Targeting OCT3 attenuates doxorubicin-induced cardiac injury.

Doxorubicin is a commonly used anticancer agent that can cause debilitating and irreversible cardiac injury. The initiating mechanisms contributing to this side effect remain unknown, and current preventative strategies offer only modest protection. Using stem-cell-derived cardiomyocytes from patients receiving doxorubicin, we probed the transcriptomic landscape of solute carriers and identified organic cation transporter 3 (OCT3) (SLC22A3) as a critical transporter regulating the cardiac accumulation of doxorubicin. Functional validation studies in heterologous overexpression models confirmed that doxorubicin is transported into cardiomyocytes by OCT3 and that deficiency of OCT3 protected mice from acute and chronic doxorubicin-related changes in cardiovascular function and genetic pathways associated with cardiac damage. To provide proof-of-principle and demonstrate translational relevance of this transport mechanism, we identified several pharmacological inhibitors of OCT3, including nilotinib, and found that pharmacological targeting of OCT3 can also preserve cardiovascular function following treatment with doxorubicin without affecting its plasma levels or antitumor effects in multiple models of leukemia and breast cancer. Finally, we identified a previously unrecognized, OCT3-dependent pathway of doxorubicin-induced cardiotoxicity that results in a downstream signaling cascade involving the calcium-binding proteins S100A8 and S100A9. These collective findings not only shed light on the etiology of doxorubicin-induced cardiotoxicity, but also are of potential translational relevance and provide a rationale for the implementation of a targeted intervention strategy to prevent this debilitating side effect.

ROR1-targeted delivery of miR-29b induces cell cycle arrest and therapeutic benefit in vivo in a CLL mouse model.

Chronic lymphocytic leukemia (CLL) occurs in 2 major forms: aggressive and indolent. Low miR-29b expression in aggressive CLL is associated with poor prognosis. Indiscriminate miR-29b overexpression in the B-lineage of mice causes aberrance, thus warranting the need for selective introduction of miR-29b into B-CLL cells for therapeutic benefit. The oncofetal antigen receptor tyrosine kinase orphan receptor 1 (ROR1) is expressed on malignant B-CLL cells, but not normal B cells, encouraging us with ROR1-targeted delivery for therapeutic miRs. Here, we describe targeted delivery of miR-29b to ROR1+ CLL cells leading to downregulation of DNMT1 and DNMT3A, modulation of global DNA methylation, decreased SP1, and increased p21 expression in cell lines and primary CLL cells in vitro. Furthermore, using an Eµ-TCL1 mouse model expressing human ROR1, we report the therapeutic benefit of enhanced survival via cellular reprograming by downregulation of DNMT1 and DNMT3A in vivo. Gene expression profiling of engrafted murine leukemia identified reprogramming of cell cycle regulators with decreased SP1 and increased p21 expression after targeted miR-29b treatment. This finding was confirmed by protein modulation, leading to cell cycle arrest and survival benefit in vivo. Importantly, SP1 knockdown results in p21-dependent compensation of the miR-29b effect on cell cycle arrest. These studies form a basis for leukemic cell-targeted delivery of miR-29b as a promising therapeutic approach for CLL and other ROR1+ B-cell malignancies.

Diverse AR-V7 cistromes in castration-resistant prostate cancer are governed by HoxB13.

The constitutively active androgen receptor (AR) splice variant 7 (AR-V7) plays an important role in the progression of castration-resistant prostate cancer (CRPC). Although biomarker studies established the role of AR-V7 in resistance to AR-targeting therapies, how AR-V7 mediates genomic functions in CRPC remains largely unknown. Using a ChIP-exo approach, we show AR-V7 binds to distinct genomic regions and recognizes a full-length androgen-responsive element in CRPC cells and patient tissues. Remarkably, we find dramatic differences in AR-V7 cistromes across diverse CRPC cells and patient tissues, regulating different target gene sets involved in CRPC progression. Surprisingly, we discover that HoxB13 is universally required for and colocalizes with AR-V7 binding to open chromatin across CRPC genomes. HoxB13 pioneers AR-V7 binding through direct physical interaction, and collaborates with AR-V7 to up-regulate target oncogenes. Transcriptional coregulation by HoxB13 and AR-V7 was further supported by their coexpression in tumors and circulating tumor cells from CRPC patients. Importantly, HoxB13 silencing significantly decreases CRPC growth through inhibition of AR-V7 oncogenic function. These results identify HoxB13 as a pivotal upstream regulator of AR-V7-driven transcriptomes that are often cell context-dependent in CRPC, suggesting that HoxB13 may serve as a therapeutic target for AR-V7-driven prostate tumors.

