CpG Nucleotides Research Articles

Abstract 5872: CDK1-SOX9 axis contributes to chemotherapy resistance in gastric cancer

Abstract Gastric carcinoma (GC) is the third most prevalent cause of cancer-related mortality worldwide, presenting a formidable challenge with the rise of resistance to chemotherapeutic agents, leading to treatment failures. Cyclin-dependent kinase 1 (CDK1) has garnered attention due to its substantial overexpression in various tumors, including GC, and its association with poor overall survival and relapse-free survival. This study aimed to understand the molecular and functional mechanisms underlying the CDK1-SOX9 axis in GC chemotherapy resistance. Through a comprehensive analysis of data from The Cancer Genome Atlas (TCGA) and our own integrated gene expression study, we found a positive correlation between CDK1 and SOX9 in both human and mouse GC tissues (p<0.001). Additionally, conserved alterations in miRNAs have been identified in the context of gastric carcinogenesis, evident in our local cohort and the TCGA dataset. Our in-depth investigation of premiR-145 has shed light on the existence of several CpG nucleotides. Notably, treatment with a DNMT inhibitor (5-Aza) has triggered the upregulation of miR-145 expression. Furthermore, the ectopic expression of CDK1 has been associated with increased DNMT1 phosphorylation and enhanced enzymatic activity. Conversely, silencing CDK1 reversed this effect, and the inhibition of DNMT1 reduced SOX9 protein expression. Using cisplatin-resistant cell lines revealed a significant increase in CDK1 and SOX9 expression where silencing CDK1 and SOX9 sensitized these cells to chemotherapy. This study provides valuable insights into the intricate mechanisms governing the CDK1-SOX9 axis in GC chemotherapy resistance, providing potential avenues for therapeutic interventions in this challenging malignancy. Citation Format: Marwah M. Al-Mathkour, Shoumin Zhu, Melanie Genoula, Wael El-Rifai. CDK1-SOX9 axis contributes to chemotherapy resistance in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5872.

Abstract 6009: An end-to-end workflow for improved methylation detection

Abstract Introduction: DNA methylation at CpG nucleotide sites in eukaryotes is a key epigenetic mark that can help regulate gene expression. Specific changes in CpG methylation occur in many human cancers, making them a promising biomarker for early cancer detection. However, existing assays can be costly, lack specificity to regions of interest and often provide only semi-quantitative estimates of the methylation fraction. Here, we present a targeted methylome panel to decrease costs associated with next generation sequencing (NGS), methylation controls to calibrate quantitative assays and UMIs for accurate deduplication in low-diversity samples. Experimental Procedures: Genomic DNA (gDNA) was prepared for sequencing using the Twist Methylation Detection System consisting of enzymatic methylation conversion and hybrid capture using the Twist Human Methylome Panel. Twist’s synthetic CpG methylation level controls were spiked-in to gDNA and taken through the Twist Methylation Detection System to demonstrate their utility in calibrating methylation assays. Additionally, libraries were generated using cell free DNA (cfDNA) and either conventional or UMI-containing adapters to investigate the impact on quantitative detection and total unique coverage. Results: Using the Twist Human Methylome Panel at 150x raw coverage achieves uniform coverage with low off-bait Picard metrics. 6.59 million CpG sites were detected using a minimum depth of 10X. Target capture with the Human Methylome Panel allows for informative CpG calling of up to 82 samples on a single Illumina Novaseq S4 flowcell, compared to 3 samples per flowcell with a tradition whole-genome bisulfite sequencing (WGBS) assay. The Twist CpG methylation specific controls are constructed of 48 unique contrived sequences that contain a total of 8 different levels of methylation, ranging from 100% to 0%. Including these controls allows for quantitation of methylation levels in the experimental samples and qualification of the enzymatic conversion process. Conclusions: Our study leverages the Twist methylation detection portfolio to interrogate genome-wide methylation patterns for various applications. In combination, these tools can be used to design cost effective end-to-end assays. *For Research Use Only. Not for use in diagnostic procedures. Citation Format: Lydia Bonar, Kristin Butcher, Michael Bocek, Holly Corbitt, Bryan Hoglund, Cibelle Nassif, Patrick Cherry, Derek Murphy, Jean Challacombe, Esteban Toro. An end-to-end workflow for improved methylation detection. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6009.

Epigenetic interactions.

