Activation of IGF signaling is a major oncogenic event in diverse cancers, including hepatocellular carcinoma (HCC). In this setting, the insulin-like growth factor binding protein IGFBP7 inhibits ...IGF signaling by binding the IGF1 receptor (IGF1R), functioning as a candidate tumor suppressor. IGFBP7 abrogates tumors by inhibiting angiogenesis and inducing cancer-specific senescence and apoptosis. Here, we report that Igfbp7-deficient mice exhibit constitutively active IGF signaling, presenting with proinflammatory and immunosuppressive microenvironments and spontaneous liver and lung tumors occurring with increased incidence in carcinogen-treated subjects. Igfbp7 deletion increased proliferation and decreased senescence of hepatocytes and mouse embryonic fibroblasts, effects that were blocked by treatment with IGF1 receptor inhibitor. Significant inhibition of genes regulating immune surveillance was observed in Igfbp7
murine livers, which was associated with a marked inhibition in antigen cross-presentation by Igfbp7
dendritic cells. Conversely, IGFBP7 overexpression inhibited growth of HCC cells in syngeneic immunocompetent mice. Depletion of CD4
or CD8
T lymphocytes abolished this growth inhibition, identifying it as an immune-mediated response. Our findings define an immune component of the pleiotropic mechanisms through which IGFBP7 suppresses HCC. Furthermore, they offer a genetically based preclinical proof of concept for IGFBP7 as a therapeutic target for immune management of HCC.
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Subcutaneous patient-derived xenografts (PDXs) are an important tool for childhood cancer research. Here, we describe a resource of 68 early passage PDXs established from 65 pediatric solid tumor ...patients. Through genomic profiling of paired PDXs and patient tumors (PTs), we observe low mutational similarity in about 30% of the PT/PDX pairs. Clonal analysis in these pairs show an aggressive PT minor subclone seeds the major clone in the PDX. We show evidence that this subclone is more immunogenic and is likely suppressed by immune responses in the PT. These results suggest interplay between intratumoral heterogeneity and antitumor immunity may underlie the genetic disparity between PTs and PDXs. We further show that PDXs generally recapitulate PTs in copy number and transcriptomic profiles. Finally, we report a gene fusion LRPAP1-PDGFRA. In summary, we report a childhood cancer PDX resource and our study highlights the role of immune constraints on tumor evolution.
Humans show sex differences related to alcohol use disorders (AUD). Animal model research has the potential to provide important insight into how sex differences affect alcohol consumption, ...particularly because female animals frequently drink more than males. In previous work, inbred strains of the selectively bred alcohol-preferring (P) and non-preferring (NP) rat lines revealed a highly significant quantitative trait locus (QTL) on rat chromosome 4, with a logarithm of the odds score of 9.2 for alcohol consumption. Recently, interval-specific congenic strains (ISCS) were developed by backcrossing the congenic P.NP line to inbred P (iP) rats to further refine the chromosome 4 QTL region. Two ISCS sub-strains, ISCS-A and ISCS-B, were obtained with a narrowed QTL, where the smallest region of overlap consisted of 8.9 Mb in ISCS-B. Interestingly, we found that females from both ISCS lines consumed significantly less alcohol than female iP controls (
< 0.05), while no differences in alcohol consumption were observed between male ISCS and iP controls. RNA-sequencing was performed on the nucleus accumbens of alcohol-naïve female ISCS-B and iP rats, which revealed differentially expressed genes (DEG) with greater than 2-fold change and that were functionally relevant to behavior. These DEGs included down-regulation of
, and
, and up-regulation of
, and
. Pathway analysis identified significant alterations in gene networks controlling nervous system development and function, as well as cell signaling, GABA and serotonin receptor signaling and G-protein coupled receptor signaling. In addition, β-estradiol was identified as the most significant upstream regulator. The expression levels of estrogen-responsive genes that mapped to the QTL interval and have been previously associated with alcohol consumption were measured using RT-qPCR. We found that expression of the
gene, encoding the pituitary adenylate cyclase-activating polypeptide type 1 (PAC
) receptor, was upregulated in female ISCS-B compared to female iP controls, while no differences were exhibited in males. In addition, sequence variants in the
promoter region showed a differential response to estrogen stimulation
. These findings demonstrate that rat chromosome 4 QTL contains genetic variants that respond to estrogen and are associated with female alcohol consumption.
