P1 Promoter Region Research Articles

Identification of RUNX1T1 as a potential epigenetic modifier in small-cell lung cancer.

Small-cell lung cancer (SCLC) can be subgrouped into common 'pure' and rare 'combined' SCLC (c-SCLC). c-SCLC features a mixed tumor histology of both SCLC and non-small-cell lung cancer (NSCLC). We performed targeted exome sequencing on 90 patients with SCLC, including two with c-SCLC, and discovered RUNX1T1 amplification specific to small cell tumors of both patients with c-SCLC, but in only 2 of 88 'pure' SCLC patients. RUNX1T1 was first identified in the fusion transcript AML1/ETO, which occurs in 12%-15% of acute myelogenous leukemia (AML). We further show higher expression of RUNX1T1 in the SCLC component of another c-SCLC tumor by in situ hybridization. RUNX1T1 expression was enriched in SCLC compared with all other cancers, including NSCLC, in both cell lines and tumor specimens, as shown by mRNA level and western blotting. Transcriptomic analysis of hallmark genes decreased by stable RUNX1T1 overexpression revealed a significant change in E2F targets. Validation experiments in multiple lung cancer cell lines showed that RUNX1T1 overexpression consistently decreased CDKN1A (p21) expression and increased E2F transcriptional activity, which is commonly altered in SCLC. Chromatin immunoprecipitation (ChIP) in these overexpressing cells demonstrated that RUNX1T1 interacts with the CDKN1A (p21) promoter region, which displayed parallel reductions in histone 3 acetylation. Furthermore, reduced p21 expression could be dramatically restored by HDAC inhibition using Trichostatin A. Reanalysis of ChIP-seq data in Kasumi-1 AML cells showed that knockdown of the RUNX1T1 fusion protein was associated with increased global acetylation, including the CDKN1A (p21) promoter. Thus, our study identifies RUNX1T1 as a biomarker and potential epigenetic regulator of SCLC.

Regulation of human β-galactoside α2,6-sialyltransferase (hST6Gal I) gene expression during differentiation of human osteoblastic MG-63 cells.

In this study, we found that gene expression of the human β-galactoside α2,6-sialyltransferase (hST6Gal I) was specifically increased during differentiation of human MG-63 osteoblastic cells by serum starvation (SS). In parallel, a distinct increase in binding to SNA, the α2,6-sialyl-specific lectin, was observed in serum-starved cells, as demonstrated by FACS analysis. 5'-Rapid amplification of cDNA ends analysis demonstrated that the increase of hST6Gal I transcript by SS is mediated by P1 promoter. To elucidate transcriptional regulation of hST6Gal I in SS-induced MG-63 cells, we functionally characterized the P1 promoter region of the hST6Gal I gene. The 5'-deletion analysis of P1 promoter region revealed that the 189bp upstream region of transcription start site is critical for transcriptional activity of hST6Gal I gene in SS-induced MG-63 cells. This region contains the predicted binding sites for several transcription factors, including AREB6, FOXP1, SIX3, HNF1, YY2, and MOK2. The mutagenesis analysis for these sites and chromatin immunoprecipitation assay demonstrated that the YY2 binding site at -98 to -77 was essential for the SS-induced hST6Gal I gene expression during differentiation of MG-63 cells.

Moderately high folate level may offset the effects of aberrant DNA methylation of P16 and P53 genes in esophageal squamous cell carcinoma and precancerous lesions

BackgroundThis study evaluated gene-nutrition interactions between folate and the aberrant DNA methylation of tumor suppressor genes in different stages of carcinogenesis of esophageal squamous cell carcinoma (ESCC).MethodsTwo hundred ESCC cases, 200 esophageal precancerous lesion (EPL) cases, and 200 controls matched by age (± 2 years) and gender were used for this study. Baseline data and dietary intake information was collected via questionnaire. The serum folate levels and methylation status of promoter regions of p16 and p53 were detected.ResultsThe interactions of increased serum folate level with unmethylated p16 and p53 promoter regions were significantly associated with a reduced risk of both EPL and ESCC (p for interaction < 0.05). The interactions of the lowest quartile of serum folate level with p16 or p53 methylation was significantly associated with an increased risk of ESCC (OR = 2.96, 95% CI, 1.45–6.05; OR = 2.34, 95% CI, 1.15–4.75). An increased serum folate level was also related to a decreasing trend of EPL and ESCC risks when p16 or p53 methylation occurred. The interaction of spinach, Chinese cabbage, liver and bean intake with unmethylated p16 and p53 was significantly associated with a reduced risk of EPL or ESCC (p for interaction < 0.05).ConclusionsThe interactions between a high folate level and unmethylated p16 and p53 promoter regions may have a strong preventive effect on esophageal carcinogenesis. Additionally, a high folate level may offset the tumor-promoting effects of aberrant DNA methylation of the genes, but it is also noteworthy that a very high level of folate may not have a protective effect on EPL in some cases.

