Downregulation Of DNMT3A Research Articles

IL-1 signaling in aging and cancer: An inflammaging feedback loop unveiled

In a Science paper, Park etal. identified interleukin (IL)-1α as a key driver of positive feedback in inflammaging, linking aging-associated downregulation of DNMT3A to increased IL-1α production in lung myeloid cells. This triggers emergency myelopoiesis in the bone marrow, amplifying myeloid-mediated intratumoral immunosuppression for tumor progression in aged mice.

Truncation mutations in MYRF underlie primary angle closure glaucoma.

Mutations in myelin regulatory factor (MYRF), a gene mapped to 11q12-q13.3, are responsible for autosomal dominant high hyperopia and seem to be associated with angle closure glaucoma, which is one of the leading causes of irreversible blindness worldwide. Whether there is a causal link from the MYRF mutations to the pathogenesis of primary angle-closure glaucoma (PACG) remains unclearat this time. Six truncation mutations, including five novel and onepreviously reported, in MYRF are identified in seven new probands with hyperopia, of whom all six adults have glaucoma, further confirming the association of MYRF mutations with PACG. Immunofluorescence microscopy demonstrates enriched expression of MYRF in the ciliary body and ganglion cell layer in humans and mice. Myrfmut/+ mice have elevated IOP and fewer ganglion cells along with thinner retinal nerve fiber layer with ganglion cell layer than wild-type. Transcriptome sequencing of Myrfmut/+ retinas shows downregulation of Dnmt3a, a gene previously associated with PACG. Co-immunoprecipitation demonstrates a physical association of DNMT3A with MYRF. DNA methylation sequencing identifies several glaucoma-related cell events in Myrfmut/+ retinas. The interaction between MYRF and DNMT3A underlies MYRF-associated PACG and provides clues for pursuing further investigation into the pathogenesis of PACG and therapeutic target.

Downregulation of DNMT3A Attenuates the Warburg Effect, Proliferation, and Invasion via Promoting the Inhibition of miR-603 on HK2 in Ovarian Cancer.

Background: Ovarian cancer is a highly malignant gynecological cancer. Aerobic glycolysis is one of the features of cancer cell metabolism. Studying the molecular modulation of the Warburg effect in ovarian cancer is significantly valuable for understanding the progression mechanism of ovarian cancer. Materials and Methods: The expression level and prognostic significance of DNMT3A were analyzed using public databases. DNMT3A was overexpressed by plasmid transfection, and DNMT3A was interfered with specific siRNAs transfection. miR-603 was overexpressed by mimic transfection or inhibited by inhibitor transfection. The expression of the molecules was detected by qPCR or western blotting. CCK-8 and transwell assays were used to determine the cell proliferation, migration, and invasion abilities of ovarian cancer. Results: We found that the DNMT3A protein level was higher in ovarian cancer tissues than in normal ovary tissues, but the mRNA level had no significant difference in ovarian cancer tissues and normal ovary tissues. The higher the RNA level of DNMT3A, the poorer prognosis of patients. DNMT3A knocking down impeded the Warburg effect, cell proliferation, migration, and invasion of ovarian cancer cells. Further investigations discovered that DNMT3A promoted ovarian cancer cell malignancy via silencing miR-603. Conclusion: We found that patients who overexpressed DNMT3A showed a poor prognosis. DNMT3A was found to promote the Warburg effect, cell proliferation, migration, and invasion of ovarian cancer by inhibiting the expression of miR-603. As a result, the research revealed that DNMT3A/miR-603/HK2 axis contributed to the Warburg effect of ovarian cancer and DNMT3A may be a potential therapeutic target for ovarian cancer.

Sesamol Epigenetically Induces Estrogen Receptor α Re-expression by Upregulating miR-370-3p in Estrogen Receptor α-Negative Breast Cancer.

