P21 Promoter Research Articles

Non-cytotoxic levels of resveratrol enhance the anticancer effects of cisplatin by increasing the methyltransferase activity of CARM1 in human cancer cells

BackgroundResveratrol (RSVL) is a plant-derived polyhydroxyphenolic compound with excellent anticancer properties, alone or in combination with other chemotherapeutic drugs. However, the anticancer mechanism of RSVL is diverse and high concentrations are often required for RSVL to exert its anticancer effect, which would also adversely affect normal cells. PurposeThe main objective of this study is to investigate the molecular mechanism of how non-cytotoxic concentrations of RSVL enhance the anticancer effect of cisplatin involving a newly identified RSVL-binding protein. MethodsCell viability of cell lines from three cancer types exposed to RSVL and/or cisplatin was measured by NBB staining assay. RSVL-binding proteins were identified using RSVL-bound CNBr-activated Sepharose 4B beads coupled with LC-MS/MS, and the binding between RSVL and novel RSVL-binding protein was further confirmed with an in vitro pull-down assay. The expression of proteins was examined by immunoblot analysis, and the activity of methyltransferase was evaluated by in vitro methylation assay. The methylation level of H3R17 in the gene promoter was investigated using ChIP-qPCR. Bioinformatics analysis was conducted to identify pathway enrichment of genes, predict drug sensitivity, and analyze the survival of cancer patients. ResultsLow doses of RSVL might promote cancer cell growth whereas high doses of RSVL showed cytotoxic effects on normal cells. When co-treated with a lower cisplatin dose, non-cytotoxic RSVL levels showed synergistic anticancer effects. Here, coactivator-associated arginine methyltransferase 1 (CARM1) was identified as a novel RSVL-binding protein, and we showed that the upregulation of CARM1 increased the sensitivity of cancer cells to RSVL. Interestingly, we found that CARM1 was essential in the RSVL-induced sensitivity of cisplatin. Further molecular mechanistic studies revealed that RSVL could stabilize CARM1 protein, resulting in the upregulation and increased methyltransferase activity of CARM1. Additionally, we showed that the methylation levels of H3R17 in the promoter of p21, a downstream gene of CARM1 involving cell cycle arrest, were significantly increased after RSVL treatment. Finally, data from our bioinformatics analysis suggested that CARM1 could be utilized as a potential biomarker for chemotherapeutic drug sensitivity and prognosis in cancers. ConclusionsThis study identified CARM1 as a RSVL-binding protein for the first time and elucidated the potential roles of CARM1 in enhancing the efficacy of cisplatin by low doses of RSVL, which could have important clinical implications.

Regulation of Cell Viability, p53 Promoter Activity, and Expression of Interleukin-8, Matrixmetalloproteinase-1 and Tissue Inhibitor of Matrixmetalloproteinase-1 in Non-Irradiated or UV-Irradiated Fibroblasts and Melanoma Cells

Regulation of Cell Viability, p53 Promoter Activity, and Expression of Interleukin-8, Matrixmetalloproteinase-1 and Tissue Inhibitor of Matrixmetalloproteinase-1 in Non-Irradiated or UV-Irradiated Fibroblasts and Melanoma Cells

SOX4 inhibits ferroptosis and promotes proliferation of endometrial cancer cells via the p53/SLC7A11 signaling.

Sex-determining region Y-related high-mobility group box 4 (SOX4) has been reported to play a carcinogenic role in endometrial cancer (EC). However, the biological function and regulatory mechanisms of SOX4 in ferroptosis during the progression of EC are still unknown. The mRNA and protein levels were scrutinized by quantitative reverse-transcription polymerase chain reaction and western blot, respectively. The cell viability and proliferative capability were determined by cell counting kit-8 assay and 5-ethynyl-2'-deoxyuridine (EdU) assay. Transcriptional regulation of gene expression was investigated by dual-luciferase reporter assay and chromatin immunoprecipitation. Ferroptosis was evaluated by detection of reactive oxygen species, malondialdehyde, Fe2+, and ferroptosis-related proteins. The mice test was implemented to confirm the influence of SOX4 on EC tumor growth and ferroptosis in vivo. We here discovered the elevation of SOX4 in EC tissues and cells. Functionally, SOX4 knockdown hampered proliferation and promoted ferroptosis of EC cells. Mechanistically, SOX4 bound to p53 promoter and inhibited its transcriptional activity in EC cells. In addition, p53 transcriptionally suppressed SLC7A11 expression in EC cells. Downregulation of p53 reverses the effect of SOX4 knockdown on proliferation and ferroptosis of EC cells. Finally, in vivo experiments demonstrated that SOX4 depletion hindered tumor growth and triggered ferroptosis in EC. These findings collectively suggested that SOX4 inhibited ferroptosis and promoted proliferation of EC cells via the p53/SLC7A11 signaling. Our research unveiled a novel regulatory mechanism of ferroptosis in EC, offering promising perspectives for the development of EC therapies.

