Paired microbiome and metabolome analyses associate bile acid changes with colorectal cancer progression.

Colorectal cancer (CRC) is driven by genomic alterations in concert with dietary influences, with the gut microbiome implicated as an effector in disease development and progression. While meta-analyses have provided mechanistic insight into patients with CRC, study heterogeneity has limited causal associations. Using multi-omics studies on genetically controlled cohorts of mice, we identify diet as the major driver of microbial and metabolomic differences, with reductions in α diversity and widespread changes in cecal metabolites seen in high-fat diet (HFD)-fed mice. In addition, non-classic amino acid conjugation of the bile acid cholic acid (AA-CA) increased with HFD. We show that AA-CAs impact intestinal stem cell growth and demonstrate that Ileibacterium valens and Ruminococcus gnavus are able to synthesize these AA-CAs. This multi-omics dataset implicates diet-induced shifts in the microbiome and the metabolome in disease progression and has potential utility in future diagnostic and therapeutic developments.

FXR mediates ILC-intrinsic responses to intestinal inflammation.

The pleiotropic actions of the Farnesoid X Receptor (FXR) are required for gut health, and reciprocally, reduced intestinal FXR signaling is seen in inflammatory bowel diseases (IBDs). Here, we show that activation of FXR selectively in the intestine is protective in inflammation-driven models of IBD. Prophylactic activation of FXR restored homeostatic levels of pro-inflammatory cytokines, most notably IL17. Importantly, these changes were attributed to FXR regulation of innate lymphoid cells (ILCs), with both the inflammation-driven increases in ILCs, and ILC3s in particular, and the induction of Il17a and Il17f in ILC3s blocked by FXR activation. Moreover, a population of ILC precursor-like cells increased with treatment, implicating FXR in the maturation/differentiation of ILC precursors. These findings identify FXR as an intrinsic regulator of intestinal ILCs and a potential therapeutic target in inflammatory intestinal diseases.

Comprehensive identification of mRNA isoforms reveals the diversity of neural cell-surface molecules with roles in retinal development and disease.

Genes encoding cell-surface proteins control nervous system development and are implicated in neurological disorders. These genes produce alternative mRNA isoforms which remain poorly characterized, impeding understanding of how disease-associated mutations cause pathology. Here we introduce a strategy to define complete portfolios of full-length isoforms encoded by individual genes. Applying this approach to neural cell-surface molecules, we identify thousands of unannotated isoforms expressed in retina and brain. By mass spectrometry we confirm expression of newly-discovered proteins on the cell surface in vivo. Remarkably, we discover that the major isoform of a retinal degeneration gene, CRB1, was previously overlooked. This CRB1 isoform is the only one expressed by photoreceptors, the affected cells in CRB1 disease. Using mouse mutants, we identify a function for this isoform at photoreceptor-glial junctions and demonstrate that loss of this isoform accelerates photoreceptor death. Therefore, our isoform identification strategy enables discovery of new gene functions relevant to disease.

FXR Regulates Intestinal Cancer Stem Cell Proliferation.

Increased levels of intestinal bile acids (BAs) are a risk factor for colorectal cancer (CRC). Here, we show that the convergence of dietary factors (high-fat diet) and dysregulated WNT signaling (APC mutation) alters BA profiles to drive malignant transformations in Lgr5-expressing (Lgr5+) cancer stem cells and promote an adenoma-to-adenocarcinoma progression. Mechanistically, we show that BAs that antagonize intestinal farnesoid X receptor (FXR) function, including tauro-ß-muricholic acid (T-ßMCA) and deoxycholic acid (DCA), induce proliferation and DNA damage in Lgr5+ cells. Conversely, selective activation of intestinal FXR can restrict abnormal Lgr5+ cell growth and curtail CRC progression. This unexpected role for FXR in coordinating intestinal self-renewal with BA levels implicates FXR as a potential therapeutic target for CRC.

ERRγ Promotes Angiogenesis, Mitochondrial Biogenesis, and Oxidative Remodeling in PGC1α/ß-Deficient Muscle.

