Subtype mRNA Research Articles

Epithelial plasticity can generate multi-lineage phenotypes in human and murine bladder cancers

Tumor heterogeneity is common in cancer, however recent studies have applied single gene expression signatures to classify bladder cancers into distinct subtypes. Such stratification assumes that a predominant transcriptomic signature is sufficient to predict progression kinetics, patient survival and treatment response. We hypothesize that such static classification ignores intra-tumoral heterogeneity and the potential for cellular plasticity occurring during disease development. We have conducted single cell transcriptome analyses of mouse and human model systems of bladder cancer and show that tumor cells with multiple lineage subtypes not only cluster closely together at the transcriptional level but can maintain concomitant gene expression of at least one mRNA subtype. Functional studies reveal that tumor initiation and cellular plasticity can initiate from multiple lineage subtypes. Collectively, these data suggest that lineage plasticity may contribute to innate tumor heterogeneity, which in turn carry clinical implications regarding the classification and treatment of bladder cancer.

Comparison of mRNA Expression of P2X Receptor Subtypes in Different Arterial Tissues of Rats.

This study aims to compare the expression of P2X receptor subtype mRNA in different arterial tissues of rats. After the rats were sacrificed, the internal carotid, pulmonary, thoracic aorta, mesenteric and caudal arteries were dissected out. Then, the P2X receptor mRNA expression in different blood vessels was detected by reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative polymerase chain reaction. The P2X1, P2X4 and P2X7 receptor mRNA amplification products revealed specific bands of the same size as the amplified target fragment in their respective lanes, while the P2X2, P2X3, P2X5 and P2X6 receptor mRNA amplification products did not reveal significant specific bands in their respective lanes by RT-PCR. Based on the P2X1 receptor mRNA expression of the mesenteric artery, there were no significant differences in the internal carotid, pulmonary and thoracic aorta (0.64 ± 0.07, 0.17 ± 0.11 and 1.49 ± 0.65, respectively). However, the P2X1 receptor mRNA expression level in the caudal artery significantly increased (11.06 ± 1.99, P < 0.01). Furthermore, there was no difference in P2X4 receptor mRNA expression among these five blood vessels (P > 0.05). The P2X7 receptor mRNA expression level was significantly different: pulmonary artery < tail artery = thoracic aorta < internal carotid artery < mesenteric artery. The relative P2X1 receptor mRNA expression in the caudal artery was observed to be elevated when compared to that of the internal carotid, pulmonary and thoracic aorta as well as the mesenteric arteries. The P2X7 receptor mRNA expression level is pulmonary artery < caudal artery = thoracic aorta < internal carotid artery < mesenteric artery. P2X4 receptor mRNA expression was not significantly different among these five blood vessels.

&lt;i&gt;Coleus forskohlii&lt;/i&gt; Extract Attenuated the Beneficial Effect of Diet-Treatment on NASH in Mouse Model

Obesity is one of the main causes of non-alcoholic steatohepatitis (NASH), which is associated with impaired liver functions including drug metabolism. Coleus forskohlii extract (CFE) is a popular ingredient of weight loss dietary supplements in Japan. In this study, we examined the effect of CFE on the treatment of NASH. C57BL/6 mice (male, 10-wk-old) were fed a NASH diet (high-fat, low-methionine, and choline-deficient diet) for 12 wk to establish NASH. Then, we examined the effect of 0.5% (w/w) CFE in diet during diet-treatment (change to control diet) and/or treadmill-exercise (45 min at 20 m/min, 5 d/wk) to improve NASH for 3 wk. After experimental period, lipids profiles and liver functional markers in the blood, and hepatic lipid content and major CYP subtype mRNA expression and activity in liver were measured. Diet-treatment, but not exercise decreased liver weight and hepatic lipid contents in NASH induced mice. CFE attenuated the effects of diet-treatment which reduced liver weight, even though body weight and adipose tissue weight were reduced. Further, CFE significantly increased liver microsomal CYP1A1, CYP1A2, CYP2C, and CYP3A activities in each condition, and CYP inductions were greater in diet-treatment group compared to those in exercise group. These results suggest that taking CFE should be avoided during diet-treatment of NASH, especially in patients under medication.