Senataxin mediates R-loop resolution on HPV episomes.

Three-stranded DNA-RNA structures known as R-loops that form during papillomavirus transcription can cause transcription-replication conflicts and lead to DNA damage. We found that R-loops accumulated at the viral early promoter in human papillomavirus (HPV) episomal cells but were greatly reduced in cells with integrated HPV genomes. RNA-DNA helicases unwind R-loops and allow for transcription and replication to proceed. Depletion of the RNA-DNA helicase senataxin (SETX) using siRNAs increased the presence of R-loops at the viral early promoter in HPV-31 (CIN612) and HPV-16 (W12) episomal HPV cell lines. Depletion of SETX reduced viral transcripts in episomal HPV cell lines. The viral E2 protein, which binds with high affinity to specific palindromes near the promoter and origin, complexes with SETX, and both SETX and E2 are present at the viral p97 promoter in CIN612 and W12 cells. SETX overexpression increased E2 transcription activity on the p97 promoter. SETX depletion also significantly increased integration of viral genomes in CIN612 cells. Our results demonstrate that SETX resolves viral R-loops to proceed with HPV transcription and prevent genome integration.IMPORTANCEPapillomaviruses contain small circular genomes of approximately 8 kilobase pairs and undergo unidirectional transcription from the sense strand of the viral genome. Co-transcriptional R-loops were recently reported to be present at high levels in cells that maintain episomal HPV and were also detected at the early viral promoter. R-loops can inhibit transcription and DNA replication. The process that removes R-loops from the PV genome and the requisite enzymes are unknown. We propose a model in which the host RNA-DNA helicase senataxin assembles on the HPV genome to resolve R-loops in order to maintain the episomal status of the viral genome.

Caveolin-2 controls preadipocyte survival in the mitotic clonal expansion for adipogenesis

Here, we report that Caveolin-2 (Cav-2) is a cell cycle regulator in the mitotic clonal expansion (MCE) for adipogenesis. For the G2/M phase transition and re-entry into the G1 phase, dephosphorylated Cav-2 by protein tyrosine phosphatase 1B (PTP1B) controlled epigenetic activation of Ccnb1, Cdk1, and p21 in a lamin A/C-dependent manner, thereby ensuring the survival of preadipocytes. Cav-2, associated with lamin A/C, recruited the repressed promoters of Ccnb1 and Cdk1 for activation, and disengaged the active promoter of p21 from lamin A/C for inactivation through histone H3 modifications at the nuclear periphery. Cav-2 deficiency abrogated the histone H3 modifications and impeded the transactivation of Ccnb1, Cdk1, and p21, leading to a delay in mitotic entry, retardation of re-entry into G1 phase, and the apoptotic cell death of preadipocytes. Re-expression of Cav-2 restored the G2/M phase transition and G1 phase re-entry, preadipocyte survival, and adipogenesis in Cav-2-deficient preadipocytes. Our study uncovers a novel mechanism by which cell cycle transition and apoptotic cell death are controlled for adipocyte hyperplasia.

Sodium valproate affects the expression of p16INK4a and p21WAFI/Cip1 cyclin‑dependent kinase inhibitors in HeLa cells.