PGC1α is a pleiotropic co-factor that affects angiogenesis, mitochondrial biogenesis, and oxidative muscle remodeling via its association with multiple transcription factors, including the master oxidative nuclear receptor ERRγ. To decipher their epistatic relationship, we explored ERRγ gain of function in muscle-specific PGC1α/ß double-knockout (PKO) mice. ERRγ-driven transcriptional reprogramming largely rescues muscle damage and improves muscle function in PKO mice, inducing mitochondrial biogenesis, antioxidant defense, angiogenesis, and a glycolytic-to-oxidative fiber-type transformation independent of PGC1α/ß. Furthermore, in combination with voluntary exercise, ERRγ gain of function largely restores mitochondrial energetic deficits in PKO muscle, resulting in a 5-fold increase in running performance. Thus, while PGC1s can interact with multiple transcription factors, these findings implicate ERRs as the major molecular target through which PGC1α/ß regulates both innate and adaptive energy metabolism.

A Novel Ultrasensitive In Situ Hybridization Approach to Detect Short Sequences and Splice Variants with Cellular Resolution.

Investigating the expression of RNAs that differ by short or single nucleotide sequences at a single-cell level in tissue has been limited by the sensitivity and specificity of in situ hybridization (ISH) techniques. Detection of short isoform-specific sequences requires RNA isolation for PCR analysis-an approach that loses the regional and cell-type-specific distribution of isoforms. Having the capability to distinguish the differential expression of RNA variants in tissue is critical because alterations in mRNA splicing and editing, as well as coding single nucleotide polymorphisms, have been associated with numerous cancers, neurological and psychiatric disorders. Here we introduce a novel highly sensitive single-probe colorimetric/fluorescent ISH approach that targets short exon/exon RNA splice junctions using single-pair oligonucleotide probes (~ 50 bp). We use this approach to investigate, with single-cell resolution, the expression of four transcripts encoding the neuregulin (NRG) receptor ErbB4 that differ by alternative splicing of exons encoding two juxtamembrane (JMa/JMb) and two cytoplasmic (CYT-1/CYT-2) domains that alter receptor stability and signaling modes, respectively. By comparing ErbB4 hybridization on sections from wild-type and ErbB4 knockout mice (missing exon 2), we initially demonstrate that single-pair probes provide the sensitivity and specificity to visualize and quantify the differential expression of ErbB4 isoforms. Using cell-type-specific GFP reporter mice, we go on to demonstrate that expression of ErbB4 isoforms differs between neurons and oligodendrocytes, and that this differential expression of ErbB4 isoforms is evolutionarily conserved to humans. This single-pair probe ISH approach, known as BaseScope, could serve as an invaluable diagnostic tool to detect alternative spliced isoforms, and potentially single base polymorphisms, associated with disease.

Simultaneous Detection of Protein and mRNA in Jurkat and KG-1a Cells by Mass Cytometry.

Mass cytometry uniquely enables high-dimensional single-cell analysis of complex populations. This recently developed technology is based on inductively coupled time-of-flight mass spectrometry for multiplex proteomic analysis of more than 40 markers per cell. The ability to characterize the transcriptome is critical for the understanding of disease pathophysiology, medical diagnostics, and drug discovery. Current techniques allowing the in situ detection of transcripts in single cells are limited to a small number of simultaneous targets and are generally tedious and labor-intensive. In this report, we present the development of a multiplex method for targeted RNA detection by combining the mass cytometry and RNAscope® platforms. This novel assay, called Metal In Situ Hybridization (MISH), includes the hybridization of RNA-specific target probes followed by signal amplification achieved through a cascade of hybridization events, ending with the binding of amplifier-specific detector probes. The detector probes are tagged with isotopically pure metal atoms used for detection by mass cytometry. Proof-of-principle experiments show the simultaneous detection of three mRNA targets in Jurkat cells in suspension cell assay mode. The localization of transcripts was also investigated using the imaging mass cytometry platform in Jurkat and KG-1a cells. In addition, we optimized the antibody staining procedure to allow the co-detection of mRNA and cell surface markers. Our data demonstrate that MISH can be used to complement protein detection by mass cytometry as well as to investigate gene transcription and translation in single cells. © 2017 International Society for Advancement of Cytometry.

