Knockdown Of Beclin Research Articles

Autophagy inhibition attenuates hyperoxaluria-induced renal tubular oxidative injury and calcium oxalate crystal depositions in the rat kidney

Hyperoxaluria-induced oxidative injury of renal tubular epithelial cell is a casual and essential factor in kidney calcium oxalate (CaOx) stone formation. Autophagy has been shown to be critical for the regulation of oxidative stress-induced renal tubular injury; however, little is known about its role in kidney CaOx stone formation. In the present study, we found that the autophagy antagonist chloroquine could significantly attenuate oxalate-induced autophagy activation, oxidative injury and mitochondrial damage of renal tubular cells in vitro and in vivo, as well as hyperoxaluria-induced CaOx crystals depositions in rat kidney, whereas the autophagy agonist rapamycin exerted contrasting effects. In addition, oxalate-induced p38 phosphorylation was significantly attenuated by chloroquine pretreatment but was markedly enhanced by rapamycin pretreatment, whereas the protective effect of chloroquine on rat renal tubular cell oxidative injury was partly reversed by a p38 protein kinase activator anisomycin. Furthermore, the knockdown of Beclin1 represented similar effects to chloroquine on oxalate-induced cell oxidative injury and p38 phosphorylation in vitro. Taken together, our results revealed that autophagy inhibition could attenuate oxalate-induced oxidative injury of renal tubular cell and CaOx crystal depositions in the rat kidney via, at least in part, inhibiting the activation of p38 signaling pathway, thus representing a novel role of autophagy in the regulation of oxalate-induced renal oxidative injury and CaOx crystal depositions for the first time.

Enterococcus faecalis lipoteichoic acid regulates macrophages autophagy via PI3K/Akt/mTOR pathway

Enterococcus faecalis (E. faecalis) infection is considered an important etiological factor for the development of persistent apical periodontitis (PAP), but the exact mechanisms of autophagy between E. faecalis and immune cells remain unknown. In this study, we elucidated how E. faecalis lipoteichoic acid (LTA) is associated with macrophages autophagy. We found that E. faecalis LTA apparently activated macrophage autophagy with significant increase of autophagosomes and autophagy relative protein. Meanwhile, we noticed significantly decreasing expression of p-Akt and p-mTOR. However, these effect were absent in macrophages knockdown of Beclin1. In summary, these findings suggested E. faecalis LTA may increased macrophages autophagy via inhibiting PI3K/Akt/mTOR pathway and this process was Beclin1 dependent.

Ginsenoside Rg1 inhibits apoptosis by increasing autophagy via the AMPK/mTOR signaling in serum deprivation macrophages.

Ginsenoside Rg1 (Rg1) has been widely used in a broad range of cardiovascular and cerebral-vascular diseases because of its unique therapeutic properties. However, the underlying mechanisms of Rg1 in the treatment of atherosclerosis have not been fully explored. This study sought to determine the precise molecular mechanisms on how Rg1 might be involved in regulating apoptosis in serum deprivation-induced Raw264.7 macrophages and primary bone marrow-derived macrophages. Results demonstrated that Rg1 treatment effectively suppressed apoptosis and the expression of phosphorylation level of mTOR induced by serum deprivation in Raw264.7 macrophages; the expressions of autophagic flux-related proteins including Atg5, Beclin1, microtubule-associated protein 1 light chain 3 (LC3), p62/SQSMT1, and the phosphorylation level of AMPK were concomitantly up-regulated. 3-Methyl-adennine (3-MA), the most widely used autophagy inhibitor, strongly up-regulated the expression of cleaved caspase-3, and blocked the anti-apoptosis function of Rg1 in macrophages. Importantly, autophagic flux was activated by Rg1, while Beclin1 knockdown partially abolished the anti-apoptosis of Rg1. Moreover, compound C, an AMPK inhibitor, partially decreased the expressions of phosphorylation of mTOR, Atg5, Beclin1, LC3, and p62/SQSMT1, which were increased by Rg1. AICAR, an AMPK inducer, promoted the protein expressions of phosphorylation of mTOR, Atg5, Beclin1, LC3, and p62/SQSMT1. In conclusion, Rg1 significantly suppressed apoptosis induced by serum deprivation in macrophages. Furthermore, Rg1 could effectively induce the autophagic flux by attenuating serum deprivation-induced apoptosis in Raw264.7 macrophages through activating the AMPK/mTOR signaling pathway.

