Genome-wide Screening Identifies SFMBT1 as an Oncogenic Driver in Cancer with VHL Loss.

von Hippel-Lindau (VHL) is a critical tumor suppressor in clear cell renal cell carcinomas (ccRCCs). It is important to identify additional therapeutic targets in ccRCC downstream of VHL loss besides hypoxia-inducible factor 2α (HIF2α). By performing a genome-wide screen, we identified Scm-like with four malignant brain tumor domains 1 (SFMBT1) as a candidate pVHL target. SFMBT1 was considered to be a transcriptional repressor but its role in cancer remains unclear. ccRCC patients with VHL loss-of-function mutations displayed elevated SFMBT1 protein levels. SFMBT1 hydroxylation on Proline residue 651 by EglN1 mediated its ubiquitination and degradation governed by pVHL. Depletion of SFMBT1 abolished ccRCC cell proliferation in vitro and inhibited orthotopic tumor growth in vivo. Integrated analyses of ChIP-seq, RNA-seq, and patient prognosis identified sphingosine kinase 1 (SPHK1) as a key SFMBT1 target gene contributing to its oncogenic phenotype. Therefore, the pVHL-SFMBT1-SPHK1 axis serves as a potential therapeutic avenue for ccRCC.

USP37 promotes deubiquitination of HIF2α in kidney cancer.

Clear cell renal cell carcinoma (ccRCC) is characterized by loss of tumor suppressor Von Hippel Lindau (VHL) function, which leads to accumulation of hypoxia inducible factor α (including HIF1α and HIF2α). HIF2α was previously reported to be one of the major oncogenic drivers in ccRCC, however, its therapeutic targets remain challenging. Here we performed a deubiquitinase (DUB) complementary DNA (cDNA) library binding screen and discovered that ubiquitin-specific peptidase 37 (USP37) is a DUB that binds HIF2α and promotes HIF2α deubiquitination. As a result, USP37 promotes HIF2α protein stability in an enzymatically dependent manner, and depletion of USP37 leads to HIF2α down-regulation in ccRCC. Functionally, USP37 depletion causes decreased cell proliferation measured by MTS, two-dimensional (2D) colony formation as well as three-dimensional (3D) anchorage- independent growth. USP37 is also essential for maintaining kidney tumorigenesis in an orthotopic xenograft model and its depletion leads to both decreased primary kidney tumorigenesis and spontaneous lung metastasis. Our results suggest that USP37 is a potential therapeutic target in ccRCC.

A conditional mouse expressing an activating mutation in NRF2 displays hyperplasia of the upper gastrointestinal tract and decreased white adipose tissue.

Activation of the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2 or NRF2) transcription factor is a critical and evolutionarily conserved cellular response to oxidative stress, metabolic stress, and xenobiotic insult. Deficiency of NRF2 results in hypersensitivity to a variety of stressors, whereas its aberrant activation contributes to several cancer types, most commonly squamous cell carcinomas of the esophagus, oral cavity, bladder, and lung. Between 10% and 35% of patients with squamous cell carcinomas display hyperactive NRF2 signaling, harboring activating mutations and copy number amplifications of the NFE2L2 oncogene or inactivating mutations or deletions of KEAP1 or CUL3, the proteins of which co-complex to ubiquitylate and degrade NRF2 protein. To better understand the role of NRF2 in tumorigenesis and more broadly in development, we engineered the endogenous Nfe2l2 genomic locus to create a conditional mutant LSL-Nrf2E79Q mouse model. The E79Q mutation, one of the most commonly observed NRF2-activating mutations in human squamous cancers, codes for a mutant protein that does not undergo KEAP1/CUL3-dependent degradation, resulting in its constitutive activity. Expression of NRF2 E79Q protein in keratin 14 (KRT14)-positive murine tissues resulted in hyperplasia of squamous cell tissues of the tongue, forestomach, and esophagus, a stunted body axis, decreased weight, and decreased visceral adipose depots. RNA-seq profiling and follow-up validation studies of cultured NRF2E79Q murine esophageal epithelial cells revealed known and novel NRF2-regulated transcriptional programs, including genes associated with squamous cell carcinoma (e.g. Myc), lipid and cellular metabolism (Hk2, Ppard), and growth factors (Areg, Bmp6, Vegfa). These data suggest that in addition to decreasing adipogenesis, KRT14-restricted NRF2 activation drives hyperplasia of the esophagus, forestomach, and tongue, but not formation of squamous cell carcinoma. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Altered DNA Methylation in the Developing Brains of Rats Genetically Prone to High versus Low Anxiety.