Suppression of FIP200 and autophagy by tumor-derived lactate promotes naïve T cell apoptosis and affects tumor immunity.

Naïve T cells are poorly studied in cancer patients. We report that naïve T cells are prone to undergo apoptosis due to a selective loss of FAK family-interacting protein of 200 kDa (FIP200) in ovarian cancer patients and tumor-bearing mice. This results in poor antitumor immunity via autophagy deficiency, mitochondria overactivation, and high reactive oxygen species production in T cells. Mechanistically, loss of FIP200 disables the balance between proapoptotic and antiapoptotic Bcl-2 family members via enhanced argonaute 2 (Ago2) degradation, reduced Ago2 and microRNA1198-5p complex formation, less microRNA1198-5p maturation, and consequently abolished microRNA1198-5p-mediated repression on apoptotic gene Bak1 Bcl-2 overexpression and mitochondria complex I inhibition rescue T cell apoptosis and promoted tumor immunity. Tumor-derived lactate translationally inhibits FIP200 expression by down-regulating the nicotinamide adenine dinucleotide level while potentially up-regulating the inhibitory effect of adenylate-uridylate-rich elements within the 3' untranslated region of Fip200 mRNA. Thus, tumors metabolically target naïve T cells to evade immunity.

PPARδ Promotes Running Endurance by Preserving Glucose.

Management of energy stores is critical during endurance exercise; a shift in substrate utilization from glucose toward fat is a hallmark of trained muscle. Here we show that this key metabolic adaptation is both dependent on muscle PPARδ and stimulated by PPARδ ligand. Furthermore, we find that muscle PPARδ expression positively correlates with endurance performance in BXD mouse reference populations. In addition to stimulating fatty acid metabolism in sedentary mice, PPARδ activation potently suppresses glucose catabolism and does so without affecting either muscle fiber type or mitochondrial content. By preserving systemic glucose levels, PPARδ acts to delay the onset of hypoglycemia and extends running time by ∼100 min in treated mice. Collectively, these results identify a bifurcated PPARδ program that underlies glucose sparing and highlight the potential of PPARδ-targeted exercise mimetics in the treatment of metabolic disease, dystrophies, and, unavoidably, the enhancement of athletic performance.

Diagnosis of HPV driven oropharyngeal cancers: Comparing p16 based algorithms with the RNAscope HPV-test.

BACKGROUND: Accurate identification of HPV-driven oropharyngeal cancer (OPC) is a major issue and none of the current diagnostic approaches is ideal. An in situ hybridization (ISH) assay that detects high-risk HPV E6/E7 mRNA, called the RNAscope HPV-test, has been recently developed. Studies have suggested that this assay may become a standard to define HPV-status. METHODS: To further assess this test, we compared its performance against the strategies that are used in routine clinical practice: p16 immunohistochemistry (IHC) as a single test and algorithms combining p16-IHC with HPV-DNA identification by PCR (algorithm-1) or ISH (algorithm-2). RESULTS: 105 OPC specimens were analyzed. The prevalence of HPV-positive samples varied considerably: 67% for p16-IHC, 54% for algorithm-1, 61% for algorithm-2 and 59% for the RNAscope HPV-test. Discrepancies between the RNAscope HPV-test and p16-IHC, algorithm-1 and 2 were noted in respectively 13.3%, 13.1%, and 8.6%. The 4 diagnostic strategies were able to identify 2 groups with different prognosis according to HPV-status, as expected. However, the greater survival differential was observed with the RNAscope HPV-test [HR: 0.19, 95% confidence interval (CI), 0.07-0.51, p=0.001] closely followed by algorithm-1 (HR: 0.23, 95% CI, 0.08-0.66, p=0.006) and algorithm-2 (HR: 0.26, 95% CI, 0.1-0.65, p=0.004). In contrast, a weaker association was found when p16-IHC was used as a single test (HR: 0.33, 95% CI, 0.13-0.81, p=0.02). CONCLUSIONS: Our findings suggest that the RNAscope HPV-test and p16-based algorithms perform better that p16 alone to identify OPC that are truly driven by HPV-infection. The RNAscope HPV-test has the advantage of being a single test.