TET1 reprograms the epithelial ovarian cancer epigenome and reveals casein kinase 2α as a therapeutic target.

Ten-eleven translocation methylcytosine dioxygenase-1, TET1, takes part in active DNA demethylation. However, our understanding of DNA demethylation in cancer biology and its clinical significance remain limited. This study showed that TET1 expression correlated with poor survival in advanced-stage epithelial ovarian carcinoma (EOC), and with cell migration, anchorage-independent growth, cancer stemness, and tumorigenicity. In particular, TET1 was highly expressed in serous tubal intraepithelial carcinoma (STIC), a currently accepted type II EOC precursor, and inversely correlated with TP53 mutations. Moreover, TET1 could demethylate the epigenome and activate multiple oncogenic pathways, including an immunomodulation network having casein kinase II subunit alpha (CK2α) as a hub. Patients with TET1high CK2αhigh EOCs had the worst outcomes, and TET1-expressing EOCs were more sensitive to a CK2 inhibitor, both in vitro and in vivo. Our findings uncover the oncogenic and poor prognostic roles of TET1 in EOC and suggest an unexplored role of epigenetic reprogramming in early ovarian carcinogenesis. Moreover, the immunomodulator CK2α represents a promising new therapeutic target, warranting clinical trials of the tolerable CK2 inhibitor, CX4945, for precision medicine against EOC. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Agonist and antagonist switch DNA motifs recognized by human androgen receptor in prostate cancer.

Human transcription factors recognize specific DNA sequence motifs to regulate transcription. It is unknown whether a single transcription factor is able to bind to distinctly different motifs on chromatin, and if so, what determines the usage of specific motifs. By using a motif-resolution chromatin immunoprecipitation-exonuclease (ChIP-exo) approach, we find that agonist-liganded human androgen receptor (AR) and antagonist-liganded AR bind to two distinctly different motifs, leading to distinct transcriptional outcomes in prostate cancer cells. Further analysis on clinical prostate tissues reveals that the binding of AR to these two distinct motifs is involved in prostate carcinogenesis. Together, these results suggest that unique ligands may switch DNA motifs recognized by ligand-dependent transcription factors in vivo. Our findings also provide a broad mechanistic foundation for understanding ligand-specific induction of gene expression profiles.

FuSpot: a web-based tool for visual evaluation of fusion candidates.

BACKGROUND: Gene fusions often occur in cancer cells and in some cases are the main driver of oncogenesis. Correct identification of oncogenic gene fusions thus has implications for targeted cancer therapy. Recognition of this potential has led to the development of a myriad of sequencing-based fusion detection tools. However, given the same input, many of these detectors will find different fusion points or claim different sets of supporting data. Furthermore, the rate at which these tools falsely detect fusion events in data varies greatly. This discrepancy between tools underscores the fact that computation algorithms still cannot perfectly evaluate evidence; especially when provided with small amounts of supporting data as is typical in fusion detection. We assert that when evidence is provided in an easily digestible form, humans are more proficient in identifying true positives from false positives. RESULTS: We have developed a web tool that, given the genomic coordinates of a candidate fusion breakpoint, will extract fusion and non-fusion reads adjacent to the fusion point from partner transcripts, and color code reads by transcript origin and read orientation for ease of intuitive inspection by the user. Fusion partner transcript read alignments are performed using a novel variant of the Smith-Waterman algorithm. CONCLUSIONS: Combined with dynamic filtering parameters, the visualization provided by our tool introduces a powerful new investigative step that allows researchers to comprehensively evaluate fusion evidence. Additionally, this allows quick identification of false positives that may deceive most fusion detectors, thus eliminating unnecessary gene fusion validation. We apply our visualization tool to publicly available datasets and provide examples of true as well as false positives reported by open source fusion detection tools.

Expression and prognostic impact of lncRNAs in acute myeloid leukemia.