p16INK4a and p21WAF1/Cip1 are cyclin-dependent kinase inhibitors involved in cell cycle control, which can function as oncogenes or tumor suppressors, depending on the context of various extracellular and intracellular signals, and cell type. In human papillomavirus-induced cervical cancer, p16 INK4a shows oncogenic activity and functions as a diagnostic marker of cervical neoplasia, whereas p21 WAF1/Cip1 acts as a tumor suppressor and its downregulation is associated with the progression of malignant transformation. Several histone deacetylase (HDAC) inhibitors promote the positive and negative regulation of a number of genes, including p16 INK4a and p21 WAF1/Cip1; however, the effects of sodium valproate (VPA) on these genes and on the proteins they encode remain uncertain in HeLa cervical cancer cells. In the present study, these effects were investigated in HeLa cells treated with 0.5 or 2 mM VPA for 24 h, using reverse transcription-quantitative PCR, confocal microscopy and western blotting. The results revealed a decrease in the mRNA expression levels of p16 INK4a and a tendency for p16INK4a protein abundance to decrease in the presence of 2 mM VPA. By contrast, an increase in the protein expression levels of p21WAF1/Cip1 was detected in the presence of 0.5 and 2 mM VPA. Furthermore, VPA was confirmed to inhibit HDAC activity and induce global hyperacetylation of histone H3. Notably, VPA was shown to suppress p16 INK4a, a biomarker gene of cervical carcinoma, and to increase the abundance of the tumor suppressor protein p21WAF1/Cip1, thus contributing to the basic knowledge regarding the antitumorigenic potential of VPA. Exploration of epigenetic changes associated with the promoters of p16 INK4a and p21 WAF1/Cip1, such as histone H3 methylation, may provide further information and improve the understanding of these findings.

Tamarixetin ameliorates cerebral ischemia-reperfusion injury via suppressing nicotinamide adenine dinucleotide phosphate oxidase 2/nucleotide-binding oligomerization domain like receptor family pyrin domain-containing 3 inflammasome activation.

Tamarixetin, a natural dietary flavone, exhibits remarkable potential for the treatment of ischemic stroke. The present article aimed to explore the impact of tamarixetin on ischemic stroke and elucidate the underlying mechanisms. Effects of tamarixetin on ischemic stroke were evaluated in rats using the middle cerebral artery occlusion and reperfusion (MCAO/R) model, by assessing the neurological deficit scores, brain water content, brain infraction, and neuronal damage. The levels of proinflammatory cytokines, NLRP3 inflammasome activation, reactive oxygen species (ROS) production, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression were measured in MCAO/R rats and lipopolysaccharide-stimulated cells. Tamarixetin administration improved the neurological dysfunction and neuronal loss in MCAO/R rats. In addition, tamarixetin reduced microglial hyperactivation and proinflammatory cytokines expression in vivo and in vitro. Tamarixetin attenuated NF-κB p65 phosphorylation and promoter activity, reduced NLRP3 expression and caspase-1 cleavage, and downregulated IL-1β and IL-18 secretions to suppress NLRP3 inflammasome activation. The levels of superoxide anion, hydrogen peroxide, and ROS were also suppressed by tamarixetin. The downregulation of NADP+ and NADPH levels, and gp91phox expression indicated the ameliorative effects of tamarixetin on NADPH oxidase activation. In the gp91phox knockdown cells treated with lipopolysaccharide, the effects of tamarixetin on NADPH oxidase activation, ROS generation, and NLRP3 inflammasome activation were diminished. Moreover, tamarixetin protects neurons against microglial hyperactivation in vitro. Our findings support the potential of tamarixetin as a therapeutic agent for ischemic stroke, and its mechanism of action involves the inhibition of NADPH oxidase-NLRP3 inflammasome signaling.

Induced dual-target rebalance simultaneously enhances efficient therapeutical efficacy in tumors

Multiple gene abnormalities are major drivers of tumorigenesis. NF-κB p65 overactivation and cGAS silencing are important triggers and genetic defects that accelerate tumorigenesis. However, the simultaneous correction of NF-κB p65 and cGAS abnormalities remains to be further explored. Here, we propose a novel Induced Dual-Target Rebalance (IDTR) strategy for simultaneously correcting defects in cGAS and NF-κB p65. By using our IDTR approach, we showed for the first time that oncolytic adenovirus H101 could reactivate silenced cGAS, while silencing GAU1 long noncoding RNA (lncRNA) inhibited NF-κB p65 overactivation, resulting in efficient in vitro and in vivo antitumor efficacy in colorectal tumors. Intriguingly, we further demonstrated that oncolytic adenoviruses reactivated cGAS by promoting H3K4 trimethylation of the cGAS promoter. In addition, silencing GAU1 using antisense oligonucleotides significantly reduced H3K27 acetylation at the NF-κB p65 promoter and inhibited NF-κB p65 transcription. Our study revealed an aberrant therapeutic mechanism underlying two tumor defects, cGAS and NF-κB p65, and provided an alternative IDTR approach based on oncolytic adenovirus and antisense oligonucleotides for efficient therapeutic efficacy in tumors.

