Critical Differential Expression Assessment for Individual Bulk RNA-Seq Projects.

Finding the right balance of quality and quantity can be important, and it is essential that project quality does not drop below the level where important main conclusions are missed or misstated. We use knock-out and over-expression studies as a simplification to test recovery of a known causal gene in RNA-Seq cell line experiments. When single-end RNA-Seq reads are aligned with STAR and quantified with htseq-count, we found potential value in testing the use of the Generalized Linear Model (GLM) implementation of edgeR with robust dispersion estimation more frequently for either single-variate or multi-variate 2-group comparisons (with the possibility of defining criteria less stringent than |fold-change| > 1.5 and FDR < 0.05). When considering a limited number of patient sample comparisons with larger sample size, there might be some decreased variability between methods (except for DESeq1). However, at the same time, the ranking of the gene identified using immunohistochemistry (for ER/PR/HER2 in breast cancer samples from The Cancer Genome Atlas) showed as possible shift in performance compared to the cell line comparisons, potentially highlighting utility for standard statistical tests and/or limma-based analysis with larger sample sizes. If this continues to be true in additional studies and comparisons, then that could be consistent with the possibility that it may be important to allocate time for potential methods troubleshooting for genomics projects. Analysis of public data presented in this study does not consider all experimental designs, and presentation of downstream analysis is limited. So, any estimate from this simplification would be an underestimation of the true need for some methods testing for every project. Additionally, this set of independent cell line experiments has a limitation in being able to determine the frequency of missing a highly important gene if the problem is rare (such as 10% or lower). For example, if there was an assumption that only one method can be tested for "initial" analysis, then it is not completely clear to the extent that using edgeR-robust might perform better than DESeq2 in the cell line experiments. Importantly, we do not wish to cause undue concern, and we believe that it should often be possible to define a gene expression differential expression workflow that is suitable for some purposes for many samples. Nevertheless, at the same time, we provide a variety of measures that we believe emphasize the need to critically assess every individual project and maximize confidence in published results.

Targeting ITGB4/SOX2-driven lung cancer stem cells using proteasome inhibitors.

This study investigates the role of integrin ß4 (ITGB4) and stemness-associated factor SOX2 in platinum resistance in lung squamous cell carcinoma (LUSC). The expression of SOX2 and ITGB4 is found to be high in all LUSC subtypes, but the impact of ITGB4 expression on overall patient survival varies by subtype. Cancer stem cells (CSCs) isolated from LUSC patients were found to be resistant to cisplatin, but knocking down ITGB4 or SOX2 sensitized them to cisplatin. Carfilzomib (CFZ) synergized with cisplatin and suppressed CSC growth by inhibiting ITGB4 and SOX2 expression. Additionally, CFZ was found to inhibit SOX2 expression epigenetically by inhibiting histone acetylation at the SOX2 promoter site. CFZ also suppressed the growth of SOX2-dependent small cell lung cancer cells in vitro and in vivo. The study highlights the unique function of CFZ as a transcriptional suppressor of SOX2, independent of its proteasome inhibitory function.

HPV genotyping by L1 amplicon sequencing of archived invasive cervical cancer samples: a pilot study.

BACKGROUND: Human papillomavirus (HPV) is the primary cause of invasive cervical cancer (ICC). The prevalence of various HPV genotypes, ranging from oncogenically low- to high-risk, may be influenced by geographic and demographic factors, which could have critical implications for the screening and prevention of HPV infection and ICC incidence. However, many technical factors may influence the identification of high-risk genotypes associated with ICC in different populations. METHODS: We used high-throughput sequencing of a single amplicon within the HPV L1 gene to assess the influence of patient age, race/ethnicity, histological subtype, sample type, collection date, experimental factors, and computational parameters on the prevalence of HPV genotypes detected in archived DNA (n = 34), frozen tissue (n = 44), and formalin-fixed paraffin-embedded (FFPE) tissue (n = 57) samples collected in the Los Angeles metropolitan area. RESULTS: We found that the percentage of off-target human reads and the concentration of DNA amplified from each sample varied by HPV genotype and by archive type. After accounting for the percentage of human reads and excluding samples with especially low levels of amplified DNA, the HPV prevalence was 95% across all ICC samples: HPV16 was the most common genotype (in 56% of all ICC samples), followed by HPV18 (in 21%). Depending upon the genotyping parameters, the prevalence of HPV58 varied up to twofold in our cohort. In archived DNA and frozen tissue samples, we detected previously established differences in HPV16 and HPV18 frequencies based on histological subtype, but we could not reproduce those findings using our FFPE samples. CONCLUSIONS: In this pilot study, we demonstrate that sample collection, preparation, and analysis methods can influence the detection of certain HPV genotypes and must be carefully considered when drawing any biological conclusions based on HPV genotyping data from ICC samples.

