Search

Abstract Autophagy can be classified as degradative and secretory based on distinct functions. The small GTPase proteins Rab8a and Rab37 are responsible for secretory autophagy‐mediated exocytosis of IL‐1β, insulin, and TIMP1 (tissue inhibitor of 54 metalloproteinase 1). