Citations

A cancer-promoting histone protein Mutations in the chromatin protein histone H3 are found in a number of pediatric cancers. The lysine-36–to–methionine (K36M) “oncohistone” mutation is seen in almost all chondroblastomas. Fang et al. show that the K36M mutant histones inhibit the normal methylation of this same residue in wild-type H3 histones. They do so by interfering with the enzymes that normally methylate this residue. The altered chromatin methylation patterns alter the expression of known cancer-related genes and impart cancer-related characteristics to the chondrocyte cells. Science , this issue p. 1344

Abstract BACKGROUND: We previously reported that low 21 gene Recurrence Scores (RS) identify patients with ER-positive, lymph node-positive breast cancer who may not benefit from anthracycline-based adjuvant chemotherapy added to tamoxifen (SWOG-8814A NCI correlative science study; Albain et al. Lancet Oncol 2010). New exploratory and comprehensive quantitative analyses now permit whole transcriptome NGS on residual RNA extracted from FFPE blocks 12-18 years post-fixation. Herein, we report methodology details and feasibility results (see companion abstract, Albain et al., for clinical outcomes correlations). METHODS and RESULTS: Sequencing was carried out in Illumina HiSeq 2000 instruments, yielding 4.2 trillion data points. Messenger RNA expression was quantified using 3rd quartile normalization. Both Library (RNA) and Sequencing Standards showed high quality coverage as measured by median uniquely mapped reads over a 13 month window (168M and 182M, respectively, including duplicate reads). The median absolute deviation (MAD) of the relative log expression (RLE) of mapped reads for the Library and Sequencing Standards was 0.22 and 0.05, respectively. The Library Standard variation was greater than the Sequencing Standard, as library preparation was manual. Of 360 patient samples with sufficient RNA (≥ 100 ng total RNA), 354 (98.3%) were successfully sequenced and included in the final analysis data set. Average library yield was 39 ng/μL. Only 5 libraries failed yield requirements and one library failed expression quality metrics. The median insert length was 120 bp with the first and third quartiles 93 and 152 bp, respectively. After removal of duplicate reads, 82% of reads were uniquely mapped, and the median library size was 8.95M (number of unique mapped reads). Sequences with counts <10 for all 354 patients were excluded. The medians of the 1st, 2nd and 3rd quartiles for exons mapped to the RefSeq database were 20, 40 and 78 counts, and for introns, 72, 134 and 244 counts, respectively. The majority of exonic (86.7%) and intronic (95%) sequences were mapped. There were 20,101 RefSeq mapped exons with counts ≥10. Of these exons, 988 passed additional filtering criteria and were subjected to hierarchical clustering, with Gene Ontology and pathway analysis performed on selected gene expression patterns (for results, see companion abstract of Albain et al.). CONCLUSIONS: High quality whole transcriptome NGS is feasible from decades-old clinical trial FFPE specimens that have not been stored in any special fashion. Controlled laboratory, bioinformatics and biostatistics methods, with inclusion of appropriate process controls, ensure robustness and reliability of the NGS process. This in turn results in the discovery and validation of biologically and clinically relevant variations from prior landmark clinical trials. SUPPORT: NCI CA 180888, CA180819, CA180821, CA180820, CA180863; in part, Genomic Health, Inc. Citation Format: Cherbavaz DB, Hayes DF, Qu K, Crager MR, Barlow WR, Goddard AD, Beasley EM, Jeong J, Collin F, Liu M-L, Rae JM, Ravdin PM, Tripathy D, Gralow JR, Livingston RB, Osborne CK, Ingle JN, Pritchard KI, Davidson NE, Carey LA, Sing AP, Baehner FL, Hortobagyi GN, Shak S, Albain KS. Successful whole transcriptome analysis of 25-year-old breast tumor samples from the phase III trial SWOG-8814 by next generation sequencing (NGS): Standardized analytical methods for exploratory and validation studies. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-07-01.