Environmental cues influence the epigenetic state of our genomes, and thereby modulate our cellular responses to the environment. In mammals this machinery seems to act, at least in part, through reproducible and reversable changes to the methylation state of specific CpG nucleotides, although there is some debate as to whether DNA methylation is a marker for rather than the actual agent of change. It is also the case that in mammals the identity of the parent of origin of a chromosome silences, or otherwise dysregulates, the expression of about 100 imprinted genes (in humans fewer; more in mice).

Multiple Myeloma Derived Mitochondrial Damps Induce Inflammation in the Bone Marrow Adipose Tissue Which Promotes Tumour Development

Multiple myeloma (MM) is a malignancy of the antibody secreting plasma cells, characterized by the localisation and accumulation of tumour cells in the bone marrow (BM) microenvironment. The mitochondrial DNA (mtDNA) belongs to a group of mitochondrial damage associated molecular patterns (mtDAMPs) and contains islands of unmethylated CpG nucleotide motifs, which have been shown to activate and promote memory B cell antibody secretion and proliferation. Studies have indicated that mitochondrial DNA (mtDNA) is elevated in the circulation of trauma and cancer patients, highlighting a need to explore the functional purpose of mtDNA in the BM microenvironment. Moreover, myeloma relies on the BM microenvironment for survival, proliferation and drug resistance. BM adipocytes are highly secretory cells that support myeloma cells through the release of various factors. It has been shown that myeloma cells induce a shift from osteoblast progenitors towards adipogenesis. Here, we hypothesise that MM cells release mtDAMPs that leads to the promotion of a state of chronic inflammation that supports multiple myeloma disease progression and expansion by inducing adipocyte inflammation.First, we engrafted immunocompromised NSG mice with human myeloma cell line MM1S, blood serum samples were taken and analysed by RT-qPCR to detect the presence of human mtDNA. We found elevated levels of human mtDNA in the serum of engrafted mice correlating to disease progression. Next, we showed that mtDNA was higher in serum patients with MM compared to healthy controls, moreover, we discovered that BM serum from MM patients had significantly higher levels of mtDNA than blood serum. Furthermore, this observation was recapitulated in myeloma engrafted NSG mice. Next, we examined the effect of mtDNA on various cell populations of the BM. We found that in vitro both BM derived macrophages and BM derived adipocytes had increased inflammatory signatures, including upregulation of IL-6, when treated with mtDNA from myeloma cells. MtDNA induced IL-6 expression was attenuated upon blocking of the TLR9 receptor with antagonist ODN 2088. To understand the role of BM adipocyte induced inflammation in MM, we treated adipocytes with mtDNA before culturing them with MM. We found that MM cells had enhanced proliferation on mtDNA activated adipocytes. We further show that MM cells cultured with mtDNA activated adipocytes have increased uptake of free fatty acids and seahorse analysis confirmed an increase in b-oxidation derived metabolism.Here we establish that MM releases mtDNA into the microenvironment promoting the disease progression via a pro-inflammatory BM. We also show that both BM macrophages and BM adipocytes play a supportive role in multiple myeloma expansion and contribute to the pro-inflammatory bone marrow microenvironment. DisclosuresNo relevant conflicts of interest to declare.

Abstract 2799: Multiple myeloma derived mitochondrial DAMPs induce a pro-inflammatory signature in the bone marrow microenvironment to promote pro-tumoral expansion