The Transcription Factor Foxc1 Promotes Osteogenesis by Directly Regulating Runx2 in Response of Intermittent Parathyroid Hormone (1-34) Treatment.

Parathyroid hormone (PTH) is crucial for bone remodeling. Intermittent PTH (1–34) administration stimulates osteogenesis and promotes bone formation; however, the possible targets and underlying mechanisms still remain unclear. In this study, functional links between PTH and Foxc1, a transcription factor reported to be predominant in skeletal development and formation, were indicated. We determined the impacts of Foxc1 on in vitro osteogenic differentiation and in vivo bone regeneration under intermittent PTH induction, and further explored its possible targets. We found that the expression level of Foxc1 was upregulated during osteogenic induction by intermittent PTH treatment, and the elevated expression of Foxc1 induced by PTH was inhibited by PTH1R silencing, while rescued by intermittent PTH supplement. By gain- and loss-of-function strategies targeting Foxc1 in MC3T3-E1 cells, we demonstrated that Foxc1 could promote in vitro osteogenic differentiation by intermittent PTH induction. Moreover, immunofluorescence analysis indicated the nuclear co-localization of Foxc1 with Runx2. Luciferase-reporter and chromatin immunoprecipitation analysis further confirmed that Foxc1 could bind to the P1 promoter region of Runx2 directly, which plays an indispensable part in osteogenic differentiation and bone mineralization. Meanwhile, we also revealed that Foxc1 could promote bone regeneration induced by intermittent PTH treatment in vivo. Taken together, this study revealed the role and mechanism of Foxc1 on in vitro osteogenic differentiation and in vivo bone regeneration in response of intermittent PTH treatment.

ZNF746/PARIS overexpression induces cellular senescence through FoxO1/p21 axis activation in myoblasts

Various stresses, including oxidative stress, impair the proliferative capacity of muscle stem cells leading to declined muscle regeneration related to aging or muscle diseases. ZNF746 (PARIS) is originally identified as a substrate of E3 ligase Parkin and its accumulation is associated with Parkinson’s disease. In this study, we investigated the role of PARIS in myoblast function. PARIS is expressed in myoblasts and decreased during differentiation. PARIS overexpression decreased both proliferation and differentiation of myoblasts without inducing cell death, whereas PARIS depletion enhanced myoblast differentiation. Interestingly, high levels of PARIS in myoblasts or fibroblasts induced cellular senescence with alterations in gene expression associated with p53 signaling, inflammation, and response to oxidative stress. PARIS overexpression in myoblasts starkly enhanced oxidative stress and the treatment of an antioxidant Trolox attenuated the impaired proliferation caused by PARIS overexpression. FoxO1 and p53 proteins are elevated in PARIS-overexpressing cells leading to p21 induction and the depletion of FoxO1 or p53 reduced p21 levels induced by PARIS overexpression. Furthermore, both PARIS and FoxO1 were recruited to p21 promoter region and Trolox treatment attenuated FoxO1 recruitment. Taken together, PARIS upregulation causes oxidative stress-related FoxO1 and p53 activation leading to p21 induction and cellular senescence of myoblasts.

Protocatechuic Aldehyde Represses Proliferation and Migration of Breast Cancer Cells through Targeting C-terminal Binding Protein 1.

PurposeC-terminal binding protein 1 (CtBP1) is a transcriptional co-repressor that is overexpressed in many cancers. CtBP1 transcriptionally represses a broad array of tumor suppressors, which promotes cancer cell proliferation, migration, invasion, and resistance to apoptosis. Recent studies have demonstrated that CtBP1 is a potential target for cancer therapy. This study was designed to screen for compounds that potentially target CtBP1.MethodsUsing a structure-based virtual screening for CtBP1 inhibitors, we found protocatechuic aldehyde (PA), a natural compound found in the root of a traditional Chinese herb, Salvia miltiorrhiza, that directly binds to CtBP1. Microscale thermophoresis assay was performed to determine whether PA and CtBP1 directly bind to each other. Further, clustered regularly interspaced short palindromic repeats associated Cas9 nuclease-mediated CtBP1 knockout in breast cancer cells was used to validate the CtBP1 targeting specificity of PA.ResultsFunctional studies showed that PA repressed the proliferation and migration of breast cancer cells. Furthermore, PA elevated the expression of the downstream targets of CtBP1, p21 and E-cadherin, and decreased CtBP1 binding affinity for the promoter regions of p21 and E-cadherin in breast cancer cells. However, PA did not affect the expression of p21 and E-cadherin in the CtBP1 knockout breast cancer cells. In addition, the CtBP1 knockout breast cancer cells showed resistance to PA-induced repression of proliferation and migration.ConclusionOur findings demonstrated that PA directly bound to CtBP1 and inhibited the growth and migration of breast cancer cells through CtBP1 inhibition. Structural modifications of PA are further required to enhance its binding affinity and selectivity for CtBP1.

Abstract C37: Setdb1 is required for formation of pancreatic ductal adenocarcinoma by inhibiting apoptosis through regulation of p53 expression

Abstract Epigenetics is one of the representative mutation signatures in human pancreatic ductal adenocarcinoma (PDAC). Histone modification, one group of epigenetic regulators, is known to play an important role in cancer development. Setdb1, a histone methyltransferase that trimethylates histone H3 on lysine 9, has been reported to promote tumorigenesis by altering the expression of oncogenes/ tumor suppressor genes. In this study, we aimed to elucidate the functional role of Setdb1 in pancreatic acinar regeneration and PDAC formation. Setdb1 was expressed in pancreatic ductal cells and a subset of pancreatic acinar cells in wild-type mice. In pancreatic specific Setdb1 conditional KO mice, no abnormality was observed in pancreatic development. However, pancreatic deletion of Setdb1 resulted in pancreatic atrophy concomitant with increased apoptosis and upregulated p53 expression after cerulein-induced pancreatitis. To investigate the impact of pancreatic Setdb1 deletion in the context of oncogenic Kras, we next generated Ptf1a-Cre; KrasG12D; Setdb1f/f (KCS) mice and compared to control Ptf1a-Cre; KrasG12D (KC) mice. Pancreatic deletion of Setdb1 significantly accelerated spontaneous development of acinar-to-ductal metaplasia (ADM) and pancreatic intraepitherial neoplasia (PanIN) in KCS mice at the age of 4 weeks. Additionally, immunostaining revealed a dramatic increase in apoptotic cells and upregulated p53 expression in KCS mice. Consistently, microarray analysis revealed upregulated expression of apoptotic signaling pathway and p53 downstream gene pathway in KCS mice compared to KC mice. Furthermore, Chromatin immunoprecipitation experiments in acinar cells isolated from wild-type mice revealed that Setdb1 bound to the promoter regions of p53 to support its expression. Given that Setdb1 loss caused increased apoptosis mediated by p53, we next examined the impact of pancreatic Setdb1 deletion on PDAC formation in the context of heterozygous p53 deletion. Surprisingly, PDAC formation was suppressed in KCS mice in the context of heterozygous p53 deletion (KPheteroCS mice). Again, apoptosis was increased and p53 expression was upregulated in KPheteroCS mice compared to KPheteroC mice. Furthermore, KPheteroCS mice showed significantly better prognosis for survival compared to control KPheteroC mice. In sharp contrast, in KCS mice in the context of homozygous p53 deletion, PDAC was formed and increased apoptosis was cancelled. Additionally, suppression of Setdb1 in human p53 wild PDAC cells led to p53-induced apoptosis. These findings suggested that Setdb1 deletion protects against PDAC formation with inducing apoptosis through regulation of p53 expression. In conclusion, Setdb1 is required for normal pancreatic acinar regeneration and is required for PDAC formation by inhibiting apoptosis through regulation of p53 expression. Thus, Setdb1 could be a therapeutic target for PDAC that retains p53 function. Citation Format: Satoshi Ogawa, Akihisa Fukuda, Makoto Sono, Yuichi Fukunaga, Tomonori Masuda, Osamu Araki, Munemasa Nagao, Takaaki Yoshikawa, Yukiko Hiramatsu, Motoyuki Tsuda, Takahisa Maruno, Yuki Nakanishi, Hiroshi Seno. Setdb1 is required for formation of pancreatic ductal adenocarcinoma by inhibiting apoptosis through regulation of p53 expression [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C37.

Autism-Associated Chromatin Remodeler CHD8 Governs the Survival and Differentiation of Hematopoietic Stem/Progenitor Cells