There is growing evidence of abnormal epigenetic processes playing a role in the neurobiology of psychiatric disorders, although the precise nature of these anomalies remains largely unknown. To study neurobiological (including epigenetic) factors that influence emotionality, we use rats bred for distinct behavioral responses to novelty. Rats bred for low novelty response (low responder [LR]) exhibit high levels of anxiety- and depressive-like behavior compared with high novelty responder (HR) rats. Prior work revealed distinct limbic brain development in HR versus LR rats, including altered expression of genes involved in DNA methylation. This led us to hypothesize that DNA methylation differences in the developing brain drive the disparate HR/LR neurobehavioral phenotypes. Here we report altered DNA methylation markers (altered DNA methyltransferase protein levels and increased global DNA methylation levels) in the early postnatal amygdala of LR versus HR male rats. Next-generation sequencing methylome profiling identified numerous differentially methylated regions across the genome in the early postnatal HR/LR amygdala. We also contrasted methylation profiles of male HRs and LRs with a control rat strain that displays an intermediate behavioral phenotype relative to the HR/LR extremes; this revealed that the LR amygdalar methylome was abnormal, with the HR profile more closely resembling that of the control group. Finally, through two methylation manipulations in early life, we found that decreasing DNA methylation in the developing male and female amygdala improves adult anxiety- and depression-like behavior. These findings suggest that inborn DNA methylation differences play important roles in shaping brain development and lifelong emotional behavior.SIGNIFICANCE STATEMENT Epigenetic changes are biological mechanisms that regulate the expression and function of genes throughout the brain and body. DNA methylation, one type of epigenetic mechanism, is known to be altered in brains of psychiatric patients, which suggests a role for DNA methylation in the pathogenesis of psychiatric disorders, such as depression and anxiety. The present study examines brains of rats that display high versus low levels of anxiety- and depression-like behavior to investigate how neural DNA methylation levels differ in these animals and how such differences shape their emotional behavioral differences. Studying how epigenetic processes affect emotional behavior may improve our understanding of the neurobiology of psychiatric disorders and lead to improved treatments.

Transcriptome-Wide Regulation of Key Developmental Pathways in the Mouse Neural Tube by Prenatal Alcohol Exposure.

BACKGROUND: Early gestational alcohol exposure is associated with severe craniofacial and CNS dysmorphologies and behavioral abnormalities during adolescence and adulthood. Alcohol exposure during the formation of the neural tube (gestational day [GD] 8 to 10 in mice; equivalent to4th week of human pregnancy) disrupts development of ventral midline brain structures such as the pituitary, septum, and ventricles. This study identifies transcriptomic changes in the rostroventral neural tube (RVNT), the region of the neural tube that gives rise to the midline structures sensitive to alcohol exposure during neurulation. METHODS: Female C57BL/6J mice were administered 2 doses of alcohol (2.9 g/kg) or vehicle 4 hours apart on GD 9.0. The RVNTs of embryos were collected 6 or 24 hours after the first dose and processed for RNA-seq. RESULTS: Six hours following GD 9.0 alcohol exposure (GD 9.25), over 2,300 genes in the RVNT were determined to be differentially regulated by alcohol. Enrichment analysis determined that PAE affected pathways related to cell proliferation, p53 signaling, ribosome biogenesis, and immune activation. In addition, over 100 genes involved in primary cilia formation and function and regulation of morphogenic pathways were altered 6 hours after alcohol exposure. The changes to gene expression were largely transient, as only 91 genes identified as differentially regulated by prenatal alcohol at GD 10 (24 hours postexposure). Functionally, the differentially regulated genes at GD 10 were related to organogenesis and cell migration. CONCLUSIONS: These data give a comprehensive view of the changing landscape of the embryonic transcriptome networks in regions of the neural tube that give rise to brain structures impacted by a neurulation-stage alcohol exposure. Identification of gene networks dysregulated by alcohol will help elucidate the pathogenic mechanisms of alcohol's actions.

