Ryne C. Ramaker

BACKGROUND: Psychiatric disorders are multigenic diseases with complex etiology that contribute significantly to human morbidity and mortality. Although clinically distinct, several disorders share many symptoms, suggesting common underlying molecular changes exist that may implicate important regulators of pathogenesis and provide new therapeutic targets. METHODS: We performed RNA sequencing on tissue from the anterior cingulate cortex, dorsolateral prefrontal cortex, and nucleus accumbens from three groups of 24 patients each diagnosed with schizophrenia, bipolar disorder, or major depressive disorder, and from 24 control subjects. We identified differentially expressed genes and validated the results in an independent cohort. Anterior cingulate cortex samples were also subjected to metabolomic analysis. ChIP-seq data were used to characterize binding of the transcription factor EGR1. RESULTS: We compared molecular signatures across the three brain regions and disorders in the transcriptomes of post-mortem human brain samples. The most significant disease-related differences were in the anterior cingulate cortex of schizophrenia samples compared to controls. Transcriptional changes were assessed in an independent cohort, revealing the transcription factor EGR1 as significantly down-regulated in both cohorts and as a potential regulator of broader transcription changes observed in schizophrenia patients. Additionally, broad down-regulation of genes specific to neurons and concordant up-regulation of genes specific to astrocytes was observed in schizophrenia and bipolar disorder patients relative to controls. Metabolomic profiling identified disruption of GABA levels in schizophrenia patients. CONCLUSIONS: We provide a comprehensive post-mortem transcriptome profile of three psychiatric disorders across three brain regions. We highlight a high-confidence set of independently validated genes differentially expressed between schizophrenia and control patients in the anterior cingulate cortex and integrate transcriptional changes with untargeted metabolite profiling.

. However, only a minority of the more than 1,600 transcription factors encoded in the human genome has been assayed. Here we present, as part of the ENCODE (Encyclopedia of DNA Elements) project, data and analyses from chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) experiments using the human HepG2 cell line for 208 chromatin-associated proteins (CAPs). These comprise 171 transcription factors and 37 transcriptional cofactors and chromatin regulator proteins, and represent nearly one-quarter of CAPs expressed in HepG2 cells. The binding profiles of these CAPs form major groups associated predominantly with promoters or enhancers, or with both. We confirm and expand the current catalogue of DNA sequence motifs for transcription factors, and describe motifs that correspond to other transcription factors that are co-enriched with the primary ChIP target. For example, FOX family motifs are enriched in ChIP-seq peaks of 37 other CAPs. We show that motif content and occupancy patterns can distinguish between promoters and enhancers. This catalogue reveals high-occupancy target regions at which many CAPs associate, although each contains motifs for only a minority of the numerous associated transcription factors. These analyses provide a more complete overview of the gene regulatory networks that define this cell type, and demonstrate the usefulness of the large-scale production efforts of the ENCODE Consortium.

BACKGROUND: There is clinical evidence that very low and safe levels of amplitude-modulated electromagnetic fields administered via an intrabuccal spoon-shaped probe may elicit therapeutic responses in patients with cancer. However, there is no known mechanism explaining the anti-proliferative effect of very low intensity electromagnetic fields. METHODS: To understand the mechanism of this novel approach, hepatocellular carcinoma (HCC) cells were exposed to 27.12 MHz radiofrequency electromagnetic fields using in vitro exposure systems designed to replicate in vivo conditions. Cancer cells were exposed to tumour-specific modulation frequencies, previously identified by biofeedback methods in patients with a diagnosis of cancer. Control modulation frequencies consisted of randomly chosen modulation frequencies within the same 100 Hz-21 kHz range as cancer-specific frequencies. RESULTS: The growth of HCC and breast cancer cells was significantly decreased by HCC-specific and breast cancer-specific modulation frequencies, respectively. However, the same frequencies did not affect proliferation of nonmalignant hepatocytes or breast epithelial cells. Inhibition of HCC cell proliferation was associated with downregulation of XCL2 and PLP2. Furthermore, HCC-specific modulation frequencies disrupted the mitotic spindle. CONCLUSION: These findings uncover a novel mechanism controlling the growth of cancer cells at specific modulation frequencies without affecting normal tissues, which may have broad implications in oncology.