Hematopoiesis is a strictly regulated process which depends on regulated proliferation, differentiation, and survival of hematopoietic stem/progenitor cells (HSPCs). Multiple signaling pathways, transcription factors and epigenetic machineries are involved in this important process. Increasing evidence from recent studies indicates that ATP-dependent chromatin-remodeling genes are involved in the control of crucial gene-expression programs in stem/progenitor cell regulation. Among them, the CHD8 gene encodes a member of chromodomain helicase DNA-binding (CHD) family of SNF2H-like ATP-dependent chromatin remodeler, mutations of which define a subtype of autism spectrum disorders. Whether CHD8 plays a role in hematopoietic cells remains unknown. In this study, to define the role of CHD8 in hematopoiesis and HSPC regulation, we carried out conditional deletion of CHD8 in the hematopoietic lineages using an interferon inducible Mx1-Cre; Chd8F/Fmouse model. As early as one week after polyI:C injection, the CHD8 knockout mice experienced drastic pancytopenia and bone marrow failure with a reduction of bone marrow (BM) cellularity to ~1/3 of that of WT controls. In the HSPC compartment, loss of CHD8 resulted in a depletion of Lin-c-Kit+ (LK) and Lin-Sca1+c-Kit+ (LSK) progenitors and a severe reduction of the GMP, MEP, MPP and CD150+CD48-LSK HSC populations. Accompanying the loss of HSPCs were dramatically increased apoptosis rate and cell cycle arrest in Chd8-/- cells. An examination of HSPC function by transplantation found that the BM cells from Chd8-/- mice could not engraft in the recipient mice that died ~20 days post-transplantation. The chimerism of Chd8-/- BM cells also reduced drastically in a competitive BM transplantation assay when induction of Chd8 deletion occurred after Mx1-Cre; Chd8F/Fcells were pre-engrafted at 1:1 ratio with WT BM cells. These results indicate that CHD8 is essential for hematopoiesis and HSPC survival and proliferation. To elucidate the underlying mechanism of CHD8 function in HSPCs, we performed complementary biochemical, genomic and genetic analyses of the WT (Chd8F/F) and Chd8-/-HSPC cells. Firstly, we found blood progenitor cells dominantly express the short form of CHD8 containing residues 1 to 751 which retains the N-terminal P53 binding domain, rather than the long form of the CHD8 protein. Secondly, we revealed by RNA-seq and subsequent RT-PCR analyses that the P53 signaling pathway signatures including P53, P21, Bax and Noxa are aberrantly activated in Chd8-/-HSPCs. At the protein level, P53 and P21 are significantly elevated in Chd8-/- LK cells. Thirdly, ATAC-seq analysis of the LSK cells revealed increased global chromatin accessibilities after CHD8 deletion, including the promoter regions of P21 and Noxa genes. By a "Cut &amp; Run" assay using H3K4me3 antibody in Chd8F/Fand Chd8-/- LSK cells we have also seen significantly enhanced epigenetic H3K4me3 mark on the promoter region of P21 and Noxa. An anti-CHD8 "Cut &amp; Run" further detected direct binding sites of CHD8 on P53 and P21, but not Noxa or Bax gene. Fourthly, we observed a direct binding interaction by co-immunoprecipitation between P53 and CHD8 proteins. Finally, genetic deletion of one allele of P53 gene in Chd8-/- HSPCs (P53+/-;Chd8-/-) was able to completely suppress the Bax/Noxa expression and apoptosis phenotype and rescue most of the HSC and early progenitor defects but not the later stage of differentiation defects and BM cellularity loss. Interestingly, deletion of both alleles of P53 gene (P53-/-;Chd8-/-) was able to further remove the differentiation block between LSK and LK cells observed in the P53+/-;Chd8-/-background which is associated with a P21 mediated G0 cell cycle phase arrest. These combined results suggest a mechanism of action that CHD8 works through a direct complex with P53 to regulate P21 gene expression which mediates cell cycle control of HSPC differentiation and an indirect CHD8 mediated P53 expression and subsequent P53 regulated Noxa/Bax expression which regulates HSPC survival. Thus, we identify the autism-associated CHD8 as an essential chromatin suppressor in regulating hematopoiesis through restricting the expression of P53 and chromatin accessibility of P53 downstream targets, Bax/Noxa and P21, to enable selective control of HSPC survival and differentiation, respectively. Disclosures No relevant conflicts of interest to declare.

LINC00978 promotes the progression of hepatocellular carcinoma by regulating EZH2-mediated silencing of p21 and E-cadherin expression

Long non-coding RNAs (lncRNAs) have been suggested as important regulators of cancer development and progression in hepatocellular carcinoma (HCC). Nevertheless, the clinical value and biological roles of LINC00978 in HCC remain unclear. In this study, we detected the expression of LINC00978 in tumor tissues and serum of HCC patients, examined the roles of LINC00978 in HCC progression and elucidated the underlying molecular mechanisms. We found that LINC00978 expression was upregulated in tumor tissues and serum of HCC patients. Higher serum levels of LINC00978 could distinguish HCC patients from hepatitis and liver cirrhosis patients and healthy controls. LINC00978 knockdown inhibited HCC cell proliferation, migration and invasion while promoted cell cycle arrest and apoptosis. Overexpression of LINC00978 led to the opposite effects. LINC00978 knockdown also inhibited HCC growth and metastasis in mouse tumor models. Mechanistically, LINC00978 bound to EZH2 and mediated its accumulation at the promoter region of p21 and E-cadherin genes, leading to the trimethylation of H27K3 and the inhibition of p21 and E-cadherin expression. Moreover, the simultaneous depletion of p21 and E-cadherin expression reversed the inhibitory effects of LINC00978 knockdown on HCC cell proliferation, migration, and invasion. Taken together, these findings suggest that LINC00978 promotes HCC progression by inhibiting p21 and E-cadherin expression via EZH2-mediated epigenetic silencing. LINC00978 may represent a novel biomarker for HCC diagnosis, prognosis, and therapy.

Resveratrol modulates epigenetic regulators of promoter histone methylation and acetylation that restores BRCA1, p53, p21CIP1 in human breast cancer cell lines.