DNMT1 regulates human erythropoiesis by modulating cell cycle and endoplasmic reticulum stress in a stage-specific manner

The dynamic balance of DNA methylation and demethylation is required for erythropoiesis. Our previous transcriptomic analyses revealed that DNA methyltransferase 1 (DNMT1) is abundantly expressed in erythroid cells at all developmental stages. However, the role and molecular mechanisms of DNMT1 in human erythropoiesis remain unknown. Here we found that DNMT1 deficiency led to cell cycle arrest of erythroid progenitors which was partially rescued by treatment with a p21 inhibitor UC2288. Mechanically, this is due to decreased DNA methylation of p21 promoter, leading to upregulation of p21 expression. In contrast, DNMT1 deficiency led to increased apoptosis during terminal stage by inducing endoplasmic reticulum (ER) stress in a p21 independent manner. ER stress was attributed to the upregulation of RPL15 expression due to the decreased DNA methylation at RPL15 promoter. The upregulated RPL15 expression subsequently caused a significant upregulation of core ribosomal proteins (RPs) and thus ultimately activated all branches of unfolded protein response (UPR) leading to the excessive ER stress, suggesting a role of DNMT1 in maintaining protein homeostasis during terminal erythroid differentiation. Furthermore, the increased apoptosis was significantly rescued by the treatment of ER stress inhibitor TUDCA. Our findings demonstrate the stage-specific role of DNMT1 in regulating human erythropoiesis and provide new insights into regulation of human erythropoiesis.

Persistent epigenetic signals propel a senescence-associated secretory phenotype and trained innate immunity in CD34+ hematopoietic stem cells from diabetic patients

BackgroundDiabetes-induced trained immunity contributes to the development of atherosclerosis and its complications. This study aimed to investigate in humans whether epigenetic signals involved in immune cell activation and inflammation are initiated in hematopoietic stem/progenitor cells (HSPCs) and transferred to differentiated progeny.Methods and resultsHigh glucose (HG)-exposure of cord blood (CB)-derived HSPCs induced a senescent-associated secretory phenotype (SASP) characterized by cell proliferation lowering, ROS production, telomere shortening, up-regulation of p21 and p27genes, upregulation of NFkB-p65 transcription factor and increased secretion of the inflammatory cytokines TNFα and IL6. Chromatin immunoprecipitation assay (ChIP) of p65 promoter revealed that H3K4me1 histone mark accumulation and methyltransferase SetD7 recruitment, along with the reduction of repressive H3K9me3 histone modification, were involved in NFkB-p65 upregulation of HG-HSPCs, as confirmed by increased RNA polymerase II engagement at gene level. The differentiation of HG-HSPCs into myeloid cells generated highly responsive monocytes, mainly composed of intermediate subsets (CD14hiCD16+), that like the cells from which they derive, were characterized by SASP features and similar epigenetic patterns at the p65 promoter. The clinical relevance of our findings was confirmed in sternal BM-derived HSPCs of T2DM patients. In line with our in vitro model, T2DM HSPCs were characterized by SASP profile and SETD7 upregulation. Additionally, they generated, after myeloid differentiation, senescent monocytes mainly composed of proinflammatory intermediates (CD14hiCD16+) characterized by H3K4me1 accumulation at NFkB-p65 promoter.ConclusionsHyperglycemia induces marked chromatin modifications in HSPCs, which, once transmitted to the cell progeny, contributes to persistent and pathogenic changes in immune cell function and composition.

Abstract 3025: Germline p53 R181 variants and DNA binding cooperativity in tumorigenesis

