Global role of IGF2BP1 in controlling the expression of Wnt/ß-catenin-regulated genes in colorectal cancer cells.

Introduction: Wnt/ß-catenin signaling controls cell division and lineage specification during embryonic development, and is crucial for stem cells maintenance and gut tissue regeneration in adults. Aberrant activation of Wnt/ß-catenin signaling is also essential for the pathogenesis of a variety of malignancies. The RNA-binding protein IGF2BP1 is a transcriptional target of Wnt/ß-catenin signaling, normally expressed during development and often reactivated in cancer cells, where it regulates the stability of oncogenic mRNA. Methods: In this study, we employed iCLIP and RNA sequencing techniques to investigate the role of IGF2BP1 in the post-transcriptional regulation of Wnt/ß-catenin-induced genes at a global level within colorectal cancer (CRC) cells characterized by constitutively active Wnt/ß-catenin signaling. Results and Discussion: In our study, we show that, in contrast to normal cells, CRC cells exhibit a much stronger dependency on IGF2BP1 expression for Wnt/ß-catenin-regulated genes. We show that both untransformed and CRC cells have their unique subsets of Wnt/ß-catenin-regulated genes that IGF2BP1 directly controls through binding to their mRNA. Our iCLIP analysis revealed a significant change in the IGF2BP1-binding sites throughout the target transcriptomes and a significant change in the enrichment of 6-mer motifs associated with IGF2BP1 binding in response to Wnt/ß-catenin signaling. Our study also revealed a signature of IGF2BP1-regulated genes that are significantly associated with colon cancer-free survival in humans, as well as potential targets for CRC treatment. Overall, this study highlights the complex and context-dependent regulation of Wnt/ß-catenin signaling target genes by IGF2BP1 in non-transformed and CRC cells and identifies potential targets for colon cancer treatment.

Antiemetic effects of baclofen in a shrew model of postoperative nausea and vomiting: Whole-transcriptome analysis in the nucleus of the solitary tract.

AIMS: The molecular genetic mechanisms underlying postoperative nausea and vomiting (PONV) in the brain have not been fully elucidated. This study aimed to determine the changes in whole transcriptome in the nucleus of the solitary tract (NTS) in an animal model of PONV, to screen a drug candidate and to elucidate the molecular genetic mechanisms of PONV development. METHODS: Twenty-one female musk shrews were assigned into three groups: the Surgery group (shrew PONV model, n = 9), the Sham group (n = 6), and the Naïve group (n = 6). In behavioral studies, the main outcome was the number of emetic episodes. In genetic experiments, changes in the transcriptome in the NTS were measured. In a separate study, 12 shrews were used to verify the candidate mechanism underlying PONV. RESULTS: A median of six emetic episodes occurred in both the Sham and Surgery groups. Whole-transcriptome analysis indicated the inhibition of the GABAB receptor-mediated signaling pathway in the PONV model. Baclofen (GABAB receptor agonist) administration eliminated emetic behaviors in the shrew PONV model. CONCLUSIONS: Our findings suggest that the GABAB receptor-mediated signaling pathway is involved in emesis and that baclofen may be a novel therapeutic or prophylactic agent for PONV.

Crucial involvement of catecholamine neurotransmission in postoperative nausea and vomiting: Whole-transcriptome profiling in the rat nucleus of the solitary tract.

The genetic mechanisms of postoperative nausea and vomiting (PONV) and the involvement of the catecholamine system in the brain have not been elucidated. Eating kaolin clay as a type of pica has been examined as an alternative behavior to emesis. Here, we evaluated changes in whole-transcriptome analysis in the nucleus of the solitary tract (NTS) in a rat pica model as a surrogate behavior of PONV to elucidate the molecular genetic mechanisms of the development of PONV and the involvement of the catecholamine system in the NTS. First, kaolin pica behaviors were investigated in 71 female Wistar rats following isoflurane anesthesia, surgical insult or morphine administration. Multiple linear regression analysis showed that 3 mg/kg morphine increased kaolin intake by 2.8 g (P = 0.0002). Next, total RNA and protein were extracted from the dissected NTS, and whole-transcriptome sequencing (RNA-seq) was performed to identify PONV-associated genes and to verify the involvement of the catecholamine system. The gene expression levels of tyrosine hydroxylase and dopamine beta-hydroxylase in the catecholamine biosynthesis pathway decreased significantly in the PONV model. Release of noradrenaline, a catecholamine pathway end product, may have increased at the synaptic terminal of the NTS neuron after pica behavior. Systematic administration of α2 adrenergic receptor agonists after surgery reduced kaolin intake from 3.2 g (control) to 1.0 g (P = 0.0014). These results indicated that catecholamine neurotransmission was involved in the development of PONV in the NTS.