Autophagy facilitates anticancer effect of 5-fluorouracil in HCT-116 cells.

The roles of autophagy performed in chemotherapy-induced cell death or proliferation inhibition were still in debate. In this study, we aimed to disclose the function of autophagy in chemotherapy of HCT-116 colon cells. Pharmacological and genetic methods were applied to induce and inhibit autophagy and elucidate the roles of autophagy performed in chemotherapy-induced proliferation inhibition and apoptosis. Autophagy was assessed by microtubule-associated protein light chain 3 (LC3) expression and monodansylcadaverine (MDC) staining. After treatment with 5-fluorouracil (5-FU), HCT-116 cells showed typical autophagy as stained by MDC. Autophagy inhibitor (3-methyladenine [3-MA]) or inducer (rapamycin) was applied in combination with 5-FU, respectively. As evidenced by our data, 3-MA inhibited while rapamycin facilitated 5-FU-induced apoptosis and proliferation inhibition of HCT-116 cells. Consistently, 3-MA inhibited, while rapamycin facilitated 5-FU-induced expressions of Beclin1 and LC3B. Moreover, 3-MA inhibited while rapamycin facilitated 5-FU-induced p53 protein expression. Using genetic method, Beclin1 overexpression increased while Beclin1 knockdown decreased 5-FU-induced cell proliferation inhibition and apoptosis. Especially, Beclin1 overexpression increased while Beclin1 knockdown decreased 5-FU-induced p53 expression. Our study provides both of pharmacological and genetic evidence to support that autophagy facilitates anticancer effect of the chemotherapeutic agent. The associated application of autophagy inducer with 5-FU would be beneficial for the chemotherapy in HCT-116 cancer cells.

Targeting LC3 and Beclin-1 autophagy genes suppresses proliferation, survival, migration and invasion by inhibition of Cyclin-D1 and uPAR/Integrin β1/ Src signaling in triple negative breast cancer cells.

Autophagy is a catabolic process for degrading dysfunctional proteins and organelles, and closely associated with cancer cell survival under therapeutic, metabolic stress, hypoxia, starvation and lack of growth factors, contributing to resistance to therapies. However, the role of autophagy in breast cancer cells is not well understood. In the present study, we investigated the role of autophagy in highly aggressive and metastatic triple negative breast cancer (TNBC) and non-metastatic breast cancer cells and demonstrated that the knockdown of autophagy-related genes (LC3 and Beclin-1) inhibited autophagy and significantly suppressed cell proliferation, colony formation, migration/invasion and induced apoptosis in MDA-MB-231 and BT-549 TNBC cells. Knockdown of LC3 and Beclin-1 led to inhibition of multiple proto-oncogenic signaling pathways, including cyclin D1, uPAR/integrin-β1/Src, and PARP1. In conclusion, our study suggests that LC3 and Beclin-1 are required for cell proliferation, survival, migration and invasion, and may contribute to tumor growth and progression of highly aggressive and metastatic TNBC cells and therapeutic targeting of autophagy genes may be a potential therapeutic strategy for TNBC in breast cancer.

Effects of autophagy on acid-sensing ion channel 1a-mediated apoptosis in rat articular chondrocytes.

Rheumatoid arthritis (RA) is a degenerative joint disease that is caused by multiple pathogenic factors. However, the precise etiology of RA is still unknown. Our previous studies demonstrated that acid-sensing ion channel 1a (ASIC1a)-mediated articular chondrocyte apoptosis played a key role in the progression of RA. In this study, we aim to explore whether ASIC1a mediates autophagy or not and the effect of autophagy on ASIC1a-mediated apoptosis. Primary articular chondrocytes, extracted from rat knee joints, were exposed to different concentrations of concentrated hydrochloric acid for different time intervals in vitro. The results indicated that extracellular acid treatment induced autophagy of rat articular chondrocytes. Moreover, inhibition of ASIC1a with either psalmotoxin 1 or ASIC1a short hairpin RNA reduced the autophagy flux. The results suggested that ASIC1a mediated acid-induced autophagy. Pretreatment with autophagy antagonist 3-methyladenine decreased the autophagy, but increased the apoptosis mediated by ASIC1a. Furthermore, knockdown of Beclin 1 by small interfering RNA attenuated autophagy but potentiated ASIC1a-mediated apoptosis of rat articular chondrocytes. Taken together, these findings suggested that both inhibition and silencing of autophagy could enhance ASIC1a-mediated apoptosis in rat articular chondrocytes, and therefore, autophagy is likely to be a new mechanism involved in ASIC1a-mediated apoptosis of articular chondrocytes during the pathogenesis of RA.

