DNA Damage-regulated Autophagy Modulator Research Articles

The p53 target DRAM1 modulates calcium homeostasis and ER stress by promoting contact between lysosomes and the ER through STIM1

It is well established that DNA Damage Regulated Autophagy Modulator 1 (DRAM1), a lysosomal protein and a target of p53, participates in autophagy. The cellular functions of DRAM1 beyond autophagy remain elusive. Here, we show p53-dependent upregulation of DRAM1 in mitochondrial damage-induced Parkinson's disease (PD) models and exacerbation of disease phenotypes by DRAM1. We find that the lysosomal location of DRAM1 relies on its intact structure including the cytosol-facing C-terminal domain. Excess DRAM1 disrupts endoplasmic reticulum (ER) structure, triggers ER stress, and induces protective ER-phagy. Mechanistically, DRAM1 interacts with stromal interacting molecule 1 (STIM1) to tether lysosomes to the ER and perturb STIM1 function in maintaining intracellular calcium homeostasis. STIM1 overexpression promotes cellular health by restoring calcium homeostasis, ER stress response, ER-phagy, and AMP-activated protein kinase (AMPK)-Unc-51 like autophagy activating kinase 1 (ULK1) signaling in cells with excess DRAM1. Thus, by promoting organelle contact between lysosomes and the ER, DRAM1 modulates ER structure and function and cell survival under stress. Our results suggest that DRAM1 as a lysosomal protein performs diverse roles in cellular homeostasis and stress response. These findings may have significant implications for our understanding of the role of the p53/DRAM1 axis in human diseases, from cancer to neurodegenerative diseases.

DNA damage-regulated autophagy modulator 1 prevents glioblastoma cells proliferation by regulating lysosomal function and autophagic flux stability

Glioblastoma (GBM) is the most aggressive and life-threatening brain tumor, characterized by its highly malignant and recurrent nature. DNA damage-regulated autophagy modulator 1 (DRAM-1) is a p53 target gene encoding a lysosomal protein that induces macro-autophagy and damage-induced programmed cell death in tumor growth. However, the precise mechanisms underlying how DRAM-1 affects tumor cell proliferation through regulation of lysosomal function and autophagic flux stability remain incompletely understood. We found that DRAM-1 expressions were evidently down-regulated in high-grade glioma and recurrent GBM tissues. The upregulation of DRAM-1 could increase mortality of primary cultured GBM cells. TEM analysis revealed an augmented accumulation of aberrant lysosomes in DRAM-1-overexpressing GBM cells. The assay for lysosomal pH and stability also demonstrated decreasing lysosomal membrane permeabilization (LMP) and impaired lysosomal acidity. Further research revealed the detrimental impact of lysosomal dysfunction, which impaired the autophagic flux stability and ultimately led to GBM cell death. Moreover, downregulation of mTOR phosphorylation was observed in GBM cells following upregulation of DRAM-1. In vivo and in vitro experiments additionally illustrated that the mTOR inhibitor rapamycin increased GBM cell mortality and exhibited an enhanced antitumor effect.

Naked Gene Delivery Induces Autophagy for Effective Treatment of Acute Lung Injury in a Mouse Model.

Acute lung injury (ALI) leads to diffuse pulmonary interstitial and alveolar edema, further developing into acute respiratory distress syndrome (ARDS). The present therapeutic approaches showed limited effects with poor clinical efficacy or severe side effects. This study aims to develop novel pharmaceutical agents to reduce lung damage with acceptable side effects for ALI. Naked gene delivery system based on epigallocatechin 3-gallate (EGCG) was synthesized to deliver plasmid expressing DNA damage regulated autophagy modulator 1 (DRAM1), designated as EGCG/DRAM1 (ED). ED was characterized by dynamic light scattering analysis and transmission electron microscope. The biodistribution of ED in mice was measured by an in vivo small animal imaging system. The therapeutic potentials of ED were evaluated in MLE12 cells and LPS-induced ALI mice. Our results showed that ED was nearly spherical with a diameter of ~100 nm and increased the stability of DRAM1 plasmid that encapsulated. The synthesized ED showed negligible toxicity at the selected experimental concentration in MLE12 cells. ED could be taken up by MLE12 cells with high efficiency and escape from the lysosome. In ALI mice, ED facilitated the accumulation and retention of DRAM1 plasmid in lung, and attenuated pulmonary edema and pulmonary vascular permeability. The therapeutic effects of ED on ALI were associated with increased autophagy and reduced oxidative stress in lung. In summary, ED attenuated pulmonary edema and pulmonary vascular permeability, and improved pulmonary dysfunction in ALI mice. This naked gene delivery system for autophagy enhancement may serve as a potential therapeutic strategy to attenuate ALI.