Abstract TP53 is the most frequently mutated gene in cancer, and its encoded protein p53 has many tumor-suppressive functions. There are several different classes of mutant p53 acquired in human cancer and inherited in cancer-prone families with Li-Fraumeni Syndrome, where individuals have an 80-90% increased risk of cancer. These include structural mutations that are generally (e.g R175H) or locally (e.g Y220C) misfolded, DNA contact mutations (e.g R273H); and oligomerization mutations (A347D). Cooperativity mutations affect binding of the p53 tetramer to DNA via disruption of a salt bridge formed by the negatively charged glutamic acid (E) 180 residue of one p53 monomer and the positively charged arginine (R) 181 residue of another. p53 “cooperativity” mutations at the R181 residue (R181H and R181C) have been increasingly identified in cancer-prone families undergoing genetic testing; however, the mechanism by which these variants disrupt p53 tumor suppression in humans is not understood. We show that the purified DNA binding domains of p53 variants R181H and R181C bind less cooperatively to the p53 binding site in the CDKN1A (p21) promoter, despite retaining wild-type levels of structural stability. RNA-sequencing of CRISPR knock-in colorectal and breast cancer cell lines shows reduced ability of R181H and R181C to transactivate a curated set of ~300 known p53 target genes. Upon treatment with p53 activating molecule Nutlin-3a, the R181-mutant cells fail to cell cycle arrest in the G1 phase and maintain high proliferative rates. Interestingly, we observe some residual apoptotic activity in R181H and R181C mutant cells treated with DNA-damaging agent 5-fluorouracil, despite losing the transactivation of proapoptotic p53 targets; this suggests that these mutants retain the p53 transcription-independent mechanism of apoptosis which is observed in other p53 variants such as P47S and A347D. These studies will define the pathogenicity of R181 variants and guide therapeutic intervention for patients that harbor these p53 mutations. Citation Format: Renyta Moses, Ryan Hausler, Gregory Kelly, Alexandra Indeglia, Sven Miller, John Karanicolas, Maureen Murphy, Kara Maxwell. Germline p53 R181 variants and DNA binding cooperativity in tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3025.

MT1G Regulates c-MYC/P53 Signal to Inhibit Proliferation, Invasion and Migration and Promote Apoptosis in Colon Cancer Cells.

Colon cancer is a common and malignant cancer featuring high morbidity and poor prognosis. This study was performed to explore the regulatory role of MT1G in colon cancer as well as its unconcealed molecular mechanism. The expressions of MT1G, c-MYC, and p53 were assessed with the application of RT-qPCR and western blot. The impacts of MT1G overexpression on the proliferative ability of HCT116 and LoVo cells were measured by CCK-8 and BrdU incorporation assays. Additionally, transwell wound healing, and flow cytometry assays were employed to evaluate the invasive and migrative capacities as well as the apoptosis level of HCT116 and LoVo cells. Moreover, the activity of the P53 promoter region was assessed with the help of a luciferase reporter assay. It was found that the expressions of MT1G at both mRNA and protein levels were greatly decreased in human colon cancer cell lines, particularly in HCT116 and LoVo cell lines. After transfection, it was discovered that the MT1G overexpression suppressed the proliferation, migration and invasion but promoted the apoptosis of HCT116 and LoVo cells, which were then partially reversed after overexpressing c-MYC. Additionally, MT1G overexpression reduced c-MYC expression but enhanced the p53 expression, revealing that the MT1G overexpression could regulate c-MYC/P53 signal. Elsewhere, it was also shown that c-MYC overexpression suppressed the regulatory effects of MT1G on P53. To conclude, MT1G was verified to regulate c-MYC/P53 signal to repress the proliferation, migration and invasion but promote the apoptosis of colon cancer cells, which might offer a novel targeted-therapy for the improvement of colon cancer.

TARGETING P21CIP1-HIGHLY-EXPRESSING SENESCENT CELLS TO IMPROVE HEALTHSPAN IN PROGEROID MICE

Abstract Aging is the leading risk factor for a variety of chronic diseases. Emerging evidence suggests that modulating fundamental aging processes such as cellular senescence may delay the onset of most chronic conditions as a group and increase healthy lifespan. Cellular senescence refers to the essentially irreversible growth arrest that occurs when cells experience stress. One common feature of senescent cells is the high expression level of p16Ink4a and/or p21Cip1. We and others have demonstrated the causal roles of p16Ink4a-highly-expressing (p16high) senescent cells in various age-related conditions. However, the role of p21high senescent cells in aging remains largely unknown. To address these questions, we have generated and validated a novel p21-Cre mouse model containing p21 promoter driving inducible Cre. By crossing with floxed mice, we managed to monitor, sort, image, eliminate, or modulate p21high cells in vivo. We investigated the maximal walking speed and grip strength of klotho (kl)-/- progeroid mice weekly starting from 5 weeks old until death. We showed that clearance of p21high cells can improve physical function in kl-/- mice for a long time period. Additionally, inactivation of NF-κB specifically in p21high cells also can improve tissue function in kl-/- mice. This study will provide invaluable research tools and pinpoint the role of cellular senescence in age-related tissue dysfunction.