Denatonium enhanced the tone of denuded rat aorta via bitter taste receptor and phosphodiesterase activation

Bitter taste receptors (Tas2rs) initiate a bitter taste signaling involving the activation of taste-specific G protein gustducin and phosphodiesterases (PDEs); it leads to the decrease of cytosolic level of cyclic adenosine monophosphate (cAMP) in taste cells. Recent studies have identified the expression of Tas2rs in a variety of non-lingual tissues including vascular smooth muscle (VSM), pulmonary smooth muscle and airway smooth muscle. The current study aims to determine the expression of Tas2rs and gustducin in rat aortic smooth muscle tissue and to investigate the effect of Tas2rs agonist denatonium on the tone of isolated denuded aorta rings. Here we reported the expression of six subtypes of Tas2r mRNA and the taste receptor-associated G proteins in endothelium-denuded aorta. Immunostaining experiments showed that the protein of gustducin expressed in vascular smooth muscle cells (VSMCs). Furthermore, denatonium increased the tone of freshly isolated denuded aorta rings in a concentration-dependent manner, and the potentiation effect of denatonium was blocked by a Tas2rs antagonist adenosine 5′-monophosphate (5′-AMP), by the cAMP-hydrolyzing PDE inhibitors, and by a cAMP-synthesizing enzyme activator forskolin, respectively. The blockade of Gβγ signaling did not have a negative impact on the denatonium-induced tonic contractions. These findings suggested that the functional Tas2rs and gustducin are expressed in rat aortic smooth muscle and that denatonium might increase the smooth muscle tone through a Tas2rs signaling pathway involving the activation of PDEs.

Estrogen Receptor β2 Oversees Germ Cell Maintenance and Gonadal Sex Differentiation in Medaka, Oryzias latipes

SummaryIn vertebrates, estrogen receptors are essential for estrogen-associated early gonadal sex development. Our previous studies revealed sexual dimorphic expression of estrogen receptor β2 (ERβ2) during embryogenesis of medaka, and here we investigated the functional importance of ERβ2 in female gonad development and maintenance using a transgenerational ERβ2-knockdown (ERβ2-KD) line and ERβ2-null mutants. We found that ERβ2 reduction favored male-biased gene transcription, suppressed female-responsive gene expression, and affected SDF1a and CXCR4b co-assisted chemotactic primordial germ cell (PGC) migration. Co-overexpression of SDF1a and CXXR4b restored the ERβ2-KD/KO associated PGC mismigration. Further analysis confirmed that curtailment of ERβ2 increased intracellular Ca2+ concentration, disrupted intra- and extracellular calcium homeostasis, and instigated autophagic germ cell degradation and germ cell loss, which in some cases ultimately affected the XX female sexual development. This study is expected improve our understanding of germ cell maintenance and sex spectrum, and hence open new avenues for reproductive disorder management.

Mirabegron elicits rat corpus cavernosum relaxation and increases in vivo erectile response

Mirabegron is the first β3-adrenoceptor agonist approved on the market and may offer beneficial pharmacological action in patients with overactive bladder and erectile dysfunction. Here, we further investigate the mechanisms by which mirabegron induces rat corpus cavernosum (CC) relaxation. Adult male Wistar rats were used. The CC were isolated for in vitro functional assays and β-adrenoceptors subtypes mRNA expression evaluation. Animals were treated orally with mirabegron (30 mg/kg, 3 h), tadalafil (10 mg/kg, 3 h) or both for intracavernous pressure (ICP). Intracellular levels of cAMP and cGMP were also determined. The β1-, β2- and β3-adrenoceptors subtypes were expressed in rat CC. Mirabegron produced concentration-dependent CC relaxations that were unaffected by the β1-, β2- or β3-adrenoceptor antagonists atenolol (1 μM), ICI-118,551 (1 μM) and L748,337 (10 μM), respectively. Mirabegron-induced relaxations were not affected by the phosphodiesterase type 4 inhibitor, rolipram, or the adenylyl cyclase selective inhibitor, SQ 22,536. Potassium channel- or calcium influx-blockade are not involved in mirabegron-induced relaxations. In contrast, mirabegron produced rightward shifts in the contractile response induced by the α1-adrenoceptor agonist, phenylephrine. Finally, cavernous nerve stimulation caused frequency-dependent ICP increases, which were significantly increased in rats treated with mirabegron in a similar degree of tadalafil-treated rat, without promoting a significant cAMP or cGMP accumulation. Together, our results demonstrate that mirabegron induced CC relaxation through α1-adrenoceptor blockade. Care should be taken to translate the effect of mirabegron into the clinic, especially when using rat as an animal model of erectile dysfunction.

Association of BCR/ABL transcript variants with different blood parameters and demographic features in Iraqi chronic myeloid leukemia patients.