The Gallus gallus RJF reference genome reveals an MHCY haplotype organized in gene blocks that contain 107 loci including 45 specialized, polymorphic MHC class I loci, 41 C-type lectin-like loci, and other loci amid hundreds of transposable elements.

MHCY is a second major histocompatibility complex-like gene region in chickens originally identified by the presence of major histocompatibility complex class I-like and class II-like gene sequences. Up to now, the MHCY gene region has been poorly represented in genomic sequence data. A high density of repetitive sequence and multiple members of several gene families prevented the accurate assembly of short-read sequence data for MHCY. Identified here by single-molecule real-time sequencing sequencing of BAC clones for the Gallus gallus Red Jungle Fowl reference genome are 107 MHCY region genes (45 major histocompatibility complex class I-like, 41 c-type-lectin-like, 8 major histocompatibility complex class IIß, 8 LENG9-like, 4 zinc finger protein loci, and a single only zinc finger-like locus) located amid hundreds of retroelements within 4 contigs representing the region. Sequences obtained for nearby ribosomal RNA genes have allowed MHCY to be precisely mapped with respect to the nucleolar organizer region. Gene sequences provide insights into the unusual structure of the MHCY class I molecules. The MHCY class I loci are polymorphic and group into 22 types based on predicted amino acid sequences. Some MHCY class I loci are full-length major histocompatibility complex class I genes. Others with altered gene structure are considered gene candidates. The amino acid side chains at many of the polymorphic positions in MHCY class I are directed away rather than into the antigen-binding groove as is typical of peptide-binding major histocompatibility complex class I molecules. Identical and nearly identical blocks of genomic sequence contribute to the observed multiplicity of identical MHCY genes and the large size (>639 kb) of the Red Jungle Fowl MHCY haplotype. Multiple points of hybridization observed in fluorescence in situ hybridization suggest that the Red Jungle Fowl MHCY haplotype is made up of linked, but physically separated genomic segments. The unusual gene content, the evidence of highly similar duplicated segments, and additional evidence of variation in haplotype size distinguish polymorphic MHCY from classical polymorphic major histocompatibility complex regions.

Loss of SIRT1 inhibits hematopoietic stem cell aging and age-dependent mixed phenotype acute leukemia.

Aging of hematopoietic stem cells (HSCs) is linked to various blood disorders and malignancies. SIRT1 has been implicated in healthy aging, but its role in HSC aging is poorly understood. Surprisingly, we found that Sirt1 knockout improved the maintenance of quiescence of aging HSCs and their functionality as well as mouse survival in serial bone marrow transplantation (BMT) recipients. The majority of secondary and tertiary BMT recipients of aging wild type donor cells developed B/myeloid mixed phenotype acute leukemia (MPAL), which was markedly inhibited by Sirt1 knockout. SIRT1 inhibition also reduced the growth and survival of human B/myeloid MPAL cells. Sirt1 knockout suppressed global gene activation in old HSCs, prominently the genes regulating protein synthesis and oxidative metabolism, which may involve multiple downstream transcriptional factors. Our results demonstrate an unexpected role of SIRT1 in promoting HSC aging and age-dependent MPAL and suggest SIRT1 may be a new therapeutic target for modulating functions of aging HSCs and treatment of MPAL.

