Tianzhou Ma

Effects of aging on circadian patterns of gene expression in the human prefrontal cortex

Significance Circadian rhythms are important in nearly all processes in the brain. Changes in rhythms that come with aging are associated with sleep problems, problems with cognition, and nighttime agitation in elderly people. In this manuscript, we identified transcripts genome-wide that have a circadian rhythm in expression in human prefrontal cortex. Moreover, we describe how these rhythms are changed during normal human aging. Interestingly, we also identified a set of previously unidentified transcripts that become rhythmic only in older individuals. This may represent a compensatory clock that becomes active with the loss of canonical clock function. These studies can help us to develop therapies in the future for older people who suffer from cognitive problems associated with a loss of normal rhythmicity. With aging, significant changes in circadian rhythms occur, including a shift in phase toward a “morning” chronotype and a loss of rhythmicity in circulating hormones. However, the effects of aging on molecular rhythms in the human brain have remained elusive. Here, we used a previously described time-of-death analysis to identify transcripts throughout the genome that have a significant circadian rhythm in expression in the human prefrontal cortex [Brodmann’s area 11 (BA11) and BA47]. Expression levels were determined by microarray analysis in 146 individuals. Rhythmicity in expression was found in ∼10% of detected transcripts (P < 0.05). Using a metaanalysis across the two brain areas, we identified a core set of 235 genes (q < 0.05) with significant circadian rhythms of expression. These 235 genes showed 92% concordance in the phase of expression between the two areas. In addition to the canonical core circadian genes, a number of other genes were found to exhibit rhythmic expression in the brain. Notably, we identified more than 1,000 genes (1,186 in BA11; 1,591 in BA47) that exhibited age-dependent rhythmicity or alterations in rhythmicity patterns with aging. Interestingly, a set of transcripts gained rhythmicity in older individuals, which may represent a compensatory mechanism due to a loss of canonical clock function. Thus, we confirm that rhythmic gene expression can be reliably measured in human brain and identified for the first time (to our knowledge) significant changes in molecular rhythms with aging that may contribute to altered cognition, sleep, and mood in later life.

Comprehensive evaluation of fusion transcript detection algorithms and a meta-caller to combine top performing methods in paired-end RNA-seq data

Background: Fusion transcripts are formed by either fusion genes (DNA level) or trans-splicing events (RNA level). They have been recognized as a promising tool for diagnosing, subtyping and treating cancers. RNA-seq has become a precise and efficient standard for genome-wide screening of such aberration events. Many fusion transcript detection algorithms have been developed for paired-end RNA-seq data but their performance has not been comprehensively evaluated to guide practitioners. In this paper, we evaluated 15 popular algorithms by their precision and recall trade-off, accuracy of supporting reads and computational cost. We further combine top-performing methods for improved ensemble detection. Results: Fifteen fusion transcript detection tools were compared using three synthetic data sets under different coverage, read length, insert size and background noise, and three real data sets with selected experimental validations. No single method dominantly performed the best but SOAPfuse generally performed well, followed by FusionCatcher and JAFFA. We further demonstrated the potential of a meta-caller algorithm by combining top performing methods to re-prioritize candidate fusion transcripts with high confidence that can be followed by experimental validation. Conclusion: Our result provides insightful recommendations when applying individual tool or combining top performers to identify fusion transcript candidates.

Complement Pathway Is Frequently Altered in Endometriosis and Endometriosis-Associated Ovarian Cancer