The epigenetic enzymes catalyze posttranslational modifications (PTMs) of histones, which functionally determine gene expression at the chromatin level. Resveratrol (RVT) a much studied anti-cancer natural molecule is known for restoration of BRCA1, p53, and p21 in cancer cells. We aimed to investigate the role of histone methylation and acetylation on upregulation of these tumor suppressor genes. Our results suggest RVT significantly increase expression of BRCA1, p53, and p21, while decreased expression of protein arginine methyltransferase 5 (PRMT5) and enhancer of Zeste homolog 2 (EZH2) at a 20 μM concentration by 48 hr in both MCF-7 and MDA-MB-231 breast cancer cells. Also, there was an overall loss of H4R3me2s (catalytic product of PRMT5) and H3K27me3 (catalytic product of PRMT5). In contrast, RVT exposure caused a significant decrease in lysine deacetylase (KDAC) activity and expression of KDAC1-3, whereas the expression of lysine acetyltransferase KAT2A/3B was increased compared to the unexposed cells. As an outcome, RVT increased global level of H3K9ac and H3K27ac marks. The chromatin immunoprecipitation showed 20 μM RVT exposure significantly reduced the enrichment of repressive histone marks (H4R3me2s and H3K27me3) while the abundance of activating histone marks (H3K9/27ac) within the proximal promoter region of BRCA1, p53, and p21 was increased. We hypothesize RVT by affecting the expression and function of methylation and acetylation enzymes altered the epigenetic modifications on promoter histones that restored expression of these critically important tumor suppressor genes.

P300 Acetylates JHDM1A to inhibit osteosarcoma carcinogenesis

JHDM1A participates in cancer development via demethylate dimethyl histone H3 lysine 36 (H3K36me2). p300 is an intrinsic acetyltransferase. This study explored the acetyltransferase activity of p300 on JHDM1A and analyzed the JHDM1A acetylation on H3K36me2 demethylation in osteosarcoma. Co-immunoprecipitation (CoIP) and immunoblotting assay found that p300 directly acetylated JHDM1A at K409 residue in osteosarcoma MG-63 and HOS cells. Nucleosomes and mononucleosomes were prepared and found that acetylation of JHDMIA disrupted its association with nucleosomes and thereby impaired its capability to induce H3K36me2 demethylation. Moreover, chromatin immunoprecipitation (ChIP) assay discovered that the input levels of H3K36me2 in the promoter regions of p21 and puma were increased after acetylation of JHDM1A, which raised the p21 and puma mRNA levels in the cells. Finally, the analysis of JHDM1A acetylation on osteosarcoma cell proliferation and invasion, along with tumor growth pointed out that acetylation of JHDMIA inhibited the proliferation and invasion of osteosarcoma HOS cells, as well as suppressed the tumor growth of osteosarcoma. In conclusion, the outcomes of our research verified that p300 could directly acetylate JHDM1A at K409 site, which reduces the demethylation of H3K36me2, enhanced the transcription of p21 and puma, and thereby inhibited the growth and metastasis of osteosarcoma.

Abstract 3023: Clonal expansion of TP53 mutated cells is associated with chemoresistance in acute myeloid leukemia

Abstract Chemoresistance is a major burden for the treatment of many cancers, including acute myeloid leukemia (AML). AML is a clonal disease and chemoresistant cells may either evolved from the expansion of a subclone of the primary clone or from the clone gained additional mutations during therapy. We have generated chemoresistant cell lines from AML patient sample derived cell lines, OCI-AML2 and MV4-11, to study the chemoresistant mechanism. Interestingly, we found that both established resistant lines have TP53 mutations (Y220C or R248W) but not in the parental cell lines by Sanger sequencing. However, high resolution sequencing result shows that residual TP53 mutation is present in both parental cell lines. These small population of cells with TP53 mutation was expanded under the selection by chemotherapy and eventually became a dominant clone in chemoresistant lines. TCGA AML patient sample sequencing data also shows, there’re around 3% of patients have residual TP53 mutation reads. However, since the reads are low, these patients are considered TP53 wild type conventionally. Importantly, these patients have worse outcome of survival as compared to the TP53 wild type patients, indicating the residual TP53 mutation may play an important role on chemoresistance in patients. Consistent with TP53 mutation in chemoresistant cells, Gene Set Enrichment Analysis (GSEA) from RNA-seq data shows p53 target genes are repressed in resistant cells. The cyclin-dependent kinase inhibitor p21 is one of p53 target genes and is down regulated in chemoresistant cells. Knocking down of p21 in parental cells increased cell chemoresistance, indicating p21 gene repression contributes to chemoresistance. Data from TCGA also shows patients with lower p21 expression have worse outcome of survival. In chemoresistant cell line, there is reduced p53 recruitment at p21 promoter, leading to failure of p21 activation. The epigenetic drug Romidepsin can elevate histone acetylation at p21 promoter region, reactivates p21 gene expression and induces cell death. Furthermore, GSEA from RNA-seq data shows Romidepsin can reactivate genes that repressed by p53 mutation. Our data indicate that TP53 mutation is directly linked to chemoresistance. Residual TP53 mutation in patients was neglected previously, however, it can be an important driver for clonal evolution and chemoresistance in AML. In addition, epigenetic drug Romidepsin can be a potential therapeutic drug to prevent chemoresistance development especially for patients with TP53 mutation or residual TP53 mutation. Citation Format: Bowen Yan, Yi Qiu. Clonal expansion of TP53 mutated cells is associated with chemoresistance in acute myeloid leukemia [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 3023.