Balance between autophagy and cell death is maintained by Polycomb-mediated regulation during stem cell differentiation.

Autophagy is a conserved cytoprotective process, aberrations in which lead to numerous degenerative disorders. While the cytoplasmic components of autophagy have been extensively studied, the epigenetic regulation of autophagy genes, especially in stem cells, is less understood. Deciphering the epigenetic regulation of autophagy genes becomes increasingly relevant given the therapeutic benefits of small-molecule epigenetic inhibitors in novel treatment modalities. We observe that, during retinoic acid-mediated differentiation of mouse embryonic stem cells (mESCs), autophagy is induced, and identify the Polycomb group histone methyl transferase EZH2 as a regulator of this process. In mESCs, EZH2 represses several autophagy genes, including the autophagy regulator DNA damage-regulated autophagy modulator protein 1 (Dram1). EZH2 facilitates the formation of a bivalent chromatin domain at the Dram1 promoter, allowing gene expression and autophagy induction during differentiation while retaining the repressive H3K27me3 mark. EZH2 inhibition leads to loss of the bivalent domain, with consequent 'hyper-expression' of Dram1, accompanied by extensive cell death. This study shows that Polycomb group proteins help maintain a balance between autophagy and cell death during stem cell differentiation, in part, by regulating the expression of the Dram1 gene.

Ginsenoside Rb2 suppresses cellular senescence of human dermal fibroblasts by inducing autophagy

BackgroundGinsenoside Rb2, a major active component of Panax ginseng, has various physiological activities, including anticancer and anti-inflammatory effects. However, the mechanisms underlying the rejuvenation effect of Rb2 in human skin cells have not been elucidated. MethodsWe performed a senescence-associated β-galactosidase staining assay to confirm cellular senescence in human dermal fibroblasts (HDFs). The regulatory effects of Rb2 on autophagy were evaluated by analyzing the expression of autophagy marker proteins, such as microtubule-associated protein 1A/1B-light chain (LC) 3 and p62, using immunoblotting. Autophagosome and autolysosome formation was monitored using transmission electron microscopy. Autophagic flux was analyzed using tandem-labeled GFP-RFP-LC3, and lysosomal function was assessed with Lysotracker. We performed RNA sequencing to identify potential target genes related to HDF rejuvenation mediated by Rb2. To verify the functions of the target genes, we silenced them using shRNAs. ResultsRb2 decreased β-galactosidase activity and altered the expression of cell cycle regulatory proteins in senescent HDFs. Rb2 markedly induced the conversion of LC3-Ⅰ to LC3-Ⅱ and LC3 puncta. Moreover, Rb2 increased lysosomal function and red puncta in tandem-labeled GFP-RFP-LC3, which indicate that Rb2 promoted autophagic flux. RNA sequencing data showed that the expression of DNA damage-regulated autophagy modulator 2 (DRAM2) was induced by Rb2. In autophagy signaling, Rb2 activated the AMPK-ULK1 pathway and inactivated mTOR. DRAM2 knockdown inhibited autophagy and Rb2-restored cellular senescence. ConclusionRb2 reverses cellular senescence by activating autophagy via the AMPK-mTOR pathway and induction of DRAM2, suggesting that Rb2 might have potential value as an antiaging agent.

The Clinical Spectrum and Disease Course of DRAM2 Retinopathy.

