DNA-Demethylating Agents Target Colorectal Cancer Cells by Inducing Viral Mimicry by Endogenous Transcripts.

DNA-demethylating agents have shown clinical anti-tumor efficacy via an unknown mechanism of action. Using a combination of experimental and bioinformatics analyses in colorectal cancer cells, we demonstrate that low-dose 5-AZA-CdR targets colorectal cancer-initiating cells (CICs) by inducing viral mimicry. This is associated with induction of dsRNAs derived at least in part from endogenous retroviral elements, activation of the MDA5/MAVS RNA recognition pathway, and downstream activation of IRF7. Indeed, disruption of virus recognition pathways, by individually knocking down MDA5, MAVS, or IRF7, inhibits the ability of 5-AZA-CdR to target colorectal CICs and significantly decreases 5-AZA-CdR long-term growth effects. Moreover, transfection of dsRNA into CICs can mimic the effects of 5-AZA-CdR. Together, our results represent a major shift in understanding the anti-tumor mechanisms of DNA-demethylating agents and highlight the MDA5/MAVS/IRF7 pathway as a potentially druggable target against CICs.

Polycomb-repressed genes have permissive enhancers that initiate reprogramming.

Key regulatory genes, suppressed by Polycomb and H3K27me3, become active during normal differentiation and induced reprogramming. Using the well-characterized enhancer/promoter pair of MYOD1 as a model, we have identified a critical role for enhancers in reprogramming. We observed an unexpected nucleosome-depleted region (NDR) at the H3K4me1-enriched enhancer at which transcriptional regulators initially bind, leading to subsequent changes in the chromatin at the cognate promoter. Exogenous Myod1 activates its own transcription by binding first at the enhancer, leading to an NDR and transcription-permissive chromatin at the associated MYOD1 promoter. Exogenous OCT4 also binds first to the permissive MYOD1 enhancer but has a different effect on the cognate promoter, where the monovalent H3K27me3 marks are converted to the bivalent state characteristic of stem cells. Genome-wide, a high percentage of Polycomb targets are associated with putative enhancers in permissive states, suggesting that they may provide a widespread avenue for the initiation of cell-fate reprogramming.

Structure of nucleosome-bound DNA methyltransferases DNMT3A and DNMT3B.

CpG methylation by de novo DNA methyltransferases (DNMTs) 3A and 3B is essential for mammalian development and differentiation and is frequently dysregulated in cancer1. These two DNMTs preferentially bind to nucleosomes, yet cannot methylate the DNA wrapped around the nucleosome core2, and they favour the methylation of linker DNA at positioned nucleosomes3,4. Here we present the cryo-electron microscopy structure of a ternary complex of catalytically competent DNMT3A2, the catalytically inactive accessory subunit DNMT3B3 and a nucleosome core particle flanked by linker DNA. The catalytic-like domain of the accessory DNMT3B3 binds to the acidic patch of the nucleosome core, which orients the binding of DNMT3A2 to the linker DNA. The steric constraints of this arrangement suggest that nucleosomal DNA must be moved relative to the nucleosome core for de novo methylation to occur.

Efficient activation of hundreds of LTR12C elements reveals cis-regulatory function determined by distinct epigenetic mechanisms.

Long terminal repeats (LTRs), which often contain promoter and enhancer sequences of intact endogenous retroviruses (ERVs), are known to be co-opted as cis-regulatory elements for fine-tuning host-coding gene expression. Since LTRs are mainly silenced by the deposition of repressive epigenetic marks, substantial activation of LTRs has been found in human cells after treatment with epigenetic inhibitors. Although the LTR12C family makes up the majority of ERVs activated by epigenetic inhibitors, how these epigenetically and transcriptionally activated LTR12C elements can regulate the host-coding gene expression remains unclear due to genome-wide alteration of transcriptional changes after epigenetic inhibitor treatments. Here, we specifically transactivated >600 LTR12C elements by using single guide RNA-based dCas9-SunTag-VP64, a site-specific targeting CRISPR activation (CRISPRa) system, with minimal off-target events. Interestingly, most of the transactivated LTR12C elements acquired the H3K27ac-marked enhancer feature, while only 20% were co-marked with promoter-associated H3K4me3 modifications. The enrichment of the H3K4me3 signal was intricately associated with downstream regions of LTR12C, such as internal regions of intact ERV9 or other types of retrotransposons. Here, we leverage an optimized CRISPRa system to identify two distinct epigenetic signatures that define LTR12C transcriptional activation, which modulate the expression of proximal protein-coding genes.

