BECN1 Expression Research Articles

Homocysteine activates autophagy by inhibition of CFTR expression via interaction between DNA methylation and H3K27me3 in mouse liver

Elevated homocysteine (Hcy) levels have been reported to be involved in liver injury, and autophagy plays an important role in normal hepatic physiology and pathophysiology, but the mechanism underlying Hcy regulated autophagy is currently unknown. In this study, CBS+/- mice were fed with regular diet for 12 weeks to establish a hyperhomocysteinemia (HHcy) model and HL-7702 cells were treated with Hcy, we found that Hcy increases autophagy and aggravates liver injury by downregulation of cystic fibrosis transmembrane conductance regulator (CFTR) expression in vivo and in vitro. Overexpression of CFTR inhibited the formation of autophagosomes and the expression of autophagy-related proteins BECN1, LC3-II/I and Atg12, while the expression of p62 increased in Hcy-treated hepatocytes and CBS+/- mice injected with lentivirus expressing CFTR. Further study showed that CFTR expression is regulated by the interaction of DNA methyltransferase 1 (DNMT1) and enhancer of zeste homolog 2 (EZH2), which, respectively, regulate DNA methylation and histone H3 lysine 27 trimethylation (H3K27me3). In conclusion, our study showed that Hcy activates autophagy by inhibition of CFTR expression via interaction between H3K27me3 and DNA methylation in the mouse liver. These findings provide new insight into the mechanism of Hcy-induced autophagy in liver injury.

The balance between apoptosis and autophagy regulates testis regression and recrudescence in the seasonal-breeding South American plains vizcacha, Lagostomus maximus.

Mammalian testis undergoes deep changes in their architecture and function during photoregression conditions in seasonal breeders. Particularly, the testicular mechanisms that regulate the transition between the active (functional) and inactive (regression) stage vary between species. The aim of the present study was to analyze the incidence of proliferation, apoptosis and autophagy in the testicular seminiferous ephitelium of a seasonal breeder, Lagostomus maximus, during the annual reproductive cycle. We observed that proliferating spermatogonia increased from the active testis until reaching the maximum peak in the activating testis. During the annual reproductive cycle, the quantity of apoptotic-TUNEL positive spermatogonia and meiotic germ cells was constant and this might be regulated by the members of the BCL2 family. Only in the activating testis, apoptosis of germ cells was almost undetectable. The analysis of the autophagic-related proteins BECN1 and LC3 showed their localization in Leydig cells and the germ cells in the active and activating testis. In the inactive testis, BECN1 and LC3 ceased to be immunolocalized within the seminiferous tubules and the mRNA expression of both regulators decreased. Moreover, the expression of BECN1 and LC3 and also the apoptotic index were up regulated in testicular cultures subjected to nutritional stress. These results suggest a possible interaction between apoptosis and autophagy in the active and activating testis (characterized by high metabolic requirement and nutrient), where autophagy could promote survival over cell death. In the inactive testis, the absence of autophagic-related proteins might explain the massive loss of germ cells, suggesting that autophagy plays new and key role in the alterations of the seminiferous epithelium during photoregression.

Melatonin reduces oxidative damage in mouse granulosa cells via restraining JNK-dependent autophagy.

Oxidative stress-induced granulosa cell (GCs) injury is believed to be a common trigger for follicular atresia. Emerging evidence indicates that excessive autophagy occurs in mammalian cells with oxidative damage. N-acetyl-5-methoxytrypamine (melatonin) has been shown to prevent GCs from oxidative injury, although the exact mechanism remains to be elucidated. Here, we first demonstrated that the suppression of autophagy through the JNK/BCL-2/BECN1 signaling is engaged in melatonin-mediated GCs protection against oxidative damage. Melatonin inhibited the loss of GCs viability, formation of GFP-MAP1LC3B puncta, accumulation of MAP1LC3B-II blots, degradation of SQSTM1 and the expression of BECN1, which was correlated with impaired activation of JNK during oxidative stress. On the other hand, blocking of autophagy and/or JNK also reduced the level of H2O2-induced GCs death, but failed to further restore GCs viability in the presence of melatonin. Particularly, the suppression of autophagy provided no additional protective effects when GCs were pretreated with JNK inhibitor and/or melatonin. Importantly, we found that the enhanced interaction between BCL-2 and BECN1 might be a responsive mechanism for autophagy suppression via the melatonin/JNK pathway. Moreover, blocking the downstream antioxidant system of melatonin using specific inhibitors further confirmed a direct role of melatonin/JNK/autophagy axis in preserving GCs survival without scavenging reactive oxygen species (ROS). Taken together, our findings uncover a novel function of melatonin in preventing GCs from oxidative damage by targeting JNK-mediated autophagy, which might contribute to develop therapeutic strategies for patients with ovulation failure-related disorders.

Overexpression of microRNA-506-3p aggravates the injury of vascular endothelial cells in patients with hypertension by downregulating Beclin1 expression.

The aim of the present study was to measure the expression of microRNA (miRNA)-506-3p in the peripheral blood of patients with hypertension and to determine the biological functions and mechanisms of action of miR-506-3p. A total of 61 patients with primary hypertension were included in the present study. Peripheral blood was collected from all patients, as well as 31 healthy subjects who were included in a control group. The expression of miR-506-3p in peripheral blood was determined by reverse transcription-quantitative polymerase chain reaction. Human umbilical vein endothelial cells (HUVECs) were transfected with miR-506-3p mimics or miR-506-3p inhibitor. The proliferation and migration of HUVECs were determined using cell-counting kit 8 and Transwell assays, respectively. The cell cycle and apoptosis of HUVECs were detected by flow cytometry. The expression of Beclin1 (BECN1) protein, a potential target of miR-506-3p, was measured using western blotting. A dual-luciferase reporter assay was performed to determine the interaction between BECN1 and miR-506-3p. It was demonstrated that miR-506-3p expression in the peripheral blood of patients with patients was upregulated and dependent on the severity of hypertension. miR-506-3p overexpression inhibited the proliferation and migration of HUVECs. In addition, miR-506-3p inhibited the transition from the G1 phase to the S-phase in HUVECs. Overexpression of miR-506-3p promoted the apoptosis of HUVECs. Notably, miR-506-3p downregulated the expression of BECN1 by directly binding to its 3′-untranslated region. The present study demonstrated that miR-506-3p expression is elevated in the peripheral blood of patients with hypertension and is associated with the severity of hypertension. By downregulating BECN1 expression, miR-506-3p aggravates injury in vascular endothelial cells by inhibiting the proliferation and migration of HUVECs, as well as promoting their apoptosis.

Inhibition of Autophagy Attenuated Ethylene Glycol Induced Crystals Deposition and Renal Injury in a Rat Model of Nephrolithiasis

Background/Aims: Nephrolithiasis is a common and frequently occurring disease, its exact pathogenesis is remains unclear. Emerging data suggest that autophagy plays a vital role in the pathophysiological processes of kidney diseases. Therefore, this study was designed to investigate the potential role of autophagy in the formation of calcium oxalate (CaOx) kidney stones in rat model. Methods: Thirty-two rats were randomly divided into four groups (eight rats/group): untreated control group, stone model group, rapamycin-treated group, chloroquine-treated group. Rat models of CaOx nephrolithiasis was administration of 0.75% ethylene glycol (EG) in their drinking water for 4 weeks. Western blot and transmission electron microscope (TEM) were used to detect the expression of autophagy related protein LC3-II, BECN1 and p62 and autophagic vacuoles respectively. Renal function was evaluated by measuring the levels of serum CRE and BUN. Renal tubular injury markers NGAL and Kim-1 was determined by ELISA kits. Von Kossa staining was used to assess crystal deposits and histological tissue injury. TUNEL staining was employed to assess apoptosis of the renal tubular cell. Results: Compare with the controls, the expression of autophagy related protein LC3-II, BECN1 and number of autophagic vacuoles were increased significantly, whereas the p62 protein level was decreased in the stone model group. The levels of apoptosis, serum CRE and BUN, NGAL and Kim-1 in the stone model group were increased compared with the control group and crystals deposition and renal injury were increased significantly. However, the levels of autophagy, kidney injury and crystal deposition were decreased by chloroquine but increased by rapamycin. Conclusion: These findings suggested that rats were administration of ethylene glycol could lead to the formation of CaOx nephrolithiasis and autophagy activation. Inhibiting autophagy could be an effective therapeutic approach for decreasing the formation of nephrolithiasis.

Inhibition of autophagy-attenuated calcium oxalate crystal-induced renal tubular epithelial cell injury in vivo and in vitro.