Pathogenic variants in DNA-damage regulated autophagy modulator 2 gene (DRAM2) cause a rare autosomal recessive retinal dystrophy and its disease course is not well understood. We present two Slovenian patients harboring a novel DRAM2 variant and a detailed review of all 23 other patients described to date. Whole exome and whole genome sequencing were performed in the two patients, and both underwent ophthalmological examination with a 2-year follow-up. PubMed was searched for papers with clinical descriptions of DRAM2 retinopathy. Patient 1 was homozygous for a novel variant, p.Met1?, and presented with the acute onset of photopsia and retina-wide retinopathy at the age of 35 years. The patient was first thought to have an autoimmune retinopathy and was treated with mycophenolate mofetil, which provided some symptomatic relief. Patient 2 was compound heterozygous for p.Met1? and p.Leu246Pro and presented with late-onset maculopathy at the age of 59 years. On review, patients with DRAM2 retinopathy usually present in the third decade with central visual loss, outer retinal layer loss on optical coherence tomography and a hyperautofluorescent ring on fundus autofluorescence. Either cone–rod or rod–cone dystrophy phenotype is observed on electroretinography, reflecting the importance of DRAM2 in both photoreceptor types. Non-null variants can result in milder disease.

EGFR-AS1 Promotes Nonsmall Cell Lung Cancer (NSCLC) Progression via Downregulating the miR-524-5p/DRAM1 Axis and Inhibiting Autophagic Lysosomal Degradation.

Nonsmall cell lung cancer (NSCLC) accounts for the majority of lung cancers. Studies have revealed the regulatory role of lncRNAs in cancer pathogenesis and their potential use as diagnostic and prognostic biomarkers. The epidermal growth factor receptor antisense RNA 1 (EGFR-AS1) has been reported to be upregulated in NSCLC tissues, while its detailed mechanism in lung cancer needs to be explored. DNA damage-regulated autophagy modulator 1 (DRAM1) has been known to act as a tumor suppressor in NSCLC, and miR-524-5p has been reported to be a biomarker in idiopathic pulmonary fibrosis and different lung disorders. Our investigation revealed that EGFR-AS1 is highly expressed in lung cancer tissues, and its knockdown inhibited lung cancer cell invasion and viability and reduced tumor growth in vivo. We also found that EGFR-AS1 targets miR-524-5p, and there was a negative correlation between their expressions in lung cancer tissues. Simultaneously, miR-524-5p has been found to promote DRAM1 expression. In addition, the inhibition of miR-524-5p diminished DRAM1 protein expression and promoted lung cancer cell invasion. Our study has revealed that EGFR-AS1 contributes to the pathogenesis of NSCLC by inhibiting autophagic-lysosomal degradation via targeting the miR-524-5p/DRAM1 axis. This finding elucidated for the first time the role of EGFR-AS1 in lung cancer progression and the positive regulatory function of miR-524-5p in regulating DRAM1 protein and suppressing lung cancer progression. This novel mechanism provided a better insight into the pathogenesis of lung cancer and presented a better strategy for the treatment of lung cancer.

Identifying gene network patterns and associated cellular immune responses in children with or without nut allergy.

BackgroundAlthough evidence suggests that the immune system plays a key role in the pathophysiology of nut allergy, the precise immunological mechanisms of nut allergy have not been systematically investigated. The aim of the present study was to identify gene network patterns and associated cellular immune responses in children with or without nut allergy.MethodsTranscriptome profiling of whole blood cells was compared between children with and without nut allergy. Three genes were selected to be validated on a larger cohort of samples (n = 86) by reverse transcription-polymerase chain reactions (RT-qPCR). The composition of immune cells was inferred from the transcriptomic data using the CIBERSORTx algorithm. A co-expression network was constructed employing weighted gene co-expression network analysis (WGCNA) on the top 5000 most variable transcripts. The modules were interrogated with pathway analysis tools (InnateDB) and correlated with clinical phenotypes and cellular immune responses.ResultsProportions of neutrophils were positively correlated and CD4+ T-cells and regulatory T-cells (Tregs) were negatively correlated with modules of nut allergy. We also identified 2 upregulated genes, namely Interferon Induced With Helicase C Domain 1 (IFIH1), DNA damage-regulated autophagy modulator 1 (DRAM1) and a downregulated gene Zinc Finger Protein 512B (ZNF512B) as hub genes for nut allergy. Further pathway analysis showed enrichment of type 1 interferon signalling in nut allergy.ConclusionsOur findings suggest that upregulation of type 1 interferon signalling and neutrophil responses and downregulation of CD4+ T-cells and Tregs are features of the pathogenesis of nut allergy.

