DNA Damage-regulated Autophagy Modulator 1 Research Articles

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.

DRAM1 plays a tumor suppressor role in clear cell renal cell carcinoma through modulating Akt signaling

BackgroundClear cell renal carcinoma (ccRCC) is one of the most common malignant tumors worldwide. DNA damage-regulated autophagy modulator1 (DRAM1) plays an important roles in apoptosis and tumor progression. However, the role of DRAM1 in ccRCC is still unknown. In our study, we aimed to investigate the effect of DRAM1 in the progression of ccRCC. MethodsThe expression and prognostic information of DRAM1 in ccRCC were obtained by immunohistochemistry staining and bioinformatics database. Cell proliferation, migration, invasion were detected by CCK-8 assay, wound-healing and transwell assays, and the cell apoptosis was examined by tunel assay and flow cytometry analysis. Western blot was used to detect the expression of DRAM1, Bax, Bcl2, Akt, p53,E-cadherin, N-cadherin of ccRCC cells. ResultsDecreased expression of DRAM1 was found in ccRCC tissues, which predicted a shorter survival rate in ccRCC patient. We confirmed that DRAM1 inhibited the proliferation, migration, invasion and epithelial mesenchymal transformation (EMT), while enhanced the apoptosis of ccRCC cells. In addition, the results of inhibition of Akt signaling were consistent with the above. We further proved that DRAM1 over-expression decreased the phosphorylation of Akt signaling, and overexpression of DRAM1 could reverse oncogenic function induced by the over-activating of Akt in ccRCC cells. Conclusionoverexpression of DRAM1 plays a tumor suppressive role in ccRCC through inactivation of Akt and highlights the potential role of DRAM1 as a prognostic biomarker in ccRCC.

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.

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.

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.

Role of DRAM1 in mitophagy contributes to preeclampsia regulation in mice.

Preeclampsia (PE) is a complication during pregnancy that is diagnosed by a new onset of hypertension and proteinuria. Although the pathogenesis of PE is not fully understood, a growing body of evidence indicates that oxidative stress and mitochondrial dysfunction might contribute to the progression of PE. Therefore, the aim of the present study was to determine the role of mitophagy in mitochondrial dysfunction and oxidative stress in PE, and to evaluate the role of DNA damage-regulated autophagy modulator 1 (DRAM1) in the development of PE. First, a mouse model of PE induced by hypoxia-inducible factor 1α was established, and high levels of oxidative stress, apoptosis and mitochondrial dysfunction were found in the placentas of PE mice. Additionally, the placentas of PE mice exhibited decreased mitophagy and significantly decreased DRAM1 expression. To further explore the role of DRAM1 in mitophagy, DRAM1 was overexpressed in the placental tissues of PE mice, and this overexpression effectively improved the symptoms of PE mice and significantly reduced blood lipid and urine protein levels. DRAM1 overexpression also improved mitochondrial function and reduced oxidative stress in the placentas of PE mice. In addition, the overexpression of DRAM1 improved mitochondrial fusion and fission, and enhanced mitophagy. Altogether, these results indicated a key role for DRAM1 in mitophagy that contributed to the regulation of PE. To the best of the authors' knowledge, the present study provided the first evidence of a role for DRAM1 in PE, and offered novel insight into the pathophysiological mechanisms of PE.

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.

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.

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.

DRAM1 regulates the migration and invasion of hepatoblastoma cells via autophagy-EMT pathway.