Accumulating evidence suggests that autophagy is involved in the pathophysiological processes of kidney diseases. However, the role of autophagy in the formation of calcium oxalate (CaOx) nephrolithiasis remains unclear. In this study, we investigated the effects of autophagy on renal tubular epithelial cell injury induced by CaOx crystals in vivo and in vitro. We first observed that the expression levels of LC3-II and BECN1 and number of autophagic vacuoles were markedly increased in the renal tissue of CaOx stone patients. We subsequently found that exposure of HK-2 cells to CaOx crystals could increase LC3-II and BECN1 expression as well as the number of GFP-LC3 dots and autophagic vacuoles in a dose- and time-dependent manner. In addition, our results suggest that CaOx crystals induced autophagy, at least in part, via activation of the reactive oxygen species (ROS) pathway in HK-2 cells. Furthermore, inhibition of autophagy using 3-methyladenine or siRNA knockdown of BECN1 attenuated CaOx crystal-induced HK-2 cells injury. However, enhancing autophagic activity with rapamycin exerted an opposite effect. Taken together, our results demonstrate that autophagy is essential for CaOx crystal-induced renal tubular epithelial cell injury and that inhibition of autophagy could be a novel therapeutic strategy for CaOx nephrolithiasis.

Polygonatum odoratum lectin promotes BECN1 expression and induces autophagy in malignant melanoma by regulation of miR1290.

Autophagy is not only a survival response to growth-factor or nutrient deprivation but also an important mechanism for tumor-cell suicide, including melanoma. Polygonatum odoratum lectin (POL) displays apoptosis- and autophagy-inducing effects in many human tumors. POL also inhibits the growth of melanoma cells, but its role and molecular mechanism in malignant melanoma remain unclear. In this study, we found that POL suppressed proliferation and induced autophagy in melanoma cells. miR1290 was upregulated and inhibited autophagy in melanoma. BECN1 is the direct functional effector of miR1290. Furthermore, we found that POL promoted BECN1 expression though inhibition of miR1290, thus inducing melanoma-cell autophagy. This finding elucidates a new role and mechanism for POL in melanoma, and provides a potential antineoplastic agent for melanoma treatment.

Blockage of the lysosome-dependent autophagic pathway contributes to complement membrane attack complex-induced podocyte injury in idiopathic membranous nephropathy

Dysregulation of autophagy-mediated podocyte homeostasis is proposed to play a role in idiopathic membranous nephropathy (IMN). In the present study, autophagic activity and lysosomal alterations were investigated in podocytes of IMN patients and in cultured podocytes exposed to sublytic terminal complement complex, C5b-9. C5b-9 upregulated the number of LC3 positive puncta and the expression of p62 in patient podocytes and in C5b-9 injuried podocyte model. The lysosomal turnover of LC3-II was not influenced, although the BECN1 expression level was upregulated after exposure of podocytes to C5b-9. C5b-9 also caused a significant increase in the number of autophagosomes but not autolysosomes, suggesting that C5b-9 impairs the lysosomal degration of autophagosomes. Moreover, C5b-9 exacerbated the apoptosis of podocytes, which could be mimicked by chloroquine treatment, indicating that C5b-9 triggered podocyte injury, at least partially through inhibiting autophagy. Subsequent studies revealed that C5b-9 triggered lysosomal membrane permeabilization, which likely caused the decrease in enzymatic activity, defective acidification of lysosomes, and suppression of DQ-ovalbumin degradation. Taken together, our results suggest that the lysosomal-dependent autophagic pathway is blocked by C5b-9, which may play a key role in podocyte injury during the development of IMN.

ADIPOQ/adiponectin induces cytotoxic autophagy in breast cancer cells through STK11/LKB1-mediated activation of the AMPK-ULK1 axis