Long noncoding RNAs (lncRNAs) are transcripts longer than 200 nucleotides, located within the intergenic stretches or overlapping antisense transcripts of protein coding genes. LncRNAs are involved in numerous biological roles including imprinting, epigenetic regulation, apoptosis, and cell cycle. To determine whether lncRNAs are associated with clinical features and recurrent mutations in older patients (aged ≥60 y) with cytogenetically normal (CN) acute myeloid leukemia (AML), we evaluated lncRNA expression in 148 untreated older CN-AML cases using a custom microarray platform. An independent set of 71 untreated older patients with CN-AML was used to validate the outcome scores using RNA sequencing. Distinctive lncRNA profiles were found associated with selected mutations, such as internal tandem duplications in the FLT3 gene (FLT3-ITD) and mutations in the NPM1, CEBPA, IDH2, ASXL1, and RUNX1 genes. Using the lncRNAs most associated with event-free survival in a training cohort of 148 older patients with CN-AML, we derived a lncRNA score composed of 48 lncRNAs. Patients with an unfavorable compared with favorable lncRNA score had a lower complete response (CR) rate [P < 0.001, odds ratio = 0.14, 54% vs. 89%], shorter disease-free survival (DFS) [P < 0.001, hazard ratio (HR) = 2.88] and overall survival (OS) (P < 0.001, HR = 2.95). The validation set analyses confirmed these results (CR, P = 0.03; DFS, P = 0.009; OS, P = 0.009). Multivariable analyses for CR, DFS, and OS identified the lncRNA score as an independent marker for outcome. In conclusion, lncRNA expression in AML is closely associated with recurrent mutations. A small subset of lncRNAs is correlated strongly with treatment response and survival.

Tcl1 protein functions as an inhibitor of de novo DNA methylation in B-cell chronic lymphocytic leukemia (CLL).

B-cell chronic lymphocytic leukemia (CLL) is the most common human leukemia. Deregulation of the T-cell leukemia/lymphoma 1 oncogene (TCL1) in mouse B cells causes a CD5(+) leukemia similar to aggressive human CLL. To examine the mechanisms by which Tcl1 protein exerts its oncogenic activity in B cells, we performed proteomics experiments to identify its interacting partners. We found that Tcl1 physically interacts with de novo DNA methylthansferases Dnmt3A and Dnmt3B. We further investigated the effects of Tcl1 up-regulation on the enzymatic activity of Dnmt3A and found that Tcl1 overexpression drastically inhibits Dnmt3A function. In addition, B cells from TCL1 transgenic mice showed a significant decrease in DNA methylation compared with WT controls. Similarly, CLL samples with high Tcl1 expression showed a decrease in DNA methylation compared with CLL samples with low Tcl1 expression. Given the previous reports of inactivating mutations of DNMT3A in acute myelogenous leukemia and myelodysplastic syndrome, our results suggest that inhibition of de novo DNA methylation may be a common oncogenic mechanism in leukemogenesis.

A new method for stranded whole transcriptome RNA-seq.

This report describes an improved protocol to generate stranded, barcoded RNA-seq libraries to capture the whole transcriptome. By optimizing the use of duplex specific nuclease (DSN) to remove ribosomal RNA reads from stranded barcoded libraries, we demonstrate improved efficiency of multiplexed next generation sequencing (NGS). This approach detects expression profiles of all RNA types, including miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (long non-coding RNA), mtRNA (mitochondrial RNA) and mRNA (messenger RNA) without the use of gel electrophoresis. The improved protocol generates high quality data that can be used to identify differential expression in known and novel coding and non-coding transcripts, splice variants, mitochondrial genes and SNPs (single nucleotide polymorphisms).

Estrogen-mediated epigenetic repression of large chromosomal regions through DNA looping.

The current concept of epigenetic repression is based on one repressor unit corresponding to one silent gene. This notion, however, cannot adequately explain concurrent silencing of multiple loci observed in large chromosome regions. The long-range epigenetic silencing (LRES) can be a frequent occurrence throughout the human genome. To comprehensively characterize the influence of estrogen signaling on LRES, we analyzed transcriptome, methylome, and estrogen receptor alpha (ESR1)-binding datasets from normal breast epithelia and breast cancer cells. This "omics" approach uncovered 11 large repressive zones (range, 0.35 approximately 5.98 megabases), including a 14-gene cluster located on 16p11.2. In normal cells, estrogen signaling induced transient formation of multiple DNA loops in the 16p11.2 region by bringing 14 distant loci to focal ESR1-docking sites for coordinate repression. However, the plasticity of this free DNA movement was reduced in breast cancer cells. Together with the acquisition of DNA methylation and repressive chromatin modifications at the 16p11.2 loci, an inflexible DNA scaffold may be a novel determinant used by breast cancer cells to reinforce estrogen-mediated repression.