Long non-coding RNA GClnc1 promotes progression of colorectal cancer by inhibiting p53 signaling pathway.

The aim of this study was to investigate whether long non-coding RNA (lncRNA) GClnc1 was involved in the development of colorectal cancer, and to explore its possible mechanisms. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was conducted to detect GClnc1 expression in 48 colorectal cancer tissues and normal colon tissues. The Kaplan-Meier method was used to analyze the relationship between GClnc1 expression and survival rate of patients with colorectal cancer. In addition, GClnc1 expression in colorectal cancer cell lines and normal colonic epithelial cell lines were analyzed. After knockdown and over-expression of GClnc1 in colorectal cancer cells, Cell Counting Kit-8 (CCK-8) and colony formation assay were performed to detect the viability and proliferation of cells, respectively. RNA pull-down and RNA-binding protein immunoprecipitation (RIP) were applied to examine the specific interaction between GClnc1 and p53. After over-expression of GClnc1 in colorectal cancer cells, qPCR and Western blot were performed to evaluate the expression levels of p53, p21 and BAX. Meanwhile, the Luciferase reporter gene assay was established to reveal the activity of p53 after over-expression of GClnc1. ChIP assay was applied to figure out whether GClnc1 could affect the binding ability of p53 to the promoter region of p21. After p53 or GClnc1 knock-down in colorectal cancer cells, the protein level of p53 was analyzed using Western blot. Finally, qRT-PCR, CCK-8 and colony formation assay were used to detect the levels of p21 and BAX, the viability, as well as the proliferation ability of cells, respectively. The expression of GClnc1 in colorectal cancer tissues was significantly higher than that of para-cancerous tissues. Meanwhile, GClnc1 expression in T3 and T4 tumors was markedly higher than that of T1 and T2. The survival analysis revealed that patients with a higher level of GClnc1 showed remarkably lower overall survival than those with lower expression of GClnc1. QRT-PCR results indicated that GClnc1 expression in colorectal cancer cells (including SW620 and HCT116) was conspicuously higher than that of normal colonic epithelial cells (NCM640). After knocking down GClnc1 in SW620 cells, the viability and proliferation abilities were conspicuously decreased. Meanwhile, the expression level of GClnc1, as well as the viability and colony formation ability of cells, were significantly increased after over-expression of GClnc1 in HCT116 cells. Subsequently, the qRT-PCR assay demonstrated that GClnc1 was mainly localized in the nucleus. RNA pull-down and RIP experiments revealed that there was a specific interaction between GClnc1 and p53. Moreover, qRT-PCR and Western blot analysis indicated that the expression level of p53 was not affected after over-expression of GClnc. However, the expressions of p21 and BAX were remarkably decreased. The Luciferase reporter gene assay revealed that GClnc1 over-expression markedly weakened the Luciferase activity of p53. Meanwhile, ChIP experiments demonstrated that GClnc1 up-regulation affected the binding condition of p53 to p21. Western blot analysis showed that knockdown of p53 reversed the increased mRNA level of p21 as well as BAX. Furthermore, p53 down-regulation significantly weakened cell viability and colony formation ability caused by knockdown of GClnc1. LncRNA GClnc1 was highly expressed in colorectal cancer tissues. Meanwhile, it could increase the proliferation of colorectal cancer cells by reducing the expression of p21 as well as BAX via p53 signaling pathway, thereby promoting the progression of colorectal cancer.

Metformin Regulates the Expression of CD133 Through the AMPK-CEBPβ Pathway in Hepatocellular Carcinoma Cell Lines

CD133 is a cellular surface protein, which has been reported to be a cancer stem cell marker, and thus is considered a potential target for cancer treatment. Metformin, one of the biguanides used for the treatment of diabetes, is also known to reduce the risk of cancer development and cancer stem-like cells (CSCs), including the expression of CD133. However, the mechanism underlying the reduction of the expression of CD133 by metformin is not yet understood. This study shows that metformin suppressed CD133 expression mainly by affecting the CD133 P1 promoter via adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling but not the mammalian target of rapamycin (mTOR). AMPK inhibition rescued the reduction of CD133 by metformin. Further experiments demonstrated that CCAAT/enhancer-binding protein beta (CEBPβ) was upregulated by metformin and that two isoforms of CEBPβ reciprocally regulated the expression of CD133. Specifically, the liver-enriched activator protein (LAP) isoform increased the expression of CD133 by directly binding to the P1 promoter region, whereas the liver-enriched inhibitory protein (LIP) isoform suppressed the expression of CD133. Consistent with these findings, a three dimensional (3D) culture assay and drug sensitivity assay demonstrated that LAP-overexpressing cells formed large spheroids and were more resistant to 5-fluorouracil (5-FU) treatment, whereas LIP-overexpressing cells were more sensitive to 5-FU and showed combined effects with metformin. Our results indicated that metformin-AMPK-CEBPβ signaling plays a crucial role in regulating the gene expression of CD133. Additionally, regulating the ratio of LAP/LIP may be a novel strategy for targeting CSCs for the treatment of cancer.