Beclin-1 knockdown decreases proliferation, invasion and migration of Ewing sarcoma SK-ES-1 cells via inhibition of MMP-9.

Although Beclin-1, a well-known key regulator of autophagy, has been demonstrated to serve a function in a number of disorders, including cancer, aging and degenerative diseases, its biological function in Ewing sarcoma (ES) remains unresolved. The objective of the present study was to determine the in vitro effect of Beclin-1 knockdown on the growth and malignant phenotype of ES SK-ES-1 cells, which have increased endogenous expression of Beclin-1 compared with RD-ES cells, and to investigate the underlying molecular mechanism. Cell proliferation, invasion and migration were investigated using CCK-8, Boyden chamber Transwell, and wound healing assays, respectively. Western blot analysis was used to detect expression levels of matrix metalloproteinase (MMP)-2 and MMP-9, which are associated with the malignant phenotype. Beclin-1 knockdown significantly inhibited proliferation, invasion and migration of SK-ES-1 cells. Western blot analysis revealed that Beclin-1 knockdown caused a significant reduction in the expression of MMP-9; no marked changes in MMP-2 expression were observed in the si-Beclin-1 group compared with the control group. The results of the present study suggest that Beclin-1 serves a function in proliferation, tumor progression and inhibition of autophagy in ES, and demonstrates it's potential as a target to increase the efficacy of anticancer agents.

Inactivation of Beclin-1-dependent autophagy promotes ursolic acid-induced apoptosis in hypertrophic scar fibroblasts.

A hypertrophic scar (HS) is caused by abnormal proliferation of dermal fibroblasts. Thus, promoting hypertrophic scar fibroblast (HSFB) apoptosis is an effective strategy for HS therapy. Ursolic acid (UA) has been widely used as an inducer of apoptosis in diverse cancers. However, whether UA plays an inhibitory role in HS formation is still unknown. In our study, UA was used to treat HSFBs and the cell viability, apoptosis, and collagen synthesis were determined by a Cell Counting Kit 8 assay, flow cytometry, and an H3 -proline incorporation assay, respectively. Autophagy activity was detected by LC3 immunoblotting and electron microscopy, and siRNAs targeting Beclin-1 were used to inhibit autophagy. Western blotting was performed to investigate the molecular changes in HSFBs after various treatments. We found that UA inhibited collagen synthesis and induced cell apoptosis in HSFBs, evidenced by the deregulated expression of Bim, Bcl-2 and Cyto C. Furthermore, we demonstrated that UA induced autophagy and inactivation of autophagy promoted UA-induced apoptosis and collagen synthesis inhibition in HSFBs. Molecular investigation indicated that UA-induced autophagy through upregulation of Beclin-1 and knockdown of Beclin-1 prevent UA-induced autophagy. Overexpression of Bcl-2 prevents UA-induced autophagy, Beclin-1 upregulation, apoptosis and collagen synthesis inhibition in HSFBs. Collectively, our study demonstrated that UA is a novel agent for inhibiting HS formation by promoting apoptosis, especially in combination with an autophagy inhibitor. Our results provide strong evidence of the application of UA in clinical HS treatment.