Dietary omega-3 fatty acid intake impacts peripheral blood DNA methylation -anti-inflammatory effects and individual variability in a pilot study.

Omega-3 or n-3 polyunsaturated fatty acids (PUFAs) are widely studied for health benefits that may relate to anti-inflammatory activity. However, mechanisms mediating an anti-inflammatory response to n-3 PUFA intake are not fully understood. Of interest is the emerging role of fatty acids to impact DNA methylation (DNAm) and thereby modulate mediating inflammatory processes. In this pilot study, we investigated the impact of n-3 PUFA intake on DNAm in inflammation-related signaling pathways in peripheral blood mononuclear cells (PBMCs) of women at high risk of breast cancer. PBMCs of women at high risk of breast cancer (n=10) were obtained at baseline and after 6 months of n-3 PUFA (5 g/d EPA+DHA dose arm) intake in a previously reported dose finding trial. DNA methylation of PBMCs was assayed by reduced representation bisulfite sequencing (RRBS) to obtain genome-wide methylation profiles at the single nucleotide level. We examined the impact of n-3 PUFA on genome-wide DNAm and focused upon a set of candidate genes associated with inflammation signaling pathways and breast cancer. We identified 24,842 differentially methylated CpGs (DMCs) in gene promoters of 5507 genes showing significant enrichment for hypermethylation in both the candidate gene and genome-wide analyses. Pathway analysis identified significantly hypermethylated signaling networks after n-3 PUFA treatment, such as the Toll-like Receptor inflammatory pathway. The DNAm pattern in individuals and the response to n-3 PUFA intake are heterogeneous. PBMC DNAm profiling suggests a mechanism whereby n-3 PUFAs may impact inflammatory cascades associated with disease processes including carcinogenesis.

Leukemia-initiating HSCs in chronic lymphocytic leukemia reveal clonal leukemogenesis and differential drug sensitivity.

The existence of "leukemia-initiating cells" (LICs) in chronic lymphocytic leukemia (CLL) remains controversial due to the difficulty in isolating and identifying the tumor-initiating cells. Here, we demonstrate a microchannel electroporation (MEP) microarray that injects RNA-detecting probes into single live cells, allowing the imaging and characterization of heterogeneous LICs by intracellular RNA expression. Using limited-cell FACS sequencing (LC-FACSeq), we can detect and monitor rare live LICs during leukemogenesis and characterize their differential drug sensitivity. Disease-associated mutation accumulation in developing B lymphoid but not myeloid lineage in CLL patient hematopoietic stem cells (CLL-HSCs), and development of independent clonal CLL-like cells in murine patient-derived xenograft models, suggests the existence of CLL LICs. Furthermore, we identify differential protein ubiquitination and unfolding response signatures in GATA2high CLL-HSCs that exhibit increased sensitivity to lenalidomide and resistance to fludarabine compared to GATA2lowCLL-HSCs. These results highlight the existence of therapeutically targetable disease precursors in CLL.

Identifying hypermethylated CpG islands using a quantile regression model.

BACKGROUND: DNA methylation has been shown to play an important role in the silencing of tumor suppressor genes in various tumor types. In order to have a system-wide understanding of the methylation changes that occur in tumors, we have developed a differential methylation hybridization (DMH) protocol that can simultaneously assay the methylation status of all known CpG islands (CGIs) using microarray technologies. A large percentage of signals obtained from microarrays can be attributed to various measurable and unmeasurable confounding factors unrelated to the biological question at hand. In order to correct the bias due to noise, we first implemented a quantile regression model, with a quantile level equal to 75%, to identify hypermethylated CGIs in an earlier work. As a proof of concept, we applied this model to methylation microarray data generated from breast cancer cell lines. However, we were unsure whether 75% was the best quantile level for identifying hypermethylated CGIs. In this paper, we attempt to determine which quantile level should be used to identify hypermethylated CGIs and their associated genes. RESULTS: We introduce three statistical measurements to compare the performance of the proposed quantile regression model at different quantile levels (95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%), using known methylated genes and unmethylated housekeeping genes reported in breast cancer cell lines and ovarian cancer patients. Our results show that the quantile levels ranging from 80% to 90% are better at identifying known methylated and unmethylated genes. CONCLUSIONS: In this paper, we propose to use a quantile regression model to identify hypermethylated CGIs by incorporating probe effects to account for noise due to unmeasurable factors. Our model can efficiently identify hypermethylated CGIs in both breast and ovarian cancer data.