BackgroundChronic myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the presence of BCR‐ABL fusion gene (GenBank accession NC_000022.11). In the vast majority of CML patients, the typical subtype of BCR‐ABL transcript are b3a2, b2a2 or both. The aim of this study was to determine the different subtypes of BCR‐ABL transcript and their impact on the demographic and hematological parameters in Iraqi patients with CML.MethodsOne hundred patients with chronic phase CML (11 newly diagnosed and 89 imatinib‐resistant) were enrolled in this study. Ribonucleic acid (RNA) was extracted from leukocytes, and complementary DNA was created using reverse transcriptase polymerase chain reaction technique. A multiplex polymerase chain reaction with four specific primers was used to determine the BCR‐ABL fusion subtypes in each patient.ResultsMale to female ratio was 1.38:1. Fifty‐nine patients expressed b3a2 transcript, whereas 39 of the remaining cases were positive for b2a2 variant. One case expressed b2a3 transcript, while the last case coexpressed the two subtypes of mRNA b3a2/b2a2. Male and female were significantly associated with b3a2 and b2a2 subtypes, respectively. The b3a2 subtype showed higher total leukocyte count than b2a2 subgroup, while b2a2 variant demonstrated significantly elevated platelet counts compared to those with b3a2 transcript. A significantly higher plateletcrit percentage (PCT%) was found in patients with b2a2 transcript whereas.ConclusionsThe testified Iraqi group expressed M‐BCR‐ABL type with preponderance of b3a2 over b2a2 subtype. There was a gender‐skewed distribution in BCR‐ABL transcript types with b3a2 transcript more prevalent in males. The type of BCR‐ABL transcript is reflected by different leukocyte and platelet counts at diagnosis, which might represent a distinct phenotype and disease biology.

Connective tissue growth factor (CTGF) methylation status is associated with prognosis of patients with head and neck squamous cell carcinoma (HNSCC) treated with radiochemotherapy (RCHT): A multicenter study of the German Cancer Consortium Radiation Oncology Group (DKTK-ROG).

6050 Background: CTGF plays a central role in tissue remodeling and has emerged as an attractive novel therapeutic target. We sought to investigate the impact of CTGF methylation status (CTGF-M) in predicting outcome of HNSCC patients undergoing RCHT. Methods: CTGF-M was discovered by applying Illumina 450K/850K arrays to DNA extracted from FFPE material of patients homogeneously treated with surgery followed by adjuvant cisplatin-based RCHT in frame of the DKTK-ROG multicenter retrospective trial (n = 194, training cohort). Methylation probes correlating with overall survival (OS) and progression were identified using random forest. Validation was done in 4 cohorts including DKTK-ROG definitive RCHT (n = 110) and 3 RCHT cohorts from Heidelberg, Dresden and Munich (n = 222). CTGF-M and RNAseq data from The Cancer Genome Atlas (TCGA) (n = 206) were analyzed to identify differentially expressed genes (DEG) as a function of CTGF-M. Results: Increased methylation of 2 probes (mapping to CTGF 3’UTR and gene body) was associated with significantly improved OS in the training cohort (HR = 0.51, p = 0.044) and the validation cohort (HR = 0.67, p = 0.016), in multivariate cox regressions adjusting for HPV status, age, T and N stages, location and treatment (adjuvant vs definitive RCHT). In the TCGA, probes’ methylation was significantly inversely correlated with CTGF gene expression (r = -0.18 and -0.51, p &lt; 0.05). 1843 DEGs were found at FDR &lt; 0.05 as a function of CTGF-M. Pathways mapping to tissue remodeling were significantly enriched for among downregulated genes in CTGF hypermethylated tumors. Increased CTGF methylation was inversely correlated with the mesenchymal subtype mRNA gene signature (r = -0.21, p = 0.0026) and correlated with the atypical subtype (r = 0.32, p = 0.000002). Conclusions: Implementation of CTGF-M in routine diagnostic is feasible and correlates well with CTGF gene-expression levels and activation of tissue remodeling pathways. Therefore, CTGF-M might be instructive for stratifying HNSCC patients for CTGF targeting therapies. CTGF emerges as a promising prognostic marker independently of HPV status.