Abstract Multiple myeloma (MM) is an incurable malignancy of antibody (Ig) secreting differentiated B cells (plasma cells) characterized by the accumulation and localization of tumor cells in the bone marrow microenvironment (BMM). Mitochondrial DNA (mtDNA) is a damage associated molecular pattern (DAMP), as the mitochondrial genome contains islands of unmethylated CpG nucleotide motifs that have been shown to activate and promote memory B cell proliferation and antibody secretion. Recent studies have indicated that mtDNA is elevated in the circulation of trauma and cancer patients. Here we investigate the functional purpose of elevated mtDNA within the BM microenvironment of MM.We hypothesize that multiple myeloma cells secrete mitochondrial DAMPs into the bone marrow microenvironment promoting a state of chronic inflammation that drives the progression and expansion of multiple myeloma. NSG immunocompromised mice engrafted with human MM1S myeloma cell line showed elevated levels of MM derived mtDNA in the serum, detected by real-time PCR. Next we engrafted C57BL/6 mice with murine 5TGM1 myeloma cell line to establish a syngeneic mouse model. Flow cytometry analysis of the haematopoietic stem and progenitor cell (HSPC) populations showed that 5TGM1 induced an inflammatory expansion of the stem cell niche To determine the role of mtDNA in HSPC expansion we treated C57BL/6 mice with multiple doses of CpG oligodeoxynucleotides to mimic mtDNA export by MM. Results showed similar expansion of haematopoietic stem and progenitor cell populations. To understand the effects of mtDAMPs on the inflammatory cells of the BMM, we show that bone marrow derived macrophages treated with mtDNA and CpG had increased expression of pro-inflammatory cytokines including IL-6. In vivo, isolated F4/80+ bone marrow macrophages from 5TGM1 and CpG treated mice also showed increased expression of pro-inflammatory cytokines. Finally, to understand the role of mtDAMPs in regulating HSPC expansion we used blocking antibodies to TLR9 (toll-like receptor 9 for mtDNA) and FPR1 (receptor for formylated mitochondrial proteins) in 5TGM1 engrafted animals. Blocking these receptors resulted in reduced myeloma tumor burden compared to control animals. Here we establish that MM releases mtDNA into the microenvironment and highlight the involvement of mtDAMPs in creating a pro-inflammatory BMM that aids in MM disease progression. This data suggests the potential for the targeting of TLR9 or FPR1 signaling pathways as a novel therapeutic approach for MM. Citation Format: Aisha Jibril, Prakrit Kumar, Charlotte Hellmich, Jamie A. Moore, Jayna J. Mistry, Victoria Willimott, Kristian M. Bowles, Stuart A. Rushworth. Multiple myeloma derived mitochondrial DAMPs induce a pro-inflammatory signature in the bone marrow microenvironment to promote pro-tumoral expansion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2799.

The Long-Term Evolutionary History of Gradual Reduction of CpG Dinucleotides in the SARS-CoV-2 Lineage.

Simple SummarySevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused the coronavirus disease 2019 (COVID-19), a pandemic that infected over 81 million people worldwide. This has led the scientific community to characterize the genome of this virus, including its nucleotide composition. Investigation of the dinucleotide frequency revealed that the proportion of CG dinucleotides (CpG) is highly reduced in the viral genomes. Since CpG dinucleotides is the target site for the host antiviral zinc finger protein, it has been suggested that the reduction in the proportion of CpG is the viral response to escape from the host defense machinery. In the present study, we investigated the time of origin of reduction in the CpG content. Whole genome analyses based on all representative viral genomes of the group Betacoronavirus revealed that the CpG content in the lineage of SARS-CoV-2 has been progressively declining over the past 1213 years. The depletion of CpG was found to occur at neutral—as well as selectively constrained—positions of the viral genomes.Recent studies suggested that the fraction of CG dinucleotides (CpG) is severely reduced in the genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The CpG deficiency was predicted to be the adaptive response of the virus to evade degradation of the viral RNA by the antiviral zinc finger protein that specifically binds to CpG nucleotides. By comparing all representative genomes belonging to the genus Betacoronavirus, this study examined the potential time of origin of CpG depletion. The results of this investigation revealed a highly significant correlation between the proportions of CpG nucleotide (CpG content) of the betacoronavirus species and their times of divergence from SARS-CoV-2. Species that are distantly related to SARS-CoV-2 had much higher CpG contents than that of SARS-CoV-2. Conversely, closely related species had low CpG contents that are similar to or slightly higher than that of SARS-CoV-2. These results suggest a systematic and continuous reduction in the CpG content in the SARS-CoV-2 lineage that might have started since the Sarbecovirus + Hibecovirus clade separated from Nobecovirus, which was estimated to be 1213 years ago. This depletion was not found to be mediated by the GC contents of the genomes. Our results also showed that the depletion of CpG occurred at neutral positions of the genome as well as those under selection. The latter is evident from the progressive reduction in the proportion of arginine amino acid (coded by CpG dinucleotides) in the SARS-CoV-2 lineage over time. The results of this study suggest that shedding CpG nucleotides from their genome is a continuing process in this viral lineage, potentially to escape from their host defense mechanisms.