Ribonucleic Acid In Situ Hybridization Is a More Sensitive Method Than Immunohistochemistry in Detection of Thyroid Transcription Factor 1 and Napsin A Expression in Lung Adenocarcinomas.

CONTEXT: TTF-1 and napsin A immunomarkers have a crucial role in differentiating lung adenocarcinoma from lung squamous cell carcinoma and in identifying a primary lung adenocarcinoma when working on a tumor of unknown origin. OBJECTIVES: To investigate the diagnostic sensitivity of ribonucleic acid in situ hybridization (RNAscope) in the detection of expression of these biomarkers in lung adenocarcinomas and to compare RNAscope to immunohistochemical techniques. DESIGN: Both RNAscope and the immunohistochemical assays for TTF-1 and napsin A were performed on tissue microarray sections containing 80 lung adenocarcinomas and 80 lung squamous cell carcinomas. The RNAscope assay for both TTF-1 and napsin A was also performed on 220 adenocarcinomas from various organs. RESULTS: The RNAscope assay for TTF-1 gave positive results in 92.5% (74 of 80) of the lung adenocarcinomas; in contrast, immunohistochemistry gave positive results in 82.5% (66 of 80) of those cases. The RNAscope assay for napsin A gave positive results in 90% (72 of 80) of lung adenocarcinomas; immunohistochemistry results were positive in 77.5% (62 of 80) of those cases. Napsin A expression was not seen in lung squamous cell carcinomas by either method. In contrast, TTF-1 expression was seen in 3.8% (3 of 80) (1(+)) and 10% (8 of 80) (1(+)) of the squamous cell carcinomas by immunochemistry and the RNAscope, respectively. All nonpulmonary adenocarcinoma results were negative for TTF-1 by the RNAscope assay. CONCLUSIONS: Preliminary data suggest that RNAscope is superior to immunohistochemistry in detecting TTF-1 and napsin A expression in primary lung adenocarcinomas. Therefore, performing an RNAscope assay may be considered for both TTF-1(-) and napsin A(-) cases with a clinical suspicion of lung adenocarcinoma. The TTF-1 results should be interpreted with caution because a small percentage of squamous cell carcinomas can be focally positive by either assay.

BRD4 is a novel therapeutic target for liver fibrosis.

Liver fibrosis is characterized by the persistent deposition of extracellular matrix components by hepatic stellate cell (HSC)-derived myofibroblasts. It is the histological manifestation of progressive, but reversible wound-healing processes. An unabated fibrotic response results in chronic liver disease and cirrhosis, a pathological precursor of hepatocellular carcinoma. We report here that JQ1, a small molecule inhibitor of bromodomain-containing protein 4 (BRD4), a member of bromodomain and extraterminal (BET) proteins, abrogate cytokine-induced activation of HSCs. Cistromic analyses reveal that BRD4 is highly enriched at enhancers associated with genes involved in multiple profibrotic pathways, where BRD4 is colocalized with profibrotic transcription factors. Furthermore, we show that JQ1 is not only protective, but can reverse the fibrotic response in carbon tetrachloride-induced fibrosis in mouse models. Our results implicate that BRD4 can act as a global genomic regulator to direct the fibrotic response through its coordinated regulation of myofibroblast transcription. This suggests BRD4 as a potential therapeutic target for patients with fibrotic complications.

Autophagy regulates T lymphocyte proliferation through selective degradation of the cell-cycle inhibitor CDKN1B/p27Kip1.