Mesenchymal stem cells internalize Mycobacterium tuberculosis through scavenger receptors and restrict bacterial growth through autophagy

Human mesenchymal stem cells (MSCs) express scavenger receptors that internalize lipids, including oxidized low-density lipoprotein (oxLDL). We report that MSCs phagocytose Mycobacterium tuberculosis (Mtb) through two types of scavenger receptors (SRs; MARCO and SR-B1), as blockade of the receptors with antibodies or siRNA knockdown decreased the uptake of Mtb. MSCs also expressed mannose receptor (MR) that was found to endocytose rhodamine-labeled mannosylated BSA (rMBSA), though the receptor was not involved in the uptake of Mtb. Dil-oxLDL and rMBSA taken up into MSC endosomes colocalized with Mtb phagosomes, thus suggesting that the latter were fusion competent. Phagocytosed Mtb did not replicate within MSCs, thus suggesting an intrinsic control of bacterial growth. Indeed, MSCs exhibited intrinsic autophagy, which was up-regulated after activation with rapamycin. SiRNA knockdown of autophagy initiator beclin-1 enhanced Mtb survival, whereas rapamycin-induced autophagy increased intracellular killing of Mtb. In addition, MSCs secreted nitric oxide after Mtb infection, and inhibition of NO by N(G)-monomethyl-L-arginine enhanced intracellular survival of Mtb. MSCs can be grown in large numbers in vitro, and autologous MSCs transfused into tuberculosis patients have been found to be safe and improve lung immunity. Thus, MSCs are novel phagocytic cells with a potential for immunotherapy in treating multidrug-resistant tuberculosis.

Retinoic acid regulates endometriotic stromal cell growth through upregulation of Beclin1.

To elucidate the role of retinoic acid (RA) in autophagy-mediated endometriosis. The mRNA and protein expressions of autophagy markers were examined in Ishikawa cells and endometriotic stromal cells (ESCs) after RA treatment. Beclin1 expression was specifically analyzed in clinical samples of endometriosis. The effect of Beclin1 knockdown on ESC growth was assessed, and the effect of autophagy inhibition on the sensitivity of endometriotic cells to RA was analyzed. RA treatment enhanced the autophagy in ESCs, and Beclin1 expression showed a negative correlation with the clinical stage of endometriosis. Beclin1 knockdown enhanced ESC growth, whereas RA treatment reversed this effect. Furthermore, inhibition of autophagy by chloroquine (CQ) and Beclin1 knockdown did not show any positive effect on the sensitivity of endometriotic cells to RA. RA treatment induces autophagy and Beclin1 may play an important role in endometriosis progression.

The preclinical analysis of TW-37 as a potential anti-colorectal cancer cell agent.

TW-37 is a novel, potent and non-peptide Bcl-2 small-molecule inhibitor. Its activity in colorectal cancer (CRC) cells is studied. In both HCT-116 cells and primary human colon cancer cells, treatment with TW-37 at only nM concentration efficiently inhibited cell survival and proliferation. TW-37 also induced caspase-3/9 and apoptosis activation in CRC cells. Feedback autophagy activation was observed in TW-37-treated CRC cells. Reversely pharmacological autophagy inhibition or Beclin-1 knockdown by targeted-shRNA potentiated TW-37-induced apoptosis and killing of CRC cells. In vivo, intravenous injection of TW-37 inhibited HCT-116 tumor growth in mice. TW-37’s anti-tumor activity was further potentiated against Beclin-1-silenced HCT-116 tumors. Together, targeting Bcl-2 family protein by TW-37 efficiently inhibits CRC cell growth in vitro and in vivo. Inhibition of feedback autophagy activation could further sensitize TW-37.

Administration of follicle-stimulating hormone induces autophagy via upregulation of HIF-1\u03b1 in mouse granulosa cells