DNA-damage regulated autophagy modulator 1 (DRAM1) is known as a target of TP53-mediated autophagy, and has been reported to promote the migration and invasion abilities of glioblastoma stem cells. However, the precise contribution of DRAM1 to cancer cell invasion and migration, and the underlying mechanisms remain unclear. In the present study, small interfering (si)RNA or short hairpin RNA mediated knockdown of DRAM1 was performed in hepatoblastoma cells and the migration and invasion abilities were detected in vitro and in vivo. To investigate the underlying mechanisms, western blotting and immunofluorescence were used to detect the expression of autophagy-associated proteins and epithelial-mesenchymal-transition (EMT)-associated markers. The results showed that DRAM1 knockdown by specific siRNA abrogated cell autophagy, as well as inhibited the migration and invasion of HepG2 cells in Transwell assays, which may be reversed by rapamycin treatment. In addition, DRAM1 knockdown increased the expression of E-Cadherin while decreased the expression of vimentin in HepG2 cells, which was also be reversed by rapamycin treatment. Taken together, these results suggest that DRAM1 is involved in the regulation of the migration and invasion of HepG2 cells via autophagy-EMT pathway.

TP53-dependent autophagy links the ATR-CHEK1 axis activation to proinflammatory VEGFA production in human bronchial epithelial cells exposed to fine particulate matter (PM2.5)

ABSTARCTEpidemiological and clinical studies have increasingly shown that fine particulate matter (PM2.5) is associated with a number of pathological respiratory diseases, such as bronchitis, asthma, and chronic obstructive pulmonary disease, which share the common feature of airway inflammation induced by particle exposure. Thus, understanding how PM2.5 triggers inflammatory responses in the respiratory system is crucial for the study of PM2.5 toxicity. In the current study, we found that exposing human bronchial epithelial cells (immortalized Beas-2B cells and primary cells) to PM2.5 collected in the winter in Wuhan, a city in southern China, induced a significant upregulation of VEGFA (vascular endothelial growth factor A) production, a signaling event that typically functions to control chronic airway inflammation and vascular remodeling. Further investigations showed that macroautophagy/autophagy was induced upon PM2.5 exposure and then mediated VEGFA upregulation by activating the SRC (SRC proto-oncogene, non-receptor tyrosine kinase)-STAT3 (signal transducer and activator of transcription 3) pathway in bronchial epithelial cells. By exploring the upstream signaling events responsible for autophagy induction, we revealed a requirement for TP53 (tumor protein p53) activation and the expression of its downstream target DRAM1 (DNA damage regulated autophagy modulator 1) for the induction of autophagy. These results thus extend the role of TP53-DRAM1-dependent autophagy beyond cell fate determination under genotoxic stress and to the control of proinflammatory cytokine production. Moreover, PM2.5 exposure strongly induced the activation of the ATR (ATR serine/threonine kinase)-CHEK1/CHK1 (checkpoint kinase 1) axis, which subsequently triggered TP53-dependent autophagy and VEGFA production in Beas-2B cells. Therefore, these findings suggest a novel link between processes regulating genomic integrity and airway inflammation via autophagy induction in bronchial epithelial cells under PM2.5 exposure.

Abstract 1678: DRAM1 regulates EMT of lung cancer A549 cells via SQSTM1/p62 in vitro