Astrocyte elevated gene‐1 (AEG‐1) and c‐Myc are overexpressed in human hepatocellular carcinoma (HCC) functioning as oncogenes. AEG‐1 is transcriptionally regulated by c‐Myc, and AEG‐1 itself induces ...c‐Myc by activating the Wnt/β‐catenin–signaling pathway. We now document the cooperation of AEG‐1 and c‐Myc in promoting hepatocarcinogenesis by analyzing hepatocyte‐specific transgenic mice expressing either AEG‐1 (albumin Alb/AEG‐1), c‐Myc (Alb/c‐Myc), or both (Alb/AEG‐1/c‐Myc). Wild‐type and Alb/AEG‐1 mice did not develop spontaneous HCC. Alb/c‐Myc mice developed spontaneous HCC without distant metastasis, whereas Alb/AEG‐1/c‐Myc mice developed highly aggressive HCC with frank metastasis to the lungs. Induction of carcinogenesis by N‐nitrosodiethylamine significantly accelerated the kinetics of tumor formation in all groups. However, in Alb/AEG‐1/c‐Myc, the effect was markedly pronounced with lung metastasis. In vitro analysis showed that Alb/AEG‐1/c‐Myc hepatocytes acquired increased proliferation and transformative potential with sustained activation of prosurvival and epithelial‐mesenchymal transition–signaling pathways. RNA‐sequencing analysis identified a unique gene signature in livers of Alb/AEG‐1/c‐Myc mice that was not observed when either AEG‐1 or c‐Myc was overexpressed. Specifically, Alb/AEG‐1/c‐Myc mice overexpressed maternally imprinted noncoding RNAs (ncRNAs), such as Rian, Meg‐3, and Mirg, which are implicated in hepatocarcinogenesis. Knocking down these ncRNAs significantly inhibited proliferation and invasion by Alb/AEG‐1/c‐Myc hepatocytes. Conclusion: Our studies reveal a novel cooperative oncogenic effect of AEG‐1 and c‐Myc that might explain the mechanism of aggressive HCC. Alb/AEG‐1/c‐Myc mice provide a useful model to understand the molecular mechanism of cooperation between these two oncogenes and other molecules involved in hepatocarcinogenesis. This model might also be of use for evaluating novel therapeutic strategies targeting HCC. (Hepatology 2015;61:915–929)
High levels of alcohol intake alter brain gene expression and can produce long-lasting effects. FK506-binding protein 51 (FKBP51) encoded by
is a physical and cellular stress response gene and has ...been associated with alcohol consumption and withdrawal severity.
has been previously linked to neurite outgrowth and hippocampal morphology, sex differences in stress response, and epigenetic modification. Presently, primary cultured
KO and WT mouse neurons were examined for neurite outgrowth and mitochondrial signal with and without alcohol. We found neurite specification differences between KO and WT; particularly, mesh-like morphology was observed after alcohol treatment and confirmed higher MitoTracker signal in cultured neurons of
KO compared to WT at both naive and alcohol-treated conditions. Brain regions that express FKBP51 protein were identified, and hippocampus was confirmed to possess a high level of expression. RNA-seq profiling was performed using the hippocampus of naïve or alcohol-injected (2 mg EtOH/Kg) male and female
KO and WT mice. Differentially expressed genes (DEGs) were identified between
KO and WT at baseline and following alcohol treatment, with female comparisons possessing a higher number of DEGs than male comparisons. Pathway analysis suggested that genes affecting calcium signaling, lipid metabolism, and axon guidance were differentially expressed at naïve condition between KO and WT. Alcohol treatment significantly affected pathways and enzymes involved in biosynthesis (Keto, serine, and glycine) and signaling (dopamine and insulin receptor), and neuroprotective role. Functions related to cell morphology, cell-to-cell signaling, lipid metabolism, injury response, and post-translational modification were significantly altered due to alcohol. In summary,
plays a critical role in the response to acute alcohol treatment by altering metabolism and signaling-related genes.
Comprehensive mRNA sequencing is a powerful tool for conducting unbiased, quantitative differential gene expression analysis. However, the reliability of these data is contingent on the extraction of ...high-quality RNA from samples. Preserving RNA integrity during extraction can be problematic, especially in tissues such as skin with dense, connective matrices and elevated ribonuclease expression. This is a major barrier to understanding the influences of altered gene expression in many preclinical pain models and clinical pain disorders where skin is the site of tissue injury.
This study developed and evaluated extraction protocols for skin and other tissues to maximize recovery of high-integrity RNA needed for quantitative mRNA sequencing.
Rodent and human tissue samples underwent one of the several different protocols that combined either RNA-stabilizing solution or snap-freezing with bead milling or cryosectioning. Indices of RNA integrity and purity were assessed for all samples.
Extraction of high-integrity RNA is highly dependent on the methods used. Bead-milling skin collected in RNA-stabilizing solution resulted in extensive RNA degradation. Snap-freezing in liquid nitrogen was required for skin and highly preferable for other tissues. Skin also required cryosectioning to achieve effective penetration of RNA-stabilizing solution to preserve RNA integrity, whereas bead milling could be used instead with other tissues. Each method was reproducible across multiple experimenters. Electrophoretic anomalies that skewed RNA integrity value assignment required manual correction and often resulted in score reduction.