The highly conserved cellular degradation pathway, macroautophagy, regulates the homeostasis of organelles and promotes the survival of T lymphocytes. Previous results indicate that Atg3-, Atg5-, or Pik3c3/Vps34-deficient T cells cannot proliferate efficiently. Here we demonstrate that the proliferation of Atg7-deficient T cells is defective. By using an adoptive transfer and Listeria monocytogenes (LM) mouse infection model, we found that the primary immune response against LM is intrinsically impaired in autophagy-deficient CD8(+) T cells because the cell population cannot expand after infection. Autophagy-deficient T cells fail to enter into S-phase after TCR stimulation. The major negative regulator of the cell cycle in T lymphocytes, CDKN1B, is accumulated in autophagy-deficient naïve T cells and CDKN1B cannot be degraded after TCR stimulation. Furthermore, our results indicate that genetic deletion of one allele of CDKN1B in autophagy-deficient T cells restores proliferative capability and the cells can enter into S-phase after TCR stimulation. Finally, we found that natural CDKN1B forms polymers and is physiologically associated with the autophagy receptor protein SQSTM1/p62 (sequestosome 1). Collectively, autophagy is required for maintaining the expression level of CDKN1B in naïve T cells and selectively degrades CDKN1B after TCR stimulation.

Diagnosis of HPV-driven head and neck cancer with a single test in routine clinical practice.

Accurate screening of HPV-driven head and neck squamous cell carcinoma is a critical issue. Although there are commercial direct and indirect assays for HPV-related head and neck squamous cell carcinoma, none are ideal. Recently, a novel RNA in situ hybridization test (the RNAscope HPV-test) has been developed for the detection of high-risk HPV E6/E7 mRNA in formalin-fixed paraffin-embedded tissue. However, validation of this assay against the 'gold standard' (identification of high-risk HPV E6/E7 mRNA in fresh-frozen tissue by quantitative real-time (qRT)-PCR) has only been reported by one team. Formalin-fixed paraffin-embedded samples from 50 patients with tonsil or tongue base carcinoma were tested using the RNAscope HPV-test, p16 immunohistochemistry, and chromogenic in situ hybridization for high-risk HPV-DNA. The results were compared with those of qRT-PCR on matched fresh-frozen samples. Compared with the reference test, the sensitivity, specificity, positive, and negative predictive values of the RNAscope HPV-test and of p16 immunohistochemistry were 93%, 94%, 96%, 88% and 96%, 93%, 96%, and 93%, respectively. Five cases were discrepant between the RNAscope HPV-test and p16-immunohistochemisrty. The RNAscope HPV-test demonstrated excellent analytical performance against the 'gold standard' and is easier to interpret than chromogenic in situ hybridization. p16-immunohistochemistry also performed very well, however its main weakness is that it is an indirect marker of the presence of HPV. These data suggest that the RNAscope HPV-test is a promising test that could be developed as a clinical standard for the precise identification of HPV-driven oropharyngeal squamous cell carcinoma.

c-FLIP protects T lymphocytes from apoptosis in the intrinsic pathway.

Apoptosis can be induced by either death receptors on the plasma membrane (extrinsic pathway) or the damage of the genome and/or cellular organelles (intrinsic pathway). Previous studies suggest that cellular caspase 8 (FLICE)-like inhibitory protein (c-FLIP) promotes cell survival in death receptor-induced apoptosis pathway in T lymphocytes. Independent of death receptor signaling, mitochondria sense apoptotic stimuli and mediate the activation of effector caspases. Whether c-FLIP regulates mitochondrion-dependent apoptotic signals remains unknown. In this study, c-FLIP gene was deleted in mature T lymphocytes in vitro, and the role of c-FLIP protein in intrinsic apoptosis pathway was studied. In resting T cells treated with the intrinsic apoptosis inducer, c-FLIP suppressed cytochrome c release from mitochondria. Bim-deletion rescued the enhanced apoptosis in c-FLIP-deficient T cells, whereas inhibition of caspase 8 did not. Different from activated T cells, there was no necroptosis or increase in reactive oxygen species in c-FLIP-deficient resting T cells. These data suggest that c-FLIP is a negative regulator of intrinsic apoptosis pathway in T lymphocytes.

Autophagy, a novel pathway to regulate calcium mobilization in T lymphocytes.