Purpose: Mechanisms of immune dysregulation associated with advanced tumors are relatively well understood. Much less is known about the role of immune effectors against cancer precursor lesions. Endometrioid and clear-cell ovarian tumors partly derive from endometriosis, a commonly diagnosed chronic inflammatory disease. We performed here a comprehensive immune gene expression analysis of pelvic inflammation in endometriosis and endometriosis-associated ovarian cancer (EAOC). Experimental Design: RNA was extracted from 120 paraffin tissue blocks comprising of normal endometrium (n = 32), benign endometriosis (n = 30), atypical endometriosis (n = 15), and EAOC (n = 43). Serous tumors (n = 15) were included as nonendometriosis-associated controls. The immune microenvironment was profiled using Nanostring and the nCounter GX Human Immunology Kit, comprising probes for a total of 511 immune genes. Results: One third of the patients with endometriosis revealed a tumor-like inflammation profile, suggesting that cancer-like immune signatures may develop earlier, in patients classified as clinically benign. Gene expression analyses revealed the complement pathway as most prominently involved in both endometriosis and EAOC. Complement proteins are abundantly present in epithelial cells in both benign and malignant lesions. Mechanistic studies in ovarian surface epithelial cells from mice with conditional (Cre-loxP) mutations show intrinsic production of complement in epithelia and demonstrate an early link between Kras- and Pten-driven pathways and complement upregulation. Downregulation of complement in these cells interferes with cell proliferation. Conclusions: These findings reveal new characteristics of inflammation in precursor lesions and point to previously unknown roles of complement in endometriosis and EAOC. Clin Cancer Res; 20(23); 6163–74. ©2014 AACR.

The molecular landscape of premenopausal breast cancer

IntroductionBreast cancer in premenopausal women (preM) is frequently associated with worse prognosis compared to that in postmenopausal women (postM), and there is evidence that preM estrogen receptor-positive (ER+) tumors may respond poorly to endocrine therapy. There is, however, a paucity of studies characterizing molecular alterations in premenopausal tumors, a potential avenue for personalizing therapy for this group of women.MethodsUsing TCGA and METABRIC databases, we analyzed gene expression, copy number, methylation, somatic mutation, and reverse-phase protein array data in breast cancers from >2,500 preM and postM women.ResultsPreM tumors showed unique gene expression compared to postM tumors, however, this difference was limited to ER+ tumors. ER+ preM tumors showed unique DNA methylation, copy number and somatic mutations. Integrative pathway analysis revealed that preM tumors had elevated integrin/laminin and EGFR signaling, with enrichment for upstream TGFβ-regulation. Finally, preM tumors showed three different gene expression clusters with significantly different outcomes.ConclusionTogether these data suggest that ER+ preM tumors have distinct molecular characteristics compared to ER+ postM tumors, particularly with respect to integrin/laminin and EGFR signaling, which may represent therapeutic targets in this subgroup of breast cancers.

Changes in Hysterectomy Trends After the Implementation of a Clinical Pathway.

OBJECTIVE: To investigate the effect of hysterectomy pathway implementation on the proportion of total abdominal hysterectomy (TAH) procedures performed between fiscal years 2012 and 2014. METHODS: We conducted a retrospective medical record review. All hysterectomy surgeries performed for benign indications at University of Pittsburgh Medical Center hospitals from fiscal year 2012 to fiscal year 2014 were identified through various systems including Medipac and EpicCare. We analyzed the cases by surgical approach (TAH compared with minimally invasive hysterectomy), age, and indication of surgery. Changes over time were analyzed using Cochran-Armitage test for linear trends. RESULTS: A total number of 6,544 patients were included in this study. The mean age of the participants was 48.6 years (standard deviation 11.69). In fiscal year 2012, of 1,934 hysterectomies performed as a result of noncancerous conditions, 538 were TAH procedures (27.8%). However, this number declined in fiscal year 2013 to 22% (485 TAH procedures of 2,186 hysterectomies) and further declined in fiscal year 2014 to 17% (413 TAH surgeries of 2,424 hysterectomies). Overall, there was a significant reduction in the proportion of TAH procedures, from 27.8% in fiscal year 2012 to 17% in fiscal year 2014 (P for trend <.001). After adjusting for surgery indication, the decreasing trend of TAH procedures still persisted (P for trend <.001). CONCLUSION: Implementation of a hysterectomy pathway has been associated with a decrease in the proportion of TAH hysterectomy procedures.

