Secretory autophagy-promoted cargo exocytosis requires active RAB37.

RAB37 GTPase regulates cargo exocytosis by cycling between an inactive GDP-bound form and an active GTP-bound form. We reveal that RAB37 simultaneously regulates autophagy activation and tissue inhibitor of metalloproteinase 1 (TIMP1) secretion in lung cancer cells under starvation conditions. TIMP1, an inflammatory cytokine, is a known inhibitory molecule of matrix metalloproteinases matrix metalloproteinase 9 and suppresses the mobility of lung cancer cells both in vitro and in vivo through conventional exocytosis under serum-free conditions. Notably, we disclosed that secretory autophagy participates in TIMP1 secretion in a RAB37- and Sec22b-dependent manner. Sec22b, a SNARE family protein, participates in vesicle and membrane fusion of secretory autophagy. Knockdown of Sec22b decreased TIMP1 secretion and cell motility but did not affect cell proliferation under starvation conditions. We confirmed that starvation-activated RAB37 accompanied by Sec22b is essential for secretory autophagy to further enhance TIMP1 exocytosis. We further use an off-label drug amiodarone to demonstrate that autophagy induction facilitates TIMP1 secretion and suppresses the motility and metastasis of lung cancer cells in a RAB37-dependent manner in the lung-to-lung mouse model. In conclusion, we demonstrated that the RAB37 activation plays a pivotal regulatory role in secretory autophagy for TIMP1 secretion in lung cancer.Abbreviations: ATG: autophagy-related gene; GDP: guanosine diphosphate; GTP: guanosine triphosphate; LC3: microtubule-associated protein 1A/1B-light chain 3; SNARE: soluble N-ethylmaleimide-sensitive-factor attachment protein receptor; TIMP1: tissue inhibitor matrix metalloproteinase 1.

Secretory autophagy promotes RAB37-mediated insulin secretion under glucose stimulation both in vitro and in vivo.

High blood glucose is one of the risk factors for metabolic disease and INS (insulin) is the key regulatory hormone for glucose homeostasis. Hypoinsulinemia accompanied with hyperglycemia was diagnosed in mice with pancreatic ß-cells exhibiting autophagy deficiency; however, the underlying mechanism remains elusive. The role of secretory autophagy in the regulation of metabolic syndrome is gaining more attention. Our data demonstrated that increased macroautophagic/autophagic activity leads to induction of insulin secretion in ß-cells both in vivo and in vitro under high-glucose conditions. Moreover, proteomic analysis of purified autophagosomes from ß-cells identified a group of vesicular transport proteins participating in insulin secretion, implying that secretory autophagy regulates insulin exocytosis. RAB37, a small GTPase, regulates vesicle biogenesis, trafficking, and cargo release. We demonstrated that the active form of RAB37 increased MAP1LC3/LC3 lipidation (LC3-II) and is essential for the promotion of insulin secretion by autophagy, but these phenomena were not observed in rab37 knockout (rab37-/-) cells and mice. Unbalanced insulin and glucose concentration in the blood was improved by manipulating autophagic activity using a novel autophagy inducer niclosamide (an antihelminthic drug) in a high-fat diet (HFD)-obesity mouse model. In summary, we reveal that secretory autophagy promotes RAB37-mediated insulin secretion to maintain the homeostasis of insulin and glucose both in vitro and in vivo.

Secretory autophagy promotes Rab37-mediated exocytosis of tissue inhibitor of metalloproteinase 1.

BACKGROUND: Rab37-mediated exocytosis of tissue inhibitor of metalloproteinase 1 (TIMP1), an inflammatory cytokine, under serum-depleted conditions which leads to suppression of lung cancer cell metastasis has been reported. Starvation is also a stimulus of autophagic activity. Herein, we reveal that starvation activates Rab37 and induces autophagy. METHODS: We used an overexpression/knockdown system to determine the relationship between autophagy and Rab37 in vitro and in vivo. The autophagy activity was detected by immunoblotting, transmission electron microscope, autophagosome purification, and immunofluorescence under the confocal microscope. Lung-to-lung metastasis mouse model was used to clarify the role of autophagy and Rab37 in lung cancer. Clinical lung cancer patient specimens and an online big database were analyzed. RESULTS: Initially, we demonstrated that active-form Rab37 increased LC3-II protein level (the marker of autophagosome) and TIMP1 secretion. Accordingly, silencing of Rab37 gene expression alleviated Rab37 and LC3-II levels as well as TIMP1 secretion, and induction of autophagy could not increase TIMP1 exocytosis under such conditions. Moreover, silencing the Atg5 or Atg7 gene of lung cancer cells harboring active-mutant Rab37 (Q89L) led to decreased autophagy activity and TIMP1 secretion. In the lung-to-lung metastasis mouse model, increased TIMP1 expression accompanied by amiodarone-induced autophagy led to decreased tumor nodules and cancer cell metastasis. These phenomena were reversed by silencing the Atg5 or Atg7 gene. Notably, increasing autophagy activity alone showed no effect on TIMP1 secretion under either Rab37 or Sec22b silencing conditions. We further detected colocalization of LC3 with either Rab37 or TIMP1, identified Rab37 and Sec22b proteins in the purified autophagosomes of the lung cancer cells harboring the active-form Rab37 gene, and confirmed that these proteins are involved in the secretion of TIMP1. We reveal that autophagic activity was significantly lower in the tumors compared to the non-tumor parts and was associated with the overall lung cancer patient survival rate. CONCLUSIONS: We are the first to report that autophagy plays a promoting role in TIMP1 secretion and metastasis in a Rab37-dependent manner in lung cancer cells and the lung-to-lung mouse model.