ABSTRACT ADIPOQ/adiponectin, an adipocytokine secreted by adipocytes in the breast tumor microenvironment, negatively regulates cancer cell growth hence increased levels of ADIPOQ/adiponectin are associated with decreased breast cancer growth. However, its mechanisms of action remain largely elusive. We report that ADIPOQ/adiponectin induces a robust accumulation of autophagosomes, increases MAP1LC3B-II/LC3B-II and decreases SQSTM1/p62 in breast cancer cells. ADIPOQ/adiponectin-treated cells and xenografts exhibit increased expression of autophagy-related proteins. LysoTracker Red-staining and tandem-mCherry-GFP-LC3B assay show that fusion of autophagosomes and lysosomes is augmented upon ADIPOQ/adiponectin treatment. ADIPOQ/adiponectin significantly inhibits breast cancer growth and induces apoptosis both in vitro and in vivo, and these events are preceded by macroautophagy/autophagy, which is integral for ADIPOQ/adiponectin-mediated cell death. Accordingly, blunting autophagosome formation, blocking autophagosome-lysosome fusion or genetic-knockout of BECN1/Beclin1 and ATG7 effectively impedes ADIPOQ/adiponectin induced growth-inhibition and apoptosis-induction. Mechanistic studies show that ADIPOQ/adiponectin reduces intracellular ATP levels and increases PRKAA1 phosphorylation leading to ULK1 activation. AMPK-inhibition abrogates ADIPOQ/adiponectin-induced ULK1-activation, LC3B-turnover and SQSTM1/p62-degradation while AMPK-activation potentiates ADIPOQ/adiponectin's effects. Further, ADIPOQ/adiponectin-mediated AMPK-activation and autophagy-induction are regulated by upstream master-kinase STK11/LKB1, which is a key node in antitumor function of ADIPOQ/adiponectin as STK11/LKB1-knockout abrogates ADIPOQ/adiponectin-mediated inhibition of breast tumorigenesis and molecular analyses of tumors corroborate in vitro mechanistic findings. ADIPOQ/adiponectin increases the efficacy of chemotherapeutic agents. Notably, high expression of ADIPOQ receptor ADIPOR2, ADIPOQ/adiponectin and BECN1 significantly correlates with increased overall survival in chemotherapy-treated breast cancer patients. Collectively, these data uncover that ADIPOQ/adiponectin induces autophagic cell death in breast cancer and provide in vitro and in vivo evidence for the integral role of STK11/LKB1-AMPK-ULK1 axis in ADIPOQ/adiponectin-mediated cytotoxic autophagy.

Promoter Variant Alters Expression of the Autophagic BECN1 Gene: Implications for Clinical Manifestations of Machado-Joseph Disease.

Autophagy is especially important in disorders where accumulation of the mutant protein is a hallmark, such as the Machado-Joseph disease/spinocerebellar ataxia type 3 (MJD/SCA3). We analyzed the promoter of the BECN1 gene, whose overexpression has been reported to exert neuroprotective effects in MJD, with the aim of finding variants that could be associated with expression levels of beclin-1 and could be tested as modifiers of onset and disease severity. A fragment encompassing the BECN1 promoter was sequenced in 95 MJD subjects and 120 controls. The impact of the variation detected on transcription factors (TFs) binding affinity was evaluated in silico and inferences concerning levels of expression were confirmed by fluorescence-based quantitative real-time PCR in a subset of 28 MJD subjects and 26 controls. Four previously described (rs60221525, rs116943570, rs34882610, and rs34037822) and one novel (c.-933delG) variants were identified. In silico analysis performed for the most frequent variants-rs60221525 C allele and rs116943570 T allele-predicted an impact of the presence of these alleles on TF binding affinity. BECN1 expression levels were in agreement with the in silico predictions, showing a tendency for decreased levels in samples with the rs60221525 C allele and for increased levels in samples with the rs116943570 T allele. MJD patients carrying the rs60221525 C allele presented a tendency for earlier estimated age at onset. Moreover, patients with the rs60221525 C allele presented a more severe clinical picture, compared to patients without this allele. The analysis of a larger number of patients from different cohorts, currently unavailable, would be required to confirm these results.

Abstract 3319: Elevating adipokine adiponectin level can induce cytotoxic autophagy in breast cancer cells and potentiate the efficacy of chemotherapeutic regimens: preclinical studies