To achieve the potential of quantitative differential gene expression analysis requires verification of tissue-dependent extraction methods that yield high-integrity RNA.
Patient-derived xenografts (PDX) remain valuable models for understanding the biology and for developing novel therapeutics. To expand current PDX models of childhood leukemia, we have developed new ...PDX models from Hispanic patients, a subgroup with a poorer overall outcome. Of 117 primary leukemia samples obtained, successful engraftment and serial passage in mice were achieved in 82 samples (70%). Hispanic patient samples engrafted at a rate (51/73, 70%) that was similar to non-Hispanic patient samples (31/45, 70%). With a new algorithm to remove mouse contamination in multi-omics datasets including methylation data, we found PDX models faithfully reflected somatic mutations, copy-number alterations, RNA expression, gene fusions, whole-genome methylation patterns, and immunophenotypes found in primary tumor (PT) samples in the first 50 reported here. This cohort of characterized PDX childhood leukemias represents a valuable resource in that germline DNA sequencing has allowed the unambiguous determination of somatic mutations in both PT and PDX.
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•Patient-derived xenografts (PDXs) are important preclinical models•50 pediatric leukemia PDXs are largely derived from Hispanic ethnicity•Paired patient tumor, PDX, and germline for sequencing analysis•This cohort uncovers genomic, transcriptomic, and epigenomic features
Cancer; Omics; Transcriptomics
Oncoprotein staphylococcal nuclease and tudor domain containing 1 (SND1) regulates gene expression at a posttranscriptional level in multiple cancers, including hepatocellular carcinoma (HCC). ...Staphylococcal nuclease (SN) domains of SND1 function as a ribonuclease (RNase), and the tudor domain facilitates protein–oligonucleotide interaction. In the present study, we aimed to identify RNA interactome of SND1 to obtain enhanced insights into gene regulation by SND1. RNA interactome was identified by immunoprecipitation (IP) of RNA using anti‐SND1 antibody from human HCC cells followed by RNA immunoprecipitation sequencing (RIP‐Seq). Among RNA species that showed more than 10‐fold enrichment over the control, we focused on the tumor suppressor protein tyrosine phosphatase nonreceptor type 23 (PTPN23) because its regulation by SND1 and its role in HCC are not known. PTPN23 levels were down‐regulated in human HCC cells versus normal hepatocytes and in human HCC tissues versus normal adjacent liver, as revealed by immunohistochemistry. In human HCC cells, knocking down SND1 increased and overexpression of SND1 decreased PTPN23 protein. RNA binding and degradation assays revealed that SND1 binds to and degrades the 3′‐untranslated region (UTR) of PTPN23 messenger RNA (mRNA). Tetracycline‐inducible PTPN23 overexpression in human HCC cells resulted in significant inhibition in proliferation, migration, and invasion and in vivo tumorigenesis. PTPN23 induction caused inhibition in activation of tyrosine‐protein kinase Met (c‐Met), epidermal growth factor receptor (EGFR), Src, and focal adhesion kinase (FAK), suggesting that, as a putative phosphatase, PTPN23 inhibits activation of these oncogenic kinases. Conclusion: PTPN23 is a novel target of SND1, and our findings identify PTPN23 as a unique tumor suppressor for HCC. PTPN23 might function as a homeostatic regulator of multiple kinases, restraining their activation.
The oncoprotein SND1 binds to and degrades the RNA of the tumor suppressor gene PTPN23. PTPN23 expression is downregulated in hepatocellular carcinoma and forced overexpression of PTPN23 inhibits proliferation, migration and invasion of HCC cells.
Many chronic pain conditions show sex differences in their epidemiology. This could be attributed to sex-dependent differential expression of genes (DEGs) involved in nociceptive pathways, including ...sensory neurons. This study aimed to identify sex-dependent DEGs in estrous female versus male sensory neurons, which were prepared by using different approaches and ganglion types. RNA-seq on non-purified sensory neuronal preparations, such as whole dorsal root ganglion (DRG) and hindpaw tissues, revealed only a few sex-dependent DEGs. Sensory neuron purification increased numbers of sex-dependent DEGs. These DEG sets were substantially influenced by preparation approaches and ganglion types DRG vs trigeminal ganglia (TG). Percoll-gradient enriched DRG and TG neuronal fractions produced distinct sex-dependent DEG groups. We next isolated a subset of sensory neurons by sorting DRG neurons back-labeled from paw and thigh muscle. These neurons have a unique sex-dependent DEG set, yet there is similarity in biological processes linked to these different groups of sex-dependent DEGs. Female-predominant DEGs in sensory neurons relate to inflammatory, synaptic transmission and extracellular matrix reorganization processes that could exacerbate neuro-inflammation severity, especially in TG. Male-selective DEGs were linked to oxidative phosphorylation and protein/molecule metabolism and production. Our findings catalog preparation-dependent sex differences in neuronal gene expressions in sensory ganglia.
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