DOT1L modulates the senescence-associated secretory phenotype through epigenetic regulation of IL1A.

Oncogene-induced senescence (OIS) is a stable cell cycle arrest that occurs in normal cells upon oncogene activation. Cells undergoing OIS express a wide variety of secreted factors that affect the senescent microenvironment termed the senescence-associated secretory phenotype (SASP), which is beneficial or detrimental in a context-dependent manner. OIS cells are also characterized by marked epigenetic changes. We globally assessed histone modifications of OIS cells and discovered an increase in the active histone marks H3K79me2/3. The H3K79 methyltransferase disruptor of telomeric silencing 1-like (DOT1L) was necessary and sufficient for increased H3K79me2/3 occupancy at the IL1A gene locus, but not other SASP genes, and was downstream of STING. Modulating DOT1L expression did not affect the cell cycle arrest. Together, our studies establish DOT1L as an epigenetic regulator of the SASP, whose expression is uncoupled from the senescence-associated cell cycle arrest, providing a potential strategy to inhibit the negative side effects of senescence while maintaining the beneficial inhibition of proliferation.

Tracheal aspirate transcriptomic and miRNA signatures of extreme premature birth with bronchopulmonary dysplasia.

OBJECTIVE: Extreme preterm infants are a growing population in neonatal intensive care units who carry a high mortality and morbidity. Multiple factors play a role in preterm birth, resulting in major impact on organogenesis leading to complications including bronchopulmonary dysplasia (BPD). The goal of this study was to identify biomarker signatures associated with prematurity and BPD. STUDY DESIGN: We analyzed miRNA and mRNA profiles in tracheal aspirates (TAs) from 55 infants receiving invasive mechanical ventilation. Twenty-eight infants were extremely preterm and diagnosed with BPD, and 27 were term babies receiving invasive mechanical ventilation for elective procedures. RESULT: We found 22 miRNAs and 33 genes differentially expressed (FDR < 0.05) in TAs of extreme preterm infants with BPD vs. term babies without BPD. Pathway analysis showed associations with inflammatory response, cellular growth/proliferation, and tissue development. CONCLUSIONS: Specific mRNA-miRNA signatures in TAs may serve as biomarkers for BPD pathogenesis, a consequence of extreme prematurity.

Identification of regulators of poly-ADP-ribose polymerase inhibitor response through complementary CRISPR knockout and activation screens.

Inhibitors of poly-ADP-ribose polymerase 1 (PARPi) are highly effective in killing cells deficient in homologous recombination (HR); thus, PARPi have been clinically utilized to successfully treat BRCA2-mutant tumors. However, positive response to PARPi is not universal, even among patients with HR-deficiency. Here, we present the results of genome-wide CRISPR knockout and activation screens which reveal genetic determinants of PARPi response in wildtype or BRCA2-knockout cells. Strikingly, we report that depletion of the ubiquitin ligase HUWE1, or the histone acetyltransferase KAT5, top hits from our screens, robustly reverses the PARPi sensitivity caused by BRCA2-deficiency. We identify distinct mechanisms of resistance, in which HUWE1 loss increases RAD51 levels to partially restore HR, whereas KAT5 depletion rewires double strand break repair by promoting 53BP1 binding to double-strand breaks. Our work provides a comprehensive set of putative biomarkers that advance understanding of PARPi response, and identifies novel pathways of PARPi resistance in BRCA2-deficient cells.