TARGETING OF P21CIP1-HIGHLY-EXPRESSING CELLS ALLEVIATES NEUROPSYCHIATRIC DISORDERS WITH OBESITY

Abstract Obesity remains a major factor for diabetes associated with insulin resistance and increases the risk of neuropsychiatric disorders, such as anxiety, depression, and cognitive deficits, resulting in a shortened in lifespan and healthspan. Cellular senescence, a state of permanent cell-cycle arrest, has been recognized as an important biological process underlying obesity and obesity-associated neurological disorders. My project aims to alleviate metabolic and neuropsychiatric dysfunction in obesity by targeting of p21Cip1-highly-expressing (p21high) cells, a rarely examined senescent cell population. By leveraging a novel transgene containing a p21 promoter driving Cre, we demonstrate the accumulation of p21high cells in brain with obesity, whereas, intermittently clearing p21high cells alleviates anxiety- and depression-related behavior and improve the cognitive function for obese mice. Of note, clearance of p21high cells decreased key factors of the senescence transcriptional program in hippocampus and amygdala. A survival study shows the intermittent clearance of p21high cells extends the lifespan of obese mice fed with high-fat diet. Our findings indicate p21high cells serve as a potential therapeutic target for neuropsychiatric disorders.

SET8 is a novel negative regulator of TGF-β signaling in a methylation-independent manner

Transforming growth factor β (TGF-β) is a multifunctional cytokine that induces a diverse set of cellular processes principally through Smad-dependent transcription. Transcriptional responses induced by Smads are tightly regulated by Smad cofactors and histone modifications; however, the underlying mechanisms have not yet been elucidated in detail. We herein report lysine methyltransferase SET8 as a negative regulator of TGF-β signaling. SET8 physically associates with Smad2/3 and negatively affects transcriptional activation by TGF-β in a catalytic activity-independent manner. The depletion of SET8 results in an increase in TGF-β-induced plasminogen activator inhibitor-1 (PAI-1) and p21 expression and enhances the antiproliferative effects of TGF-β. Mechanistically, SET8 occupies the PAI-1 and p21 promoters, and a treatment with TGF-β triggers the replacement of the suppressive binding of SET8 with p300 on these promoters, possibly to promote gene transcription. Collectively, the present results reveal a novel role for SET8 in the negative regulation of TGF-β signaling.

LncRNA DCST1-AS1 drives the malignant progression of pediatric acute myeloid leukemia via regulating p53 methylation.

To uncover the biological role of lncRNA DCST1-AS1 in the process of pediatric AML and its regulatory effect on p53 methylation. Serum level of DCST1-AS1 in AML children and healthy participants was detected by qRT-PCR. The role of DCST1-AS1 in mediating biological functions of AML193 and U937 cells was assessed by functional experiments. p53 methylation level was examined. Through BSP, MSP and dual-luciferase reporter assay, the regulatory effect of DCST1-AS1 on p53 methylation was explored. The correlation between DCST1-AS1 and p53 in the serum of AML children was assessed. Serum level of DCST1-AS1 was higher in AML children than in healthy subjects. Knockdown of DCST1-AS1 decreased proliferative and migratory rates in AML193 and U937 cells. DCST1-AS1 was able to induce methylation of p53 promoter. P53 was markedly upregulated by the knockdown of DCST1-AS1, presenting a negative correlation. LncRNA DCST1-AS1 drives the malignant progression of pediatric AML through inducing methylation of the p53 promoter.

CREB3 facilitates Donafenib resistance in hepatocellular carcinoma cells via the LSD1/CoREST/p65 axis by transcriptionally activating long noncoding RNA ZFAS1

ObjectiveDrug resistance greatly limits the therapeutic effect of a drug. This study aimed to explore the role of long noncoding RNA ZFAS1 in Donafenib resistance of hepatocellular carcinoma (HCC) cells. MethodsThe expression of CREB3, ZFAS1, and p65 in HCC cell lines was measured by RT-qPCR and western blotting. After transfection with sh-ZFAS1, sh-CREB3, or sh-CREB3 + oe-p65 in Donafenib-resistent (DR) HCC cell lines, the transfection efficiency was evaluated by RT-qPCR and western blotting. The proliferation and IC50 to Donafenib of HCC cell lines was examined by MTT assay. Cell proliferation and apoptosis were examined by colony formation and flow cytometry assays. Then, the correlation amongst CREB3, ZFAS1, LSD1/CoREST, and p65 was analysed by ChIP, dual-luciferase reporter gene, and RIP assays. ResultsZFAS1, CREB3, and p65 were upregulated in HepG2-DR and Huh7-DR cells. Silencing of ZFAS1 or CREB3 enhanced the sensitivity of HCC cells to Donafenib, inhibited cell proliferation and IC50, and increased cell apoptosis, which were reversed by p65 overexpression. Mechanistically, CREB3 bound to ZFAS1 promoter to augment ZFAS1 transcriptional expression, and ZFAS1 recruited LSD1/CoREST to the p65 promoter region to decrease H3K4 methylation and elevate p65 transcriptional expression. ConclusionCREB3 overexpression contributed to Donafenib resistance in HCC cells by activating the ZFAS1/p65 axis.