Myeloma Derived Mitochondrial Damage Associated Molecular Patterns Promote Pro-Tumoral Expansion By Inducing a Pro-Inflammatory Signature in the Bone Marrow Microenvironment

Multiple Myeloma (MM) is a malignancy of antibody producing B cells (plasma cells), leading to the accumulation of tumour cells within the bone marrow (BM) microenvironment. Mitochondrial DNA (mtDNA) is a damage associated molecular pattern (DAMP), the mitochondrial genome contains islands of unmethylated CpG nucleotide motifs that have been shown to activate and promote memory B cell proliferation and antibody secretion. Recent studies have indicated that mtDNA is elevated in the circulation of trauma and cancer patients and have highlighted the need to determine the functional purpose of elevated mtDNA within the BM microenvironment. Here we investigate the hypothesis that multiple myeloma cells secrete mtDNA and other mtDAMPs to induce a pro-inflammatory bone marrow microenvironment, supportive of the progression and survival of MM. As well as investigating how MM affects the haematopoietic stem cell niche through establishing a syngeneic myeloma mouse model. We show that immunocompromised NSG mice transplanted with human MM cell lines released higher levels of MM derived mtDNA detected in the plasma by real-time PCR. Furthermore, in vitro experiments showed that pro-inflammatory cytokines IL-1B, IL-6, and IL-8 were elevated in both primary and cell line myeloma samples upon treatment with CpG oligonucleotides. Murine bone marrow derived macrophages (BMDMs) treated with mtDNA or mtDAMPs showed increased expression of pro-inflammatory cytokines. To understand the significance of the mtDAMP proinflammatory induction, C57BL/6 mice were successfully engrafted with murine 5TGM1 myeloma cell line to establish a syngeneic mouse model. Analysis of the haematopoietic stem and progenitor cell populations showed that 5TGM1 induced the expansion of these cell populations when compared to control WT animals. To understand the significance of mtDAMPs in regulating this HSPC expansion we used blocking antibodies to TLR9 (receptor for mtDNA) and FPR1 (receptor for formylated mitochondrial proteins) in 5TGM1 engrafted animals. Results show that myeloma tumour burden was reduced when FPR1 and TLR9 are blocked compared to control animals. Here we highlight the involvement of mtDAMPs in MM to create a favourable proinflammatory microenvironment which provides the conditions for MM survival and proliferation. This data highlights the importance of mtDAMPs in MM disease progression and suggests targeting these activating receptor pathways may provide a novel therapeutic intervention. Disclosures Bowles: Janssen: Research Funding; AbbVie: Research Funding. Rushworth:Janssen: Research Funding; AbbVie: Research Funding.

Mitoepigenetics: Methylation of mitochondrial DNA is strand-biased in bovine oocytes and embryos.

Global mitochondrial DNA (mtDNA) methylation has been recently described in bovine and showed particular signatures in both gametes and embryos. Here, we investigated the distribution of mtDNA methylation through strand-specific mapping of methylation sites to gain perspective on how epigenetic mechanisms can be involved in mitochondrial function. We demonstrate that in both oocytes and embryos, the frequency of methylation is biased towards the light strand (L-strand), particularly in the gene bodies and in the region containing the L-strand promoter (LSP). Methylation is not restricted to CpG nucleotides and is not symmetrical on both strands. This configuration reinforces the hypothesis of a specific epigenetic regulation of mtDNA, which is an important observation for the understanding of how mitochondrial function is regulated.

Discrimination of CpG Methylation Status and Nucleotide Differences in Tissue Specimen DNA by Oligoribonucleotide Interference-PCR.

Oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) is a method in which PCR amplification of a target sequence is inhibited in a sequence-specific manner by the hybridization of an ORN with the target sequence. Previously, we reported that ORNi-PCR could detect nucleotide mutations in DNA purified from cultured cancer cell lines or genome-edited cells. In this study, we investigated whether ORNi-PCR can discriminate nucleotide differences and CpG methylation status in damaged DNA, such as tissue specimen DNA and bisulfite-treated DNA. First, we showed that ORNi-PCR could discriminate nucleotide differences in DNA extracted from acetone-fixed paraffin-embedded rat liver specimens or formalin-fixed paraffin-embedded human specimens. Rat whole blood specimens were compatible with ORNi-PCR for the same purpose. Next, we showed that ORNi-PCR could discriminate CpG methylation status in bisulfite-treated DNA. These results demonstrate that ORNi-PCR can discriminate nucleotide differences and CpG methylation status in multiple types of DNA samples. Thus, ORNi-PCR is potentially useful in a wide range of fields, including molecular biology and medical diagnosis.