Glucocorticoids enhance the antileukemic activity of FLT3 inhibitors in FLT3-mutant acute myeloid leukemia.

FLT3 is a frequently mutated gene that is highly associated with a poor prognosis in acute myeloid leukemia (AML). Despite initially responding to FLT3 inhibitors, most patients eventually relapse with drug resistance. The mechanism by which resistance arises and the initial response to drug treatment that promotes cell survival is unknown. Recent studies show that a transiently maintained subpopulation of drug-sensitive cells, so-called drug-tolerant "persisters" (DTPs), can survive cytotoxic drug exposure despite lacking resistance-conferring mutations. Using RNA sequencing and drug screening, we find that treatment of FLT3 internal tandem duplication AML cells with quizartinib, a selective FLT3 inhibitor, upregulates inflammatory genes in DTPs and thereby confers susceptibility to anti-inflammatory glucocorticoids (GCs). Mechanistically, the combination of FLT3 inhibitors and GCs enhances cell death of FLT3 mutant, but not wild-type, cells through GC-receptor-dependent upregulation of the proapoptotic protein BIM and proteasomal degradation of the antiapoptotic protein MCL-1. Moreover, the enhanced antileukemic activity by quizartinib and dexamethasone combination has been validated using primary AML patient samples and xenograft mouse models. Collectively, our study indicates that the combination of FLT3 inhibitors and GCs has the potential to eliminate DTPs and therefore prevent minimal residual disease, mutational drug resistance, and relapse in FLT3-mutant AML.

Understanding the Action of RARγ Agonists on Human Osteochondroma Explants.

Osteochondromas are cartilage-capped growths located proximate to the physis that can cause skeletal deformities, pain, limited motion, and neurovascular impingement. Previous studies have demonstrated retinoic acid receptor gamma (RARγ) agonists to inhibit ectopic endochondral ossification, therefore we hypothesize that RARγ agonists can target on established osteochondromas. The purpose of this study was to examine the action of RARγ agonist in human osteochondromas. Osteochondroma specimens were obtained during surgery, subjected to explant culture and were treated with RARγ agonists or vehicles. Gene expression analysis confirmed the up-regulation of RARγ target genes in the explants treated with NRX 204647 and Palovarotene and revealed strong inhibition of cartilage matrix and increased extracellular matrix proteases gene expression. In addition, immunohistochemical staining for the neoepitope of protease-cleaved aggrecan indicated that RARγ agonist treatment stimulated cartilage matrix degradation. Interestingly, cell survival studies demonstrated that RARγ agonist treatment stimulated cell death. Moreover, RNA sequencing analysis indicates changes in multiple molecular pathways due to RARγ agonists treatment, showing similarly to human growth plate chondrocytes. Together, these findings suggest that RARγ agonist may exert anti-tumor function on osteochondromas by inhibiting matrix synthesis, promoting cartilage matrix degradation and stimulating cell death.

YAP1 subgroup supratentorial ependymoma requires TEAD and nuclear factor I-mediated transcriptional programmes for tumorigenesis.

YAP1 fusion-positive supratentorial ependymomas predominantly occur in infants, but the molecular mechanisms of oncogenesis are unknown. Here we show YAP1-MAMLD1 fusions are sufficient to drive malignant transformation in mice, and the resulting tumors share histo-molecular characteristics of human ependymomas. Nuclear localization of YAP1-MAMLD1 protein is mediated by MAMLD1 and independent of YAP1-Ser127 phosphorylation. Chromatin immunoprecipitation-sequencing analyses of human YAP1-MAMLD1-positive ependymoma reveal enrichment of NFI and TEAD transcription factor binding site motifs in YAP1-bound regulatory elements, suggesting a role for these transcription factors in YAP1-MAMLD1-driven tumorigenesis. Mutation of the TEAD binding site in the YAP1 fusion or repression of NFI targets prevents tumor induction in mice. Together, these results demonstrate that the YAP1-MAMLD1 fusion functions as an oncogenic driver of ependymoma through recruitment of TEADs and NFIs, indicating a rationale for preclinical studies to block the interaction between YAP1 fusions and NFI and TEAD transcription factors.