Autophagy Upregulates miR-449a Expression to Suppress Progression of Colorectal Cancer.

Many studies reported that microRNAs (miRNAs) target autophagy-related genes to affect carcinogenesis, however, autophagy-deficiency-related miRNA dysfunction in cancer development remains poorly explored. During autophagic progression, we identified miR-449a as the most up-regulated miRNA. MiR-449a expression was low in the tumor parts of CRC patient specimens and inversely correlated with tumor stage and metastasis with the AUC (area under the curve) of 0.899 and 0.736 as well as poor overall survival rate, indicating that miR-449a has the potential to be a prognostic biomarker. In the same group of CRC specimens, low autophagic activity (low Beclin 1 expression and high p62 accumulation) was detected, which was significantly associated with miR-449a expression. Mechanistic studies disclosed that autophagy upregulates miR-449a expression through degradation of the coactivator p300 protein which acetylates the transcription factor Forkhead Box O1 (FoxO1). Unacetylated FoxO1 translocated to the nucleus and bound to the miR-449a promoter to drive gene expression. Either activation of autophagy by the inducer or overexpression of exogenous miR-449a decreases the expression of target gene LEF-1 and cyclin D1, which lead to decreased proliferation, colony formation, migration, and invasion of CRC cells. Autophagy-miR-449a-tartet genes mediated suppression of tumor formation was further confirmed in the xenograft mouse model. In conclusion, this study reveals a novel mechanism wherein autophagy utilizes miR-449a-LEF1-cyclin D1 axis to suppress CRC tumorigenesis. Our findings open a new avenue toward prognosis and treatment of CRC patients by manipulating autophagy-miR-449a axis.

The Autophagosomes Containing Dengue Virus Proteins and Full-Length Genomic RNA Are Infectious.

Autophagic machinery is involved in selective and non-selective recruitment as well as degradation or exocytosis of cargoes, including pathogens. Dengue virus (DENV) infectioninduces autophagy that enhances virus replication and vesicle release to evade immune systemsurveillance. This study reveals that DENV2 induces autophagy in lung and liver cancer cells andshowed that DENV2 capsid, envelope, NS1, NS3, NS4B and host cell proinflammatory high mobilitygroup box 1 (HMGB1) proteins associated with autophagosomes which were purified by gradientcentrifugation. Capsid, NS1 and NS3 proteins showing high colocalization with LC3 protein in thecytoplasm of the infected cells were detected in the purified double-membrane autophagosome byimmunogold labeling under transmission electron microscopy. In DENV infected cells, the levels ofcapsid, envelope, NS1 and HMGB1 proteins are not significantly changed compared to the dramaticaccumulation of LC3-II and p62/SQSTM1 proteins when autophagic degradation was blocked bychloroquine, indicating that these proteins are not regulated by autophagic degradation machinery.We further demonstrated that purified autophagosomes were infectious when co-cultured withuninfected cells. Notably, these infectious autophagosomes contain DENV2 proteins, negativestrandand full-length genomic RNAs, but no viral particles. It is possible that the infectivity ofthe autophagosome originates from the full-length DENV RNA. Moreover, we reveal that DENV2promotes HMGB1 exocytosis partially through secretory autophagy. In conclusion, we are the firstto report that DENV2-induced double-membrane autophagosomes containing viral proteins andfull-length RNAs are infectious and not undergoing autophagic degradation. Our novel findingwarrants further validation of whether these intracellular vesicles undergo exocytosis to becomeinfectious autophagic vesicles.

Diallyl sulfide promotes cell-cycle arrest through the p53 expression and triggers induction of apoptosis via caspase- and mitochondria-dependent signaling pathways in human cervical cancer Ca Ski cells.

Diallyl sulfide (DAS) is a component of garlic (Alliaceae family). Although diallyl polysulfide has been shown to exhibit anticancer activities, no report explored DAS-affected cell death in human cervical cancer cells in vitro. This study investigated DAS affected on cell-cycle regulation and apoptosis in human cervical cancer Ca Ski cells. DAS at 25-100 µM decreased the viability of Ca Ski cells by increasing G0/G1 phase arrest followed by induction of apoptosis in concentration- and time-dependent effects. Flow cytomteric assay indicated that DAS (75 µM) promoted the production of Ca(2+) accumulation and decreased the level of mitochondrial membrane potential in Ca Ski cells. Western blotting showed that 75 µM of DAS-induced G0/G1 phase arrest was mediated through the increased expression of p21, p27, and p53 with a simultaneous decrease in CDK2, CDK6, and CHK2 expression. The characteristics of apoptosis, such as morphological changes and DNA condensation, altered the ratio of Bax/Bcl-2 and sub-G1 phase occurred in Ca Ski cells after exposure to DAS. Furthermore, DAS induced mitochondrial dysfunction, leading to the release of cytochrome c for causing apoptosis in Ca Ski cells. These findings suggest that DAS might be a potential chemotherapeutic agent for the treatment of cervical cancer.