Correction: 2'-Hydroxyflavanone effectively targets RLIP76-mediated drug transport and regulates critical signaling networks in breast cancer.

[This corrects the article DOI: 10.18632/oncotarget.24720.].

Single-cell RNA-sequencing analysis of estrogen- and endocrine-disrupting chemical-induced reorganization of mouse mammary gland.

Menopause is a critical window of susceptibility for its sensitivity to endocrine disrupting chemicals due to the decline of endogenous estrogen. Using a surgical menopausal (ovariectomized) mouse model, we assessed how mammary tissue was affected by both 17ß-estradiol (E2) and polybrominated diphenyl ethers (PBDEs). As flame retardants in household products, PBDEs are widely detected in human serum. During physiologically-relevant exposure to E2, PBDEs enhanced E2-mediated regrowth of mammary glands with terminal end bud-like structures. Analysis of mammary gland RNA revealed that PBDEs both augmented E2-facilitated gene expression and modulated immune regulation. Through single-cell RNA sequencing (scRNAseq) analysis, E2 was found to induce Pgr expression in both Esr1+ and Esr1- luminal epithelial cells and Ccl2 expression in Esr1+ fibroblasts. PBDEs promote the E2-AREG-EGFR-M2 macrophage pathway. Our findings support that E2 + PBDE increases the risk of developing breast cancer through the expansion of estrogen-responsive luminal epithelial cells and immune modulation.

A Human Embryonic Stem Cell Model of Aß-Dependent Chronic Progressive Neurodegeneration.

We describe the construction and phenotypic analysis of a human embryonic stem cell model of progressive Aß-dependent neurodegeneration (ND) with potential relevance to Alzheimer's disease (AD). We modified one allele of the normal APP locus to directly express a secretory form of Aß40 or Aß42, enabling expression from this edited allele to bypass the normal amyloidogenic APP processing pathway. Following neuronal differentiation, edited cell lines specifically accumulate intracellular aggregated/oligomeric Aß, exhibit a synaptic deficit, and have an abnormal accumulation of endolysosomal vesicles. Edited cultures progress to a stage of overt ND. All phenotypes appear at earlier culture times for Aß42 relative to Aß40. Whole transcriptome RNA-Seq analysis identified 23 up and 70 down regulated genes (differentially expressed genes) with similar directional fold change but larger absolute values in the Aß42 samples suggesting common underlying pathogenic mechanisms. Pathway/annotation analysis suggested that down regulation of extracellular matrix and cilia functions is significantly overrepresented. This cellular model could be useful for uncovering mechanisms directly linking Aß to neuronal death and as a tool to screen for new therapeutic agents that slow or prevent human ND.

Identification of multiple potent neutralizing and non-neutralizing antibodies against Epstein-Barr virus gp350 protein with potential for clinical application and as reagents for mapping immunodominant epitopes.

Prevention of Epstein-Barr virus (EBV) infection has focused on generating neutralizing antibodies (nAbs) targeting the major envelope glycoprotein gp350/220 (gp350). In this study, we generated 23 hybridomas producing gp350-specific antibodies. We compared the candidate gp350-specific antibodies to the well-characterized nAb 72A1 by: (1) testing their ability to detect gp350 using enzyme-linked immunosorbent assay, flow cytometry, and immunoblot; (2) sequencing their heavy and light chain complementarity-determining regions (CDRs); (3) measuring the ability of each monoclonal antibody (mAb) to neutralize EBV infection in vitro; and (4) mapping the gp350 amino acids bound by the mAbs using competitive cell and linear peptide binding assays. We performed sequence analysis to identify 15 mAbs with CDR regions unique from those of murine 72A1 (m72A1). We observed antigen binding competition between biotinylated m72A1, serially diluted unlabeled gp350 nAbs (HB1, HB5, HB11, HB20), and our recently humanized 72A1, but not gp350 non-nAb (HB17) or anti-KSHV gH/gL antibody.

Corrigendum to "Herpesvirus BACs: Past, Present, and Future".

[This corrects the article DOI: 10.1155/2011/124595.].