Gut microbiota composition associated with alterations in cardiorespiratory fitness and psychosocial outcomes among breast cancer survivors.

PURPOSE: In this proof-of-concept pilot study, our purpose was to determine correlations between gut microbiota composition and alterations in cardiorespiratory fitness and psychosocial outcomes among post-primary treatment breast cancer survivors (BCS). METHODS: Composition of the gut microbiota in BCS (n = 12) was assessed at baseline (M0) and at the end of 3 months (M3) using Illumina MiSeq DNA Sequencing of the 16S rRNA gene. Gut microbiota composition was analyzed using the QIIME bioinformatics software and represented through diversity metrics and taxa analyses. Cardiorespiratory fitness, fatigue, anxiety, depression, and sleep dysfunction were assessed at M0 and M3 via the submaximal treadmill test, Fatigue Symptom Inventory, Hospital Anxiety and Depression Scale, and Pittsburgh Sleep Quality Index, respectively. RESULTS: Increased fatigue interference in BCS was associated with increased mean within-sample Shannon diversity (organism richness and evenness) (p = 0.009). Weighted UniFrac analysis (shifts in taxa relative abundance) revealed significant differences in between-sample (beta) diversity for changes in fatigue interference (p = 0.01) and anxiety (p = 0.022), with a trend observed for fatigue intensity and sleep dysfunction (p < 0.1). Unweighted UniFrac analysis (shifts in taxa types) found significant beta diversity differences for cardiorespiratory fitness (p = 0.026). Prior to false discovery correction (FDR), changes in fitness, fatigue, anxiety, and sleep dysfunction were associated with the frequency of certain gut bacteria genera (e.g., Faecalibacterium, Prevotella, Bacteroides) (p < 0.05). CONCLUSIONS: Correlations may exist between alterations in gut microbiota composition and longitudinal changes in cardiorespiratory fitness, fatigue, and anxiety in BCS. Further research examining the role of the microbiota-gut-brain axis in exercise-induced effects on psychosocial outcomes in BCS is warranted.

Feasibility of Providing Safe Mouth Care and Collecting Oral and Fecal Microbiome Samples from Nursing Home Residents with Dysphagia: Proof of Concept Study.

Individuals with dysphagia who reside in nursing homes often receive inadequate mouth care and experience poor oral health. From a policy perspective, the combination of absent evidence-based mouth care protocols coupled with insufficient dental coverage create a pool of individuals at great risk for preventable infectious illnesses that contribute to high health care costs. The purpose of the current study was to determine (a) the safety of a mouth care protocol tailored for individuals with dysphagia residing in nursing homes without access to suction equipment, and (b) the feasibility of collecting oral and fecal samples for microbiota analyses. The mouth care protocol resulted in improved oral hygiene without aspiration, and oral and fecal samples were safely collected from participants. Policies supporting ongoing testing of evidence-based mouth care protocols for individuals with dysphagia are important to improve quality, demonstrate efficacy, and save health care costs. [Journal of Gerontological Nursing, 43(9), 9-15.].

Colonization potential to reconstitute a microbe community in patients detected early after fecal microbe transplant for recurrent C. difficile.