Effects of ginsenoside Rg3 on epigenetic modification in ovarian cancer cells.

Epigenetic modifications are closely related to oncogene activation and tumor suppressor gene inactivation. The aim of this study was to determine the effects of ginsenoside Rg3 on epigenetic modification in ovarian cancer cells. Cell proliferation, metastasis, invasion and apoptosis were respectively determined using Cell Counting Kit-8 (CCK-8), wound healing, Transwell and flow cytometric assays. Methylation levels were determined using methylation specific PCR (MSP). Related-factor expression was detected by conducting real-time-qPCR (RT-qPCR) and western blotting. The results revealed that cell proliferation was inhibited by ginsenoside Rg3 (0, 25, 50, 100 and 200 µg/ml) in a time-dependent manner (12, 24 and 48 h). Ginsenoside Rg3 (50, 100 and 200 µg/ml) was selected to treat cells in various experiments. When ovarian cells were treated with ginsenoside Rg3, cell apoptosis was observed to be promoted, while cell metastasis and invasion were inhibited at 48 h. The results of the present study revealed that in the promoter regions of p53, p16 and hMLH1, the methylation levels decreased, while the mRNA and protein levels significantly increased. The activities of DNMTs and mRNA as well as protein levels of DNMT1, DNMT3a and DNMT3b were decreased by Rg3. The data also demonstrated that the mRNA and protein levels of acetyl-H3 K14/K9 and acetyl-H4 K12/K5/K16 were increased by Rg3. Hence, ginsenoside Rg3 inhibited ovarian cancer cell viability, migration and invasion as well as promoted cell apoptosis.

DMBX1 promotes tumor proliferation and regulates cell cycle progression via repressing OTX2-mediated transcription of p21 in lung adenocarcinoma cell.

Lung adenocarcinoma (LUAD) was the predominant histological subtype of lung cancer, with poor prognosis. By analyzing the TCGA dataset, we found that DMBX1 (diencephalon/mesencephalon homeobox 1), a member of the bicoid sub-family of homeodomain-containing transcription factors, was overexpressed in LUAD and correlated with poorer prognosis and more advanced clinicopathological features of LUAD patients. Silencing of DMBX1 inhibited proliferation of LUAD and induced G1/S cell cycle arrest, whereas ectopic expression of DMBX1 enhanced tumor growth of LUAD and promoted G1/S cell cycle exit. Furtherly we found that the function of DMBX1 was dependent on p21 (CDKN1A), a key regulator of G1/S cell cycle progression. Co-IP assay revealed that DMBX1 directly bound to another homeobox transcription factor, OTX2. ChIP and luciferase reporter assay confirmed that OTX2 directly interacted with the promoter region of p21 to enhance its transcription, and DMBX1 repressed OTX2-mediated transcription of p21. Our study reveals that DMBX1 plays an oncogenic role in LUAD by repressing OTX2-mediated transcription of p21 and the results may provide new therapeutic targets for LUAD patients.

Fra-1 plays a critical role in angiotensin II-induced vascular senescence.

Vascular aging has a strong relationship with cardiovascular disease. Fos-related antigen 1 (Fra-1), also referred to as Fos-like antigen 1, is a transcription factor and has been reported to be involved in many pathologic processes. Here, we demonstrate that Fra-1 plays a critical role in angiotensin II (Ang II)-induced vascular senescence. Fra-1 expression is increased significantly in Ang II-induced rat aortic endothelial cell (RAEC) senescence and the arteries from Ang II-infused mice. Interestingly, silencing Fra-1 blocks Ang II-induced senescence phenotypes in RAECs, including decreased senescence-associated β-galactosidase staining, and mitigated proliferation suppression and senescence-associated secretory phenotype. Further, knocking down Fra-1 inhibits vascular aging phenotypes in an Ang II-infused mice model. The up-regulated Fra-1 also exists in human atherosclerotic plaques and Ang II-induced vascular smooth muscle cells as well as in replicated senescence RAECs. Mechanistic studies reveal that Fra-1 preferentially associates with c-Jun and binds to the cyclin-dependent kinase inhibitor 1a (p21) and cyclin-dependent kinase inhibitor 2a (p16) promoter region, leading to elevated gene expression, which causes senescence-related phenotypes. In conclusion, our results identify that Fra-1 plays a novel and key role in promoting vascular aging by directly binding and transcriptionally activating p21 and p16 signaling, suggesting intervention of Fra-1 is a potential strategy for preventing aging-associated cardiovascular disorders.-Yang, D., Xiao, C., Long, F., Wu, W., Huang, M., Qu, L., Liu, X., Zhu, Y. Fra-1 plays a critical role in angiotensin II-induced vascular senescence.