Increased burden of rare deleterious variants of the KCNQ1 gene in patients with large­vessel ischemic stroke.

The impact of rare and damaging variants in genes associated with platelet function in large­vessel ischemic stroke (LVIS) remains unknown. The aim of this study was to investigate the contribution of some of these variants to the genetic susceptibility to LVIS in Polish patients using a deep re­sequencing of 54 selected genes, coding for proteins associated with altered platelet function. Targeted pooled re­sequencing (Illumina HiSeq 2500) was performed on genomic DNA of 500 cases (patients with history of clinically proven diagnosis of LVIS) and 500 age­, smoking status­, and sex­matched controls (no history of any type of stroke), and from the same population as patients with LVIS. After quality control and prioritization based on allele frequency and damaging probability, individual genotyping of all deleterious rare variants was performed in patients from the original cohort, and stratified to concomitant cardiac conditions differing between the study and stroke groups. We demonstrated a statistically significant increase in the number of rare and potentially damaging variants in some of the investigated genes in the LVIS pool (an increase in the genomic variants burden). Furthermore, we identified an association between LVIS and 6 rare functional and damaging variants in the Kv7.1 potassium channel gene (KCNQ1). The predicted functional properties (partial loss­of function) for the three most damaging variants in KCNQ1 coding locus were further confirmed in vitro by analyzing the membrane potential changes in cell lines co­transfected heterogeneously with human muscarinic type 1 receptor and wild­type or mutated KCNQ1 cDNA constructs using fluorescence imaging plate reader. The study demonstrated an increased rare variants burden for 54 genes associated with platelet function, and identified a putative role for rare damaging variants in the KCNQ1 gene on LVIS susceptibility in the Polish population.

PARP10 promotes cellular proliferation and tumorigenesis by alleviating replication stress.

During carcinogenesis, cells are exposed to increased replication stress due to replication fork arrest at sites of DNA lesions and difficult to replicate genomic regions. Efficient fork restart and DNA repair are important for cancer cell proliferation. We previously showed that the ADP-ribosyltransferase PARP10 interacts with the replication protein proliferating cell nuclear antigen and promotes lesion bypass by recruiting specialized, non-replicative DNA polymerases. Here, we show that PARP10 is overexpressed in a large proportion of human tumors. To understand the role of PARP10 in cellular transformation, we inactivated PARP10 in HeLa cancer cells by CRISPR/Cas9-mediated gene knockout, and overexpressed it in non-transformed RPE-1 cells. We found that PARP10 promotes cellular proliferation, and its overexpression alleviates cellular sensitivity to replication stress and fosters the restart of stalled replication forks. Importantly, mouse xenograft studies showed that loss of PARP10 reduces the tumorigenesis activity of HeLa cells, while its overexpression results in tumor formation by non-transformed RPE-1 cells. Our findings indicate that PARP10 promotes cellular transformation, potentially by alleviating replication stress and suggest that targeting PARP10 may represent a novel therapeutic approach.

Isolation and Identification of the Follicular Microbiome: Implications for Acne Research.

Our understanding of the microbiome and the role of Propionibacterium acnes in skin homeostasis and acne pathogenesis is evolving. Multiple methods for sampling and identifying the skin's microbiome exist, and understanding the differences between the abilities of various methods to characterize the microbial landscape is warranted. This study compared the microbial diversity of samples obtained from the cheeks of 20 volunteers, collected by surface swab, pore strips, and cyanoacrylate glue follicular biopsy, all sequenced with 16S rRNA sequencing (V1-V3) and whole-genome metagenomic sequencing. The sequencing method of choice influenced the detection of microbial profiles as whole-genome sequencing captured more species diversity, including viruses, compared with 16S sequencing. The relative abundance of bacterial or fungal species and overall diversity did not differ between sampling methods. However, the viral composition of the skin's surface is unique compared with the follicle, suggesting distinct viral niches within the skin. P. acnes bacteria, ribotypes, and bacteriophages were identified equally by all sampling methods indicating that the sampling method, whether for the skin's surface or follicle, does not impact P. acnes-related characterization and that all may be equally useful for acne-related research studies.