BACKGROUND: Fecal microbiota transplants (FMT) are an effective treatment for patients with gut microbe dysbiosis suffering from recurrent C. difficile infections. To further understand how FMT reconstitutes the patient's gut commensal microbiota, we have analyzed the colonization potential of the donor, recipient and recipient post transplant fecal samples using transplantation in gnotobiotic mice. RESULTS: A total of nine samples from three human donors, recipient's pre and post FMT were transplanted into gnotobiotic mice. Microbiome analysis of three donor fecal samples revealed the presence of a high relative abundance of commensal microbes from the family Bacteriodaceae and Lachnospiraceae that were almost absent in the three recipient pre FMT fecal samples (<0.01%). The microbe composition in gnotobiotic mice transplanted with the donor fecal samples was similar to the human samples. The recipient samples contained Enterobacteriaceae, Lactobacillaceae, Enterococcaceae in relative abundance of 43, 11, 8%, respectively. However, gnotobiotic mice transplanted with the recipient fecal samples had an average relative abundance of unclassified Clostridiales of 55%, approximately 7000 times the abundance in the recipient fecal samples prior to transplant. Microbiome analysis of fecal samples from the three patients early (2-4 weeks) after FMT revealed a microbe composition with the relative abundance of both Bacteriodaceae and Lachnospiraceae that was approximately 7% of that of the donor. In contrast, gnotobioitc mice transplanted with the fecal samples obtained from the three at early times post FMT revealed increases in the relative abundance of Bacteriodaceae and Lachnospiraceae microbe compositions to levels similar to the donor fecal samples. Furthermore, the unclassified Clostridiales in the recipient samples post FMT was reduced to an average of 10%. CONCLUSION: We have used transplantation into gnotobiotic mice to evaluate the colonization potential of microbiota in FMT patients early after transplant. The commensal microbes present at early times post FMT out competed non-commensal microbes (e.g. such as unclassified Clostridiales) for niche space. The selective advantage of these commensal microbes to occupy niches in the gastrointestinal tract helps to explain the success of FMT to reconstitute the gut microbe community of patients with recurrent C. difficile infections.

Feasibility outcomes of a presurgical randomized controlled trial exploring the impact of caloric restriction and increased physical activity versus a wait-list control on tumor characteristics and circulating biomarkers in men electing prostatectomy for prostate cancer.

BACKGROUND: Obesity is associated with tumor aggressiveness and disease-specific mortality for more than 15 defined malignancies, including prostate cancer. Preclinical studies suggest that weight loss from caloric restriction and increased physical activity may suppress hormonal, energy-sensing, and inflammatory factors that drive neoplastic progression; however, exact mechanisms are yet to be determined, and experiments in humans are limited. METHODS: We conducted a randomized controlled trial among 40 overweight or obese, newly-diagnosed prostate cancer patients who elected prostatectomy to explore feasibility of a presurgical weight loss intervention that promoted a weight loss of roughly one kg. week(-1) via caloric restriction and physical activity, as well as to assess effects on tumor biology and circulating biomarkers. Measures of feasibility (accrual, retention, adherence, and safety) were primary endpoints. Exploratory aims were directed at the intervention's effect on tumor proliferation (Ki-67) and other tumor markers (activated caspase-3, insulin and androgen receptors, VEGF, TNFß, NFκB, and 4E-BP1), circulating biomarkers (PSA, insulin, glucose, VEGF, TNFß, leptin, SHBG, and testosterone), lymphocytic gene expression of corresponding factors and cellular bioenergetics in neutrophils, and effects on the gut microbiome. Consenting patients were randomized in a 1:1 ratio to either: 1) weight loss via a healthful, guidelines-based diet and exercise regimen; or 2) a wait-list control. While biological testing is currently ongoing, this paper details our methods and feasibility outcomes. RESULTS: The accrual target was met after screening 101 cases (enrollment rate: 39.6%). Other outcomes included a retention rate of 85%, excellent adherence (95%), and no serious reported adverse events. No significant differences by age, race, or weight status were noted between enrollees vs. non-enrollees. The most common reasons for non-participation were "too busy" (30%), medical exclusions (21%), and "distance" (16%). CONCLUSIONS: Presurgical trials offer a means to study the impact of diet and exercise interventions directly on tumor tissue, and other host factors that are feasible and safe, though modifications are needed to conduct trials within an abbreviated period of time and via distance medicine-based approaches. Pre-surgical trials are critical to elucidate the impact of lifestyle interventions on specific mechanisms that mediate carcinogenesis and which can be used subsequently as therapeutic targets. TRIAL REGISTRATION: NCT01886677.