Age-Related Gene Expression in the Frontal Cortex Suggests Synaptic Function Changes in Specific Inhibitory Neuron Subtypes

Genome-wide expression profiling of the human brain has revealed genes that are differentially expressed across the lifespan. Characterizing these genes adds to our understanding of both normal functions and pathological conditions. Additionally, the specific cell-types that contribute to the motor, sensory and cognitive declines during aging are unclear. Here we test if age-related genes show higher expression in specific neural cell types. Our study leverages data from two sources of murine single-cell expression data and two sources of age-associations from large gene expression studies of postmortem human brain. We used nonparametric gene set analysis to test for age-related enrichment of genes associated with specific cell-types; we also restricted our analyses to specific gene ontology groups. Our analyses focused on a primary pair of single-cell expression data from the mouse visual cortex and age-related human post-mortem gene expression information from the orbitofrontal cortex. Additional pairings that used data from the hippocampus, prefrontal cortex, somatosensory cortex and blood were used to validate and test specificity of our findings. We found robust age-related up-regulation of genes that are highly expressed in oligodendrocytes and astrocytes, while genes highly expressed in layer 2/3 glutamatergic neurons were down-regulated across age. Genes not specific to any neural cell type were also down-regulated, possibly due to the bulk tissue source of the age-related genes. A gene ontology-driven dissection of the cell-type enriched genes highlighted the strong down-regulation of genes involved in synaptic transmission and cell-cell signaling in the Somatostatin (Sst) neuron subtype that expresses the cyclin dependent kinase 6 (Cdk6) and in the vasoactive intestinal peptide (Vip) neuron subtype expressing myosin binding protein C, slow type (Mybpc1). These findings provide new insights into cell specific susceptibility to normal aging, and suggest age-related synaptic changes in specific inhibitory neuron subtypes.

Effects of Kras activation and Pten deletion alone or in combination on MUC1 biology and epithelial-to-mesenchymal transition in ovarian cancer.

Mucin1 (MUC1) is an epithelial glycoprotein overexpressed in ovarian cancer and actively involved in tumor cell migration and metastasis. Using novel in vitro and in vivo MUC1-expressing conditional (Cre-loxP) ovarian tumor models, we focus here on MUC1 biology and the roles of Kras activation and Pten deletion during cell transformation and epithelial-to-mesenchymal transition (EMT). We generated several novel murine ovarian cancer cell lines derived from the ovarian surface epithelia (OSE) of mice with conditional mutations in Kras, Pten or both. In addition, we also generated several tumor-derived new cell lines that reproduce the original tumor phenotype in vivo and mirror late stage metastatic disease. Our results demonstrate that de novo activation of oncogenic Kras does not trigger increased proliferation, cellular transformation or EMT, and prevents MUC1 upregulation. In contrast, Pten deletion accelerates cell proliferation, triggers cellular transformation in vitro and in vivo, and stimulates MUC1 expression. Ovarian tumor-derived cell lines MKP-Liver and MKP-Lung cells reproduce in vivo EMT and represent the first immune competent mouse model for distant hematogenous spread. Whole genome microarray expression analysis using tumor and OSE-derived cell lines reveal a 121 gene signature associated with EMT and metastasis. When applied to n=542 cases from The Cancer Genome Atlas (TCGA) ovarian cancer dataset, the gene signature identifies a patient subset with decreased survival (P=0.04). Using an extensive collection of novel murine cell lines we have identified distinct roles for Kras and Pten on MUC1 and EMT in vivo and in vitro. The data has implications for future design of combination therapies targeting Kras mutations, Pten deletions and MUC1 vaccines.