DNA strand asymmetry generated by CpG hemimethylation has opposing effects on CTCF binding.

CpG methylation generally occurs on both DNA strands and is essential for mammalian development and differentiation. Until recently, hemimethylation, in which only one strand is methylated, was considered to be simply a transitory state generated during DNA synthesis. The discovery that a subset of CCCTC-binding factor (CTCF) binding sites is heritably hemimethylated suggests that hemimethylation might have an unknown biological function. Here we show that the binding of CTCF is profoundly altered by which DNA strand is methylated and by the specific CTCF binding motif. CpG methylation on the motif strand can inhibit CTCF binding by up to 7-fold, whereas methylation on the opposite strand can stimulate binding by up to 4-fold. Thus, hemimethylation can alter binding by up to 28-fold in a strand-specific manner. The mechanism for sensing methylation on the opposite strand requires two critical residues, V454 and S364, within CTCF zinc fingers 7 and 4. Similar to methylation, CpG hydroxymethylation on the motif strand can inhibit CTCF binding by up to 4-fold. However, hydroxymethylation on the opposite strand removes the stimulatory effect. Strand-specific methylation states may therefore provide a mechanism to explain the transient and dynamic nature of CTCF-mediated chromatin interactions.

Bivalent Regions of Cytosine Methylation and H3K27 Acetylation Suggest an Active Role for DNA Methylation at Enhancers.

The role of cytosine methylation in the structure and function of enhancers is not well understood. In this study, we investigate the role of DNA methylation at enhancers by comparing the epigenomes of the HCT116 cell line and its highly demethylated derivative, DKO1. Unlike promoters, a portion of regular and super- or stretch enhancers show active H3K27ac marks co-existing with extensive DNA methylation, demonstrating the unexpected presence of bivalent chromatin in both cultured and uncultured cells. Furthermore, our findings also show that bivalent regions have fewer nucleosome-depleted regions and transcription factor-binding sites than monovalent regions. Reduction of DNA methylation genetically or pharmacologically leads to a decrease of the H3K27ac mark. Thus, DNA methylation plays an unexpected dual role at enhancer regions, being anti-correlated focally at transcription factor-binding sites but positively correlated globally with the active H3K27ac mark to ensure structural enhancer integrity.

A Urine-based DNA Methylation Marker Test to Detect Upper Tract Urothelial Carcinoma: A Prospective Cohort Study.

PURPOSE: We explored the accuracy of a urine-based epigenetic test for detecting upper tract urothelial carcinoma. MATERIALS AND METHODS: Under an Institutional Review Board-approved protocol, urine samples were prospectively collected from primary upper tract urothelial carcinoma patients before radical nephroureterectomy, ureterectomy, or ureteroscopy between December 2019 and March 2022. Samples were analyzed with Bladder CARE, a urine-based test that measures the methylation levels of 3 cancer biomarkers (TRNA-Cys, SIM2, and NKX1-1) and 2 internal control loci using methylation-sensitive restriction enzymes coupled with quantitative polymerase chain reaction. Results were reported as the Bladder CARE Index score and quantitatively categorized as positive (>5), high risk (2.5-5), or negative (<2.5). The findings were compared with those of 1:1 sex/age-matched cancer-free healthy individuals. RESULTS: Fifty patients (40 radical nephroureterectomy, 7 ureterectomy, and 3 ureteroscopy) with a median (IQR) age of 72 (64-79) years were included. Bladder CARE Index results were positive in 47, high risk in 1, and negative in 2 patients. A significant correlation was found between Bladder CARE Index values and tumor size. Urine cytology was available for 35 patients, of whom 22 (63%) results were false-negative. Upper tract urothelial carcinoma patients had significantly higher Bladder CARE Index values compared to the controls (mean 189.3 vs 1.6, P < .001). The sensitivity, specificity, positive predictive value, and negative predictive value of the Bladder CARE test for detecting upper tract urothelial carcinoma were 96%, 88%, 89%, and 96%, respectively.Conclusions:Bladder CARE is an accurate urine-based epigenetic test for the diagnosis of upper tract urothelial carcinoma, with much higher sensitivity than standard urine cytology.