Due to lack of estrogen receptor α (ERα, gene name: ESR1), ERα-negative breast carcinoma is insensitive to endocrine therapy, and restoration of ERα has become a promising strategy for ERα-negative breast cancer treatment. Sesamol, a naturally occurring phenolic compound, is usually extracted from sesame seeds. Previous investigations have unmasked its anti-oxidant and anti-inflammation properties. In this study, sesamol induced ERα functional re-expression followed by upregulation of its downstream pS2 and GREB1 genes in ERα-negative breast carcinoma. Moreover, it endowed responsiveness of ERα-negative breast carcinoma to the endocrine treatment drug 4-hydroxytamoxifen without influencing the viability of normal human umbilical vein endothelial cells. Mechanistically, sesamol induced ESR1 gene promoter demethylation by downregulating the expression of the DNA methyltransferases DNMT3A and DNMT3B, without affecting DNMT1. Moreover, the non-coding RNA miR-370-3p directly targeted DNMT3A and DNMT3B mRNA, and its expression increased upon treatment with sesamol. Artificial abrogation of miR-370-3p expression with an antagomir abolished the inhibition of DNMT3A and DNMT3B expression by sesamol, resulting in a fallback in ERα reactivation. In mice, sesamol significantly induced ERα re-expression via miR-370-3p-mediated downregulation of DNMT3A and DNMT3B. Sesamol may be a safe and effective option for clinical adjuvant therapy in patients with ERα-negative breast cancer.

SAT-725 Alterations in the Phenotype and Epigenomic Landscape of Luminal Breast Cancer Following Long-Term Nanomolar Exposure to Bisphenol a (BPA)

Breast cancer (BCa) is the leading cause of female cancer-related death worldwide. Luminal BCa accounts for at least 70% of all BCa and is characterized by its hormone dependence, particularly to estrogen. Endocrine disruptors may function to exert the pro-tumorigenic effects of estrogen in proxy following environmental exposure. Bisphenol A (BPA) is a ubiquitous endocrine disruptor shown to increase risk towards developing BCa in multiple in vivo mammalian models and in vitro human breast models. Despite this, there is limited information on the phenotypic and epigenetic effects of nanomolar BPA following long-term exposure particularly in the context of BCa stem cell population, which accumulate the ‘cellular insult’ and are likely to pass such information down through epigenetic mechanisms. We hypothesize that BPA affects the epigenome, in part, by altering the regulation of DNA methyltransferases (DNMTs) since BPA disrupts transcriptional and translational regulation of DNMTs in various other tissue types such as the brain, testis and prostate. In this study, MCF7 cells were chronically (>4 weeks) cultured with nanomolar doses of BPA, and subsequently subjected to phenotypic assays and gene expression analysis in monolayer or mammosphere culture. Gene expression analysis revealed a downregulation of DNMT3A and ESR1 following long-term exposure to BPA in mammosphere culture of MCF7 cells, but not in monolayer culture. This is accompanied by alterations in mammosphere morphology, reduction in mammosphere size, an increase in the mammosphere number, and an increase in the CD49f+ population, indicating a positive contribution of BPA to stemness. However, there are no significant changes in proliferation, apoptotic rate, and metastasis in both monolayer and mammosphere culture. Future analysis includes assessment of genome-wide alterations in DNA methylation patterns in these chronically exposed MCF7 mammospheres, as well as identifying estrogen responsiveness, chemotherapeutic response, and self-renewal properties of these cancer cells.

Down-regulation of lncRNA NEAT1 regulated by miR-194-5p/DNMT3A facilitates acute myeloid leukemia

ObjectivemiR-194-5p and NEAT1 have been reported to be associated with multiple malignancies, but their roles in acute myeloid leukemia (AML) remains not fully understood. MethodsBone marrow samples were collected for monocyte separation. qRT-PCR assay was performed to investigate the expression patterns of NEAT1 and miR-194-5p in AML. CCK-8, soft agar colony formation, flow cytometry and transwell assays were employed to explore the biological functions of NEAT1 or miR-194-5p. Methylation PCR was performed to monitor the methylation of NEAT1. Luciferase reporter assay was subjected to verify the relationship between miR-194-5p and DNMT3A. Immunofluorescence and western blotting were performed to detect the alterations of protein expression. ResultsNEAT1 and miR-194-5p were both down-regulated in AML. Overexpression of either NEAT1 or miR-194-5p repressed proliferation, induced apoptosis and restrained migration and invasion of AML cells. There was a negative correlation between NEAT1 and DNMT3A in AML. Knockdown of DNMT3A dramatically decreased the methylation of NEAT1. Moreover, DNMT3A was identified as a downstream target of miR-194-5p. Furthermore, down-regulation of DNMT3A rescued the impacts on the malignant phenotypes of NEAT1 inhibition by miR-194-5p inhibitor. ConclusionAltogether, down-regulation of NEAT1 mediated by miR-194-5p/DNMT3A axis promotes AML progression, which might provide therapeutic targets in AML treatment.