Novel neurosteroid hypnotic blocks T-type calcium channel-dependent rebound burst firing and suppresses long-term potentiation in the rat subiculum

BackgroundHypnotics and general anaesthetics impair memory by altering hippocampal synaptic plasticity. We recently reported on a neurosteroid analogue with potent hypnotic activity [(3β,5β,17β)-3-hydroxyandrostane-17-carbonitrile; 3β-OH], which does not cause developmental neurotoxicity in rat pups. Here, we investigated the effects of 3β-OH on neuronal excitability in the subiculum, the major output structure of the hippocampal formation, and synaptic plasticity at two key hippocampal synapses in juvenile rats. MethodsBiophysical properties of isolated T-type calcium currents (T-currents) in the rat subiculum were investigated using acute slice preparations. Subicular T-type calcium channel (T-channel) subtype mRNA expression was compared using qRT–PCR. Using electrophysiological recordings, we examined the effects of 3β-OH and an endogenous neuroactive steroid, allopregnanolone (Allo), on T-currents and burst firing properties of subicular neurones, and on the long-term potentiation (LTP) in CA3-CA1 and CA1-subiculum pathways. ResultsBiophysical and molecular studies confirmed that CaV3.1 channels represent the dominant T-channel isoform in the subiculum of juvenile rats. 3β-OH and Allo inhibited rebound burst firing by decreasing the amplitude of T-currents in a voltage-dependent manner with similar potency, with 30–80% inhibition. Both neurosteroids suppressed LTP at the CA1-subiculum, but not at the CA3-CA1 Schaffer collateral synapse. ConclusionsNeurosteroid effects on T-channels modulate hippocampal output and provide possible molecular mechanisms for the amnestic action of the novel hypnotic 3β-OH. Effects on T-channels in the subiculum provide a novel target for amnestic effects of hypnotics.

Abstract P6-17-05: Independent validation of a combined biomarker based on the PAM50 HER2-enriched subtype and ERBB2 mRNA levels following HER2 blockade without chemotherapy in the PerELISA phase II trial

Abstract Background: A combined biomarker based on HER2-enriched subtype (HER2-E) and ERBB2 mRNA predicts response and survival in HER2+ breast cancer following trastuzumab +/- lapatinib in the absence of chemotherapy (Prat et al. ASCO 2018). Here, we tested the ability of the combined biomarker to predict pathological complete response (pCR) following neoadjuvant trastuzumab, pertuzumab and endocrine therapy. Methods: RNA from 40 baseline tumor samples from the phase II PerELISA trial were evaluated. PerELISA evaluated the efficacy of a de-escalated, chemotherapy-free neoadjuvant regimen based on dual HER2 blockade with trastuzumab and pertuzumab in combination with letrozole in HER2+/hormone receptor-positive breast cancer selected on the basis of Ki67 response after short course letrozole-alone (Guarneri ASCO 2018). Ki67 response was defined by protocol as relative Ki67 reduction ≥20% from baseline at day 14. Gene-expression was measured using the nCounter platform. Intrinsic subtypes and ERBB2 levels were determined by the PAM50 gene expression predictor. A pre-specified ERBB2 cutoff was determined to define ERBB2-high. Univariate and multivariable logistic regression analyses were performed. Results: The proportion of HER2-E disease within the ERBB2-high and ERBB2-low groups was 46.2% (6/13) and 18.5% (5/27), respectively. The discordance rate at the individual level was 30% (12/40). A total of 6 (15%) and 34 (85%) samples were HER2-E/ERBB2-high and others, respectively. The magnitude of Ki67 reduction of the HER2-E/ERBB2-high and others groups was 64.8% and 63.2%, respectively (p=0.88). The pCR rate of HER2-E/ERBB2-high was 66.7%. The pCR rate of the others group was 14.7%. The univariate odds ratio between HER2-E/ERBB2-high tumors and the others groups was 11.60 (95% CI 1.66-81.10; p=0.014). No other clinical-pathological variable was significantly associated with pCR. Conclusion: The combined HER2-E/ERBB2-high biomarker can identify patients who might be good candidates to receive dual HER2 blockade alone without chemotherapy. Citation Format: Prat A, Griguolo G, Dieci MV, Bisagni G, Frassoldati A, Bianchi GV, Pascual T, Pare L, Galvan P, Urso L, Conte P, Guarneri V. Independent validation of a combined biomarker based on the PAM50 HER2-enriched subtype and ERBB2 mRNA levels following HER2 blockade without chemotherapy in the PerELISA phase II trial [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-17-05.

Mechanisms underlying the relaxation by A484954, a eukaryotic elongation factor 2 kinase inhibitor, in rat isolated mesenteric artery

Eukaryotic elongation factor 2 kinase (eEF2K) is a calmodulin-related protein kinase which regulates protein translation. A484954 is an inhibitor of eEF2K. In the present study, we investigated the acute effects of A484954 on contractility of isolated blood vessels. Isometric contraction of rat isolated aorta and main branch of superior mesenteric artery (MA) was measured. Expression of an inward rectifier K+ (Kir) channel subtype mRNA and protein was examined. A484954 caused relaxation in endothelium-intact [E (+)] and -denuded [E (−)] aorta or MA precontracted with noradrenaline (NA). The relaxation was higher in MA than aorta. The relaxation was partially inhibited by a nitric oxide (NO) synthase inhibitor, NG-nitro-l-arginine methyl ester (300 μM) in E (+) MA. The relaxation was significantly smaller in MA precontracted with high K+ than NA. The A484954-induced relaxation was significantly inhibited by a Kir channel blocker, BaCl2 (1 mM) compared with vehicle control in E (−) MA. Expression of Kir2.2 mRNA and protein was significantly higher in MA than aorta. We for the first time revealed that A484954 induces relaxation through opening smooth muscle Kir (Kir2.2) channel and through endothelium-derived NO in MA.