ABSTRACTPreservation of osteochondral allografts used for transplantation is critical to ensure favorable outcomes for patients after surgical treatment of cartilage defects. To study the biological effects of protocols currently used for cartilage storage, we investigated differences in gene expression between stored allograft cartilage and fresh cartilage from living donors using high throughput molecular screening strategies. We applied next generation RNA sequencing (RNA‐seq) and real‐time reverse transcription quantitative polymerase chain reaction (RT‐qPCR) to assess genome‐wide differences in mRNA expression between stored allograft cartilage and fresh cartilage tissue from living donors. Gene ontology analysis was used to characterize biological pathways associated with differentially expressed genes. Our studies establish reduced levels of mRNAs encoding cartilage related extracellular matrix (ECM) proteins (i.e., COL1A1, COL2A1, COL10A1, ACAN, DCN, HAPLN1, TNC, and COMP) in stored cartilage. These changes occur concomitantly with increased expression of “early response genes” that encode transcription factors mediating stress/cytoprotective responses (i.e., EGR1, EGR2, EGR3, MYC, FOS, FOSB, FOSL1, FOSL2, JUN, JUNB, and JUND). The elevated expression of “early response genes” and reduced levels of ECM‐related mRNAs in stored cartilage allografts suggests that tissue viability may be maintained by a cytoprotective program that reduces cell metabolic activity. These findings have potential implications for future studies focused on quality assessment and clinical optimization of osteochondral allografts used for cartilage transplantation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1950–1959, 2016.

Diabetes-induced cognitive decline has been recognized in human patients of type 2 diabetes mellitus and mouse model of obesity, but the underlying mechanisms or therapeutic targets are not clearly identified. We investigated the effect of caloric restriction on diabetes-induced memory deficits and searched a molecular mechanism of caloric restriction-mediated neuroprotection. C57BL/6 mice were fed a high-fat diet for 40 weeks and RNA-seq analysis was performed in the hippocampus of high-fat diet-fed mice. To investigate caloric restriction effect on differential expression of genes, mice were fed high-fat diet for 20 weeks and continued on high-fat diet or subjected to caloric restriction (2 g/day) for 12 weeks. High-fat diet-fed mice exhibited insulin resistance, glial activation, blood–brain barrier leakage, and memory deficits, in that we identified neurogranin, a down-regulated gene in high-fat diet-fed mice using RNA-seq analysis; neurogranin regulates Ca 2+ /calmodulin-dependent synaptic function. Caloric restriction increased insulin sensitivity, reduced high-fat diet-induced blood–brain barrier leakage and glial activation, and improved memory deficit. Furthermore, caloric restriction reversed high-fat diet-induced expression of neurogranin and the activation of Ca 2+ /calmodulin-dependent protein kinase II and calpain as well as the downstream effectors. Our results suggest that neurogranin is an important factor of high-fat diet-induced memory deficits on which caloric restriction has a therapeutic effect by regulating neurogranin-associated calcium signaling.

Transforming growth factor β (TGFβ) is a pleiotropic protein secreted from essentially all cell types and primary tissues. While TGFβ’s actions reflect the activity of a number of signaling networks, the primary mediator of TGFβ responses are the Smad proteins. Following receptor activation, these cytoplasmic proteins form hetero-oligomeric complexes that translocate to the nucleus and affect gene transcription. Here, through biological, biochemical, and immunofluorescence approaches, sorting nexin 9 (SNX9) is identified as being required for Smad3-dependent responses. SNX9 interacts with phosphorylated (p) Smad3 independent of Smad2 or Smad4 and promotes more rapid nuclear delivery than that observed independent of ligand. Although SNX9 does not bind nucleoporins Nup153 or Nup214 or some β importins (Imp7 or Impβ), it mediates the association of pSmad3 with Imp8 and the nuclear membrane. This facilitates nuclear translocation of pSmad3 but not SNX9.