Recent studies reported the important role of autophagy in follicular development. However, the underlying molecular mechanisms remain elusive. In this study, we investigated the effect of follicle-stimulating hormone (FSH) on mouse granulosa cells (MGCs). Results indicated that autophagy was induced by FSH, which is known to be the dominant hormone regulating follicular development and granulosa cell (GC) proliferation. The activation of mammalian target of rapamycin (mTOR), a master regulator of autophagy, was inhibited during the process of MGC autophagy. Moreover, MHY1485 (an agonist of mTOR) significantly suppressed autophagy signaling by activating mTOR. The expression of hypoxia-inducible factor 1-alpha (HIF-1α) was increased after FSH treatment. Blocking hypoxia-inducible factor 1-alpha attenuated autophagy signaling. In vitro, CoCl2-induced hypoxia enhanced cell autophagy and affected the expression of beclin1 and BCL2/adenovirus E1B interacting protein 3 (Bnip3) in the presence of FSH. Knockdown of beclin1 and Bnip3 suppressed autophagy signaling in MGCs. Furthermore, our in vivo study demonstrated that the FSH-induced increase in weight was significantly reduced after effectively inhibiting autophagy with chloroquine, which was correlated with incomplete mitophagy process through the PINK1-Parkin pathway, delayed cell cycle, and reduced cell proliferation rate. In addition, chloroquine treatment decreased inhibin alpha subunit, but enhanced the expression of 3 beta-hydroxysteroid dehydrogenase. Blocking autophagy resulted in a significantly lower percentage of antral and preovulatory follicles after FSH stimulation. In conclusion, our results indicate that FSH induces autophagy signaling in MGCs via HIF-1α. In addition, our results provide evidence that autophagy induced by FSH is related to follicle development and atresia.

MTOR-Notch3 signaling mediates pulmonary hypertension in hypoxia-exposed neonatal rats independent of changes in autophagy.

Mammalian target of rapamycin (mTOR) is a pivotal regulator of cell proliferation, survival, and autophagy. Autophagy is increased in adult experimental chronic pulmonary hypertension (PHT), but its contributory role to pulmonary vascular disease remains uncertain and has yet to be explored in the neonatal animal. Notch is a major pro-proliferative pathway activated by mTOR. A direct relationship between autophagy and Notch signaling has not been previously explored. Our aim was to examine changes in mTOR-, Notch-, and autophagy-related pathways and the therapeutic effects of autophagy modulators in experimental chronic neonatal PHT secondary to chronic hypoxia. Rat pups were exposed to normoxia or hypoxia (13% O2 ) from postnatal days 1-21, while receiving treatment with temsirolimus (mTOR inhibitor), DAPT (Notch inhibitor), or chloroquine (inhibitor of autophagic flux). Exposure to hypoxia up-regulated autophagy and Notch3 signaling markers in lung, pulmonary artery (PA), and PA-derived smooth muscle cells (SMCs). Temsirolimus prevented chronic PHT and attenuated PA and SMC signaling secondary to hypoxia. These effects were replicated by DAPT. mTOR or Notch inhibition also down-regulated smooth muscle content of platelet-derived growth factor β-receptor, a known contributor to vascular remodeling. In contrast, chloroquine had no modifying effects on markers of chronic PHT. Knockdown of Beclin-1 in SMCs had no effect on hypoxia-stimulated Notch3 signaling. mTOR-Notch3 signaling plays a critical role in experimental chronic neonatal PHT. Inhibition of autophagy did not suppress Notch signaling and had no effect on markers of chronic PHT.

Docetaxel Reverses Pulmonary Vascular Remodeling by Decreasing Autophagy and Resolves Right Ventricular Fibrosis

Pulmonary arterial hypertension remains a fatal disease despite the availability of approved vasodilators. Since vascular remodeling contributes to increased pulmonary arterial pressure, new agents that reduce the thickness of pulmonary vascular walls have therapeutic potential. Thus, antitumor agents that are capable of killing cells were investigated. Testing of various antitumor drugs identified that docetaxel is a superior drug for killing proliferating pulmonary artery smooth muscle cells compared with other drugs, including gemcitabine, methotrexate, and ifosfamide. The administration of docetaxel to rats with severe pulmonary arterial hypertension reversed pulmonary vascular remodeling and reduced right ventricular pressure. Docetaxel was found to decrease autophagy as monitored by LC3B-II and p62 expression. The small interfering RNA knockdown of Beclin-1 or LC3B potentiated docetaxel-induced cell death, and knocking down p62 inhibited the docetaxel effects. The suppressed autophagic process is due to the ability of docetaxel to decrease Beclin-1 protein expression in a proteasome-dependent manner. Mass spectrometry identified a novel docetaxel-inducible Beclin-1 binding protein, namely, myosin-9. Knocking down myosin-9 inhibited docetaxel-induced cell death. In damaged right ventricles of pulmonary arterial hypertension rats, docetaxel remarkably promoted the resolution of fibrosis and the regeneration of myocardium. Thus, docetaxel is capable of reversing pulmonary vascular remodeling and resolving right ventricle fibrosis and is a promising therapeutic agent for the treatment of pulmonary arterial hypertension and right heart failure.