Epigenetic regulation of AURKA by miR-4715-3p in upper gastrointestinal cancers

Aurora kinase A (AURKA) is frequently overexpressed in several cancers. miRNA sequencing and bioinformatics analysis indicated significant downregulation of miR-4715-3p. We found that miR-4715-3p has putative binding sites on the 3UTR region of AURKA. Upper gastrointestinal adenocarcinoma (UGC) tissue samples and cell models demonstrated significant overexpression of AURKA with downregulation of miR-4715-3p. Luciferase reporter assays confirmed binding of miR-4715-3p on the 3UTR region of AURKA. miR-4715-3p mediated a reduction in AURKA levels leading to G2/M delay, chromosomal polyploidy, and cell death. We also detected a remarkable decrease in GPX4, an inhibitor of ferroptosis, with an increase in cleaved PARP and caspase-3. Inhibition of AURKA using siRNA produced similar results, suggesting a possible link between AURKA and GPX4. Analysis of UGC samples and cell models demonstrated increased methylation levels of several CpG nucleotides upstream of miR-4715-3p. 5-Aza-2′-deoxycytidine induced demethylation of several CpG nucleotides, restoring miR-4715-3p expression, leading to downregulation of AURKA. In conclusion, our data identified a novel epigenetic mechanism mediating silencing of miR-4715-3p and induction of AURKA in UGCs. Inhibition of AURKA or reconstitution of miR-4715-3p inhibited GPX4 and induced cell death, suggesting a link between AURKA and ferroptosis.

Clinical significance and biological role of KLF17 as a tumour suppressor in colorectal cancer.

It has been reported that kruppel‑like factor17 (KLF17) acts as a tumour suppressor in several tissues and cancer cells, however, the molecular roles, the underlying mechanisms and clinical significance of KLF17 in colorectal cancer (CRC) have not been completely elucidated. In the present study, KLF17 protein expression was detected in 140primary CRCs and paired adjacent non‑tumour tissues using immunohistochemistry with tissue microarrays. The KLF17 mRNA expression was determined in 4 CRC cell lines and 20pairs of the aforementioned tissues using reverse transcription quantitative polymerase chain reaction. The correlation between KLF17 expression and clinicopathologic characteristics was determined. Next, the functions of KLF17 in CRC were examined by cell proliferation, colony formation, adhesion, invasion and mouse xenograft assays. Methylation‑specific PCR and bisulfite sequencing PCR were also carried out to investigate the promoter methylation status of KLF17 in CRC cells and tissues and explore the effects of lentiviral‑mediated RNAi of UHRF1 on the methylation and expression of KLF17. The results revealed that KLF17 expression was abnormally decreased in CRC and associated with lymph node metastasis and unfavorable overall survival. Moreover, ectopic KLF17 expression suppressed CRC cell growth and invasion invitro and invivo. In addition, the downregulation of KLF17 was associated with the hypermethylation of the CpG nucleotides on the KLF17 promoter. The knockdown of the epigenetic regulator UHRF1 reduced the methylation level of the KLF17 promoter and inhibited CRC cell adhesion, invasion and epithelial‑mesenchymal transition by upregulating KLF17. The present findings indicated that KLF17 may act as a tumour suppressor gene in CRC and a potential independent prognostic biomarker in CRC patients. UHRF1 can suppress KLF17 expression through the hypermethylation of its promoter in CRC. These results offer insights into the KLF17 expression regulation in CRC and suggest an inhibitory effect of KLF17 on tumourigenesis.

Abstract 832: Comparison of single step to two step PCR for DNA methylation status determination utilizing a system that continuously contains PCR products in plastic consumables

Abstract DNA methylation specific PCR is a commonly used method to determine DNA methylation status. However, a well-known risk for PCR, and particularly methylation specific PCR, is carryover contamination from amplification products that can result in false positive PCR results in subsequent tests. This risk can be mitigated using careful technique, facility design, and biochemical methods such as uracil incorporation and subsequent uracil N-glycosylase (UNG) negative selection. Unfortunately, careful technique is highly user dependent, facility design can be cumbersome and expensive, and uracil incorporation is effective for only one step of two-step (nested) PCR reactions and cannot be used with the DNA bisulfite treatment methods used for methylation specific PCR. Our company has developed a system that mitigates PCR product contamination by continuously containing PCR products in plastic consumables and can interface with existing laboratory robotics, real time thermocycling instruments, and cassette electrophoresis gels. Here we utilize this system to compare the sensitivity and specificity of two-step multiplex nested PCR vs. single-step PCR for DNA methylation status detection applications using both qPCR and gel electrophoresis analysis. Published methylation status specific PCR primers that target CpG nucleotides in promotor regions were used to PCR amplify commercially available fully methylated and fully unmethylated DNA. The results demonstrate that a multiplex preamplification using amplicons for the CHAD, GFI1, MX2, NEU1, VWCE genes followed by monoplex nested methylation specific PCRs resulted in a Limit Of Detection (LOD) of 1 to 10 DNA template copies whereas performing just the monoplex methylation specific PCR (no preamplification) resulted in a LOD of 10 to 300 DNA template copies. If DNA isolated from macro dissected FFPE tissue was utilized, positive signals were identified using multiplex nested PCR whereas no signals were identified using single-step PCR. When mixtures of methylated and unmethylated DNA were utilized along with the GSTP gene amplicons, two-step nested PCR using were able to distinguish as low as 1% unmethylated DNA or 2% methylated DNA in a background of the other form, whereas single-step PCR was able to distinguish just 10% unmethylated or 33% methylated DNA under the same conditions. Furthermore, no PCR product contamination was observed upon amplification of samples collected from the external portion of the device demonstrating the PCR products were well contained. Citation Format: Wanyuan Ao, Derek Bosh, Dale Emery, Robert Parry, Nils Adey. Comparison of single step to two step PCR for DNA methylation status determination utilizing a system that continuously contains PCR products in plastic consumables [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 832.

