCa therapy is recommended. Nevertheless, postsurgical recurrence is often observed in advanced disease.A study revealed that lipoprotein protein/receptor‐route‐mediated cholesterol import and the resulting steroidogenesis play crucial roles in GCa progression.The mechanism of action (MOA) of ARIs involves inhibiting Ar‐enzymatic functionIn this study, we used a bioinformatics approach to explore the suitability of Ar as a targeting agent for treating GCa, thus meeting clinical needs. We also tested the value of targeting Ar with ARIs in GCa preclinical models and provide new insight into ablating Ar with a non‐irreversible inhibitor in GCa treatment.22.1This study was approved by the Ethics Committee of the Institutional Review Board of Kaohsiung Medical University Hospital (KMUH‐IRB‐20120176). Informed consent was obtained from all participants in accordance with the Declaration of Helsinki. Primary tumour tissues were obtained from 220 GCa patients undergoing surgical resection at Kaohsiung Medical University Hospital between 2007 and 2014. Patient characteristics and clinical outcome were followed until death, censorship or loss to follow‐up. Gastric tumour tissue cores were collected from each patient and used to construct a tissue microarray (TMA).2.2http://kmplot.com/analysis/index.php?p=service&cancer=gastric) was used and a log‐rank test was used to assess the differences between patient groups stratified according to the median of gene expression. A P‐value of <.05 was considered statistically significant.To analyse the association of survival with gene expression, a web‐based Kaplan‐Meier plotter  are in the online 33.1P = .0017). The GCa death rate was higher (P = .0283) in women (46%) than it was in men (27%). Notably, the association of tumour Ar positivity with death rate was higher in men . We observed comparable death rates between high  and low  Ar expressions among women. In normal parental lesions, the effect of Ar expression was significant (P = .0283) in women, where low expression was associated with a high death rate  and high expression was associated with a low death rate . These data indicate a notable effect of Ar expression on sex, prognosis and microenvironmental regulations.To assess the effect of Ar expression in clinical settings, we implemented two strategies to evaluate the roles of Ar in human GCa progression: one of the strategies entailed conducting a single‐hospital cohort TMA immunohistochemistry study on an Asian population Tables  and 2; tAssessing a gene expression database in the Caucasian population revealed that high Ar expression was associated with poor prognosis (OS and free progression [FP]) in patients with GCa stratified according to multiple clinical classifications and therapy modalities. The positive association of Ar expression with treatment modalities implies that Ar has potential to serve as a targeting agent to fulfil unmet medical needs. Figure 3.2As mentioned, two MOAs of ARIs were considered to test whether ARIs can be used for GCa therapy. Three ARIs  were introduced. Cytotoxicity Figure A,2B and To test this hypothesis, we introduced gene‐silencing technology with shRNA targeting Ar mRNA expression Figure A. We comOverall, our findings provide proof‐of‐concept evidence that ablating Ar expression using exemestane or shRNA is potentially therapeutic.3.3As shown in Figure 3. Subsequently, various drugs were intraperitoneally injected three times per week for four consecutive weeks. The tumour size decreased with exemestane treatment in a dose‐dependent manner , but mice receiving a low dose of 5‐FU alone did not show significant tumour growth inhibition (P = .3895). In addition, exemestane and 5‐FU could synergistically promote anticancer efficacy (from P = .0263 to P = .007). Considering the effects of the treatments on the general wellness of the experimental mice, we divided tumour weight by bodyweight  in a xenograft mouse model of GCa in a 4‐week therapeutic term. As shown in Figure In sum, targeting Ar with exemestane (ARI‐II) might be a new effective therapeutic approach for GCa. Single therapy or combination usage with 5‐FU is worthwhile to be tested in clinical settings.4Gastric cancer is a complex malignancy because patients are usually diagnosed at an advanced stage and have a poor prognosis.4.1The contribution of female factors to GCa development is controversial. For example, a large‐scale epidemiological survey indicated female factors, such as reproductive age, ovariectomy surgery, breastfeeding, pregnancy and contraceptive agents, were suggested that the oestrogenic signal suppresses GCa incidence.4.2Two types of ARIs are approved for treating cancer: ARI‐I, comprising reversible nonsteroidal inhibitors (eg anastrozole and letrozole), and ARI‐II, comprising irreversible steroidal inhibitors (eg exemestane and formestane). Both types of ARIs can inhibit oestrogen synthesis by targeting the catalytic binding site of Ar4.3Exemestane inhibits Ar expression, which is considered an essential MOA of GCa cell suppression. We propose several mechanisms of Ar silencing. The first mechanism is transcriptional regulation; for example, Foxl2  binds to the sequence ACAAATA in the promoter region of the Ar gene through its forkhead domain. Foxl2 could also interact with the ligand‐binding domain of Ad4BP/SF‐1  through the forkhead domain to form a heterodimer and enhance Ad4BP/SF‐1‐mediated Ar transcription.5In this study, we observed that Ar is a crucial GCa prognostic biomarker. Suppressing Ar expression by using ARI‐II could be an excellent therapeutic strategy, particularly when ARI‐II is used in combination with 5‐FU. Additional pharmaceutical studies and human trials are encouraged.There is no conflict of interests in this work.JC Yang and N Chang were responsible for the execution of experiments, pharmacological verification and drafting of the article. DC Wu, IC Wu and CJ Liu provided the clinical sample and performed statistical analyses. WC Cheng, WM Chung, FJ Lei, WC Chang and HC Lai assisted with immunoblot, IHC, statistical analyses, clinical consulting, compound validation, translational study design and article drafting. WL Ma initiated, supported and designed the study. HC Lai and WL Ma supported the study and finally approved the article. Click here for additional data file.