DRAM1 increases the secretion of PKM2-enriched EVs from hepatocytes to promote macrophage activation and disease progression in ALD

DNA damage-regulated autophagy modulator 1 (DRAM1) could play important roles in inflammation and hepatic apoptosis, while its roles in alcohol-related liver disease (ALD), which is characterized by hepatic inflammation and apoptosis, are still unclear. In this study, we explored the expression, role, and mechanism of DRAM1 in ALD. Firstly, our results showed that DRAM1 was significantly increased in liver tissues of mice at the early stage of alcohol treatment. In addition, DRAM1 knockout reduced, and liver-specific overexpression of DRAM1 aggravated, alcohol-induced hepatic steatosis, injury, and expressions of M1 macrophage markers in mice. Furthermore, ethanol-induced DRAM1 of hepatic cells increased pyruvate kinase M2 (PKM2)-enriched extracellular vesicles (EVs), and ectosomes derived from hepatic cells with DRAM1 overexpression promoted macrophage activation. Mechanistic investigations showed that DRAM1 interacted with PKM2 and increased the PKM2 level in plasma membrane. At last, DRAM1 was significantly increased in liver tissues of ALD patients, and it was positively correlated with M1 macrophage markers. Taken together, this study revealed that ethanol-induced DRAM1 of hepatic cells could increase the PKM2-enriched EVs, promote macrophage activation, and aggravate the disease progression of ALD. These findings suggested that DRAM1 might be a potentially promising target for the therapy of ALD.

Abstract 2383: Mir-7974: An oncogenic miRNA that perpetuates gastric cancer

Abstract The global incidence of gastric cancer exceeds 1 million people per year and has a 5-year survival rate of 31%. Current chemotherapies are not effective because of genetic and epigenetic heterogeneity and late stage detection. Previously, we showed that exportin 1 (XPO1) is a therapeutic target in gastric cancer, as it facilitates the nuclear export of various cancer related tumor suppressor proteins (TSPs) through direct interaction with their nuclear export sequences (NES). XPO1 is upregulated in a variety of tumors including gastric cancer, leading to increased cytoplasmic localization of TSPs supporting malignant transformation and cancer cell proliferation, Knockdown of XPO1 leads to anti-tumor effects and pharmacologic targeting of XPO1 is achievable with selective inhibitors of nuclear export (SINE) compounds. In 2019 and 2020, the first generation XPO1 inhibitor KPT-330 was FDA approved for the use in penta-refractory multiple myeloma and non-Hodgkin's Lymphoma. Besides protein, mRNA and non-coding RNAs are also transported across the nuclear membrane. Non-coding RNA transport is generally attributed to another nuclear export protein, exportin 5 (XPO5), which plays a critical role in microRNA (miRNA) biogenesis. Nevertheless, there is emerging evidence supporting the role of XPO1 in non-coding RNA transport. In order to elucidate the role of XPO1 in non-coding RNA biology, we performed small RNA sequencing on gastric cancer cell lines treated with the XPO1 inhibitor selinexor. Our results revealed statistically significant changes in miR-7974 and miR-129-1-3p. These two miRs are recognized to influence protein modifications and cell survival. Administration of a miR-7974 mimic and a miR-129-1-3p inhibitor reduced proliferation of gastric cancer cells. Next, we evaluated the miR target genes of interest. At the mRNA level, we found upregulation of RasGEF Domain Family Member 1a (RASGEF1A), downregulation of DNA damage-regulated autophagy modulator 1 (DRAM1), downregulation of TIMP2 and downregulation of Cyclin G2 protein encoding gene CCNG2. Western Blots confirmed inhibition of AKT, RAS and Wnt signaling. Our results also showed that miR-7974 overexpression correlated with cellular XPO1 levels through the enhancement of XPO1 and its cargoes RCC1 and RANBP2. Finally inhibition of XPO1 by SINE compounds reversed the observed oncogenic phenotypes caused by these microRNAs. Our results unveil a novel interaction network between XPO1 and small non-coding RNAs and show that XPO1 induced small-noncoding microRNAs influence gastric cancer proliferation, and can be reversed by SINE compounds. This implies that modulation of microRNAs might be explored as a biomarker for XPO1 inhibitor therapy and warrants further investigation. Citation Format: Rachel E. Sexton, Mohammad Najeeb Al-Hallak, Bayan Al-Share, Yiwei Li, Amro Aboukameel, Yosef Landesman, Trinayan Kashyap, Steve Kim, Ramzi M. Mohammad, Philip A. Philip, Asfar S. Azmi. Mir-7974: An oncogenic miRNA that perpetuates gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2383.