Hypomethylated Fgf3 is a potential biomarker for early detection of oral cancer in mice treated with the tobacco carcinogen dibenzo[def,p]chrysene.

Genetic and epigenetic alterations observed at end stage OSCC formation could be considered as a consequence of cancer development and thus changes in normal or premalignant tissues which had been exposed to oral carcinogens such as Dibenzo[def,p]chrysene (DBP) may better serve as predictive biomarkers of disease development. Many types of DNA damage can induce epigenetic changes which can occur early and in the absence of evident morphological abnormalities. Therefore we used ERRBS to generate genome-scale, single-base resolution DNA methylomes from histologically normal oral tissues of mice treated with DBP under experimental conditions known to induce maximum DNA damage which is essential for the development of OSCC induced by DBP in mice. After genome-wide correction, 30 and 48 differentially methylated sites (DMS) were identified between vehicle control and DBP treated mice using 25% and 10% differences in methylation, respectively. RT-PCR was further performed to examine the expressions of nine selected genes. Among them, Fgf3, a gene frequently amplified in head and neck cancer, showed most prominent and significant gene expression change (2.4× increases), despite the hypomethylation of Fgf3 was identified at >10kb upstream of transcription start site. No difference was observed in protein expression between normal oral tissues treated with DBP or vehicle as examined by immunohistochemistry. Collectively, our results indicate that Fgf3 hypomethylation and gene overexpression, but not protein expression, occurred in the early stage of oral carcinogenesis induced by DBP. Thus, Fgf3 hypomethylation may serve as a potential biomarker for early detection of OSCC.

"Stealth dissemination" of macrophage-tumor cell fusions cultured from blood of patients with pancreatic ductal adenocarcinoma.

Here we describe isolation and characterization of macrophage-tumor cell fusions (MTFs) from the blood of pancreatic ductal adenocarcinoma (PDAC) patients. The MTFs were generally aneuploidy, and immunophenotypic characterizations showed that the MTFs express markers characteristic of PDAC and stem cells, as well as M2-polarized macrophages. Single cell RNASeq analyses showed that the MTFs express many transcripts implicated in cancer progression, LINE1 retrotransposons, and very high levels of several long non-coding transcripts involved in metastasis (such as MALAT1). When cultured MTFs were transplanted orthotopically into mouse pancreas, they grew as obvious well-differentiated islands of cells, but they also disseminated widely throughout multiple tissues in "stealth" fashion. They were found distributed throughout multiple organs at 4, 8, or 12 weeks after transplantation (including liver, spleen, lung), occurring as single cells or small groups of cells, without formation of obvious tumors or any apparent progression over the 4 to 12 week period. We suggest that MTFs form continually during PDAC development, and that they disseminate early in cancer progression, forming "niches" at distant sites for subsequent colonization by metastasis-initiating cells.

HUWE1 interacts with PCNA to alleviate replication stress.

Defects in DNA replication, DNA damage response, and DNA repair compromise genomic stability and promote cancer development. In particular, unrepaired DNA lesions can arrest the progression of the DNA replication machinery during S-phase, causing replication stress, mutations, and DNA breaks. HUWE1 is a HECT-type ubiquitin ligase that targets proteins involved in cell fate, survival, and differentiation. Here, we report that HUWE1 is essential for genomic stability, by promoting replication of damaged DNA We show that HUWE1-knockout cells are unable to mitigate replication stress, resulting in replication defects and DNA breakage. Importantly, we find that this novel role of HUWE1 requires its interaction with the replication factor PCNA, a master regulator of replication fork restart, at stalled replication forks. Finally, we provide evidence that HUWE1 mono-ubiquitinates H2AX to promote signaling at stalled forks. Altogether, our work identifies HUWE1 as a novel regulator of the replication stress response.

Macrophage-tumor cell fusions from peripheral blood of melanoma patients.