Molecular Mechanisms of Polybrominated Diphenyl Ethers (BDE-47, BDE-100, and BDE-153) in Human Breast Cancer Cells and Patient-Derived Xenografts.

Polybrominated diphenyl ethers (PBDEs) have been used as flame retardants in household materials. Their environmental persistence has led to continuous human exposure and significant tissue levels. Three PBDE congeners (BDE-47, BDE-100, and BDE-153) have been frequently detected in human serum. Although these compounds appear to possess endocrine disrupting activity, studies are largely missing to determine the biological mechanisms of PBDEs in breast cancer cells. Here, we assessed PBDE bioactivities with three complementary strategies: receptor binding/activity assays; nonbiased RNA-sequencing analysis using an estrogen-dependent breast cancer cell line MCF-7aroERE; and in vivo assessments using patient-derived xenograft (PDX) models of human breast cancer. According to the results from in vitro experiments, the PBDE congeners regulate distinct nuclear receptor signaling pathways. BDE-47 acts as a weak agonist of both estrogen receptor α (ERα) and estrogen-related receptor α (ERRα); it could stimulate proliferation of MCF-7aroERE and induced expression of ER-regulated genes (including cell cycle genes). BDE-153 was found to act as a weak antagonist of ERα. BDE-100 could act as (1) an agonist of aryl hydrocarbon receptor (AhR), inducing expression of CYP1A1 and CYP1B1 and (2) as a very weak agonist/antagonist of ERα. In vivo, a mixture of the three congeners with ratios detected in human serum was tested in an ER+ PDX model. The mixture exhibited estrogenic activity through apoptosis/cell cycle regulation and increased the expression of a proliferation marker, Ki-67. These results advance our understanding of the mechanisms of PBDE exposure in breast cancer cells.

Molecular subtypes of triple-negative breast cancer in women of different race and ethnicity.

Molecular subtypes of triple negative breast cancer (TNBC) are associated with variation in survival and may assist in treatment selection. However, the association of patient race or ethnicity with subtypes of TNBC and clinical outcome has not been addressed. Using nCounter Gene Expression Codesets, we classified TNBCs into subtypes: basal-like immune-activated (BLIA), basal-like immunosuppressed (BLIS), luminal androgen receptor (LAR), and mesenchymal (MES) in 48 Hispanic, 12 African-American, 21 Asian, and 34 White patients. Mean age at diagnosis was significantly associated with subtype, with the youngest mean age (50 years) in MES and the oldest mean age (64 years) in LAR (p < 0.0005). Subtype was significantly associated with tumor grade (p = 0.0012) and positive lymph nodes (p = 0.021), with a marginally significant association of tumor stage (p = 0.076). In multivariate Cox-proportional hazards modeling, BLIS was associated with worst survival and LAR with best survival. Hispanics had a significantly higher proportion of BLIS (53%, p = 0.03), whereas Asians had a lower proportion of BLIS (19%, p = 0.05) and a higher proportion of LAR (38%, p = 0.06) compared to the average proportion across all groups. These differences in proportions of subtype across racial and ethnic groups may explain differences in their outcomes. Determining subtypes of TNBC facilitates understanding of the heterogeneity of the TNBCs and provides a foundation for developing subtype-specific therapies and better predictors of TNBC prognosis for all races and ethnicities.

CCNE1 amplification is associated with poor prognosis in patients with triple negative breast cancer.