Changes in inflammatory endometrial cancer risk biomarkers in individuals undergoing surgical weight loss

OBJECTIVE Obesity has been strongly linked to endometrial cancer (EC) risk. A number of potential EC risk biomarkers have been proposed, including heightened pro-inflammatory cytokines and adipokines. To evaluate if bariatric surgery can serve as a means for altering levels of such EC risk biomarkers, we investigated changes in these biomarkers after weight loss. METHODS Blood samples were collected pre-operatively and 6months post-operatively in 107 female bariatric surgery patients aged 18-72years. Wilcoxon signed-rank tests were used to compare biomarker levels (measured using xMAP immunoassays) pre- and post-surgery. Normative comparisons were implemented to contrast 6-month post-surgery biomarker levels to levels in a sample of 74 age-matched non-obese women. Linear regression was used to evaluate the relationship between biomarker expression at baseline and 6months post-surgery and the relationship between race and biomarker levels. RESULTS On average, participants lost 30.15kg (SD: 12.26) after the bariatric intervention. Levels of C-peptide, insulin, CRP, leptin, IL-1Rα, and IL-6 significantly decreased, while levels of SHBG, IGFBP1, and adiponectin significantly increased with weight loss. Normative comparisons showed the levels of SHBG, C-peptide, insulin, IGFBP1, adiponectin, CRP, and TNFα after bariatric intervention approached the level of markers in comparison group. Multiple regression analyses revealed significant relationships between changes in BMI and changes in biomarker levels. The changes in IL-1Rα were significantly associated with race. CONCLUSIONS Our findings demonstrate that normalization of EC risk biomarkers can be achieved with bariatric surgery. Improved understanding of biological mechanisms associated with weight loss may inform preventive strategies for EC.

Active estrogen receptor-alpha signaling in ovarian cancer models and clinical specimens

Purpose: High-grade serous ovarian cancer (HGSOC) is an aggressive disease with few available targeted therapies. Despite high expression of estrogen receptor-alpha (ERα) in approximately 80% of HGSOC and some small but promising clinical trials of endocrine therapy, ERα has been understudied as a target in this disease. We sought to identify hormone-responsive, ERα-dependent HGSOC. Experimental Design: We characterized endocrine response in HGSOC cells across culture conditions [ two-dimensional (2D), three-dimensional (3D), forced suspension] and in patient-derived xenograft (PDX) explants, assessing proliferation and gene expression. Estrogen-regulated transcriptome data were overlapped with public datasets to develop a comprehensive panel of ERα target genes. Expression of this panel and ERα H-score were assessed in HGSOC samples from patients who received endocrine therapy. Time on endocrine therapy was used as a surrogate for clinical response. Results: Proliferation is ERα-regulated in HGSOC cells in vitro and in vivo, and is partly dependent on 3D context. Transcriptomic studies identified genes shared by cell lines and PDX explants as ERα targets. The selective ERα downregulator (SERD) fulvestrant is more effective than tamoxifen in blocking ERα action. ERα H-score is predictive of efficacy of endocrine therapy, and this prediction is further improved by inclusion of target gene expression, particularly IGFBP3. Conclusions: Laboratory models corroborate intertumor heterogeneity of endocrine response in HGSOC but identify features associated with functional ERα and endocrine responsiveness. Assessing ERα function (e.g., IGFBP3 expression) in conjunction with H-score may help select patients who would benefit from endocrine therapy. Preclinical data suggest that SERDs might be more effective than tamoxifen. Clin Cancer Res; 23(14); 3802–12. ©2017 AACR.

Incorporating functional annotation information in prioritizing disease associated SNPs from genome wide association studies

With recent advances in genotyping and sequencing technologies, many disease susceptibility loci have been identified. However, much of the genetic heritability remains unexplained and the replication rate between independent studies is still low. Meanwhile, there have been increasing efforts on functional annotations of the entire human genome, such as the Encyclopedia of DNA Elements (ENCODE) project and other similar projects. It has been shown that incorporating these functional annotations to prioritize genome wide association signals may help identify true association signals. However, to our knowledge, the extent of the improvement when functional annotation data are considered has not been studied in the literature. In this article, we propose a statistical framework to estimate the improvement in replication rate with annotation data, and apply it to Crohn’s disease and DNase I hypersensitive sites. The results show that with cell line specific functional annotations, the expected replication rate is improved, but only at modest level.