Studying DNA Methylation in Single-Cell Format with scBS-seq.

DNA methylation at cytosine is a major epigenetic mark, heavily implicated in controlling key cellular processes such as development and differentiation, cellular memory, or carcinogenesis. Bisulfite treatment in conjunction with next generation sequencing has been a powerful tool for studying this modification in a quantitative manner in the context of the whole genome and with a single nucleotide resolution. This chapter describes a protocol for bisulfite sequencing adapted to a single-cell format that allows for capturing the methylation signal from up to 50% CpG nucleotides in each cell.

Non-CpG Methylation Revised

Textbook and scientific papers addressing DNA methylation usually still cite “DNA methylation occurs at CpG cytosines”. Methylation at cytosines outside the CpG nucleotide, the so-called “non-CpG methylation”, is usually considered a minor and not biologically relevant process. However, the technical improvements and additional studies in epigenetics have demonstrated that non-CpG methylation is present with frequency higher than previously thought and retains biological activity, potentially relevant to the understanding and the treatment of human diseases.

Big data analysis of human mitochondrial DNA substitution models: a regression approach

BackgroundWe study Phylotree, a comprehensive representation of the phylogeny of global human mitochondrial DNA (mtDNA) variations, to better understand the mtDNA substitution mechanism and its most influential factors. We consider a substitution model, where a set of genetic features may predict the rate at which mtDNA substitutions occur. To find an appropriate model, an exhaustive analysis on the effect of multiple factors on the substitution rate is performed through Negative Binomial and Poisson regressions. We examine three different inclusion options for each categorical factor: omission, inclusion as an explanatory variable, and by-value partitioning. The examined factors include genes, codon position, a CpG indicator, directionality, nucleotide, amino acid, codon, and context (neighboring nucleotides), in addition to other site based factors. Partitioning a model by a factor’s value results in several sub-models (one for each value), where the likelihoods of the sub-models can be combined to form a score for the entire model. Eventually, the leading models are considered as viable candidates for explaining mtDNA substitution rates.ResultsInitially, we introduce a novel clustering technique on genes, based on three similarity tests between pairs of genes, supporting previous results regarding gene functionalities in the mtDNA. These clusters are then used as a factor in our models.We present leading models for the protein coding genes, rRNA and tRNA genes and the control region, showing it is disadvantageous to separate the models of transitions/transversions, or synonymous/non-synonymous substitutions. We identify a context effect that cannot be attributed solely to protein level constraints or CpG pairs.For protein-coding genes, we show that the substitution model should be partitioned into sub-models according to the codon position and input codon; additionally we confirm that gene identity and cluster have no significant effect once the above factors are accounted for.ConclusionsWe leverage the large, high-confidence Phylotree mtDNA phylogeny to develop a new statistical approach. We model the substitution rates using regressions, allowing consideration of many factors simultaneously. This admits the use of model selection tools helping to identify the set of factors best explaining the mutational dynamics when considered in tandem.

Methylation status of KLF4 and HS3ST2 genes as predictors of endometrial cancer and hyperplastic endometrial lesions.