TP53/p53-FBXO22-TFEB controls basal autophagy to govern hormesis

ABSTRACT Preconditioning with a mild stressor such as fasting is a promising way to reduce severe side effects from subsequent chemo- or radiotherapy. However, the underlying mechanisms have been largely unexplored. Here, we demonstrate that the TP53/p53-FBXO22-TFEB (transcription factor EB) axis plays an essential role in this process through upregulating basal macroautophagy/autophagy. Mild stress-activated TP53 transcriptionally induced FBXO22, which in turn ubiquitinated KDM4B (lysine-specific demethylase 4B) complexed with MYC-NCOR1 suppressors for degradation, leading to transcriptional induction of TFEB. Upregulation of autophagy-related genes by increased TFEB dramatically enhanced autophagic activity and cell survival upon following a severe stressor. Mitogen-induced AKT1 activation counteracted this process through the phosphorylation of KDM4B, which inhibited FBXO22-mediated ubiquitination. Additionally, fbxo22−/− mice died within 10 h of birth, and their mouse embryonic fibroblasts (MEFs) showed a lowered basal autophagy, whereas FBXO22-overexpressing mice were resistant to chemotherapy. Taken together, these results suggest that TP53 upregulates basal autophagy through the FBXO22-TFEB axis, which governs the hormetic effect in chemotherapy. Abbreviations: BBC3/PUMA: BCL2 binding component 3; CDKN1A/p21: cyclin dependent kinase inhibitor 1A; ChIP-seq: chromatin immunoprecipitation followed by sequencing; DDB2: damage specific DNA binding protein 2; DRAM: DNA damage regulated autophagy modulator; ESR/ER: estrogen receptor 1; FMD: fasting mimicking diet; HCQ: hydroxychloroquine; KDM4B: lysine-specific demethylase 4B; MAP1LC3/LC3: microtubule associated protein 1 light chain 3 alpha; MEFs: mouse embryonic fibroblasts; MTOR: mechanistic target of rapamycin kinase; NCOR1: nuclear receptor corepressor 1; SCF: SKP1-CUL-F-box protein; SQSTM1: sequestosome 1; TFEB: transcription factor EB

DNA Damage-Regulated Autophagy Modulator 1 (DRAM1) Mediates Autophagy and Apoptosis of Intestinal Epithelial Cells in Inflammatory Bowel Disease.

DNA damage-regulated autophagy modulator 1 (DRAM1)is required for induction of autophagy and apoptosis. However, the influence of DRAM1 on the pathogenesis of inflammatory bowel disease (IBD) has not been explored. DRAM1 expression was examined in the intestinal mucosa of patients with IBD and colons of colitis mice. We used a recombinant adeno-associated virus carrying small hairpain DRAM1 to knock down the DRAM1 gene to treat colitis in the mice. The effect of DRAM1 on autophagy and apoptosis of intestinal epithelial cells was explored. DRAM1-mediated interaction with the c-Jun N-terminal kinase (JNK) pathway was also examined. DRAM1 expression in the intestinal mucosa of the IBD patients was higher than that in the control participates. DRAM1 expression in the inflammatory cells in patients with Crohn's disease (CD) was lower than that in patients with ulcerative colitis (UC). Additionally, DRAM1 expression was correlated with the Simple Endoscopic Score for CD and the Mayo endoscopic score for UC. Serum levels of DRAM1 in the IBD group were substantially higher than those in the normal group. The knockdown of DRAM1 could alleviate colitis symptoms in mice. In in vitro experiments, knocking down DRAM1 could reduce autophagy and apoptosis levels. Mechanistically, DRAM1 may participate in the regulation of these two processes by positively regulating JNK activation. During intestinal inflammation, the upregulation of DRAM1 may promote the activation of JNK and further aggravate intestinal epithelium damage.