DNMT3L facilitates DNA methylation partly by maintaining DNMT3A stability in mouse embryonic stem cells.

DNMT3L (DNMT3-like), a member of the DNMT3 family, has no DNA methyltransferase activity but regulates de novo DNA methylation. While biochemical studies show that DNMT3L is capable of interacting with both DNMT3A and DNMT3B and stimulating their enzymatic activities, genetic evidence suggests that DNMT3L is essential for DNMT3A-mediated de novo methylation in germ cells but is dispensable for de novo methylation during embryogenesis, which is mainly mediated by DNMT3B. How DNMT3L regulates DNA methylation and what determines its functional specificity are not well understood. Here we show that DNMT3L-deficient mouse embryonic stem cells (mESCs) exhibit downregulation of DNMT3A, especially DNMT3A2, the predominant DNMT3A isoform in mESCs. DNA methylation analysis of DNMT3L-deficient mESCs reveals hypomethylation at many DNMT3A target regions. These results confirm that DNMT3L is a positive regulator of DNA methylation, contrary to a previous report that, in mESCs, DNMT3L regulates DNA methylation positively or negatively, depending on genomic regions. Mechanistically, DNMT3L forms a complex with DNMT3A2 and prevents DNMT3A2 from being degraded. Restoring the DNMT3A protein level in DNMT3L-deficient mESCs partially recovers DNA methylation. Thus, our work uncovers a role for DNMT3L in maintaining DNMT3A stability, which contributes to the effect of DNMT3L on DNMT3A-dependent DNA methylation.

Double‐negative feedback interaction between DNA methyltransferase 3A and microRNA‐145 in the Warburg effect of ovarian cancer cells

Ovarian cancer is the most lethal gynecological malignancy because of its poor prognosis. The Warburg effect is one of the key mechanisms mediating cancer progression. Molecules targeting the Warburg effect are therefore of significant therapeutic value for the treatment of cancers. Many microRNAs (miR) are dysregulated in cancers, and aberrant miR expression patterns have been suggested to correlate with the Warburg effect in cancer cells. In our study, we found that miR‐145 negatively correlated with DNA methyltransferase (DNMT)3A expression at cellular/histological levels. miR‐145 inhibited the Warburg effect by targeting HK2. Luciferase reporter assays confirmed that miR‐145‐mediated downregulation of DNMT3A occurred through direct targeting of its mRNA 3′‐UTRs, whereas methylation‐specific PCR (MSP) assays found that knockdown of DNMT3A increased mRNA level of miR‐145 and decreased methylation levels of promoter regions in the miR‐145 precursor gene, thus suggesting a crucial crosstalk between miR‐145 and DNMT3A by a double‐negative feedback loop. DNMT3A promoted the Warburg effect through miR‐145. Coimmunoprecipitation assays confirmed no direct binding between DNMT3A and HK2. In conclusion, a feedback loop between miR‐145 and DNMT3A is a potent signature for the Warburg effect in ovarian cancer, promising a potential target for improved anticancer treatment.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Downregulation of DNMT3A by miR-708-5p Inhibits Lung Cancer Stem Cell-like Phenotypes through Repressing Wnt/β-catenin Signaling.

Purpose: Lung cancer is the leading cause of cancer-related death in the world, and emerging evidences suggest that lung cancer stem cells (CSC) are associated with its poor prognosis, tumor recurrence, and therapy resistance. Here we reveal a novel role for miR-708-5p in inhibiting lung CSC-like features.Experimental Design: Phenotypic effects of miR-708-5p on the lung CSC-like properties were examined by in vitro sphere formation assay and in xenografted animal models. Immunoblotting, dual luciferase reporter, and immunocytochemistry were performed to determine the target of miR-708-5p. DNA methylation of CDH1 promoter region was tested using bisulfate sequencing. Genome-wide miRNA sequencing data of 990 patients from The Cancer Genome Atlas (TCGA) dataset and 148 patients from China cohort were analyzed to excavate the pathogenic implications of miR-708-5p.Results: Expression of miR-708-5p inhibits the CSC traits of NSCLC cells in vitro while antagonizing miR-708-5p promotes tumorigenesis in vivo miR-708-5p directly suppresses the translation of DNMT3A, which results in a substantial reduction of global DNA methylation and the upregulated expression of tumor suppressor CDH1. The upregulation of CDH1 decreased the activity of Wnt/β-catenin signaling and then impaired the stemness characteristics of NSCLC cells. Clinically, patients with high miR-708-5p expression show significantly better survival and lower recurrence. Furthermore, miR-708-5p has a promising potential to apply to differentiating histologic subtypes in NSCLC.Conclusions: Our findings support that miR-708-5p suppresses NSCLC initiation, development, and stemness through interfering DNMT3A-dependent DNA methylation. miR-708-5p may function as a novel diagnostic and prognostic biomarker in NSCLC. Clin Cancer Res; 24(7); 1748-60. ©2017 AACR.