BACKGROUND: Triple negative breast cancer (TNBC) is aggressive with limited treatment options upon recurrence. Molecular discordance between primary and metastatic TNBC has been observed, but the degree of biological heterogeneity has not been fully explored. Furthermore, genomic evolution through treatment is poorly understood. In this study, we aim to characterize the genomic changes between paired primary and metastatic TNBCs through transcriptomic and genomic profiling, and to identify genomic alterations which may contribute to chemotherapy resistance. METHODS: Genomic alterations and mRNA expression of 10 paired primary and metastatic TNBCs were determined through targeted sequencing, microarray analysis, and RNA sequencing. Commonly mutated genes, as well as differentially expressed and co-expressed genes were identified. We further explored the clinical relevance of differentially expressed genes between primary and metastatic tumors to patient survival using large public datasets. RESULTS: Through gene expression profiling, we observed a shift in TNBC subtype classifications between primary and metastatic TNBCs. A panel of eight cancer driver genes (CCNE1, TPX2, ELF3, FANCL, JAK2, GSK3B, CEP76, and SYK) were differentially expressed in recurrent TNBCs, and were also overexpressed in TCGA and METABRIC. CCNE1 and TPX2 were co-overexpressed in TNBCs. DNA mutation profiling showed that multiple mutations occurred in genes comprising a number of potentially targetable pathways including PI3K/AKT/mTOR, RAS/MAPK, cell cycle, and growth factor receptor signaling, reaffirming the wide heterogeneity of mechanisms driving TNBC. CCNE1 amplification was associated with poor overall survival in patients with metastatic TNBC. CONCLUSIONS: CCNE1 amplification may confer resistance to chemotherapy and is associated with poor overall survival in TNBC.

GFAP Mutations in Astrocytes Impair Oligodendrocyte Progenitor Proliferation and Myelination in an hiPSC Model of Alexander Disease.

Alexander disease (AxD) is a leukodystrophy that primarily affects astrocytes and is caused by mutations in the astrocytic filament gene GFAP. While astrocytes are thought to have important roles in controlling myelination, AxD animal models do not recapitulate critical myelination phenotypes and it is therefore not clear how AxD astrocytes contribute to leukodystrophy. Here, we show that AxD patient iPSC-derived astrocytes recapitulate key features of AxD pathology such as GFAP aggregation. Moreover, AxD astrocytes inhibit proliferation of human iPSC-derived oligodendrocyte progenitor cells (OPCs) in co-culture and reduce their myelination potential. CRISPR/Cas9-based correction of GFAP mutations reversed these phenotypes. Transcriptomic analyses of AxD astrocytes and postmortem brains identified CHI3L1 as a key mediator of AxD astrocyte-induced inhibition of OPC activity. Thus, this iPSC-based model of AxD not only recapitulates patient phenotypes not observed in animal models, but also reveals mechanisms underlying disease pathology and provides a platform for assessing therapeutic interventions.

ERα-mediated cell cycle progression is an important requisite for CDK4/6 inhibitor response in HR+ breast cancer.

While ER has multiple biological effects, ER-cyclin D1-CDK4/6-RB is a critical pathway for the action of estrogen on the cell cycle, especially for breast cancers that rely on estrogen for growth. The latest and most efficient CDK4/6 inhibitors target the phosphorylation of retinoblastoma (RB) tumor suppressor gene; thus, altering levels of many cell cycle molecules. Estrogen receptor (ER)+/HER2- breast cancers have shown great progression free survival when CDK4/6 inhibitors are combined with endocrine therapies. Here we report the mechanism of antiestrogen (fulvestrant) combination with CDK4/6 inhibitors is due to synergism in the suppression of ER-mediated cell cycle progression. Furthermore, we performed single cell analysis of cells from an estrogen dependent/hormone receptor-positive patient derived xenograft (PDX) tumor model treated with palbociclib. These single cells expressed various levels of ER and RB which are involved in cell cycle regulation; and the response to palbociclib treatment relies not only on the ER-cyclin D1-CDK4/6-RB pathway but it is also dependent on elevated levels of ER and/or RB. Our preclinical studies show that palbociclib response is dependent on cells with ER, which is directly involved in cell cycle progression in hormone receptor positive (HR+) breast cancer.

2'-Hydroxyflavanone effectively targets RLIP76-mediated drug transport and regulates critical signaling networks in breast cancer.