Abstract Adiponectin, an adipocytokine secreted by adipocytes in the breast tumor microenvironment, negatively regulates cancer cell growth hence increased levels of adiponectin are associated with decreased breast cancer growth. However, its mechanisms of action remain largely elusive. We report that adiponectin induces a robust accumulation of autophagosomes, increases LC3II and decreases p62/SQSTM1 in breast cancer cells. Adiponectin-treated cells and xenografts exhibit increased expression of autophagy-related proteins. Lysotracker-Red-staining and tandem-mCherry-GFP-LC3 assay show that autophagosomes/lysosomes fusion is augmented upon adiponectin treatment. Adiponectin significantly inhibits breast cancer growth and induces apoptosis both in vitro and in vivo, and these events are preceded by autophagy, which is integral for adiponectin-mediated cell death. Accordingly, blunting autophagosomes-formation, blocking autophagosomes-autolysosome fusion or genetic-knockout of BECN1/Beclin1/ATG7 effectively impedes adiponectin induced growth-inhibition and apoptosis-induction. Mechanistic studies show that adiponectin reduces intracellular ATP levels and increases AMPK phosphorylation leading to ATG1 activation. AMPK-inhibition abrogates adiponectin-induced ATG1-activation, LC3II-turnover and p62-degradation while AMPK-activation potentiates adiponectin’s effects. Further, adiponectin-mediated AMPK-activation and autophagy-induction are regulated by upstream master-kinase LKB1, which is a key node in anti-tumor function of adiponectin as LKB1-knockout abrogates adiponectin-mediated inhibition of breast tumorigenesis and molecular analyses of tumors corroborate in vitro mechanistic findings. Adiponectin increases the efficacy of chemotherapeutic agents. Notably, high expression of adiponectin receptor, adiponectin and BECN1 significantly correlates with increased overall survival in chemotherapy-treated breast cancer patients. Collectively, these data uncover that adiponectin induces autophagic cell death in breast cancer and provide in vitroa and in vivo evidence for the integral role of LKB1-AMPK-ATG1 axis in adiponectin-mediated cytotoxic-autophagy. Citation Format: Seung J Chung, Ganji Purnachandra Nagaraju, Arumugam Nagalingam, Nethaji Muniraj, Panjamurthy Kuppusamy, Alyssa Walker, Juhyung woo, Balázs Győrffy, Edward Gabrielson, Neeraj K. Saxena, Dipali Sharma. Elevating adipokine adiponectin level can induce cytotoxic autophagy in breast cancer cells and potentiate the efficacy of chemotherapeutic regimens: preclinical studies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3319. doi:10.1158/1538-7445.AM2017-3319

Lactobacillus fermentum Postbiotic-induced Autophagy as Potential Approach for Treatment of Acetaminophen Hepatotoxicity.

The aim of this study was to investigate the potential of postbiotics originated from Lactobacillus fermentum BGHV110 strain (HV110) to counteract acetaminophen (APAP)-induced hepatotoxicity in HepG2 cells. This strain was selected according to its autophagy inducing potential, based on previous studies reporting protective role of autophagy in APAP caused cellular damage. Cell viability was assessed using MTT and LDH assays, while autophagy was monitored by qPCR analysis of BECN1, Atg5, p62/SQSTM1, and PINK1 mRNA expression and by Western blot analysis of p62/SQSTM1 and lipidated LC3 accumulation. Our results showed that detrimental effect of APAP on cell viability was suppressed in the presence of HV110 which was linked with increased conversion of LC3 protein and p62/SQSTM1 protein degradation. Additionally, higher p62/SQSTM1 and PINK1 mRNA transcription were noticed in cells co-treated with APAP/HV110, simultaneously. In conclusion, this study suggests that HV110 enhances activation of PINK1-dependent autophagy in HepG2 cells and its eventual co-supplementation with APAP could be potentially used for alleviation of hepatotoxic side effects caused by APAP overdose.

Mir30c Is Involved in Diabetic Cardiomyopathy through Regulation of Cardiac Autophagy via BECN1.

Multiple factors have been shown to promote the progression of diabetic cardiomyopathy. A link has previously been found between Mir30 and autophagy in cancer cells and in the heart, but the role of Mir30 in diabetic heart has not been studied. Using in vitro and in vivo approaches, we found that the depletion of Mir30c and induction of BECN1 enhanced autophagy in diabetic (db/db) hearts and in cardiomyocytes treated with the fatty acid palmitate. We verified that Mir30c repressed BECN1 expression by direct binding to the BECN1 3′ UTRs. Mir30c overexpression inhibited the induction of BECN1 and subsequent autophagy in diabetic hearts and improved cardiac function and structure in diabetic mice. However, these effects were abrogated by BECN1 overexpression. Similarly, Mir30c knockdown resulted in increased BECN1 levels and autophagic flux, aggravating cardiac abnormalities. We also show that SP1, an important transcriptional factor in energy metabolism regulation, is a key upstream activator of Mir30c that binds the promoter region of Mir30c. Our findings indicate that downregulation of Mir30c and subsequent activation of BECN1 promotes autophagy, contributing to the pathogenesis of diabetic cardiomyopathy. This observation suggests a theoretical ground for developing microRNA-based therapeutics against diabetic cardiomyopathy by inhibiting autophagy.

Synthesis and in vitro anticancer activity of new 2-thioxo-oxazolidin-4-one derivatives.