Midtrimester Cervicovaginal Microbiota: Identification of Microbial Variations Associated with Puerperal Infection at Term.

Objective This study aims to evaluate differences in the midtrimester cervicovaginal microbiota between women who developed puerperal infections at term and those who did not, and whether obesity modulates this relationship. Methods Previously, cervicovaginal swabs were collected at 21 to 25 weeks gestation (stored at -80°C). Samples were identified from Black women with normal vaginal flora (Nugent score: 0-2) delivering term singletons. Patients were in one of four equally sized groups (total n = 120) characterized by absence or presence of puerperal infection and maternal obesity. Samples were thawed, DNA extracted, and polymerase chain reaction with primers targeting the 16S rDNA V4 region was used to prepare an amplicon library sequenced and analyzed using Quantitative Insights into Microbial Ecology (QIIME) suite. Microbiota differences were assessed using permutation-based anodis over three ß-diversity measures; Kruskal-Wallis test was used for taxa level analysis. Results After quality control measures, 113 samples were analyzed. Overall, there was significant clustering by puerperal infection (p = 0.03), but not by obesity (p > 0.05). Detailed taxa level analysis revealed approximately 66% less Proteobacteria phylum and 400% more BVAB1 genera in the second-trimester microbiota of women who had puerperal infections at term (p < 0.05). Conclusion Women who develop puerperal infections at term have a significantly altered midtrimester cervicovaginal microbiome with less Proteobacteria and greater BVAB1. This finding may represent a potential method to identify women at an increased risk of puerperal infection.

Comparative genome analysis of Mycoplasma pneumoniae.

BACKGROUND: Mycoplasma pneumoniae is a common pathogen that causes upper and lower respiratory tract infections in people of all ages, responsible for up to 40% of community-acquired pneumonias. It also causes a wide array of extrapulmonary infections and autoimmune phenomena. Phylogenetic studies of the organism have been generally restricted to specific genes or regions of the genome, because whole genome sequencing has been completed for only 4 strains. To better understand the physiology and pathogenicity of this important human pathogen, we performed comparative genomic analysis of 15 strains of M. pneumoniae that were isolated between the 1940s to 2009 from respiratory specimens and cerebrospinal fluid originating from the USA, China and England. RESULTS: Illumina MiSeq whole genome sequencing was performed on the 15 strains and all genome sequences were completed. Results from the comparative genomic analysis indicate that although about 1500 SNP and indel variants exist between type1 and type 2 strains, there is an overall high degree of sequence similarity among the strains (>99% identical to each other). Within the two subtypes, conservation of most genes, including the CARDS toxin gene and arginine deiminase genes, was observed. The major variation occurs in the P1 and ORF6 genes associated with the adhesin complex. Multiple hsdS genes (encodes S subunit of type I restriction enzyme) with variable tandem repeat copy numbers were found in all 15 genomes. CONCLUSIONS: These data indicate that despite conclusions drawn from 16S rRNA sequences suggesting rapid evolution, the M. pneumoniae genome is extraordinarily stable over time and geographic distance across the globe with a striking lack of evidence of horizontal gene transfer.

Proteobacteria impair anti-tumor immunity in the omentum by consuming arginine.