Maternal stress and the MPOA: Activation of CRF receptor 1 impairs maternal behavior and triggers local oxytocin release in lactating rats

Maternal behavior and anxiety are potently modulated by the brain corticotropin-releasing factor (CRF) system postpartum. Downregulation of CRF in limbic brain regions is essential for appropriate maternal behavior and an adaptive anxiety response. Here, we focus our attention on arguably the most important brain region for maternal behavior, the hypothalamic medial preoptic area (MPOA).Within the MPOA, mRNA for CRF receptor subtype 1 (protein: CRFR1, gene: Crhr1) was more abundantly expressed than for subtype 2 (protein: CRFR2, gene: Crhr2), however expression of Crhr1, Crhr2 and CRF-binding protein (protein: CRFBP, gene: Crhbp) mRNA was similar between virgin and lactating rats. Subtype-specific activation of CRFR, predominantly CRFR1, in the MPOA decreased arched back nursing and total nursing under non-stress conditions. Following acute stressor exposure, only CRFR1 inhibition rescued the stress-induced reduction in arched back nursing while CRFR1 activation prolonged the decline in nursing. Furthermore, inhibition of CRFR1 strongly increased maternal aggression in the maternal defense test. CRFR1 activation had anxiogenic actions and reduced locomotion on the elevated plus-maze, however neither CRFR1 nor R2 manipulation affected maternal motivation. In addition, activation of CRFR1, either centrally or locally in the MPOA, increased local oxytocin release. Finally, inhibition of CRFBP (a potent regulator of CRFR activity) in the MPOA did not affect any of the maternal parameters investigated.In conclusion, activity of CRFR in the MPOA, particularly of subtype 1, needs to be dampened during lactation to ensure appropriate maternal behavior. Furthermore, oxytocin release in the MPOA may provide a regulatory mechanism to counteract the negative impact of CRFR activation on maternal behavior.

Therapeutic effects of tyrosine kinase inhibitors and subtypes of BCR-ABL1 transcripts in Japanese chronic myeloid leukemia patients with three-way chromosomal translocations

We analyzed the clinical responses to thyrosine kinase inhibitors (TKIs) and the molecular and cytogenetic characteristics of 18 chronic myeloid leukemia (CML) patients with 3-way chromosomal translocations. The patients were 14 men and 4 women, aged 23–75 years (median 57 years). The Sokal risk was low in 12 patients, intermediate in 4 patients, and high in 2 patients. Newly identified translocation breakpoints were seen in 7 of the 18 patients. Three patients had the same breakpoints of t(9;22;11)(q34;q11.2;q23).The best responses to TKIs were partial cytogenic response (PCyR) in 2 patients, complete cytogenic response (CCyR) in 3 patients, molecular response (MR) 3.0 in 7 patients, MR 4.0 in 3 patients, and MR 4.5 or higher in 3 patients. A total of 66.7% of patients did not achieve MR 4.0 or higher. In 3 patients in whom TKIs resulted in MR 4.5 or higher for more than 2 years, TKI treatment was discontinued. However, all of them exhibited a loss of MR3.0, at 2, 6, and 20 months after the discontinuation of treatment, respectively, and TKI treatment needed to be restarted. According to Kaplan-Meier survival curve analysis, the overall survival (OS) was 100 months in 56% of the patients. The 60-months cumulative incidences of CCyR, MR3.0, MR4.0 and MR4.5 were 88.9%, 72.2%, 33.3%, and 16.7%, respectively. In the 11 analyzable patients, the BCR-ABL1 mRNA subtype was e14a2 type in 4 patients and e13a2 type in 7 patients.