Abstract Epithelial-to-mesenchymal transition (EMT) has been linked to the regulation of non-small cell lung cancer (NSCLC) progression, migration, invasion, metastasis, and chemoresistance as well. Recent studies showed that autophagy was a novel target to mediate EMT of cancer cells through regulating the migration and invasion in cancer. The DNA Damage-Regulated Autophagy Modulator 1 (DRAM1) is an evolutionarily conserved transmembrane protein and localizes predominantly to lysosomes, while also colocalizing with the autophagosome marker LC3. Previous investigation discovered that DRAM1 played an important role in regulating autophagy, inducing apoptosis of NSCLC cells. However, the function and mechanism of DRAM1 mediated autophagy in regulating EMT in NSCLC remains poorly understood. In this study, transforming growth factor-β (TGF-β) and starvation were employed to induce autophagy for investigating EMT in NSCLC A549 cells. The results showed that after exposure to TGF-β 24h, the protein levels of DRAM1, autophagy marker SQSTM1/p62, LC3I/II and EMT marker vimentin significantly increased while another EMT marker E-cadherin protein decreased obviously compared with the control by western blot. Also the similar results were observed after starvation 48h in this study. To further illustrate the role of DRAM1 in regulation of autophagy and EMT, wildtype DRAM1 overexpression plasmid and DRAM1 siRNA was transfected into A549 cells respectively to check the autophagy markers and EMT markers expression, meanwhile wound healing test and cell invasion transwell chamber test were assayed after the gene manipulation. These experiments results demonstrated that DRAM1 overexpression dramatically increased the SQSTM1/p62, LC3I/II and vimentin protein levels and inhibited the expression of E-cadherin, promoting the migration and invasion abilities of A549 cells. Conversely, down-regulation of DRAM1 restrained SQSTM1/p62, LC3 I/II and vimentin protein levels and elevated the expression of E-cadherin, leading to the inhibition of migration and invasion abilities of A549 cells. Interestingly, our further study showed that after p62 siRNA transfected into A549 cells E-cadherin upregulated while vimentin reduced compared to the control group. Consistently, knockdown of p62 suppressed the migration and invasion abilities of A549 cells and down regulated the expression of twist, which is an EMT related transcription factor. These data suggested that DRAM1 played an important role in regulating the EMT of lung cancer A549 cells through SQSTM1/p62 modulating, which might be a novel potential target for lung cancer treatment. Citation Format: Binfeng He, Jiajing Cai, Guangcheng Huang, Fan Chang, Lei Xu, Dongsheng Wang, Yibin Deng, Xiaolan Guo. DRAM1 regulates EMT of lung cancer A549 cells via SQSTM1/p62 in vitro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1678.

N-Myc and STAT Interactor regulates autophagy and chemosensitivity in breast cancer cells.

We have previously reported that expression of NMI (N-myc and STAT interactor) is compromised in invasive breast cancers. We also demonstrated that loss of NMI expression promotes epithelial-mesenchymal-transition and results in enhanced invasive ability of breast cancer cells. Additionally we had demonstrated that restoration of NMI expression reduced breast cancer xenograft growth and downregulated Wnt and TGFβ/SMAD signaling. Here we present our observations that NMI expression drives autophagy. Our studies were promoted by our observation that NMI expressing breast cancer cells showed autophagic vacuoles and LC3 processing. Additionally, we found that NMI expression increased the cisplatin sensitivity of the breast cancer cells. Our mechanistic investigations show that NMI prompts activation of GSK3-β. This multifunctional kinase is an upstream effector of the TSC1/TSC2 complex that regulates mTOR signaling. Inhibition of GSK3-β activity in NMI expressing cells activated mTOR signaling and decreased the cells’ autophagic response. Additionally we demonstrate that a key component of autophagy, DNA-damage regulated autophagy modulator 1 (DRAM1), is regulated by NMI. Our TCGA database analysis reveals concurrent expression of NMI and DRAM1 in breast cancer specimens. We present evidence that NMI sensitizes breast cancer cells to cisplatin treatment through DRAM1 dependent autophagy.

DRAM1 regulates apoptosis through increasing protein levels and lysosomal localization of BAX.

DRAM1 (DNA damage-regulated autophagy modulator 1) is a TP53 target gene that modulates autophagy and apoptosis. We previously found that DRAM1 increased autophagy flux by promoting lysosomal acidification and protease activation. However, the molecular mechanisms by which DRAM1 regulates apoptosis are not clearly defined. Here we report a novel pathway by which DRAM1 regulates apoptosis involving BAX and lysosomes. A549 or HeLa cells were treated with the mitochondrial complex II inhibitor, 3-nitropropionic acid (3NP), or an anticancer drug, doxorubicin. Changes in the protein and mRNA levels of BAX and DRAM1 and the role of DRAM1 in BAX induction were determined. The interaction between DRAM1 and BAX and its effect on BAX degradation, BAX lysosomal localization, the release of cathepsin B and cytochrome c by BAX and the role of BAX in 3NP- or doxorubicin-induced cell death were studied. The results showed that BAX, a proapoptotic protein, was induced by DRAM1 in a transcription-independent manner. BAX was degraded by autophagy under basal conditions; however, its degradation was inhibited when DRAM1 expression was induced. There was a protein interaction between DRAM1 and BAX and this interaction prolonged the half-life of BAX. Furthermore, upregulated DRAM1 recruited BAX to lysosomes, leading to the release of lysosomal cathepsin B and cleavage of BID (BH3-interacting domain death agonist). BAX mediated the release of mitochondrial cytochrome c, activation of caspase-3 and cell death partially through the lysosome-cathepsin B-tBid pathway. These results indicate that DRAM1 regulates apoptosis by inhibiting BAX degradation. In addition to mitochondria, lysosomes may also be involved in BAX-initiated apoptosis.