DNA methylation enables transposable element-driven genome expansion.

Multicellular eukaryotic genomes show enormous differences in size. A substantial part of this variation is due to the presence of transposable elements (TEs). They contribute significantly to a cell's mass of DNA and have the potential to become involved in host gene control. We argue that the suppression of their activities by methylation of the C-phosphate-G (CpG) dinucleotide in DNA is essential for their long-term accommodation in the host genome and, therefore, to its expansion. An inevitable consequence of cytosine methylation is an increase in C-to-T transition mutations via deamination, which causes CpG loss. Cytosine deamination is often needed for TEs to take on regulatory functions in the host genome. Our study of the whole-genome sequences of 53 organisms showed a positive correlation between the size of a genome and the percentage of TEs it contains, as well as a negative correlation between size and the CpG observed/expected (O/E) ratio in both TEs and the host DNA. TEs are seldom found at promoters and transcription start sites, but they are found more at enhancers, particularly after they have accumulated C-to-T and other mutations. Therefore, the methylation of TE DNA allows for genome expansion and also leads to new opportunities for gene control by TE-based regulatory sites.

Diagnostic Aqueous Humor Proteome Predicts Metastatic Potential in Uveal Melanoma.

Gene expression profiling (GEP) is clinically validated to stratify the risk of metastasis by assigning uveal melanoma (UM) patients to two highly prognostic molecular classes: class 1 (low metastatic risk) and class 2 (high metastatic risk). However, GEP requires intraocular tumor biopsy, which is limited by small tumor size and tumor heterogeneity; furthermore, there are small risks of retinal hemorrhage, bleeding, or tumor dissemination. Thus, ocular liquid biopsy has emerged as a less-invasive alternative. In this study, we seek to determine the aqueous humor (AH) proteome related to the advanced GEP class 2 using diagnostic AH liquid biopsy specimens. Twenty AH samples were collected from patients with UM, grouped by GEP classes. Protein expression levels of 1472 targets were analyzed, compared between GEP classes, and correlated with clinical features. Significant differentially expressed proteins (DEPs) were subjected to analysis for cellular pathway and upstream regulator identification. The results showed that 45 DEPs detected in the AH could differentiate GEP class 1 and 2 at diagnosis. IL1R and SPRY2 are potential upstream regulators for the 8/45 DEPs that contribute to metastasis-related pathways. AH liquid biopsy offers a new opportunity to determine metastatic potential for patients in the absence of tumor biopsy.

KRT13 promotes stemness and drives metastasis in breast cancer through a plakoglobin/c-Myc signaling pathway.

BACKGROUND: Keratins (KRTs) are intermediate filament proteins that interact with multiple regulatory proteins to initiate signaling cascades. Keratin 13 (KRT13) plays an important role in breast cancer progression and metastasis. The objective of this study is to elucidate the mechanism by which KRT13 promotes breast cancer growth and metastasis. METHODS: The function and mechanisms of KRT13 in breast cancer progression and metastasis were assessed by overexpression and knockdown followed by examination of altered behaviors in breast cancer cells and in xenograft tumor formation in mouse mammary fat pad. Human breast cancer specimens were examined by immunohistochemistry and multiplexed quantum dot labeling analysis to correlate KRT13 expression to breast cancer progression and metastasis. RESULTS: KRT13-overexpressing MCF7 cells displayed increased proliferation, invasion, migration and in vivo tumor growth and metastasis to bone and lung. Conversely, KRT13 knockdown inhibited the aggressive behaviors of HCC1954 cells. At the molecular level, KRT13 directly interacted with plakoglobin (PG, γ-catenin) to form complexes with desmoplakin (DSP). This complex interfered with PG expression and nuclear translocation and abrogated PG-mediated suppression of c-Myc expression, while the KRT13/PG/c-Myc signaling pathway increased epithelial to mesenchymal transition and stem cell-like phenotype. KRT13 expression in 58 human breast cancer tissues was up-regulated especially at the invasive front and in metastatic specimens (12/18) (p < 0.05). KRT13 up-regulation in primary breast cancer was associated with decreased overall patient survival. CONCLUSIONS: This study reveals that KRT13 promotes breast cancer cell growth and metastasis via a plakoglobin/c-Myc pathway. Our findings reveal a potential novel pathway for therapeutic targeting of breast cancer progression and metastasis.