Estrogen-mediated epigenetic repression of the imprinted gene cyclin-dependent kinase inhibitor 1C in breast cancer cells.

While tumor suppressor genes frequently undergo epigenetic silencing in cancer, how the instructions directing this transcriptional repression are transmitted in cancer cells remain largely unclear. Expression of cyclin-dependent kinase inhibitor 1C (CDKN1C), an imprinted gene on chromosomal band 11 p15.5, is reduced or lost in the majority of breast cancers. Here, we report that CDKN1C is suppressed by estrogen through epigenetic mechanisms involving the chromatin-interacting noncoding RNA KCNQ1OT1 and CCCTC-binding factor (CTCF). Activation of estrogen signaling reduced CDKN1C expression 3-fold (P < 0.001) and established repressive histone modifications at the 5' regulatory region of the locus. These events were concomitant with induction of KCNQ1OT1 expression as well as increased recruitment of CTCF to both the distal KCNQ1OT1 promoter-associated imprinting control region (ICR) and the CDKN1C locus. Transient depletion of CTCF by small interfering RNA increased CDKN1C expression and significantly reduced the estrogen-mediated repression of CDKN1C. Further studies in breast cancer cell lines indicated that the epigenetic silencing of CDKN1C occurs in part as the result of genetic loss of the inactive methylated 11p15.5 ICR allele (R(2) = 0.612, P < 0.001). We also found a novel cis-encoded antisense transcript, CDKN1C-AS, which is induced by estrogen signaling following pharmacologic inhibition of DNA methyltransferase and histone deacetylase activity. Forced expression of CDKN1C-AS was capable of repressing endogenous CDKN1C in vivo. Our findings suggest that in addition to promoter hypermethylation, epigenetic repression of tumor suppressor genes by CTCF and noncoding RNA transcripts could be more common and important than previously understood.

Functional characterization of Trip10 in cancer cell growth and survival.

BACKGROUND: The Cdc42-interacting protein-4, Trip10 (also known as CIP4), is a multi-domain adaptor protein involved in diverse cellular processes, which functions in a tissue-specific and cell lineage-specific manner. We previously found that Trip10 is highly expressed in estrogen receptor-expressing (ER+) breast cancer cells. Estrogen receptor depletion reduced Trip10 expression by progressively increasing DNA methylation. We hypothesized that Trip10 functions as a tumor suppressor and may be involved in the malignancy of ER-negative (ER-) breast cancer. To test this hypothesis and evaluate whether Trip10 is epigenetically regulated by DNA methylation in other cancers, we evaluated DNA methylation of Trip10 in liver cancer, brain tumor, ovarian cancer, and breast cancer. METHODS: We applied methylation-specific polymerase chain reaction and bisulfite sequencing to determine the DNA methylation of Trip10 in various cancer cell lines and tumor specimens. We also overexpressed Trip10 to observe its effect on colony formation and in vivo tumorigenesis. RESULTS: We found that Trip10 is hypermethylated in brain tumor and breast cancer, but hypomethylated in liver cancer. Overexpressed Trip10 was associated with endogenous Cdc42 and huntingtin in IMR-32 brain tumor cells and CP70 ovarian cancer cells. However, overexpression of Trip10 promoted colony formation in IMR-32 cells and tumorigenesis in mice inoculated with IMR-32 cells, whereas overexpressed Trip10 substantially suppressed colony formation in CP70 cells and tumorigenesis in mice inoculated with CP70 cells. CONCLUSIONS: Trip10 regulates cancer cell growth and death in a cancer type-specific manner. Differential DNA methylation of Trip10 can either promote cell survival or cell death in a cell type-dependent manner.

Epigenetic Silencing of STAT3-Targeted miR-193a, by Constitutive Activation of JAK/STAT Signaling, Leads to Tumor Progression Through Overexpression of YWHAZ in Gastric Cancer.