BACKGROUND: Pancreatic adenocarcinoma patients have low survival rates due to late-stage diagnosis and high rates of cancer recurrence even after surgical resection. It is important to understand the molecular characteristics associated with survival differences in pancreatic adenocarcinoma tumors that may inform patient care. RESULTS: RNA sequencing was performed for 51 patient tumor tissues extracted from patients undergoing surgical resection, and expression was associated with overall survival time from diagnosis. Our analysis uncovered 323 transcripts whose expression correlates with survival time in our pancreatic patient cohort. This genomic signature was validated in an independent RNA-seq dataset of 68 additional patients from the International Cancer Genome Consortium. We demonstrate that this transcriptional profile is largely independent of markers of cellular division and present a 19-transcript predictive model built from a subset of the 323 transcripts that can distinguish patients with differing survival times across both the training and validation patient cohorts. We present evidence that a subset of the survival-associated transcripts is associated with resistance to gemcitabine treatment in vitro, and reveal that reduced expression of one of the survival-associated transcripts, Angiopoietin-like 4, impairs growth of a gemcitabine-resistant pancreatic cancer cell line. CONCLUSIONS: Gene expression patterns in pancreatic adenocarcinoma tumors can distinguish patients with differing survival outcomes after undergoing surgical resection, and the survival difference could be associated with the intrinsic gemcitabine sensitivity of primary patient tumors. Thus, these transcriptional differences may impact patient care by distinguishing patients who would benefit from a non-gemcitabine based therapy.

// Joy M. McDaniel 1, 2 , Katherine E. Varley 3 , Jason Gertz 3 , Daniel S. Savic 1 , Brian S. Roberts 1 , Sarah K. Bailey 4 , Lalita A. Shevde 4, 6 , Ryne C. Ramaker 1, 7 , Brittany N. Lasseigne 1 , Marie K. Kirby 1 , Kimberly M. Newberry 1 , E. Christopher Partridge 1 , Angela L. Jones 1 , Braden Boone 1 , Shawn E. Levy 1 , Patsy G. Oliver 5 , Katherine C. Sexton 6 , William E. Grizzle 6 , Andres Forero 6 , Donald J. Buchsbaum 5 , Sara J. Cooper 1 , Richard M. Myers 1 1 HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA 2 The University of Alabama in Huntsville, Huntsville, AL 35899, USA 3 Department of Oncological Sciences, Huntsman Cancer Institute, University of Utah, Salt Lake City, UT, 84112, USA 4 Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA 5 Department of Radiation Oncology, University of Alabama at Birmingham, Birmingham, AL, 35294, USA 6 University of Alabama at Birmingham Comprehensive Cancer Center, Birmingham, AL 35294, USA 7 Department of Genetics, University of Alabama at Birmingham, Birmingham, AL, 35294, USA Correspondence to: Richard M. Myers, email: rmyers@hudsonalpha.org Keywords: TNBC, STAT3, ChIP-seq, RNA-seq, invasion Received: August 15, 2016      Accepted: November 14, 2016      Published: December 24, 2016 ABSTRACT Breast cancer is a heterogeneous disease comprised of four molecular subtypes defined by whether the tumor-originating cells are luminal or basal epithelial cells. Breast cancers arising from the luminal mammary duct often express estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth receptor 2 (HER2). Tumors expressing ER and/or PR are treated with anti-hormonal therapies, while tumors overexpressing HER2 are targeted with monoclonal antibodies. Immunohistochemical detection of ER, PR, and HER2 receptors/proteins is a critical step in breast cancer diagnosis and guided treatment. Breast tumors that do not express these proteins are known as “triple negative breast cancer” (TNBC) and are typically basal-like. TNBCs are the most aggressive subtype, with the highest mortality rates and no targeted therapy, so there is a pressing need to identify important TNBC tumor regulators. The signal transducer and activator of transcription 3 (STAT3) transcription factor has been previously implicated as a constitutively active oncogene in TNBC. However, its direct regulatory gene targets and tumorigenic properties have not been well characterized. By integrating RNA-seq and ChIP-seq data from 2 TNBC tumors and 5 cell lines, we discovered novel gene signatures directly regulated by STAT3 that were enriched for processes involving inflammation, immunity, and invasion in TNBC. Functional analysis revealed that STAT3 has a key role regulating invasion and metastasis, a characteristic often associated with TNBC. Our findings suggest therapies targeting STAT3 may be important for preventing TNBC metastasis.