Oxazolidinones derivatives exhibit different biological properties, including anticancer activity. This work aimed to investigate the anticancer potential of five novel 2-Thioxo-oxazolidin-4-one derivatives. Cytotoxicity assays were performed in human peripheral blood mononuclear cells (PBMCs) from healthy individuals and seven tumor cell lines. Apoptosis detection and cell cycle were evaluated by flow cytometry and the expression of genes involved in cell death processes by Real-Time PCR. All oxazolinedione derivatives were not cytotoxic in PBMCs. NB-5 showed the best results in cancer cells, inhibiting the growth of all tumor cell lines tested. NB-4 exhibited the highest cytotoxicity in Jurkat cells (IC50=15.19μM) and NB-3 showed better anticancer effects in HL-60 (17.84μM). Only NB-4 significantly induced apoptosis in acute leukemia cells (p=0.001). All compounds caused a significant increase in expression of pro-apoptotic gene BID (p<0.05) and BECN1 (p<0.05). NB-3 significantly modulated the expression of RIPK3 (p=0.02) and DDIT3 (p=0.014), while NB-2 induced an increase of CDKN1A (p=0.03) and NB-4 induced PPARγ gene (p=0.0006). NB-5 showed antitumor effects in solid and hematopoietic cancer cells, while other derivatives produced higher activity against hematopoietic cells. In acute leukemia cells, oxazolidinone derivatives modulated the expression of genes involved in apoptosis, ER stress, necroptosis and inflammation.

Haploinsufficiency networks identify targetable patterns of allelic deficiency in low mutation ovarian cancer

Identification of specific oncogenic gene changes has enabled the modern generation of targeted cancer therapeutics. In high-grade serous ovarian cancer (OV), the bulk of genetic changes is not somatic point mutations, but rather somatic copy-number alterations (SCNAs). The impact of SCNAs on tumour biology remains poorly understood. Here we build haploinsufficiency network analyses to identify which SCNA patterns are most disruptive in OV. Of all KEGG pathways (N=187), autophagy is the most significantly disrupted by coincident gene deletions. Compared with 20 other cancer types, OV is most severely disrupted in autophagy and in compensatory proteostasis pathways. Network analysis prioritizes MAP1LC3B (LC3) and BECN1 as most impactful. Knockdown of LC3 and BECN1 expression confers sensitivity to cells undergoing autophagic stress independent of platinum resistance status. The results support the use of pathway network tools to evaluate how the copy-number landscape of a tumour may guide therapy.

Lutein Induces Autophagy via Beclin-1 Upregulation in IEC-6 Rat Intestinal Epithelial Cells.

Lutein is a carotenoid with anti-oxidant properties. Autophagy, an evolutionarily conserved catabolic cellular pathway for coping with stress conditions, is responsive to reactive oxygen species (ROS) and degrades damaged organelles. We previously demonstrated that lutein can induce anti-oxidant enzymes to relieve methotrexate-induced ROS stress. We therefore hypothesized that lutein, which activates ROS-scavenging enzymes, can also induce autophagy for cell survival. In this study, we demonstrated that lutein treatment attenuated the reduction in cell viability caused by H2O2. Lutein dose-dependently induced the processing of microtubule-associated protein light chain 3 (LC3)-II, an autophagy marker protein, and accumulation of LC3-positive puncta in rat intestinal IEC-6 cells. Furthermore, (a) direct observation of autophagosome formation through transmission electron microscopy, (b) upregulation of autophagy-related genes including ATG4A, ATG5, ATG7, ATG12, and beclin-1 (BENC1), and (c) increased BECN1/Bcl-2 ratio confirmed the induction of autophagy by lutein. The results revealed that bafilomycin-A1-induced inhibition of autophagy reduced cell viability and increased apoptosis in lutein-treated cells, indicating a protective role of lutein-induced autophagy. Lutein treatment also activated adenosine monophosphate-activated protein kinase (AMPK), c-Jun N-terminal kinase (JNK), and p-38, but had no effects on the induction of extracellular signal-related kinase or inhibition of mTOR; however, the inhibition of activated AMPK, JNK, or p-38 did not attenuate lutein-induced autophagy. Finally, increased BECN1 expression levels were detected in lutein-treated cells, and BECN1 knockdown abolished autophagy induction. These results suggest that lutein-induced autophagy was mediated by the upregulation of BECN1 in IEC-6 cells. We are the first to demonstrate that lutein induces autophagy. Elevated autophagy in lutein-treated IEC-6 cells may have a protective role against various stresses, and this warrants further investigation.