Gut microbiota influence anti-tumor immunity, often by producing immune-modulating metabolites. However, microbes consume a variety of metabolites that may also impact host immune responses. We show that tumors grow unchecked in the omenta of microbe-replete mice due to immunosuppressive Tregs. By contrast, omental tumors in germ-free, neomycin-treated mice or mice colonized with altered Schaedler's flora (ASF) are spontaneously eliminated by CD8+ T cells. These mice lack Proteobacteria capable of arginine catabolism, causing increases in serum arginine that activate the mammalian target of the rapamycin (mTOR) pathway in Tregs to reduce their suppressive capacity. Transfer of the Proteobacteria, Escherichia coli (E. coli), but not a mutant unable to catabolize arginine, to ASF mice reduces arginine levels, restores Treg suppression, and prevents tumor clearance. Supplementary arginine similarly decreases Treg suppressive capacity, increases CD8+ T cell effectiveness, and reduces tumor burden. Thus, microbial consumption of arginine alters anti-tumor immunity, offering potential therapeutic strategies for tumors in visceral adipose tissue.

FcγR gene copy number in Kawasaki disease and intravenous immunoglobulin treatment response.

OBJECTIVE: Kawasaki disease (KD), response to intravenous immunoglobulin (IVIG) therapy, and associated coronary artery disease progression have been associated with genetic polymorphisms in Fc gamma receptor (FcγR) genes. However, it is not known whether the existing gene copy number (GCN) variability relates to KD treatment response, susceptibility, or associated sequelae. METHODS: The copy number of individuals with KD (n=510) and their family members (n=808) for three variable FcγRs was assessed using pyrosequencing. We performed the transmission disequilibrium test to examine the association of GCN for FcγRs (FcγR2C, FcγR3A, and FcγR3B) with susceptibility and used logistic regression models to determine its association with IVIG treatment outcomes. RESULTS: FcγR2C and FcγR3B GCN were significantly associated with KD susceptibility. IVIG response was associated with GCN variations of FcγR3B in Whites and FcγR2C in Hispanics, and gene risk score based on single nucleotide polymorphism and GCN in FcγRs were significantly different between IVIG responders and nonresponders among Whites. We found no significant associations between coronary artery disease and any of the FcγR copy numbers. CONCLUSION: GCN of FcγR2C and FcγR3B influences IVIG treatment response and predisposes individuals to KD, providing potential insights into understanding the mechanism of the FcγR gene family in the IVIG pathway.

Biomimetic cardiac tissue chip and murine arteriovenous fistula models for recapitulating clinically relevant cardiac remodeling under volume overload conditions.

Cardiovascular events are the primary cause of death among dialysis patients. While arteriovenous fistulas (AVFs) are the access of choice for hemodialysis patients, AVF creation can lead to a volume overload (VO) state in the heart. We developed a three-dimensional (3D) cardiac tissue chip (CTC) with tunable pressure and stretch to model the acute hemodynamic changes associated with AVF creation to complement our murine AVF model of VO. In this study, we aimed to replicate the hemodynamics of murine AVF models in vitro and hypothesized that if 3D cardiac tissue constructs were subjected to "volume overload" conditions, they would display fibrosis and key gene expression changes seen in AVF mice. Mice underwent either an AVF or sham procedure and were sacrificed at 28 days. Cardiac tissue constructs composed of h9c2 rat cardiac myoblasts and normal adult human dermal fibroblasts in hydrogel were seeded into devices and exposed to 100 mg/10 mmHg pressure (0.4 s/0.6 s) at 1 Hz for 96 h. Controls were exposed to "normal" stretch and experimental group exposed to "volume overload". RT-PCR and histology were performed on the tissue constructs and mice left ventricles (LVs), and transcriptomics of mice LVs were also performed. Our tissue constructs and mice LV both demonstrated cardiac fibrosis as compared to control tissue constructs and sham-operated mice, respectively. Gene expression studies in our tissue constructs and mice LV demonstrated increased expression of genes associated with extracellular matrix production, oxidative stress, inflammation, and fibrosis in the VO conditions vs. control conditions. Our transcriptomics studies demonstrated activated upstream regulators related to fibrosis, inflammation, and oxidative stress such as collagen type 1 complex, TGFB1, CCR2, and VEGFA and inactivated regulators related to mitochondrial biogenesis in LV from mice AVF. In summary, our CTC model yields similar fibrosis-related histology and gene expression profiles as our murine AVF model. Thus, the CTC could potentially play a critical role in understanding cardiac pathobiology of VO states similar to what is present after AVF creation and may prove useful in evaluating therapies.