MicroRNA-29a inhibits cardiac fibrosis in Sprague-Dawley rats by downregulating the expression of DNMT3A.

Objective:This study aims to investigate the effect of miR-29a targeting the regulation of DNMT3A on the development of cardiac fibrosis in Sprague-Dawley (SD) rats.Methods:In vivo experiment: SD rats were randomly divided into model and control groups. The cardiac and left ventricular indices in each group were calculated. The pathological changes of the myocardium were observed. The expression levels of miR-29a, CollA1, α-SMA, and DNMT3A in the myocardium of each group were detected. In vitro experiment: The cardiac fibroblasts (CFs) of SD rats were isolated from the myocardial tissue of SD rats and cultured. The miR-29a mimics, inhibitors, DNMT3A-siRNA, and control-siRNA were transfected into CFs. The expression levels of miR-29a, DNMT3A, CollA1, and α-SMA were detected, and the proliferation of CFs after transfection was observed.Results:The heart weight index of the rats in the model group increased significantly compared with that in the control group. Obvious collagen deposition was observed in the myocardial tissue of the model group. The expression levels of CollA1, α-SMA, and DNMT3A in the model group were significantly higher than those in the control group (p<0.05).Conclusion:miR-29a reduced the activation and proliferation of CFs to improve cardiac fibrosis probably by the downregulation of DNMT3A.

DNA methyltransferase 3a modulates chemosensitivity to gemcitabine and oxaliplatin via CHK1 and AKT in p53‑deficient pancreatic cancer cells.

The aberrant expression of DNA methyltransferases (DNMTs) has been considered to be associated with pancreatic carcinogenesis and progression. DNMT3a is widely involved in cell proliferation and cell cycle progression in pancreatic ductal adenocarcinoma (PDAC) cells. However, its regulation of chemosensitivity to gemcitabine (GEM) and oxaliplatin (OXA) in p53-deficient PDAC remains unclear. In the present study, the effect and mechanisms of DNMT3a on GEM and OXA chemosensitivity in p53-deficient PDAC cells were investigated using MTT assay, cell cycle analysis, apoptosis analysis and western blotting. The treatment of GEM and OXA induced S phase arrest by DNA damage, and enhanced the activation of the AKT signaling pathway in Panc-1 cells. Downregulation of DNMT3a increased the chemosensitivity to both GEM and OXA in Panc-1 cells. DNMT3a depletion distinctly abolished S phase arrest induced by GEM and OXA. Further research demonstrated that activation inhibition of CHK1 and AKT, as well as an increase in apoptosis, were involved in DNMT3a-mediated chemosensitivity to GEM and OXA. Taken together, these data demonstrated that DNMT3a serves a crucial role in the regulation of chemosensitivity to GEM and OXA, and suggests a promising therapeutic target for p53-deficient PDAC.

The effects of methionine on TCE-induced DNA methylation and mRNA expression changes in mouse liver

Trichloroethylene (TCE) had been shown to induce aberrant DNA methylation changes in mouse liver, which could be critical in the development of hepatocellular carcinoma. To understand the effect of methionine, a methyl donor, on TCE-induced mouse liver cancer, we exposed B6C3F1 mice to TCE in the presence or absence of methionine. We found that supplementation with methionine attenuated the TCE-induced hypo- and hyper-methylation status of Cdkn1a and Ihh in mouse liver, respectively. The TCE-induced mRNA expression changes of Cdkn1a, Ihh and Jun were also alleviated by methionine. However, methionine supplementation accelerated the TCE-induced mRNA over-expression of Mki67, and enhanced the TCE-induced mRNA down-regulation of Dnmt3a and Tet2. In summary, our results showed that methionine could attenuate at least part of the TCE-induced DNA methylation changes, but the additional gene expression changes induced by supplementary of methionine does not support the assumption that methionine could prevent TCE-induced liver carcinogenesis.