The sodium salicylate affects the expression of NMDA receptor and GABAa receptor subunits in spiral ganglion neurons of the cochlea through DA receptor

Objective:To investigate mRNA expression of dopamine receptor subtypes in the rat cochlear spiral ganglion neurons (SGN) following exposure to the sodium salicylate. In addition, we observed the effect of sodium salicylate on N methyl-D-aspartic acid (NMDA) receptor subunit NR1 and gamma aminobutyric acid (GABA)a receptor subunit GABRα2 mRNA under the circumstance of DR activation or blocking. Moreover, we also focused on the the interaction between receptors mediated by SS.Method:Immunofluorescence techniques were applied to detect DR (DR1 and DR2) expression in cultured rat SGN. Moreover, RT-PCR was performed to assess NR1 and GABRα2 subunit mRNA.Result:Immunofluorescence images showed co-localization of DR1/DR2 and βⅢ-tubulin protein in SGN bodies and axons. RT-PCR results illustrated that ①DR subtypes of DRd1-5 were detected in the SGN. ②The mRNA expression of all subtypes of DR and GABRα2, NR1 subunits were obviously upregulated except DRd3 after treatment with sodium salicylate. Among them, DRd1 expression increased 34.64%(t=-5.123,P=0.007),DRd2 expression increased 34.60%(t=-5.206,P=0.006),DRd4 expression increased 20.87%(t=-3.337,P=0.029),DRd5 expression increased 26.42%(t=-6.054,P=0.004),GABRα2 expression increased 30.41%(t=-2.839,P=0.047),NR1 expression increased 39.22%(t=-6.243,P=0.003).③After exposure to sodium salicylate (5 mmol/L), dopamine (100 μmol/L), DR1 agonist (SKF38393,20 μmol/L), DR2 agonist (Quinpirole,20 μmol/L), GABRα2 expression increased 21.78%,27.45%,33.02%,33.42% respectively (F=12.399,P=0.001),and NR1 expression increased 28.70%,26.82%,29.03%,35.05%(F=50.395,P=0.000) respectively.④Compared with the group of sodium salicylate treatment alone, both sodium salicylate + DR1 antagonist (SCH23390,20 μmol/L) group and sodium salicylate + DR2 antagonists (Eticlopride,20 μmol/L) group had a suppression on GABRα2 and NR1 mRNA expression.GABRα2 mRNA reduced 29.56%,37.10%(F=22.101,P=0.000) and NR1 mRNA expression decreased 37.62%,32.83% respectively(F=72.933,P=0.000).Conclusion:Most of the DR subtypes mRNA expression in SGN were increased following exposure to sodium salicylate. DR may be involved in the effect of sodium salicylate on GABAaR and NMDAR mRNA expression.

Integrated Genomic Characterization of Pancreatic Ductal Adenocarcinoma

We performed integrated genomic, transcriptomic, and proteomic profiling of 150 pancreatic ductal adenocarcinoma (PDAC) specimens, including samples with characteristic low neoplastic cellularity. Deep whole-exome sequencing revealed recurrent somatic mutations in KRAS, TP53, CDKN2A, SMAD4, RNF43, ARID1A, TGFβR2, GNAS, RREB1, and PBRM1. KRAS wild-type tumors harbored alterations in other oncogenic drivers, including GNAS, BRAF, CTNNB1, and additional RAS pathway genes. A subset of tumors harbored multiple KRAS mutations, with some showing evidence of biallelic mutations. Protein profiling identified a favorable prognosis subset with low epithelial-mesenchymal transition and high MTOR pathway scores. Associations of non-coding RNAs with tumor-specific mRNA subtypes were also identified. Our integrated multi-platform analysis reveals a complex molecular landscape of PDAC and provides a roadmap for precision medicine.

Abstract 5405: Comprehensive analysis of long non-coding RNAs in human stomach adenocarcinoma molecular subtypes