MicroRNA expression profiling of Leishmania donovani-infected host cells uncovers the regulatory role of MIR30A-3p in host autophagy

ABSTRACTLeishmania is an obligate intracellular parasite that replicates inside phagolysosomes or parasitophorous vacuoles (PV) of the monocyte/macrophage lineage. It reprograms macrophages and produces a metabolic state conducive to successful infection and multiplication. MicroRNAs (miRNAs), a class of small 22 to 24 nucleotide noncoding regulatory RNAs alter the gene expression and consequently proteome output by targeting mRNAs, may play a regulatory role in modulating host cell functions. In the present study, we demonstrate the novel regulatory role of host microRNA, MIR30A-3p in modulation of host cell macroautophagy/autophagy after infection with L. donovani. Our in vitro studies showed that MIR30A-3p expression was significantly enhanced after L. donovani infection in a time-dependent manner. Transient transfection with a MIR30A-3p inhibitor followed by L. donovani infection promoted the autophagic response and decreased the intracellular parasite burden in both THP-1 cells and human monocyte-derived macrophages (HsMDM). BECN1/Beclin 1, the mammalian ortholog of yeast Vps30/Atg6, is a key autophagy-promoting protein that plays a key role in the regulation of cell death and survival. We report BECN1-dependent modulation of host cell autophagy in response to L. donovani infection. Pretreatment of L. donovani-infected macrophages with the MIR30A-3p mimic decreased and with antagomir increased the expression of BECN1 protein. We demonstrate that BECN1 is a potential target of MIR30A-3p and this miRNA negatively regulates BECN1 expression. Our present study reveals for the first time a novel role of MIR30A-3p in regulating autophagy-mediated L. donovani elimination by targeting BECN1. The present study has significant impact for the treatment of visceral leishmaniasis.

Abstract 3529: Immunotherapy targeting folate receptor induces autophagy in ovarian cancer

Abstract Cancer cells are highly dependent on folate metabolism mediated by the folate receptor (FRa). Gene encoding FRa (FOLR1) is overexpressed in endometrial and ovarian cancers. We aim to investigate the functional mechanisms of blocking FRa with MORAb-003 antibody, a humanized monoclonal antibody directly against FRa. We first analyzed clinical data from the Cancer Genome Atlas (TCGA) for correlation between copy numbers of FOLR1 gene and disease-free survival in patients with high-grade serous ovarian carcinoma (HGSC). Next, we determined the surface−expression of FRa on HGSC cells using flow cytometry (FACS). We then used xenograft mouse models to test tumor inhibition by MORAb-003 antibody-induced FRa blockade. We performed reverse-phase protein array (RPPA) analysis in FRa-positive and FRa-negative HGSC tumors to compare their responses to MORAb-003 treatment. To recapitulate the in vivo environment, we cultured FRa-positive and -negative epithelial ovarian cancer cells in three-dimensional (3D) condition and targeted the specific factors identified by RPPA using shRNAs knockdown. MORAb-003 monotherapy significantly reduced tumor weights and numbers of tumor nodules in both FRa-positive IGROV1 and SKOV3 models; combination of MORAb-003 and docetaxel significantly reduced both tumor weights and numbers of tumor nodules compared to docetaxel monotherapy. As expected, we did not observe these effects in the A2780 model, which has low FRa expression. Immunohistochemistry using PCNA and TUNEL staining in resulting tumors revealed that MORAb-003 reduced proliferation, but had no significant effect on apoptosis. Systematic analysis from RPPA identified a number of autophagy factors and cell-death initiators as being significantly upregulated by MORAb-003 treatment in FRa-positive tumors including Beclin-1 and PEA-15 (a factor involved in activation of autophagic cell death). cDNA microarrays further showed that MORAb-003 treatment up-regulated the expression of BECN1, ATG8, ATG3 and LC3B in FRa-positive tumors. Using FACS analysis with acridine orange (AO) staining, we observed significantly more AO positive population in 3D cultured SKOV3 cells treated with MORAb-003 alone (16.9%) or MORAb-003+docetaxel (21.2%) than in cells treated with control IgG (5.24%). Such differences were not observed in A2780 cells. We also observed significant increase of autophagy flux in SKOV3 cells but not in A2780 cells. We also found that blocking autophagy with hydroxychloroquine or bafilomycin A1, or knockdown Beclin-1 or PEA-15 with shRNAs reversed the growth inhibition induced by MORAB-003 in 3D cultured SKOV3 cells. We reported autophagy-associated cell death is a previously unrecognized mechanism for targeting of FRa in addition to the known mechanisms including antibody-dependent cellular cytotoxicity. Our studies also suggested that Beclin-1 and PEA-15 could be further explored as prognostic markers for FRa-targeted therapy. Citation Format: Yun-Fei Wen, Anil Sood. Immunotherapy targeting folate receptor induces autophagy in ovarian cancer. [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 3529.