ARID3A and ARID3B exert direct regulatory control over the long non-coding RNAs (lncRNAs) MALAT1 and NORAD within the context of non-small cell lung cancer (NSCLC)

Lung cancer, known for its high mortality rates and poor prognosis, remains one of the most prevalent cancer types. Early detection and effective treatment methods are crucial for improving survival rates. Non-small cell lung cancer (NSCLC) accounts for approximately 85 % of all lung cancer cases. Long non-coding RNAs (lncRNAs), which play vital roles in various biological processes, have been implicated in the development of cancer and can impact key therapeutic targets in different cancer types. In NSCLC, the dysregulation of specific lncRNAs, such as MALAT1 and NORAD, has been associated with neoplastic initiation, progression, metastasis, tumor angiogenesis, chemoresistance, and genomic instability. Both MALAT1 and NORAD directly regulate the expression of the transcription factor E2F1, thereby influencing cell cycle progression. Additionally, MALAT1 has been reported to affect the expression of p53 target genes, leading to cell cycle progression through the repression of p53 promoter activity. NORAD, on the other hand, is indirectly regulated by p53. The AT-rich interaction domain (ARID) family of DNA-binding proteins, particularly ARID3A and ARID3B, are involved in various biological processes such as cell proliferation, differentiation, and development. They also play significant roles in E2F-dependent transcription and are transcriptional targets of p53. The intricate balance between promoting cellular proliferation through the pRB-E2F pathway and inducing growth arrest through the p53 pathway underscores the crucial regulatory role of ARID3A, ARID3B, and their interaction with lncRNAs MALAT1 and NORAD. In this study, we aimed to investigate the potential interactive and functional connections among ARID3A, ARID3B, MALAT1, and NORAD in NSCLC, considering their involvement in the pRB-E2F and p53 pathways. Our findings strongly suggest that ARID3A and ARID3B play a regulatory role in controlling MALAT1 and NORAD in NSCLC. Specifically, our study demonstrates that the activities of MALAT1 and NORAD were markedly increased upon the overexpression of ARID3A and ARID3B. Therefore, we can conclude that ARID3A and ARID3B likely contribute significantly to the oncogenic functions of MALAT1 and NORAD in NSCLC. Consequently, targeting ARID3A and ARID3B could hold promise as a therapeutic approach in NSCLC, given their direct control over the expression of MALAT1 and NORAD.

Epigenetic control of inflammation by histone methylation in cardiometabolic heart failure with preserved ejection fraction

Abstract Background Obesity represents one of the most common comorbidities in patients with heart failure with preserved ejection fraction (HFpEF). Histone post-translational modifications by chromatin modifying enzymes (CMEs) are emerging as pivotal regulators of gene transcription in the heart. Purpose To investigate the role of chromatin remodelling in obese HFpEF (obHFpEF). Methods Unbiased gene expression profiling of CMEs (PCR array) was performed in left ventricular (LV) myocardial specimens from obHFpEF patients and age-matched control donors (n=8/group). Among myocardial CMEs, the methyltransferase SETD7 showed the highest variation in gene expression. Hence, we investigated the role of SETD7 and its chromatin mark H3K4me1 in a murine model of obHFpEF. Mice with cardiomyocyte-specific deletion of SETD7 (c-SETD7-/-) and control littermates (SETD7fl/fl) were generated and subjected to high fat diet feeding and L-NAME treatment for 15 weeks to induce obHFpEF. Echocardiography and Treadmill exhaustion test were performed. ChIP-Seq datasets were employed to determine the biological pathways regulated by SETD7, whereas chromatin immunoprecipitation assays (ChIP) were performed to investigate SETD7/H3k4me1 enrichment on target gene promoters. SETD7 gain- and loss-of-function experiments were performed in cultured neonatal rat ventricular myocytes (NRVMs) exposed to palmitic acid (200µM) for 48h. Selective inhibition of SETD7 by (R)-PFI-2 was performed in skinned cardiomyocytes isolated from left ventricular specimens of obHFpEF patients. Passive stiffness, a main feature of HFpEF, was assessed before and after RPFI-2 treatment. Results CMEs profiling showed SETD7 as the top-ranking transcript (fold change, 7.36, P < 0.01) in myocardial specimens from obHFpEF patients as compared to controls. ChIP-Seq in CMs showed a strong enrichment of SETD7 and H3k4me1 on the promoter of NF-kB p65 gene, a master regulator of inflammation. SETD7 and H3k4me1 were upregulated in HFpEF vs. control mouse hearts, showed enrichment on NF-kB p65 promoter and were associated with IL-1β and IL-6 upregulation. In HFpEF mice, cardiomyocyte-specific deletion of SETD7 protected against LV hypertrophy, diastolic dysfunction (assessed by E/E’ ratio) and lung congestion while improving exercise tolerance. At the molecular level, SETD7 deletion blunted H3K4me1 enrichment on p65 promoter thus preventing the upregulation of inflammatory genes and myocardial apoptosis. In cultured CMs exposed to PA, SETD7 inhibition by (R)-PFI-2 prevented H3k4me1-driven p65 upregulation, whereas SETD7 overexpression mimicked HFpEF features. Moreover, knockdown of NF-kB p65 prevented IL-1β/IL-6 transcription in SETD7-overexpressing CMs. Of clinical relevance, (R)-PFI-2 reduced passive stiffness in skinned CMs isolated from obHFpEF patients. Conclusions Pharmacological targeting of SETD7 may represent a new strategy to prevent myocardial inflammation in cardiometabolic HFpEF.