Abstract Objective: Long non-coding RNA (lncRNA) plays key role in cell biology including epigenetic remodeling and post-transcriptional regulation. The potential roles of lncRNAs as biomarkers and therapeutic targets have been proven in solid tumors. Molecular profiling in stomach adenocarcinoma (STAD) has been characterized using The Cancer Genome Atlas (TCGA) database. However, there are no current lncRNA comprehensive analyses and the relationships between patterns of molecular subtypes and the signature of lncRNA in in human stomach adenocarcinoma are unclear. Methods: (1) The Cancer Genome Atlas (TCGA) 312 primary STAD RNA-Seq data (bam files) and their related clinical data were obtained from the Cancer Genomics Hub and TCGA Data Portal. (2) We filtered the dataset to remove lncRNAs with low expression, defined as having an RPKM value &amp;lt;1 in at least 90% of the tumor samples. (3) We generated a correlation matrix between lncRNAs and mRNAs by computing the Pearson correlation coefficient between all pairs of significant lncRNAs and mRNAs. A matrix was constructed with entries in the ternary scale (-1, 0, 1), where the top 1% with negative correlation was assigned -1; the top 1% with positive correlation was assigned 1; and the others were assigned 0. The matrix was clustered and visualized using a Euclidian distance metric and complete linkage clustering. Results: (1) Using stringent criteria, we identified 1830 expressed lncRNAs in human genome in STAD. (2) Unsupervised clustering of lncRNA expression in STAD revealed five robust categories: clusters C1-C5. (3) We further performed concordance calculation between lncRNA-based clustering and other clusters identified in TCGA molecular, mRNA, miRNA and SCNA subtypes, respectively. We also investigated the association of lncRNA subclasses with distinct clinicopathological and genomic features of STAD. We found that C2 is significantly associated with MSI molecular subtype, MHL silencing, and mRNA C2, miRNA C2, methylation C2 subtypes. Cluster C3 is significantly associated with EBV-positive molecular subtype, CDKN2A silencing and mRNA C2, miRNA C2, methylation C1 subtypes; C4 is most significantly associated with CIN molecular subtype, p53 mutation and high SCNA, methylation C4 subtypes; C5 is significantly associated with GS molecular subtype, diffuse Lauren subtype, and mRNA C1, miRNA C4, methylation C1 subtypes. Interestingly, C5 defines the most aggressive tumors with the worst overall survival. Conclusions: Our study represents the first comprehensive analysis of lncRNAs in STAD cancers, with integrative analysis revealing that the signatures of lncRNAs we characterize here appear to have prognostic significance. The additional association with the STAD molecular subtypes and genomic features supports new approaches to developing biomarkers and setting the stage for a new framework for future research in the role of lncRNAs in STAD. Citation Format: Jian Chen, Jiun-Sheng Chen, Keping Xie, John R Stroehlein, Marta Davila, Xiaoping Su. Comprehensive analysis of long non-coding RNAs in human stomach adenocarcinoma molecular subtypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5405. doi:10.1158/1538-7445.AM2017-5405

Comprehensive molecular characterization and analysis of muscle-invasive urothelial carcinomas.

4500 Background: We reported the integrated molecular analysis of 131 tumors in 2014 (Nature 507:315, 2014) and now report on the entire cohort of 412 tumors from the TCGA project in chemotherapy-naïve, muscle-invasive urothelial bladder cancer. Methods: Following strict clinical and pathologic quality control, tumors were analyzed for DNA copy number variants, somatic mutations (WES), DNA methylation, mRNA, non-coding RNA (lncRNA and miRNA) and (phospho-) protein expression, gene fusions, viral integration, pathway perturbation, clinical correlates, outcomes, and histopathology. Results: There was a high overall somatic mutation rate (8.2/Mb), as previously reported. There were 58 significantly mutated genes (SMGs) (MutSig_2CV), increased from 32 in the original report. We identified 5 mutation signatures including APOBEC-a and b, ERCC2, C &gt; T_CpG, and a single ultra-mutated sample with a functional POLE mutation. APOBEC mutagenesis explained 70% of the mutation burden and was associated with survival (p = 0.0013). High mutation burden and neoantigen load were also associated with improved outcome (p = 0.00014 and 0.00078). The previously identified four mRNA subtypes were predicted on the larger set and also identified a novel poor-survival ‘neuronal’ subtype that nevertheless lacked small cell or neuroendocrine histology. Clustering converged for mRNA, lncRNA and miRNA expression, and for inferred activity of gene sets associated with regulator expression. We identified subsets with differential epithelial-mesenchymal transition scores, carcinoma-in-situ scores, and survival, with implications for distinct therapeutic potential. Conclusions: This integrated analysis of 412 TCGA patient samples validates and extends observations from the first 131 patients and significantly increases our power to detect additional low-frequency aberrations. The results provide unique insights into mechanisms of bladder cancer development, and identify novel subsets of MIBC that may benefit from differential treatment approaches.

Molecular cloning of ESR2 and gene expression analysis of ESR1 and ESR2 in the pituitary gland of the Chinese alligator (Alligator sinensis) during female reproductive cycle