The T lymphocyte response initiates with the recognition of MHC/peptides on antigen presenting cells by the T cell receptor (TCR). After the TCR engagement, the proximal signaling pathways are activated for downstream cellular events. Among these pathways, the calcium-signaling flux is activated through the depletion of endoplasmic reticulum (ER) calcium stores and plays pivotal roles in T cell proliferation, cell survival, and apoptosis. In studying the roles of macroautophagy (hereafter referred to as autophagy) in T cell function, we found that a pathway for intracellular degradation, autophagy, regulates calcium signaling by developmentally maintaining the homeostasis of the ER. Using mouse genetic models with specific deletion of autophagy-related genes in T lymphocytes, we found that the calcium influx is defective and the calcium efflux is increased in autophagy-deficient T cells. The abnormal calcium flux is related to the expansion of the ER and higher calcium stores in the ER. Because of this, treatment with the ER sarco/ER Ca(2+)-ATPase pump inhibitor, thapsigargin, rescues the calcium influx defect in autophagy-deficient T cells. Therefore, autophagy regulates calcium mobilization in T lymphocytes through ER homeostasis.

A vitamin D receptor/SMAD genomic circuit gates hepatic fibrotic response.

Liver fibrosis is a reversible wound-healing response involving TGFß1/SMAD activation of hepatic stellate cells (HSCs). It results from excessive deposition of extracellular matrix components and can lead to impairment of liver function. Here, we show that vitamin D receptor (VDR) ligands inhibit HSC activation by TGFß1 and abrogate liver fibrosis, whereas Vdr knockout mice spontaneously develop hepatic fibrosis. Mechanistically, we show that TGFß1 signaling causes a redistribution of genome-wide VDR-binding sites (VDR cistrome) in HSCs and facilitates VDR binding at SMAD3 profibrotic target genes via TGFß1-dependent chromatin remodeling. In the presence of VDR ligands, VDR binding to the coregulated genes reduces SMAD3 occupancy at these sites, inhibiting fibrosis. These results reveal an intersecting VDR/SMAD genomic circuit that regulates hepatic fibrogenesis and define a role for VDR as an endocrine checkpoint to modulate the wound-healing response in liver. Furthermore, the findings suggest VDR ligands as a potential therapy for liver fibrosis.

CFLAR/c-FLIPL: a star in the autophagy, apoptosis and necroptosis alliance.

Necroptosis, a caspase-independent, receptor (TNFRSF)-interacting serine-threonine kinase 1 (RIPK1)/RIPK3-dependent necrotic cell death, occurs in cells when apoptosis is blocked. A high level of macroautophagy (herein referred to as autophagy) is usually detected in necroptotic cells, although it is still controversial as to whether excessive autophagy leads to cell death or is cytoprotective. In a recently published paper, we show that the anti-apoptotic protein CFLAR (CASP8 and FADD-like apoptosis regulator) long isoform (CFLARL) plays a critical role in all three fundamental intracellular processes: autophagy, necroptosis, and apoptosis in T lymphocytes. CFLARL-deficient T cells suffer from severe cell death upon T cell receptor stimulation, in which both apoptosis and necroptosis are involved. Autophagy is enhanced in both naïve and activated CFLARL-deficient T cells and plays a cytoprotective function. Here, we summarize our findings and discuss the future direction in the study of the interplay of autophagy, apoptosis and necroptosis in T lymphocytes.

Macroautophagy in T lymphocyte development and function.

Macroautophagy (referred to as autophagy) is a fundamental intracellular process characterized by the sequestration of cytoplasmic compartments through double-membrane vesicles, termed autophagosomes. Recent studies have established important roles of autophagy in regulating T lymphocyte development and function. Resting T lymphocytes have basal levels of autophagy that is upregulated by T cell receptor stimulation. Several specific knockout or transgenic models have been developed during the past few years, and it has been revealed that autophagy plays an essential role in regulating thymocyte selection, peripheral T cell survival, and proliferation. The regulation of T cell development and function by autophagy is mediated through its role in regulating self-antigen presentation, intracellular organelle homeostasis, and energy production. Here we will review the current findings concerning how autophagy regulates T cell function, as well as compare different models in studying autophagy in T lymphocytes.