Abstract 3307: Induction of autophagy as a mechanism of therapeutic resistance in head and neck cancer

Abstract Background: In the curative setting for head and neck cancer (HNC) a commonly used treatment is radiation combined with the anti-EGFR monoclonal antibody cetuximab (CTX). However, despite decades of research into improved treatments, therapeutic resistance remains a major challenge for this malignancy with roughly 40% of patients developing recurrent disease. We are studying the molecular mechanisms by which radiation and cetuximab induce autophagy and how this process can be modulated to improve the efficacy of therapy. Methods: We evaluated a panel of both human papillomavirus (HPV) positive and negative HNC cell lines for autophagic response to both CTX treatment and ionizing radiation (XRT). Induction of autophagy was detected by immunoblot flux assays for LC3 and p62 and by immunofluorescent staining of autophagic vesicles. siRNA knockdown of EGFR, LAMPT4B, ULK1, Beclin1 and others was used to probe the involvement of different signaling molecules. The addition of specific autophagy inhibitors, such as the ULK1 inhibitor SBI-0206965, to CTX or XRT treatment was tested to determine whether the reduction of autophagic response reduced cell survival in a clonogenic survival assay. Induction of apoptosis was analyzed by immunoblot against cleaved Caspase and PARP and AnnexinV staining. Results: Flux assays revealed that both CTX and XRT induced autophagy in a time and dose dependent manner. Immunofluorescent staining of LC3 to identify autophagic vesicles showed that a relatively small fraction of the total cell population is able to induce this response. Specific siRNA knockdown of EGFR and LAMPT4B was able to abrogate the induction of autophagy in response to both CTX and XRT. Involvement of alternative downstream pathways involving either ULK1 and Beclin1 or p53 in response to either treatment is under investigation. To determine whether blockade of cytoprotective autophagy can help overcome therapeutic resistance, we tested specific autophagy inhibitors in combination with either CTX or XRT treatment. The addition of the ULK1 inhibitor to CTX or XRT induced apoptosis as shown by caspase activity, AnnexinV staining, and reduced cell survival in clonogenic assays. Conclusions: Autophagy may play a critical and protective role in how head and neck cancers respond to therapeutic stress. The addition of specific autophagy inhibitors to standard treatments may provide a way to overcome resistance to therapy. Citation Format: Jaimee C. Eckers, Adam D. Swick, Tyler Fowler, Justin Skiba, Kwangok P. Nickel, Randall J. Kimple. Induction of autophagy as a mechanism of therapeutic resistance in head and neck cancer [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 3307. doi:10.1158/1538-7445.AM2017-3307

Zinc oxide nanoparticles harness autophagy to induce cell death in lung epithelial cells

Although zinc oxide nanoparticles (ZnONPs) are widely used, they have raised concerns of toxicity in humans. Previous studies have indicated that reactive oxygen species (ROS) and autophagy are involved in the cytotoxicity of ZnONPs, but the regulatory mechanisms between autophagy and ROS remain to be elucidated. Herein, we comprehensively investigated the regulatory mechanism of autophagy and the link between autophagy and ROS in ZnONPs-treated lung epithelial cells. We demonstrated that ZnONPs could induce autophagy, and this process could enhance the dissolution of ZnONPs in lysosomes to release zinc ions. Sequentially, zinc ions released from ZnONPs were able to damage not only lysosomes, leading to impaired autophagic flux, but also mitochondria. Impaired autophagic flux resulted in the accumulation of damaged mitochondria, which could generate excessive ROS to cause cell death. We further demonstrated that the inhibition of autophagy by either pharmacological inhibitors or small interfering RNA (siRNA)-mediated knockdown of Beclin-1 and AMP-activated protein kinase could ameliorate ZnONPs-induced cell death. Moreover, we found that lysosomal-associated membrane protein 1/2 (LAMP-1/2), which were the most abundant highly glycosylated protein in late endosomes/lysosomes, exhibited aberrant expression pattern upon treatment with ZnONPs. Intriguingly, LAMP-2 knockdown, but not LAMP-1 knockdown, could exacerbate the ROS generation and cell death induced by ZnONPs treatment. Meanwhile, LAMP-2 overexpression alleviated ZnONPs-induced cell death, suggesting that LAMP-2 was linked to this toxic phenotype induced by ZnONPs. Our results indicate that autophagic dysfunction could contribute to excessive ROS generation upon treatment with ZnONPs in lung epithelial cells, suggesting that modulating the autophagy process would minimize ZnONPs-associated toxicity.