Dysregulation of DNA methylation induced by past arsenic treatment causes persistent genomic instability in mammalian cells.

The mechanisms by which arsenic-induced genomic instability is initiated and maintained are poorly understood. To investigate potential epigenetic mechanisms, in this study we evaluated global DNA methylation levels in V79 cells and human HaCaT keratinocytes at several time points during expanded growth of cell cultures following removal of arsenite exposures. We have found altered genomic methylation patterns that persisted up to 40 cell generations in HaCaT cells after the treatments were withdrawn. Moreover, mRNA expression levels were evaluated by RT-PCR for DNMT1, DNMT3A, DNMT3B, HMLH1, and HMSH2 genes, demonstrating that the down regulation of DNMT3A and DNMT3B genes, but not DNMT1, occurred in an arsenic dose-dependent manner, and persisted for many cell generations following removal of the arsenite, offering a plausible mechanism of persistently genotoxic arsenic action. Analyses of promoter methylation status of the DNA mismatch repair genes HMLH1 and HMSH2 show that HMSH2, but not HMLH1, was epigenetically regulated by promoter hypermethylation changes following arsenic treatment. The results reported here demonstrate that arsenic exposure promptly induces genome-wide global DNA hypomethylation, and some specific gene promoter methylation changes, that persist for many cell generations following withdrawal of arsenite, supporting the hypothesis that the cells undergo epigenetic reprogramming at both the gene and genome level that is durable over many cell generations in the absence of further arsenic treatment. These DNA methylation changes, in concert with other known epigenome alterations, are likely contributing to long-lasting arsenic-induced genomic instability that manifests in several ways, including aberrant chromosomal effects.

Abstract 1717: Downregulation of miR-217 correlates with resistance of ph+ leukemia cells to ABL tyrosine kinase inhibitors

Abstract This study found that long-term exposure of chronic myelogenous leukemia (CML) K562 cells to BCR/ABL thyrosine kinase inhibirors (TKIs) caused drug-resistance in association with an increase in levels of DNA methyltransferases (DNMTs) and a decrease in levels of microRNAs (miRNAs) miR-217. These observations are clinically relevant; an increase in levels of DNMT3A in association with downregulation of miR-217 were noted in leukemia cells isolated from individuals with BCR/ABL TKI-resistant philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL) and CML. Further studies with TKI-resistant K562 cells found that forced expression of miR-217 inhibited expression of DNMT3A through a miR-217-binding site within the 3′-untranslated region of DNMT3A and sensitized these cells to growth inhibition mediated by the TKI. Of note, long-term exposure of K562 cells to dasatinib (10 nM) together with 5-Aza-2′-deoxycytidine (5-AzadC) (0.1 μM) potently inhibited proliferation of these cells in association with upregulation of miR-217 and downregulation of DNMT3A in vitro. In addition, a decrease in levels of DNMT3A and an increase in levels of miR-217 were noted in K562 tumors growing in immune deficient mice that were treated by the combination of 5-AzadC and dasatinib. Taken together, Ph+ leukemia cells acquire TKI-resistance via downregulation of miR-217 and upregulation of DNMT3A. Inhibition of DNMT3A by forced expression of miR-217 or 5-AzadC may be useful to prevent drug resistance in individuals who receive TKIs. Citation Format: Takayuki Ikezoe, Chie Nishioka, Jing Yang, Akihito Yokoyama. Downregulation of miR-217 correlates with resistance of ph+ leukemia cells to ABL tyrosine kinase inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1717. doi:10.1158/1538-7445.AM2014-1717

Downregulation of miR‐217 correlates with resistance of ph+ leukemia cells to ABL tyrosine kinase inhibitors