Autism-linked UBE3A gain-of-function mutation causes interneuron and behavioral phenotypes when inherited maternally or paternally in mice.

The E3 ubiquitin ligase Ube3a is biallelically expressed in neural progenitors and glial cells, suggesting that UBE3A gain-of-function mutations might cause neurodevelopmental disorders irrespective of parent of origin. Here, we engineered a mouse line that harbors an autism-linked UBE3AT485A (T503A in mouse) gain-of-function mutation and evaluated phenotypes in animals that inherited the mutant allele paternally, maternally, or from both parents. We find that paternally and maternally expressed UBE3AT503A results in elevated UBE3A activity in neural progenitors and glial cells. Expression of UBE3AT503A from the maternal allele, but not the paternal one, leads to a persistent elevation of UBE3A activity in neurons. Mutant mice display behavioral phenotypes that differ by parent of origin. Expression of UBE3AT503A, irrespective of its parent of origin, promotes transient embryonic expansion of Zcchc12 lineage interneurons. Phenotypes of Ube3aT503A mice are distinct from Angelman syndrome model mice. Our study has clinical implications for a growing number of disease-linked UBE3A gain-of-function mutations.

Genome-wide DNA methylation study identifies significant epigenomic changes associated with internalized stigma in adults with non-specific chronic low back pain.

Non-specific chronic low back pain (cLBP) represents a common musculoskeletal condition with no identifiable cause. It cannot be diagnosed with conventional neuroimaging techniques such as computerized tomography (CT). The diagnostic uncertainty that characterizes non-specific cLBP can lead to stigmatizing responses from others that can become internalized Among individuals with non-specific cLBP, internalized stigma is associated with greater pain intensity and disability. Yet, no study has examined the biological mechanism linking high internalized stigma to worse outcomes in individuals with non-specific cLBP. We aimed to identify differentially methylated loci (DML), enrichment pathways, and associated network interactions among individuals with non-specific cLBP experiencing low vs. high internalized stigma. We examined DNA methylation in whole blood samples from 48 adults, ages 19-85, using reduced representation bisulfite sequencing (RRBS). After controlling for age, sex, race, and multiple testing, differentially methylated loci (DML) differed in adults with low vs. high internalized stigma by at least 10% and q < 0.01 in 3,665 CpG sites: 2,280 hypomethylated and 1,385 hypermethylated. Gene ontology (GO) analyses of the annotated genes from these sites revealed significant enrichment of 274 biological processes, 29 cellular components, and 24 molecular functions (adjusted p < 0.05). The top enriched molecular functions regulate protein binding and DNA binding of transcription factor activity. Pathway analyses indicated that many functional genomic pathways, including Hippo Signaling, Melanogenesis, and Pathways in Cancer, were enriched with differentially methylated genes. Also, there was a significant interaction between relevance pathways such as P53, mTOR, PI3K-Akt, and Wnt signaling pathways. These pathways have previously been associated with neuroinflammation, neurodegeneration, and stress-related conditions. Thus, findings point to possible stress-induced DNAm changes as the link between high levels of internalized stigma and worse outcomes in adults with non-specific cLBP.

Epigenome-wide DNA methylation profiling of conditioned pain modulation in individuals with non-specific chronic low back pain.