Abstract 2977: Secretory autophagy in tumor associated fibroblasts promotes head and neck squamous cell carcinoma progression and emerges as a novel therapeutic target

Abstract As the fifth most common cancer worldwide, head and neck squamous cell carcinoma (HNSCC) is a leading global health burden. Despite therapeutic advancements, the survival rate has remained relatively unchanged for the last fifty years. To improve treatment and survival, there is a need to better understand the underlying biology of this disease. The tumor microenvironment’s role in promoting cancer progression and resistance to therapy has gathered great attention. In HNSCC, the predominant microenvironment cell type is tumor associated fibroblasts (TAFs). Studies in our lab and others have demonstrated the significant tumor promoting role of TAFs; however, little is understood of the underlying biology of TAFs. In this study, we identify that TAFs have upregulated an unconventional pathway of secretion: secretory autophagy. Although paradigmatically a degradation pathway, there has been a growing appreciation for a novel role of autophagy in cellular secretion. We hypothesized HNSCC induces secretory autophagy in the TAFs, modulating secreted factors responsible for tumor progression. We assessed the role of autophagy inhibition in alleviating TAF-facilitated HNSCC progression, and uncovered a significant reduction in proliferation, migration, and invasion of the cancer cells. This was achieved by both siRNA knockdown of Beclin-1, and therapeutic inhibition of the lysosome through chloroquine. We discovered that NFs placed in co-culture with HNSCC had a significantly upregulated level of autophagy marker LC3-II. By characterizing the role of known HNSCC secreted factors in inducing TAF autophagy, we identified basic fibroblast growth factor is responsible for the LC3-II accumulation. To understand which TAF factors are secreted by an autophagy dependent mechanism, we assessed TAF-conditioned media in the presence of autophagy knockdown. Key cytokines, such as IL-6 and IL-8, were identified as autophagy dependent. Rescue of these cytokines in autophagy inhibited TAF-conditioned media restored HNSCC migration. Although autophagy has been identified as upregulated in a variety of cancer types, no one has previously characterized the role of autophagy in HNSCC, and both early pre-clinical and clinical studies of autophagy inhibition as a cancer therapy have been limited by the lack of a specifically targeted inhibitor. We demonstrate a significant decrease in HNSCC progression in preclinical models by using a new and highly specific small molecule inhibitor of autophagy, SAR405, which inhibits Vps34, an upstream autophagy pathway kinase. In summary, we uncover a novel role for secretory autophagy in the tumor microenvironment which promotes tumor progression, and can be uniquely targeted for therapy. Citation Format: Jacob New, Levi Arnold, Megha Ananth, Sameer Alvi, Wen-Xing Ding, Sufi M. Thomas. Secretory autophagy in tumor associated fibroblasts promotes head and neck squamous cell carcinoma progression and emerges as a novel therapeutic target [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 2977. doi:10.1158/1538-7445.AM2017-2977

Sphingosine kinase 2 activates autophagy and protects neurons against ischemic injury through interaction with Bcl-2 via its putative BH3 domain