This study found that long-term exposure of chronic myelogenous leukemia (CML) K562 cells to BCR/ABL thyrosine kinase inhibitors (TKI) caused drug-resistance in association with an increase in levels of DNA methyltransferases (DNMT) and a decrease in levels of microRNA miR-217. These observations are clinically relevant; an increase in levels of DNMT3A in association with downregulation of miR-217 were noted in leukemia cells isolated from individuals with BCR/ABL TKI-resistant Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL) and CML. Further studies with TKI-resistant K562 cells found that forced expression of miR-217 inhibited expression of DNMT3A through a miR-217-binding site within the 3′-untranslated region of DNMT3A and sensitized these cells to growth inhibition mediated by the TKI. Of note, long-term exposure of K562 cells to dasatinib (10 nM) together with 5-Aza-2′-deoxycytidine (5-AzadC) (0.1 μM) potently inhibited proliferation of these cells in association with upregulation of miR-217 and downregulation of DNMT3A in vitro. In addition, a decrease in levels of DNMT3A and an increase in levels of miR-217 were noted in K562 tumors growing in immune-deficient mice that were treated with the combination of 5-AzadC and dasatinib. Taken together, Ph+ leukemia cells acquire TKI resistance via downregulation of miR-217 and upregulation of DNMT3A. Inhibition of DNMT3A by forced expression of miR-217 or 5-AzadC may be useful to prevent drug resistance in individuals who receive TKI.

Naive pluripotency is associated with global DNA hypomethylation.

Naïve pluripotent embryonic stem (ESCs) cells and embryonic germ (EGCs) cells are derived from the preimplantation epiblast and primordial germ cells (PGCs), respectively. We investigated whether differences exist between ESCs and EGCs in view of their distinct developmental origins. PGCs are programmed to undergo global DNA demethylation; however we find that EGCs exhibit equivalent levels of global DNA methylation to ESCs. Importantly, inhibition of MEK and Gsk3b by 2i conditions leads to a pronounced reduction in DNA methylation in both cell types. This is driven by Prdm14 and is associated with downregulation of Dnmt3a and Dnmt3b. However, genomic imprints are maintained in 2i and we report derivation of EGCs with intact genomic imprints. Collectively, our findings establish that culture in 2i instils a naïve pluripotent state with a distinctive epigenetic configuration that parallels molecular features observed in both the preimplantation epiblast and nascent PGCs.

Alteration in methylation pattern of GATA-4 promoter region in vitamin A-deficient offspring's heart

Epigenetics might explain correlations between lifestyle and risk of disease. Maternal diet has been shown to dynamically alter epigenetic regulation, including affecting DNA methylation status. This study was designed to test the hypothesis that GATA-4 gene methylation would lead to congenital heart defects in vitamin A-deficient offspring. Ten weaning female rats (VAN group) were fed with a diet which contents 4 IU vitamin A/g diet, while 20 rats (VAD group) were maintained on a diet without vitamin A. After 10 weeks of feeding, all the female rats were mated with normal male rats. The VAN group and a portion of VAD group rats were still given the same diet as before mating, while the rest of the rats from the VAD group (VADS group) were transferred to a diet with enough added vitamin A (10 IU/g diet) for the pregnancy cycle. The embryo hearts were dissected out at embryonic day 13.5 (E13.5) for observation of cardiac development, GATA-4 gene methylation status and the expression of DNA methyltransferases (DNMTs). Embryos from vitamin A-deficient group exhibited a high incidence of cardiac defects. High methylation was present in the CpG loci of GATA-4 gene with a low expression of GATA-4 mRNA from vitamin A-deficient group embryos. Moreover, up-regulation of DNMT1 and down-regulation of DNMT3a and DNMT3b expression were found in this group embryo. These findings show that aberrant methylation is one of key mechanisms to heart defects in vitamin A-deficient offspring. DNMTs play a critical role in this process.

Restoration of full‐length APC protein in SW480 colon cancer cells induces exosome‐mediated secretion of DKK‐4

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are common in both inherited and sporadic forms of colorectal cancer (CRC), and are associated with dysregulated Wnt signaling. Colon carcinoma SW480 cells restored with stable expression of wild-type APC (SW480APC cells) exhibit attenuated Wnt signaling, and reduced tumorigenicity, including increased cell adhesion. We performed a comparative proteomic analysis of exosomes isolated from SW480 and SW480APC cells to examine the effects of restored APC on exosome protein expression. A salient finding of our study was the unique expression of the Wnt antagonist Dickkopf-related protein 4 (DKK4) in SW480APC, but not parental SW480 cell-derived exosomes. Upregulation of DKK4 in SW480APC cells was confirmed by semiquantitative RT-PCR, immunoblotting, and immunogold electron microscopy. Analysis of the DKK4 gene promoter by methylation-specific PCR revealed reduced methylation in SW480APC cells, while RT-PCR demonstrated the downregulation of DNMT-3a, compared to the parental cell line. Our discovery of exosome-mediated secretion of DKK4 opens up the possibility that exosomal DKK4 may be a mechanism used by epithelial colon cells to regulate Wnt signaling which is lost during CRC progression.