Breast cancer (BC) is the most common cancer in women. Estrogen, epidermal growth factor receptor 2 (ERBB2, HER2), and oxidative stress represent critical mechanistic nodes associated with BC. RLIP76 is a major mercapturic acid pathway transporter whose expression is increased in BC. In the quest of a novel molecule with chemopreventive and chemotherapeutic potential, we evaluated the effects of 2'-Hydroxyflavanone (2HF) in BC. 2HF enhanced the inhibitory effects of RLIP76 depletion and also inhibited RLIP76-mediated doxorubicin transport in BC cells. RNA-sequencing revealed that 2HF induces strong reversal of the gene expression pattern in ER+MCF7, HER2+ SKBR3 and triple-negative MDA-MB-231 BC cells with minimal effects on MCF10A normal breast epithelial cells. 2HF down regulated ERα and enhanced inhibitory effects of imatinib mesylate/Gleevec in MCF7 cells. 2HF also down regulated ERα and HER2 gene networks in MCF7 and SKBR3 cells, respectively. 2HF activated TP53 and inhibited TGFß1 canonical pathway in MCF7 and MDA-MB-231 BC cells. 2HF also regulated the expression of a number of critical prognostic genes of MammaPrint panel and their upstream targets including TP53, CDKN2A and MYC. The collective findings from this study provide a comprehensive, direct and integrated evidence for the benefits of 2HF in targeting major and clinically relevant mechanistic regulators of BC.

Rlip depletion prevents spontaneous neoplasia in TP53 null mice.

TP53 (p53) is a tumor suppressor whose functions are lost or altered in most malignancies. p53 homozygous knockout (p53-/-) mice uniformly die of spontaneous malignancy, typically T-cell lymphoma. RALBP1 (RLIP76, Rlip) is a stress-protective, mercapturic acid pathway transporter protein that also functions as a Ral effector involved in clathrin-dependent endocytosis. In stark contrast to p53-/- mice, Rlip-/- mice are highly resistant to carcinogenesis. We report here that partial Rlip deficiency induced by weekly administration of an Rlip-specific phosphorothioate antisense oligonucleotide, R508, strongly inhibited spontaneous as well as benzo(a)pyrene-induced carcinogenesis in p53-/- mice. This treatment effectively prevented large-scale methylomic and transcriptomic abnormalities suggestive of inflammation found in cancer-bearing p53-/- mice. The remarkable efficiency with which Rlip deficiency suppresses spontaneous malignancy in p53-/- mice has not been observed with any previously reported pharmacologic or genetic intervention. These findings are supported by cross-breeding experiments demonstrating that hemizygous Rlip deficiency also reduces the spontaneous malignancy phenotype of p53+/- mice. Rlip is found on the cell surface, and antibodies directed against Rlip were found to inhibit growth and promote apoptosis of cell lines as effectively as Rlip siRNA. The work presented here investigates several features, including oxidative DNA damage of the Rlip-p53 association in malignant transformation, and offers a paradigm for the mechanisms of tumor suppression by p53 and the prospects of suppressing spontaneous malignancy in hereditary cancer syndromes such as Li-Fraumeni.

SGK3 sustains ERα signaling and drives acquired aromatase inhibitor resistance through maintaining endoplasmic reticulum homeostasis.

Many estrogen receptor alpha (ERα)-positive breast cancers initially respond to aromatase inhibitors (AIs), but eventually acquire resistance. Here, we report that serum- and glucocorticoid-inducible kinase 3 (SGK3), a kinase transcriptionally regulated by ERα in breast cancer, sustains ERα signaling and drives acquired AI resistance. SGK3 is up-regulated and essential for endoplasmic reticulum (EnR) homeostasis through preserving sarcoplasmic/EnR calcium ATPase 2b (SERCA2b) function in AI-resistant cells. We have further found that EnR stress response down-regulates ERα expression through the protein kinase RNA-like EnR kinase (PERK) arm, and SGK3 retains ERα expression and signaling by preventing excessive EnR stress. Our study reveals regulation of ERα expression mediated by the EnR stress response and the feed-forward regulation between SGK3 and ERα in breast cancer. Given SGK3 inhibition reduces AI-resistant cell survival by eliciting excessive EnR stress and also depletes ERα expression/function, we propose SGK3 inhibition as a potential effective treatment of acquired AI-resistant breast cancer.