Estrogens play critical roles in reproductive physiology via estrogen receptors (ESRs) in vertebrates, including reptiles. Chinese alligator (Alligator sinensis) is an endemic and endangered reptile species in China. In the present study, we cloned ESR2 gene from the ovary using rapid amplification of cDNA ends (RACE), investigated the spatial expression of ESRs in various tissues and temporal expression of ESRs in the pituitary glands during the reproductive cycle in Chinese alligators by quantitative real-time PCR (qPCR). Bioinformatics and phylogenetic analysis of deduced ESR2 protein were also performed. The full-length cDNA of the ESR2 is 1647bp in length, with an open-reading frame encoding 548 amino acids. The bioinformatics analysis indicated that the deduced amino acid sequence of alligator ESR2 was highly conserved with that of other vertebrate species. In addition, compared to human ESR2, the 14 amino acids in the alligator ESR2 that are essential for specific recognition of estradiol are entirely conserved. The phylogenetic analysis showed that alligators were more closely related to birds than to other reptiles. The results of qPCR showed that the tissue distribution patterns of both ESR subtype mRNAs appeared to be different. In male tissues, the highest mRNA level of both ESRs is in the liver. While in female tissues, ESR1 and ESR2 showed the highest mRNA level in the hypothalamus and pituitary gland, respectively. During the female reproductive cycle, the expression level of ESR1 mRNA increased from the initial post-hibernation period to the reproductive period, reached its peak in the reproductive period, and then decreased in the autumn active period and hibernation period. Conversely, the highest transcription level of ESR2 was observed in the hibernation period.

Integrative clustering reveals a novel split in the luminal A subtype of breast cancer with impact on outcome

BackgroundBreast cancer is a heterogeneous disease at the clinical and molecular level. In this study we integrate classifications extracted from five different molecular levels in order to identify integrated subtypes.MethodsTumor tissue from 425 patients with primary breast cancer from the Oslo2 study was cut and blended, and divided into fractions for DNA, RNA and protein isolation and metabolomics, allowing the acquisition of representative and comparable molecular data. Patients were stratified into groups based on their tumor characteristics from five different molecular levels, using various clustering methods. Finally, all previously identified and newly determined subgroups were combined in a multilevel classification using a “cluster-of-clusters” approach with consensus clustering.ResultsBased on DNA copy number data, tumors were categorized into three groups according to the complex arm aberration index. mRNA expression profiles divided tumors into five molecular subgroups according to PAM50 subtyping, and clustering based on microRNA expression revealed four subgroups. Reverse-phase protein array data divided tumors into five subgroups. Hierarchical clustering of tumor metabolic profiles revealed three clusters. Combining DNA copy number and mRNA expression classified tumors into seven clusters based on pathway activity levels, and tumors were classified into ten subtypes using integrative clustering. The final consensus clustering that incorporated all aforementioned subtypes revealed six major groups. Five corresponded well with the mRNA subtypes, while a sixth group resulted from a split of the luminal A subtype; these tumors belonged to distinct microRNA clusters. Gain-of-function studies using MCF-7 cells showed that microRNAs differentially expressed between the luminal A clusters were important for cancer cell survival. These microRNAs were used to validate the split in luminal A tumors in four independent breast cancer cohorts. In two cohorts the microRNAs divided tumors into subgroups with significantly different outcomes, and in another a trend was observed.ConclusionsThe six integrated subtypes identified confirm the heterogeneity of breast cancer and show that finer subdivisions of subtypes are evident. Increasing knowledge of the heterogeneity of the luminal A subtype may add pivotal information to guide therapeutic choices, evidently bringing us closer to improved treatment for this largest subgroup of breast cancer.

Serotonin-mediated modulation of acetylcholine-induced intracellular calcium responses in chromaffin cells isolated from the rat adrenal medulla

We examined serotonin (5-HT)-mediated modulation of acetylcholine (ACh)-induced intracellular Ca2+ ([Ca2+]i) responses in rat adrenal chromaffin cells using calcium imaging. 5-HT did not induce any [Ca2+]i response in clustered chromaffin cells. However, the magnitude of ACh-induced [Ca2+]i increases in the same specimens was inhibited in the presence of 5-HT. ACh-induced [Ca2+]i increases in chromaffin cells were also inhibited by the 5-HT1A receptor agonist, 8-hydroxy-2-(dipropylamino) tetralin hydrobromide, but were not changed by the 5-HT1B, 5-HT2, or 5-HT3 receptor agonists, CP93129, α-methyl-5-HT, or 1-(m-chlorophenyl) biguanide, respectively. RT-PCR analysis detected the expression of all 5-HT receptor subtype mRNAs, except for 5-HT5 receptors, in extracts of the adrenal medulla. Immunohistochemistry revealed that immunoreactivity for 5-HT1A receptor was located in the chromaffin cells immunoreactive for the biosynthetic enzyme for noradrenaline, dopamine β-hydroxylase. These results suggest that 5-HT inhibits ACh-induced excitability in adrenal chromaffin cells via the 5-HT1A receptor in order to reduce catecholamine release during preganglionic sympathetic stimuli.