Leukemogenic MicroRNA-29a Causes Myeloproliferation by Inhibiting Apoptosis and Inducing Aberrant Self-Renewal in a Developmental Stage Specific Manner

Abstract 2556We recently reported that miR-29a is highly expressed in AML as well as in hematopoietic stem cells (HSC). Overexpression of miR-29a in mouse bone marrow resulted in myeloproliferative disease characterized by a granulocyte/macrophage differentiation bias and progressive disease culminating in AML. Prior to the development of AML, committed myeloid progenitors including common myeloid progenitors (CMP) and granulocyte/macrophage progenitors (GMP) aberrantly acquired the ability to self-renew, demonstrated by long-term engraftment of FACS-purified transplanted cells. In order to elucidate the mechanisms of miR-29a induced leukemogenesis, we characterized the immature hematopoietic compartment during the myeloproliferative phase of disease. Similar to our prior studies, miR-29a primary chimeras exhibited granulocytic hyperplasia as soon as 6–8 weeks after transplantation, with increased frequencies of GFP+Gr-1+Mac-1+ granulocytes in the peripheral blood of animals transplanted with miR-29a versus empty vector transduced cells (mean 34.7% vs. 9.2%, p<0.05). miR-29a expressing primary chimeras also showed increased frequencies of LSK (Lin-Sca1+c-kit+) cells and myeloid progenitors (Lin-Sca1-c-kit+) compared with empty vector controls (2.34% vs. 0.54%, p<0.05; and 6.85% vs. 14.3%, p<0.05, respectively). miR-29a expressing LSK cells also exhibited decreased apoptosis as assessed by annexin V staining (mean 10.8% vs. 5.9%, p<0.05), as well as a marked increase in the number of cells in G0/G1 (mean 85.3% vs. 64%; p<0.05); both of these effects were significantly attenuated or absent in more mature myeloid progenitors. Evaluation of miR-29a target mRNAs by qPCR revealed down-regulation of DNMT3a and DNMT3b transcripts in miR-29a progenitor cells. Secondary transplants were performed with FACS-sorted cells including myeloid-biased (My-bi, Lin-Sca1+c-kit+CD150hiCD34-) and lymphoid-biased (Ly-bi, Lin-Sca1+c-kit+CD150lowCD34-) HSC, multipotent progenitors (MPP, Lin-Sca1+c-kit+CD150negCD34+), and myeloid progenitors. Five months after transplant, mice transplanted with miR-29a expressing MPP (4/5) or myeloid progenitors (3/7) exhibited a myeloid expansion, consistent with acquisition of aberrant self-renewal and transfer of disease-initiating cells. In contrast, GFP+ chimerism was either low or undetectable in secondary recipients transplanted with My-bi HSC (1/4) or Ly-bi HSC (0/6). Finally, we measured miR-29a expression levels in AML blasts from several mouse models of AML including MLL-AF9, AML-ETO9a, and NUP98-HOXD13 and show that miR-29a expression is increased 6–12 fold over normal c-kit+ cells, consistent with our previously published results demonstrating high levels of miR-29a in primary AML blasts compared to their normal HSC/progenitor counterparts. Together, these results indicate that miR-29a induces myeloproliferation by inducing self-renewal in hematopoietic progenitors including MPP and committed myeloid progenitors. The enhanced self-renewal and expansion of LSK cells is likely due to the anti-apoptotic effect of miR-29a, which is developmental stage specific and associated with decreased cell cycling and decreased DNMT3a/b expression. These findings indicate that myeloproliferation may be due to expansions caused by increased self-renewal of progenitors and not necessarily through enhanced HSC function. Disclosures:No relevant conflicts of interest to declare.