Silencing miR-125b-5p attenuates inflammatory response and apoptosis inhibition in mycobacterium tuberculosis-infected human macrophages by targeting DNA damage-regulated autophagy modulator 2 (DRAM2)

ABSTRACT Tuberculosis is one of the most important infectious diseases worldwide and macrophage apoptosis is the major host defense mechanism against TB. We attempted to characterize the role of miRNA (miR)-125b-5p on mycobacterium tuberculosis (Mtb) infection and macrophages behaviors in vitro. According to fluorescence-activated cell separation (FACS), primary monocytes (CD14+) in TB patients were accumulated, and apoptotic monocytes were decreased. Peripheral blood mononuclear cells (PBMCs)-derived macrophages (MDMs) and monocytic cells THP-1-derived macrophage-like cells (TDMs) in vitro were used to be infected with H37Rv. After infection, colony-forming units assay revealed the increase of bacterial activity, FACS demonstrated the decrease of apoptosis rate of MDMs and TDMs, as well as promoted levels of IL-6, TNF-α, Bax, and Bim and suppressed levels of IL-10 and Bcl-2, examined by enzyme-linked immunosorbent assay (ELISA) and western blot assay. Expression of miR-125b-5p and DNA damage-regulated autophagy modulator 2 (DRAM2) was examined, and real-time PCR and western blot assay showed that miR-125b-5p was upregulated, whereas DRAM2 was downregulated in primary monocytes and H37Rv-infected macrophages (MDMs and TDMs). Moreover, blocking miR-125b-5p could attenuated H37Rv-induced bacterial activity and inflammatory response of MDMs and TDMs, accompanied with apoptosis inhibition. Whereas these effects of miR-125b-5p knockdown were abolished by downregulating DRAM2. In mechanism, DRAM2 was a downstream target of miR-125b-5p, as evidenced by dual-luciferase reporter assay. Collectively, silencing miR-125b-5p could protect human macrophages against Mtb infection through promoting apoptosis and inhibiting inflammatory response via targeting DRAM2, suggesting a novel target for Mtb eliminating. Abbreviations: TB: tuberculosis; PBMCs: peripheral blood mononuclear cells; Mtb: mycobacterium tuberculosis; AFB: acid fast bacilli; FITC: fluorescein isothiocyanate; MDMs: monocytes-derived macrophages; TDMs: THP-1-derived macrophage-like cells; ERFP: Mtb-enhanced red fluorescent protein; CFU: colony-forming units; ELISA: enzyme-linked immunosorbent assay; FACS: fluorescence-activated cell separation; PI: propidium iodide; DRAM2: DNA damage-regulated autophagy modulator 2; Real-time PCR: real-time polymerase chain reaction; in-miR-125b-5p: miR-125b-5p inhibitor; si-DRAM2: siRNA against DRAM2

DRAM1 plays a tumor suppressor role in NSCLC cells by promoting lysosomal degradation of EGFR

Lung cancer is the leading cause of cancer-associated mortality worldwide. DNA damage-regulated autophagy modulator 1 (DRAM1) plays an important roles in autophagy and tumor progression. However, the mechanisms by which DRAM1 inhibits tumor growth are not fully understood. Here, we report that DRAM1 was decreased in nonsmall-cell lung carcinoma (NSCLC) and was associated with poor prognosis. We confirmed that DRAM1 inhibited the growth, migration, and invasion of NSCLC cells in vitro. Furthermore, overexpression of DRAM1 suppressed xenografted NSCLC tumors in vivo. DRAM1 increased EGFR endocytosis and lysosomal degradation, downregulating EGFR signaling pathway. On one side, DRAM1 interacted with EPS15 to promote EGFR endocytosis, as evidence by the results of proximity labeling followed by proteomics; on the other, DRAM1 recruited V-ATP6V1 subunit to lysosomes, thereby increasing the assemble of the V-ATPase complex, resulting in decreased lysosomal pH and increased activation of lysosomal proteases. These two actions of DRAM1 results in acceleration of EGFR degradation. In summary, these in vitro and in vivo studies uncover a novel mechanism through which DRAM1 suppresses oncogenic properties of NSCLC by regulating EGFR trafficking and degradation and highlights the potential value of DRAM1 as a prognostic biomarker in lung cancers.