Our previous findings suggest that sphingosine kinase 2 (SPK2) mediates ischemic tolerance and autophagy in cerebral preconditioning. The aim of this study was to determine by which mechanism SPK2 activates autophagy in neural cells. In both primary murine cortical neurons and HT22 hippocampal neuronal cells, overexpression of SPK2 increased LC3II and enhanced the autophagy flux. SPK2 overexpression protected cortical neurons against oxygen glucose deprivation (OGD) injury, as evidenced by improvement of neuronal morphology, increased cell viability and reduced lactate dehydrogenase release. The inhibition of autophagy effectively suppressed the neuroprotective effect of SPK2. SPK2 overexpression reduced the co-immunoprecipitation of Beclin-1 and Bcl-2, while Beclin-1 knockdown inhibited SPK2-induced autophagy. Both co-immunoprecipitation and GST pull-down analysis suggest that SPK2 directly interacts with Bcl-2. SPK2 might interact to Bcl-2 in the cytoplasm. Notably, an SPK2 mutant with L219A substitution in its putative BH3 domain was not able to activate autophagy. A Tat peptide fused to an 18-amino acid peptide encompassing the native, but not the L219A mutated BH3 domain of SPK2 activated autophagy in neural cells. The Tat-SPK2 peptide also protected neurons against OGD injury through autophagy activation. These results suggest that SPK2 interacts with Bcl-2 via its BH3 domain, thereby dissociating it from Beclin-1 and activating autophagy. The observation that Tat-SPK2 peptide designed from the BH3 domain of SPK2 activates autophagy and protects neural cells against OGD injury suggest that this structure may provide the basis for a novel class of therapeutic agents against ischemic stroke.

The effect of miR-30d on apoptosis and autophagy in cultured astrocytes under oxygen-glucose deprivation

MiR-30d has been demonstrated to regulate autophagy in human cancer cells. However, the role of miR-30d in astrocytes exposed to hypoxia-ischemia is not clear. In this study, we established model of oxygen and glucose deprivation (OGD) with rat primary astrocytes and evaluated the role of miR-30d in the cross-talk between autophagy and apoptosis in astrocytes after OGD. We found that miR-30d inhibited Beclin1 expression in astrocytes. Inhibition of miR-30d by antagomir significantly increased cell autophagy but decreased cell apoptosis in astrocytes exposed to OGD. Knockdown of Beclin1 reversed the upregulation of autophagy and downregulation of apoptosis induced by miR-30d inhibition in astrocytes subjected to OGD. These results strongly indicated that miR-30d played critical roles in the cross-talk between autophagy and apoptosis of astrocytes subjected to OGD by targeting Beclin1. MiR-30d is a novel target to attenuate cell injury under hypoxia-ischemia condition.

Ochratoxin A-induced autophagy in vitro and in vivo promotes porcine circovirus type 2 replication

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium. Porcine circovirus type 2 (PCV2) is recognized as the causative agent of porcine circovirus-associated diseases. Recently, we reported that low doses of OTA promoted PCV2 replication in vitro and in vivo, but the underlying mechanism needed further investigation. The present studies further confirmed OTA-induced PCV2 replication promotion as measured by cap protein expression, viral titer, viral DNA copies and the number of infected cells. Our studies also showed that OTA induced autophagy in PK-15 cells, as assessed by the markedly increased expression of microtubule-associated protein 1 light chain 3 (LC3)-II, autophagy-related protein 5 (ATG5), and Beclin-1 and the accumulation of green fluorescent protein (GFP)-LC3 dots. OTA induced complete autophagic flux, which was detected by monitoring p62 degradation and LC3-II turnover using immunoblotting. Inhibition of autophagy by 3-methylademine (3-MA) and chloroquine (CQ) significantly attenuated OTA-induced PCV2 replication promotion. The observed phenomenon was further confirmed by the knock-down of ATG5 or Beclin-1 by specific siRNA. Further studies showed that N-acetyl-L-cysteine (NAC), an ROS scavenger could block autophagy induced by OTA, indicating that ROS may be involved in the regulation of OTA-induced autophagy. Furthermore, we observed significant increases in OTA concentrations in lung, spleen, kidney, liver and inguinal lymph nodes (ILN) and bronchial lymph nodes (BLN) of pigs fed 75 and 150 μg/kg OTA compared with controls in vivo. Administration of 75 μg/kg OTA significantly increased PCV2 replication and autophagy in the lung, spleen, kidney and BLN of pigs. Taken together, it could be concluded that OTA-induced autophagy in vitro and in vivo promotes PCV2 replication.