OCT4 maintains self-renewal and reverses senescence in human hair follicle mesenchymal stem cells through the downregulation of p21 by DNA methyltransferases

BackgroundSelf-renewal is dependent on an intrinsic gene regulatory network centered on OCT4 and on an atypical cell cycle G1/S transition, which is also regulated by OCT4. p21, a gene negatively associated with self-renewal and a senescence marker, is a member of the universal cyclin-dependent kinase inhibitors (CDKIs) and plays critical roles in the regulation of the G1/S transition. The expression of p21 can be regulated by OCT4-targeted DNA methyltransferases (DNMTs), which play distinct roles in gene regulation and maintaining pluripotency properties. The aim of this study was to determine the role of OCT4 in the regulation of self-renewal and senescence in human hair follicle mesenchymal stem cells (hHFMSCs) and to characterize the molecular mechanisms involved.MethodsA lentiviral vector was used to ectopically express OCT4. The influences of OCT4 on the self-renewal and senescence of hHFMSCs were investigated. Next-generation sequencing (NGS) was performed to identify the downstream genes of OCT4 in this process. Methylation-specific PCR (MSP) analysis was performed to measure the methylation level of the p21 promoter region. p21 was overexpressed in hHFMSCsOCT4 to test its downstream effect on OCT4. The regulatory effect of OCT4 on DNMTs was examined by ChIP assay. 5-aza-dC/zebularine was used to inhibit the expression of DNMTs, and then self-renewal properties and senescence in hHFMSCs were detected.ResultsThe overexpression of OCT4 promoted proliferation, cell cycle progression, and osteogenic differentiation capacity of hHFMSCs. The cell senescence of hHFMSCs was markedly suppressed due to the ectopic expression of OCT4. Through NGS, we identified 2466 differentially expressed genes (DEGs) between hHFMSCsOCT4 and hHFMSCsEGFP, including p21, which was downregulated. The overexpression of p21 abrogated the proliferation and osteogenic differentiation capacity of hHFMSCsOCT4 and promoted cell senescence. OCT4 enhanced the transcription of DNMT genes, leading to an elevation in the methylation of the p21 promoter. The inhibition of DNMTs reversed the OCT4-induced p21 reduction, depleted the self-renewal of hHFMSCsOCT4, and triggered cell senescence.ConclusionsOCT4 maintains the self-renewal ability of hHFMSCs and reverses senescence by suppressing the expression of p21 through the upregulation of DNMTs.

Bromate-induced Changes in p21 DNA Methylation and Histone Acetylation in Renal Cells.

Bromate (BrO3-) is a water disinfection byproduct (DBP) previously shown to induce nephrotoxicity in vitro and in vivo. We recently showed that inhibitors of DNA methyltransferase 5-aza-2'-deoxycytidine (5-Aza) and histone deacetylase trichostatin A (TSA) increased BrO3- nephrotoxicity whereas altering the expression of the cyclin-dependent kinase inhibitor p21. Human embryonic kidney cells (HEK293) and normal rat kidney (NRK) cells were sub-chronically exposed to BrO3- or epigenetic inhibitors for 18 days, followed by 9 days of withdrawal. DNA methylation was studied using a modification of bisulfite amplicon sequencing called targeted gene bisulfite sequencing. Basal promoter methylation in the human p21 promoter region was substantially lower than that of the rat DNA. Furthermore, 5-Aza decreased DNA methylation in HEK293 cells at the sis-inducible element at 3 distinct CpG sites located at 691, 855, and 895 bp upstream of transcription start site (TSS). 5-Aza also decreased methylation at the rat p21 promoter about 250 bp upstream of the p21 TSS. In contrast, sub-chronic BrO3- exposure failed to alter methylation in human or rat renal cells. BrO3- exposure altered histone acetylation in NRK cells at the p21 TSS, but not in HEK293 cells. Interestingly, changes in DNA methylation induced by 5-Aza persisted after its removal; however, TSA- and BrO3--induced histone hyperacetylation returned to basal levels after 3 days of withdrawal. These data demonstrate novel sites within the p21 gene that are epigenetically regulated and further show that significant differences exist in the epigenetic landscape between rat and human p21, especially with regards to toxicant-induced changes in histone acetylation.

Design of Novel Putative Peptide Fragments Derived from Exons of P53 Isoform G and P53 Promoter Region ORF Sequence Targeting HDM2/MDM2-P53 Interaction

p53 is a potent endogenous tumour suppressor agent which guards against DNA damage and prevents transformation of normal cells into malignant cells. In most forms of cancers except malignant melanoma, mutation occurs in p53 gene rendering it dysfunctional or suppressed. This research attempts to design novel putative bioactive oligopeptides from tumour suppressor p53 gene sequences using its promoter sequence and deriving several primer-based oligomers from the exons of p53 gene. p53 induces cells to undergo repair or apoptosis in response to DNA-damage induced stress and thus acts as the Warden of the Cell. We devise some novel methods to derive our de novo sequences from the exons of this gene and report discovery of two new oligopep-tides such as HRGRES and PAAPA (P 7 TA 7) peptide being two putative oligopeptides.