PURPOSE: The purpose of this study was to identify genes that were epigenetically silenced by STAT3 in gastric cancer. METHODS: MBDcap-Seq and expression microarray were performed to identify genes that were epigenetically silenced in AGS gastric cancer cell lines depleted of STAT3. Cell lines and animal experiments were performed to investigate proliferation and metastasis of miR-193a and YWHAZ in gastric cancer cell lines. Bisulfite pyrosequencing and tissue microarray were performed to investigate the promoter methylation of miR-193a and expression of STAT3, YWHAZ in patients with gastritis (n = 8) and gastric cancer (n = 71). Quantitative methylation-specific PCR was performed to examine miR-193a promoter methylation in cell-free DNA of serum samples in gastric cancer patients (n = 19). RESULTS: As compared with parental cells, depletion of STAT3 resulted in demethylation of a putative STAT3 target, miR-193a, in AGS gastric cancer cells. Although bisulfite pyrosequencing and epigenetic treatment confirmed that miR-193a was epigenetically silenced in gastric cancer cell lines, ChIP-PCR found that it may be indirectly affected by STAT3. Ectopic expression of miR-193a in AGS cells inhibited proliferation and migration of gastric cancer cells. Further expression microarray and bioinformatics analysis identified YWHAZ as one of the target of miR-193a in AGS gastric cancer cells, such that depletion of YWHAZ reduced migration in AGS cells, while its overexpression increased invasion in MKN45 cells in vitro and in vivo. Clinically, bisulfite pyrosequencing revealed that promoter methylation of miR-193a was significantly higher in human gastric cancer tissues (n = 11) as compared to gastritis (n = 8, p < 0.05). Patients infected with H. pylori showed a significantly higher miR-193a methylation than those without H. pylori infection (p < 0.05). Tissue microarray also showed a positive trend between STAT3 and YWHAZ expression in gastric cancer patients (n = 60). Patients with serum miR-193a methylation was associated with shorter overall survival than those without methylation (p < 0.05). CONCLUSIONS: Constitutive activation of JAK/STAT signaling may confer epigenetic silencing of the STAT3 indirect target and tumor suppressor microRNA, miR-193a in gastric cancer. Transcriptional suppression of miR-193a may led to overexpression of YWHAZ resulting in tumor progression. Targeted inhibition of STAT3 may be a novel therapeutic strategy against gastric cancer.

Promoter hypermethylation of FBXO32, a novel TGF-beta/SMAD4 target gene and tumor suppressor, is associated with poor prognosis in human ovarian cancer.

Resistance to TGF-beta is frequently observed in ovarian cancer, and disrupted TGF-beta/SMAD4 signaling results in the aberrant expression of downstream target genes in the disease. Our previous study showed that ADAM19, a SMAD4 target gene, is downregulated through epigenetic mechanisms in ovarian cancer with aberrant TGF-beta/SMAD4 signaling. In this study, we investigated the mechanism of downregulation of FBXO32, another SMAD4 target gene, and the clinical significance of the loss of FBXO32 expression in ovarian cancer. Expression of FBXO32 was observed in the normal ovarian surface epithelium, but not in ovarian cancer cell lines. FBXO32 methylation was observed in ovarian cancer cell lines displaying constitutive TGF-beta/SMAD4 signaling, and epigenetic drug treatment restored FBXO32 expression in ovarian cancer cell lines regardless of FBXO32 methylation status, suggesting that epigenetic regulation of this gene in ovarian cancer may be a common event. In advanced-stage ovarian tumors, a significant (29.3%; P<0.05) methylation frequency of FBXO32 was observed and the association between FBXO32 methylation and shorter progression-free survival was significant, as determined by both Kaplan-Meier analysis (P<0.05) and multivariate Cox regression analysis (hazard ratio: 1.003, P<0.05). Reexpression of FBXO32 markedly reduced proliferation of a platinum-resistant ovarian cancer cell line both in vitro and in vivo, due to increased apoptosis of the cells, and resensitized ovarian cancer cells to cisplatin. In conclusion, the novel tumor suppressor FBXO32 is epigenetically silenced in ovarian cancer cell lines with disrupted TGF-beta/SMAD4 signaling, and FBXO32 methylation status predicts survival in patients with ovarian cancer.

DNA methylation of the Trip10 promoter accelerates mesenchymal stem cell lineage determination.