Cross-validation of survival associated biomarkers in gastric cancer using transcriptomic data of 1,065 patients.

IntroductionMultiple gene expression based prognostic biomarkers have been repeatedly identified in gastric carcinoma. However, without confirmation in an independent validation study, their clinical utility is limited. Our goal was to establish a robust database enabling the swift validation of previous and future gastric cancer survival biomarker candidates.ResultsThe entire database incorporates 1,065 gastric carcinoma samples, gene expression data. Out of 29 established markers, higher expression of BECN1 (HR = 0.68, p = 1.5E-05), CASP3 (HR = 0.5, p = 6E-14), COX2 (HR = 0.72, p = 0.0013), CTGF (HR = 0.72, p = 0.00051), CTNNB1 (HR = 0.47, p = 4.3E-15), MET (HR = 0.63, p = 1.3E-05), and SIRT1 (HR = 0.64, p = 2.2E-07) correlated to longer OS. Higher expression of BIRC5 (HR = 1.45, p = 1E-04), CNTN1 (HR = 1.44, p = 3.5E- 05), EGFR (HR = 1.86, p = 8.5E-11), ERCC1 (HR = 1.36, p = 0.0012), HER2 (HR = 1.41, p = 0.00011), MMP2 (HR = 1.78, p = 2.6E-09), PFKB4 (HR = 1.56, p = 3.2E-07), SPHK1 (HR = 1.61, p = 3.1E-06), SP1 (HR = 1.45, p = 1.6E-05), TIMP1 (HR = 1.92, p = 2.2E- 10) and VEGF (HR = 1.53, p = 5.7E-06) were predictive for poor OS.MATERIALS AND METHODSWe integrated samples of three major cancer research centers (Berlin, Bethesda and Melbourne datasets) and publicly available datasets with available follow-up data to form a single integrated database. Subsequently, we performed a literature search for prognostic markers in gastric carcinomas (PubMed, 2012–2015) and re-validated their findings predicting first progression (FP) and overall survival (OS) using uni- and multivariate Cox proportional hazards regression analysis.ConclusionsThe major advantage of our analysis is that we evaluated all genes in the same set of patients thereby making direct comparison of the markers feasible. The best performing genes include BIRC5, CASP3, CTNNB1, TIMP-1, MMP-2, SIRT, and VEGF.

Chaperone-like protein HYPK and its interacting partners augment autophagy

To decipher the function(s) of HYPK, a huntingtin (HTT)-interacting protein with chaperone-like activity, we had previously identified 36 novel interacting partners of HYPK. Another 13 proteins were known earlier to be associated with HYPK. On the basis of analysis of the interacting partners of HYPK, it has been shown that HYPK may participate in diverse cellular functions relevant to Huntington’s disease. In the present study, we identified additional 5 proteins by co-immunoprecipitation and co-localization. As of now we have 54 primary interactors of HYPK. From the database we collected 1026 unique proteins (secondary interactors) interacting with these 54 primary HYPK interacting partners. We observed that 10 primary and 91 secondary interacting proteins of HYPK are associated with two types of autophagy processes. We next tested the hypothesis that the hub, HYPK, might itself be involved in autophagy. Using mouse striatal STHdhQ7/HdhQ7 cell lines, we observed that over expression of HYPK significantly increased background cellular autophagy, while knock down of endogenous HYPK decreased the autophagy level as detected by altered LC3I conversion, BECN1 expression, cleavage of GFP from LC3-GFP, ATG5-ATG12 conjugate formation and expression of transcription factors like Tfeb, Srebp2 and Zkscan3. This result shows that HYPK, possibly with its interacting partners, induces autophagy. We further observed that N-terminal mutant HTT reduced the cellular levels of LC3II and BECN1, which could be recovered significantly upon over expression of HYPK in these cells. This result further confirms that HYPK could also be involved in clearing mutant HTT aggregates by augmenting autophagy pathway.