Epigenetically-driven persistence of inflammatory and oxidative phenotypes in adolescents born to women with gestational diabetes

Abstract Background According to the theory of developmental origins of health and disease1, environmental changes during the in-utero period can permanently affect health and vulnerability to diseases later in life. As a matter of fact, the offspring of women with gestational diabetes (GD) exhibit a higher risk for developing obesity, type 2 diabetes (T2D), and atherosclerotic cardiovascular disease (ASCVD) than those individuals with a family history of diabetes2–4. Importantly, epigenetics has emerged as a crucial determinant for this transmission across generations5–8. Mixed Lineage Leukemia 1 (MLL1) histone methyltransferase activates inflammatory and oxidative pathways through trimethylation of histone 3 at lysine 4 amino (H3K4me3)9. Purpose To investigate whether such epigenetic mark regulates genes triggering inflammation and oxidative stress in GD women, and its potential transmission and persistence in their offspring. Methods We isolated peripheral blood mononuclear cells (PBMCs), fetal cord blood mononuclear cells (CBMCs), and umbilical cord vein endothelial cells (HUVECs) from GD and control pregnant women, as well as PBMCs and plasma samples from a retrospective cohort of adolescents (12-16 years old) born to control or GD women. Gene and protein expressions were investigated by RT-PCR, immunoblotting and/or confocal microscopy. H3K4me3 levels on NF-kB p65 and NADPH oxidase isoform 4 (NOX4) promoter regions were assessed by chromatin immunoprecipitation (ChIP) and RT-PCR. Mechanistic studies were performed in control- and GD-HUVECs using the MLL1 inhibitor MM-102 to assess downstream inflammatory mediators and superoxide anion (O2-) generation by electron spin resonance (ESR) spectroscopy. Results Our data showed upregulation of MLL1 and H3K4me3 levels in PBMCs, HUVECs, and CBMCs isolated from GD as compared to control women. ChIP analysis revealed H3K4me3 enrichment in the promoter region of the inflammatory and oxidative master regulatory genes NF-kB p65 and NOX4, respectively, in these cells. We also found an upregulation of NF-kB p65 target genes IL-6, MCP-1, ICAM-1, and VCAM-1, and increased monocyte adhesion and O2- production. The inhibition of MLL1 was able to reduce H3K4me3 levels blunting the inflammatory and oxidative phenotypes in GD-HUVECs. Interestingly enough, we demonstrated a persistent increase of H3K4me3 in the NF-kB p65 promoter region of PBMCs together with an elevation of inflammatory markers in plasma from adolescents born to GD women. Conclusions Our results suggest that GD may induce inflammation and oxidative stress through MLL1-dependent H3K4me3 mechanisms. Such epigenetic mark is transmitted to the offspring and, most importantly, can still be found in adolescence as a pro-inflammatory trigger. These findings pave the way for pharmacological reprogramming of adverse histone modifications to counteract early abnormal phenotypes in GD offspring which may accelerate ASCVD burden.