Endometrial carcinoma is one of the most common tumours in developed countries. In addition to the active role of genetic factors, epigenetic changes also have an important effect. The present study analysed the methylation status of kruppel like factor 4 (KLF4) and heparan sulfate-glucosamine 3-sulfotransferase 2 (HS3ST2) genes in three endometrial tissue types for carcinoma prediction. The sample comprised 91 women with histologically-confirmed endometrial carcinoma (64.16±9.64 years old), 36 women with hyperplasia (53.39±9.64 years old) and 45 with no signs or symptoms of malignancy (48.53±11.11 years old). The CpG dinucleotide methylation levels were examined by quantitative pyrosequencing, and the discrimination accuracy of the model was calculated using the Random Forest classification algorithm of the area under the ROC curve (AUC). The mean values of KLF4 and HS3ST2 methylation indices were 23.83±11.39 and 8.52±2.57 in the control samples; 30.40±8.52 and 33.76±20.66 in hyperplasia and 34.72±10.79 and 34.49±18.39 in the cancerous tissues. Multinomial logistic regression indicated that the HS3ST2 CpG1 methylation status is a predictor of hyperplasia (P<0.05) and that the KLF4 CpG2 dinucleotide can predict carcinoma formation (P<0.001). The AUC value of 0.95 indicates high discrimination accuracy of the CpG nucleotides methylation status model between the controls and the two other diagnoses. The results of the present study establish the likelihood that aberrations in KLF4 and HS3ST2 gene methylation levels are important in the development of endometrial hyperplasia and carcinoma, with hyperplasia an intermediate step between healthy and tumour tissues.

Abstract P1-08-11: UHRF1 promotes breast cancer progression via suppressing KLF17 expression

Abstract Background: UHRF1, also termed as ICBP90 or Np95, is reported to be an epigenetic regulator of DNA methylation and histone modifications, which takes pivotal functions in cell differentiation and tumorigenesis. Here, we show that UHRF1 is markedly increased in breast cancer tissues and cell lines. And patients with high UHRF1 expression have a poor prognosis compare with those in low UHRF1 expression as showed by Kaplan Meier plotter analysis. However, the underlying mechanisms of UHRF1 in breast cancer remain largely unknown. Thus, the aim of this study is to validate potential mechanisms of UHRF1 in breast cancer cells. Methods: The stable cell lines were constructed using interference and overexpression methods and the efficiency was confirmed by western blot. Then cell proliferation and migration were evaluated by CCK8 and transwell assay. The MDA-MB-231 pCDH and pCDH-UHRF1 cells were inoculate into mouse left flank to further testify UHRF1 function in vivo. Furthermore, microarray and ChIP-seq were implemented to detect potential mechanisms and the candidate genes were assessed by ChIP and qPCR. The BSP or MSP primers were designed by the methprimer database. Genomic DNA was prepared from breast cancer cells and then the genomic DNA was bisulfite modified using EZ DNA Methylation-Gold Kit. The methylation PCR products were cloned into pMD-18T and ten positive clones were sequenced. The data were analyzed using the QUMA analyzer software. Results: We found that UHRF1 was indeed increased in breast cancer tissues and cells. And patients with high levels of UHRF1 were likely to have a shorter disease-free survival than patients with low levels. UHRF1 overexpression can promote cell proliferation and migration while UHRF1 downregulation have inverse functions. In vivo, UHRF1 also accelerate tumor growth on mice. Mechanistic studies revealed that 9 candidate genes were obtained by analysis from ChIP-seq and microarray databases. The further verification showed that Krüppel-like factor 17 (KLF17) is the most significantly upregulated one when UHRF1 gene was depleted, with rich CpG islands on its promoter region. We also observed that an inverse relationship between the expression of UHRF1 and KLF17 both in breast cancer cell lines and tissues.What's more, cells proliferation and migration which were inhibited by UHRF1-depletion can be rescued by KLF17 silence, suggesting UHRF1 promote breast cancer proliferation and migration through downregulating KLF17 expression. Moreover, overexpression of UHRF1 increases the methylation of CpG nucleotides and reduces the expression of KLF17 while depletion of UHRF1 decreases the methylation of CpG nucleotides with elevating expression of KLF17. Conclusion: Our results demonstrate that increased UHRF1 can promote breast cancer cell proliferation and migration by epigenetic silencing of KLF17 expression through CpG islands methylation in its promoter region. These results provide insight into the breast cancer progression process and suggest that making changes in this mechanism represent new therapeutic approaches to block breast cancer development. Citation Format: Gao S-P, Sun H-F, Li L-D, Jin W. UHRF1 promotes breast cancer progression via suppressing KLF17 expression [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-08-11.

Functional Toll-Like Receptor 9 Expression and CXCR3 Ligand Release in Pulmonary Sarcoidosis.