Deficiency in the autophagy modulator Dram1 exacerbates pyroptotic cell death of Mycobacteria-infected macrophages

DNA damage regulated autophagy modulator 1 (DRAM1) is a stress-inducible regulator of autophagy and cell death. DRAM1 has been implicated in cancer, myocardial infarction, and infectious diseases, but the molecular and cellular functions of this transmembrane protein remain poorly understood. Previously, we have proposed DRAM1 as a host resistance factor for tuberculosis (TB) and a potential target for host-directed anti-infective therapies. In this study, we generated a zebrafish dram1 mutant and investigated its loss-of-function effects during Mycobacterium marinum (Mm) infection, a widely used model in TB research. In agreement with previous knockdown analysis, dram1 mutation increased the susceptibility of zebrafish larvae to Mm infection. RNA sequencing revealed major effects of Dram1 deficiency on metabolic, immune response, and cell death pathways during Mm infection, and only minor effects on proteinase and metabolic pathways were found under uninfected conditions. Furthermore, unchallenged dram1 mutants did not display overt autophagic defects, but autophagic targeting of Mm was reduced in the absence of Dram1. The phagocytic ability of macrophages in dram1 mutants was unaffected, but acidification of Mm-containing vesicles was strongly reduced, indicating that Dram1 is required for phagosome maturation. By in vivo imaging, we observed that Dram1-deficient macrophages fail to restrict Mm during early stages of infection. The resulting increase in bacterial burden could be reverted by knockdown of inflammatory caspase a (caspa) and gasdermin Eb (gsdmeb), demonstrating pyroptosis as the mechanism underlying premature cell death of Mm-infected macrophages in dram1 mutants. Collectively, these data demonstrate that dissemination of mycobacterial infection in zebrafish larvae is promoted in the absence of Dram1 due to reduced maturation of mycobacteria-containing vesicles, failed intracellular containment, and consequent pyroptotic death of infected macrophages. These results provide new evidence that Dram1 plays a central role in host resistance to intracellular infection, acting at the crossroad of autophagy and cell death.

KCNQ1OT1 accelerates gastric cancer progression via miR-4319/DRAM2 axis.

Introduction:This work was to explore the connection of KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1) and microRNA-4319 (miR-4319), and to investigate the associated underlying mechanisms in gastric cancer (GC) progression.Methods:Quantitative real-time PCR was performed to measure KCNQ1OT1, miR-4319 and DNA-damage regulated autophagy modulator 2 (DRAM2) expression levels in GC cells. Moreover, expression level of KCNQ1OT1 and DRAM2 in GC tissues was analyzed at ENCORI website (http://starbase.sysu.edu.cn/index.php). Cell proliferation, colony formation assay and flow cytometry assays were performed to analyze effects of KCNQ1OT1, miR-4319 and DRAM2 on cell growth and death. Dual-luciferase activity reporter assay and RNA immunoprecipitation assay was conducted to verify the interactions of KCNQ1OT1 or DRAM2 and miR-4319.Results and Conclusion:We found KCNQ1OT1 level was increased in tumor tissues and cells. Force the expression of KCNQ1OT1 promotes, while knockdown KCNQ1OT1 inhibits GC cell growth. Further studies indicated miR-4319 functioned as a bridge between KCNQ1OT1 and DRAM2. Finally, we showed KCNQ1OT1/miR-4319/DRAM2 axis regulates GC cell growth in vitro and in vivo. lncRNA KCNQ1OT1 promotes GC progression by sponging miR-4319 to upregulate DRAM2, indicating KCNQ1OT1 might be a promising target for GC treatment.

DRAM1 deficiency affects the organization and function of the Golgi apparatus

DRAM1 (DNA damage-regulated autophagy modulator 1) is a transmembrane protein that predominantly localizes to the lysosome but is also found in other membranous organelles; however, its function in these organelles remains largely unknown. We found that DRAM1 was partially located in the Golgi apparatus, and knockdown of DRAM1 caused fragmentation of the Golgi apparatus in cells. The phenomenon of fragmented Golgi was not related to microtubule organization, and there was no direct interaction between DRAM1 and Golgi structural proteins (ARF1, GM130, syntaxin 6 and GRASP55). Moreover, Golgi-targeting DRAM1 failed to rescue the fragmentation of Golgi in DRAM1-deficient cells. The transport of ts045-VSVG-GFP, an indicator of movement from the Golgi apparatus to the plasma membrane, was delayed in DRAM1-knockdown cells. Moreover, the trafficking of CI-MPR from the plasma membrane to the Golgi was also impeded in DRAM1-knockdown cells. These results indicated that DRAM1 regulated the structure of the Golgi apparatus and affected Golgi apparatus-associated vesicular transport.

Downregulation of microRNA-425-5p suppresses cervical cancer tumorigenesis by targeting AIFM1.