BACKGROUND: The pathoanatomic cause of chronic low back pain (cLBP) cannot be identified for up to 90% of individuals. However, dysfunctional processing of endogenous nociceptive input, measured as conditioned pain modulation (CPM), has been associated with cLBP and may involve changes in neuronal gene expression. Epigenetic-induced changes such as DNA methylation (DNAm) have been associated with cLBP. METHODS: In the present study, the relationship between CPM and DNAm changes in a sample of community-dwelling adults with nonspecific cLBP (n = 48) and pain-free controls (PFC; n = 50) was examined using reduced representation bisulfite sequencing. Gene ontology (GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were applied to identify key pathways involved in efficient versus deficient CPM. RESULTS: Based on CPM efficiency, we identified 6006 and 18,305 differentially methylated CpG sites (DMCs) with q values < 0.01 among individuals with cLBP and PFCs, respectively. Most of the DMCs were hypomethylated and annotated to genes of relevance to pain, including OPRM1, ADRB2, CACNA2D3, GNA12, LPL, NAXD, and ASPHD1. In both cLBP and PFC groups, the DMCs annotated genes enriched many GO terms relevant to pain processing, including transcription regulation by RNA polymerase II, nervous system development, generation of neurons, neuron differentiation, and neurogenesis. Both groups also enriched the pathways involved in Rap1-signaling, cancer, and dopaminergic neurogenesis. However, MAPK-Ras signaling pathways were enriched in the cLBP, not the PFC group. CONCLUSIONS: This is the first study to investigate the genome-scale DNA methylation profiles of CPM phenotype in adults with cLBP and PFCs. Based on CPM efficiency, fewer DMC enrichment pathways were unique to the cLBP than the PFCs group. Our results suggest that epigenetically induced modification of neuronal development/differentiation pathways may affect CPM efficiency, suggesting novel potential therapeutic targets for central sensitization. However, CPM efficiency and the experience of nonspecific cLBP may be independent. Further mechanistic studies are required to confirm the relationship between CPM, central sensitization, and nonspecific cLBP.

Transcriptomic analyses of gastrulation-stage mouse embryos with differential susceptibility to alcohol.

Genetics are a known contributor to differences in alcohol sensitivity in humans with fetal alcohol spectrum disorders (FASDs) and in animal models. Our study profiled gene expression in gastrulation-stage embryos from two commonly used, genetically similar mouse substrains, C57BL/6J (6J) and C57BL/6NHsd (6N), that differ in alcohol sensitivity. First, we established normal gene expression patterns at three finely resolved time points during gastrulation and developed a web-based interactive tool. Baseline transcriptional differences across strains were associated with immune signaling. Second, we examined the gene networks impacted by alcohol in each strain. Alcohol caused a more pronounced transcriptional effect in the 6J versus 6N mice, matching the increased susceptibility of the 6J mice. The 6J strain exhibited dysregulation of pathways related to cell death, proliferation, morphogenic signaling and craniofacial defects, while the 6N strain showed enrichment of hypoxia and cellular metabolism pathways. These datasets provide insight into the changing transcriptional landscape across mouse gastrulation, establish a valuable resource that enables the discovery of candidate genes that may modify alcohol susceptibility that can be validated in humans, and identify novel pathogenic mechanisms of alcohol. This article has an associated First Person interview with the first author of the paper.

ZHX2 promotes HIF1α oncogenic signaling in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease, which warrants the critical need to identify new therapeutic targets. We show that Zinc Fingers and Homeoboxes 2 (ZHX2) is amplified or overexpressed in TNBC cell lines and patients. Functionally, depletion of ZHX2 inhibited TNBC cell growth and invasion in vitro, orthotopic tumor growth, and spontaneous lung metastasis in vivo. Mechanistically, ZHX2 bound with hypoxia-inducible factor (HIF) family members and positively regulated HIF1α activity in TNBC. Integrated ChIP-seq and gene expression profiling demonstrated that ZHX2 co-occupied with HIF1α on transcriptionally active promoters marked by H3K4me3 and H3K27ac, thereby promoting gene expression. Among the identified ZHX2 and HIF1α coregulated genes, overexpression of AP2B1, COX20, KDM3A, or PTGES3L could partially rescue TNBC cell growth defect by ZHX2 depletion, suggested that these downstream targets contribute to the oncogenic role of ZHX2 in an accumulative fashion. Furthermore, multiple residues (R491, R581, and R674) on ZHX2 are important in regulating its phenotype, which correspond with their roles on controlling ZHX2 transcriptional activity in TNBC cells. These studies establish that ZHX2 activates oncogenic HIF1α signaling, therefore serving as a potential therapeutic target for TNBC.