Epigenetic regulation of gene expression by DNA methylation and histone modification controls cell fate during development and homeostasis in adulthood. Aberrant epigenetic modifications may lead to abnormal development, even diseases. We have found that Trip10 (thyroid hormone receptor interactor 10), an adaptor protein involved in diverse functions, is epigenetically regulated during lineage-specific induction of human bone marrow-derived mesenchymal stem cells (MSCs). To determine whether DNA methylation-induced gene silencing is sufficient to restrict cell fate changes, we applied an invitro method to specifically methylate the promoter of Trip10. Our hypothesis was that the methylation status of the Trip10 promoter in MSCs alters the differentiation preference of MSCs. Transfection of in vitro-methylated Trip10 promoter DNA into MSCs resulted in progressive accumulation of cytosine methylation at the endogenous Trip10 promoter, reduced Trip10 expression, and accelerated MSC-to-neuron and MSC-to-osteocyte differentiation. A two-component EGFP reporter gene system was established to confirm the level of transcriptional silencing and visualize the targeted DNA methylation. EGFP expression induced in the reporter system by targeted Trip10 methylation was reversed by adding 5-aza-2'-deoxycytidine, a DNA methyltransferase inhibitor, confirming that the suppressed Trip10 expression and disrupted MSC differentiation resulted from the in vitro-introduced methylations in the Trip10 promoter. With this targeted DNA methylation and reporter system, we are able to monitor the progression of locus-specific DNA methylation in vivo and correlate such changes with potential functional changes. Using this approach, we have established a new role for Trip10, showing that the level of Trip10 expression is associated with the maintenance and differentiation of MSCs.

Environmental Factor-Mediated Transgenerational Inheritance of Igf2r Hypomethylation and Pulmonary Allergic Response via Targeting Dendritic Cells.

The developmental origin of allergic diseases has been suggested, but the molecular basis remains enigmatic. Exposure to environmental factors, such as di-(2-ethylhexyl) phthalate (DEHP; a common plasticizer), is suggested to be associated with increased childhood allergic asthma, but the causal relationship and its underlying mechanism remain unknown. This study explored the transgenerational mechanism of DEHP on allergic asthma and dendritic cell (DC) homeostasis through epigenetic modification. In a murine model, ancestral exposure of C57BL/6 mice to low-dose DEHP led to trans-generational promoter hypomethylation of the insulin-like growth factor 2 receptor (Igf2r), concomitant with enhanced Igf2r expression and increased apoptosis prominently in CD8α+ DCs upon ligand stimulation, with consequent reduction in their IL-12 secretion and subsequent T cell-derived IFN-γ, thereby promoting a default Th2-associated pulmonary allergic response. Increased apoptosis was also noted in circulating IGF2Rhigh human DCs. Further, in human placenta, the methylation level at the orthologous IGF2R promoter region was shown to be inversely correlated with the level of maternal DEHP intake. These results support the importance of ancestral phthalate exposure in conferring the trans-generational risk of allergic phenotypes, featuring hypo-methylation of the IGF2R gene and dysregulated DC homeostasis.

Bacteria pathogens drive host colonic epithelial cell promoter hypermethylation of tumor suppressor genes in colorectal cancer.

BACKGROUND: Altered microbiome composition and aberrant promoter hypermethylation of tumor suppressor genes (TSGs) are two important hallmarks of colorectal cancer (CRC). Here we performed concurrent 16S rRNA gene sequencing and methyl-CpG binding domain-based capture sequencing in 33 tissue biopsies (5 normal colonic mucosa tissues, 4 pairs of adenoma and adenoma-adjacent tissues, and 10 pairs of CRC and CRC-adjacent tissues) to identify significant associations between TSG promoter hypermethylation and CRC-associated bacteria, followed by functional validation of the methylation-associated bacteria. RESULTS: Fusobacterium nucleatum and Hungatella hathewayi were identified as the top two methylation-regulating bacteria. Targeted analysis on bona fide TSGs revealed that H. hathewayi and Streptococcus spp. significantly correlated with CDX2 and MLH1 promoter hypermethylation, respectively. Mechanistic validation with cell-line and animal models revealed that F. nucleatum and H. hathewayi upregulated DNA methyltransferase. H. hathewayi inoculation also promoted colonic epithelial cell proliferation in germ-free and conventional mice. CONCLUSION: Our integrative analysis revealed previously unknown epigenetic regulation of TSGs in host cells through inducing DNA methyltransferase by F. nucleatum and H. hathewayi, and established the latter as CRC-promoting bacteria. Video abstract.