A novel protein family controls trafficking and metabolism of glycosphingolipids (606.9)

Glycosphingolipid (GSL) trafficking and metabolism are closely linked with cell fate determination, balancing signaling cascades controlling proliferation, apoptosis and autophagy. Altered cellular levels of complex glycosphingolipids often correlate with cancer progression, yet little is known about the principles that guide trafficking of these lipids.Here we identified a family of proteins, consisting of five mammalian homologues of the yeast Sfk1 protein that are involved in ganglioside transport. These six‐transmembrane domain proteins are widely expressed, each showing a unique cellular localization spanning the ER, Golgi, lysosomes and plasma membrane. One of these proteins, DNA‐damage regulated autophagy modulator 1 (DRAM1), was recently described as a p53‐inducible lysosomal regulator of autophagy. However, the cellular function(s) of the Sfk1 homologues remains unknown. Here we describe that mammalian Sfk1 proteins control the cellular distribution and metabolism of complex GSLs and one of them, DRAM2, specifically regulates transport of ganglioside GM3 from the plasma membrane to the Golgi and lysosomes. DRAM2‐mediated trafficking of GM3, in turn, was found to be critical for processing GM3 into higher ganglioside species.We propose that the Sfk1/DRAM family of proteins recognize GSLs within membranes and facilitate their sorting and hence their metabolic fate within the cell.Grant Funding Source: Supported by the NICHD Intramural research program

DRAM1 regulates autophagy flux through lysosomes.

We have previously reported that the mitochondria inhibitor 3-nitropropionic acid (3-NP), induces the expression of DNA damage-regulated autophagy modulator1 (DRAM1) and activation of autophagy in rat striatum. Although the role of DRAM1 in autophagy has been previously characterized, the detailed mechanism by which DRAM1 regulates autophagy activity has not been fully understood. The present study investigated the role of DRAM1 in regulating autophagy flux. In A549 cells expressing wilt-type TP53, 3-NP increased the protein levels of DRAM1 and LC3-II, whereas decreased the levels of SQSTM1 (sequestosome 1). The increase in LC3-II and decrease in SQSTM1 were blocked by the autophagy inhibitor 3-methyl-adenine. Lack of TP53 or knock-down of TP53 in cells impaired the induction of DRAM1. Knock-down of DRAM1 with siRNA significantly reduced 3-NP-induced upregulation of LC3-II and downregulation of SQSTM1, indicating DRAM1 contributes to autophagy activation. Knock-down of DRAM1 robustly decreased rate of disappearance of induced autophagosomes, increased RFP-LC3 fluorescence dots and decreased the decline of LC3-II after withdraw of rapamycin, indicating DRAM1 promotes autophagy flux. DRAM1 siRNA inhibited lysosomal V-ATPase and acidification of lysosomes. As a result, DRAM1 siRNA reduced activation of lysosomal cathepsin D. Similar to DRAM1 siRNA, lysosomal inhibitors E64d and chloroquine also inhibited clearance of autophagosomes and activation of lysosomal cathapsin D after 3-NP treatment. These data suggest that DRAM1 plays important roles in autophagy activation induced by mitochondria dysfunction. DRAM1 affects autophagy through argument of lysosomal acidification, fusion of lysosomes with autophagosomes and clearance of autophagosomes.