Although microRNA-425-5p (miR-425-5p) has been previously revealed to be upregulated in cervical cancer, the cellular function of miR-425-5p in cervical cancer remains unknown. The aim of the current study was to investigate the cellular function of miR-425-5p and its underlying mechanism in cervical cancer. Reverse transcription-quantitative polymerase chain reaction was used to measure miR-425-5p expression in several cervical cancer cell lines. TargetScan bioinformatics analysis was used to predict apoptosis-inducing factor mitochondria-associated 1 (AIFM1) as a novel target of miR-425-5p, and this was verified by dual-luciferase reporter assay. Furthermore, cell transfections were used to investigate the role of miR-425-5p in cervical cancer. The effect of miR-425-5p on cell viability and apoptosis in HeLa cells was detected by MTT assay and flow cytometry, respectively. The present study demonstrated that miR-425-5p was significantly upregulated in cervical cancer cell lines. In addition, AIFM1 was identified as a direct target of miR-425-5p and negatively regulated by miR-425-5p. Downregulation of miR-425-5p inhibited HeLa cell viability and induced cell apoptosis. Furthermore, downregulation of miR-425-5p significantly increased the protein and mRNA expression levels of cytochrome c, caspase-3, caspase-9 and DNA damage regulated autophagy modulator 1. The effects of miR-425-5p inhibition on HeLa cell viability and apoptosis were significantly reversed by AIFM1 knockdown. In conclusion, the present study demonstrated that miR-425-5p was upregulated in cervical cancer, and downregulation of miR-425-5p inhibited cervical cancer cell growth by targeting AIFM1.

Clinical significance of serum DRAM1 mRNA, ARSA mRNA, hsa-miR-2053 and lncRNA-RP1-86D1.3 axis expression in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is a lethal cancer mainly caused by chronic exposure of asbestos. In this pilot study, we aimed to assess the expression of serum RNA-based biomarker panel exploring their clinical utility as diagnostic and prognostic biomarkers for MPM. We have selected an MPM-specific RNA-based biomarker panel through bioinformatics analysis based on the integration of DNA damage regulated autophagy modulator 1 (DRAM1) and arylsulfatase A ( ARSA) gene expression with their epigenetic regulators microRNA ( miR-2053) and long noncoding RNA ( lncRNA-RP1-86D1.3). Then, quantitative real-time polymerase chain reaction (qPCR) validation in sera of 60 MPM patients, 20 chronic asbestos exposure patients, and 20 healthy volunteers was done. Lastly, the prognostic power of the selected panel was assessed. The expression of serum DRAM1 messenger RNA (mRNA), ARSA mRNA, hsa-miR-2053 and lncRNA-RP1-86D1.3 were positive in 78.3%, 90%, 85%, and 83.3% of MPM patients, respectively. The RNA-based biomarker panel was able to discriminate between MPM patients and controls with high accuracy and their combined sensitivity reached 100% for the diagnosis of MPM. Kaplan-Meier analysis showed that hsa-miR-2053 is an independent prognostic factor of MPM. Our preliminary data revealed that the chosen RNAs play an important role in driving MPM development and progression.

Ten-eleven translocation 1 regulates methylation of autophagy-related genes in human glioma.

Ten-eleven translocation 1 catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which plays an important role in epigenetics and is related to the malignant biological behavior of tumors. However, its regulatory role in glioma remains unclear. In this study, the levels of 5mC and 5hmC were detected using immunohistochemistry, dot-blot, hMeDIP-chip, and western blot in glioma tissues and normal brain tissues, whereas 5hmC differentially enriched genes were determined and further validated. The level of 5hmC in gliomas was decreased, whereas 5mC was increased. 5hmC highly enriched 10 functional protein-coding genes and 10 signaling pathways were identified using hMeGIP-chip in glioma tissues. Two autophagy-related genes, ATG13 and DNA damage-regulated autophagy modulator protein 1, with low enrichment of 5hmC in glioma tissues were verified in the promoter region, and hMeGIP-PCR further confirmed this result in U251 cells. Immunohistochemistry further confirmed that autophagy level in glioma tissues was lower than that of normal controls, and negatively correlated with WHO grade. This study indicates that ten-eleven translocation 1 may be involved in the development and progression of glioma through demethylation regulating a variety of cellular functions and signaling pathways, and autophagy is one of the regulatory mechanisms.