Sarcoidosis is a granulomatous disease characterized by a T-helper type 1 (Th1) cell-dominated alveolitis. As a role of bacteria in the pathogenesis of sarcoidosis has been discussed, Toll-like receptors (TLRs) may be involved in the initiation of a first immune reaction. We analyzed expression and functional relevance of several TLRs in bronchoalveolar lavage (BAL) cells from patients with pulmonary sarcoidosis. In parallel, we determined the release of C-X-C motif chemokine 9 (CXCL9), CXCL10, and CXCL11 by BAL cells from patients with pulmonary sarcoidosis. Nucleotide-binding oligomerization domain-containing protein (NOD) 1 and 2, TLR2, TLR6, and TLR9 expression by BAL cells was analyzed by real-time RT-PCR and cell surface expression by flow cytometry. Chemokine release was measured in BAL cell culture supernatants by ELISA. We found increased TLR9 mRNA expression in patients with sarcoidosis with chest X-ray type I and II and TLR9 protein expression in BAL cells from patients with chest X-ray type II and III. Stimulation with CpG nucleotides increased CXCL10 release by BAL cells from patients with sarcoidosis type II significantly compared with control subjects or other patients with sarcoidosis. In contrast, no increase in TNF, IL-12p40, or CXCL8 was detected. Spontaneous release of CXCL10, but not CXCL9 or CXCL11, by cultured BAL cells was also highest in cells from patients with chest X-ray type II. We found a significant association between TLR9 expression and CD4+ lymphocytes in BAL. Our data demonstrate that TLR9 ligands may contribute to the immunopathogenesis of sarcoidosis via induction of CXCL10 release in the alveolar macrophages.

Genome-Wide Estimates of Mutation Rates and Spectrum in Schizosaccharomyces pombe Indicate CpG Sites are Highly Mutagenic Despite the Absence of DNA Methylation.

We accumulated mutations for 1952 generations in 79 initially identical, haploid lines of the fission yeast Schizosaccharomyces pombe, and then performed whole-genome sequencing to determine the mutation rates and spectrum. We captured 696 spontaneous mutations across the 79 mutation accumulation (MA) lines. We compared the mutation spectrum and rate to a recently published equivalent experiment on the same species, and to another model ascomycetous yeast, the budding yeast Saccharomyces cerevisiae. While the two species are approximately 600 million years diverged from each other, they share similar life histories, genome size and genomic G/C content. We found that Sc. pombe and S. cerevisiae have similar mutation rates, but Sc. pombe exhibits a stronger insertion bias. Intriguingly, we observed an increased mutation rate at cytosine nucleotides, specifically CpG nucleotides, which is also seen in S. cerevisiae. However, the absence of methylation in Sc. pombe and the pattern of mutation at these sites, primarily C → A as opposed to C → T, strongly suggest that the increased mutation rate is not caused by deamination of methylated cytosines. This result implies that the high mutability of CpG dinucleotides in other species may be caused in part by a methylation-independent mechanism. Many of our findings mirror those seen in the recent study, despite the use of different passaging conditions, indicating that MA is a reliable method for estimating mutation rates and spectra.

Epigenetic repression of regulator of G‐protein signaling 2 by ubiquitin‐like with PHD and ring‐finger domain 1 promotes bladder cancer progression

Ubiquitin-like with PHD and ring-finger domain1 (UHRF1) binds to methylated promoters of tumor-suppressor genes and suppresses gene expression by forming complexes with DNA methyltransferases. Recent studies have shown that repression of regulator of G-protein signaling (RGS) 2 increases cancer cell growth. However, little is known about whether UHRF1 promotes bladder cancer progression by epigenetic silencing of RGS2. Here, we show that UHRF1 expression is increased in bladder cancer cell lines and in most bladder cancer tissues as compared with normal controls. UHRF1 overexpression increases bladder cancer cell proliferation, whereas inhibition of UHRF1 suppresses cell proliferation. In bladder cancer cells, UHRF1 inhibits RGS2 expression by increasing the methylation of CpG nucleotides of the RGS2 promoter. DNA methylation analysis showed tumor-specific TGS2 promoter methylation in 73% (38/52) of bladder tumors. High UHRF1 expression of correlated with aberrant TGS2 promoter methylation in bladder tumors, which results in the loss of TGS2 expression, as confirmed by demethylation analysis in cell lines. Functionally, re-expression of RGS2 partly abrogates UHRF1-induced bladder cell proliferation. Furthermore, Kaplan-Meier analysis showed that low TGS2 expression is significantly correlated with reduced overall survival in patients with bladder cancer. These results demonstrate that epigenetic repression of RGS2 by UHRF1 contributes to bladder cancer progression.