BACKGROUND: While the morbidity and mortality from cancer are largely attributable to its metastatic dissemination, the integral features of the cascade are not well understood. The widely accepted hypothesis is that the primary tumor microenvironment induces the epithelial-to-mesenchymal transition in cancer cells, facilitating their escape into the bloodstream, possibly accompanied by cancer stem cells. An alternative theory for metastasis involves fusion of macrophages with tumor cells (MTFs). Here we culture and characterize apparent MTFs from blood of melanoma patients. METHODS: We isolated enriched CTC populations from peripheral blood samples from melanoma patients, and cultured them. We interrogated these cultured cells for characteristic BRAF mutations, and used confocal microscopy for immunophenotyping, motility, DNA content and chromatin texture analyses, and then conducted xenograft studies using nude mice. FINDINGS: Morphologically, the cultured MTFs were generally large with many pseudopod extensions and lamellipodia. Ultrastructurally, the cultured MTFs appeared to be macrophages. They were rich in mitochondria and lysosomes, as well as apparent melanosomes. The cultured MTF populations were all heterogeneous with regard to DNA content, containing aneuploid and/or high-ploidy cells, and they typically showed large sheets (and/or clumps) of cytoplasmic chromatin. This cytoplasmic DNA was found within heterogeneously-sized autophagic vacuoles, which prominently contained chromatin and micronuclei. Cultured MTFs uniformly expressed pan-macrophage markers (CD14, CD68) and macrophage markers indicative of M2 polarization (CD163, CD204, CD206). They also expressed melanocyte-specific markers (ALCAM, MLANA), epithelial biomarkers (KRT, EpCAM), as well as the pro-carcinogenic cytokine MIF along with functionally related stem cell markers (CXCR4, CD44). MTF cultures from individual patients (5 of 8) contained melanoma-specific BRAF activating mutations. Chromatin texture analysis of deconvoluted images showed condensed DNA (DAPI-intense) regions similar to focal regions described in stem cell fusions. MTFs were readily apparent in vivo in all human melanomas examined, often exhibiting even higher DNA content than the cultured MTFs. When cultured MTFs were transplanted subcutaneously in nude mice, they disseminated and produced metastatic lesions at distant sites. CONCLUSIONS AND HYPOTHESIS: Apparent MTFs are present in peripheral blood of patients with cutaneous melanomas, and they possess the ability to form metastatic lesions when transplanted into mice. We hypothesize that these MTFs arise at the periphery of primary tumors in vivo, that they readily enter the bloodstream and invade distant tissues, secreting cytokines (such as MIF) to prepare "niches" for colonization by metastasis initiating cells.

Habitual sleep variability, not sleep duration, is associated with caloric intake in adolescents.

OBJECTIVE: The objective of this study was to investigate the associations between objectively measured habitual sleep duration (HSD), habitual sleep variability (HSV), and energy and snack intake in adolescents. METHODS: We used data from 324 adolescents who participated in the Penn State Child Cohort follow-up examination. Actigraphy was used over seven consecutive nights to estimate nightly sleep duration. The seven-night mean and standard deviation of sleep duration were used to represent HSD and HSV, respectively. The Youth/Adolescent Food Frequency Questionnaire was used to obtain the daily average total energy, protein, fat, and carbohydrate intake, and number of snacks consumed. Linear regression models were used to investigate the associations between habitual sleep patterns and caloric, protein, fat, and carbohydrate intake. Proportional odds models were used to associate habitual sleep patterns with snack consumption. RESULTS: After adjusting for age, sex, race, body mass index (BMI) percentile, and smoking status, an increased HSV was associated with a higher energy intake, particularly from fat and carbohydrate. For example, with a 1-h increase in HSV, there was a 170 (66)-kcal increase in the daily total energy intake. An increased HSV was also related to increased snack consumption, especially snacks consumed after dinner. For instance, a 1-h increase in HSV was associated with 65% and 94% higher odds of consuming more snacks after dinner during school/workdays and weekends/vacation days, respectively. Neither energy intake nor snack consumption was significantly related to HSD. CONCLUSION: High habitual sleep variability, not habitual sleep duration, is related to increased energy and food consumption in adolescents. Maintaining a regular sleep pattern may decrease the risk of obesity in adolescents.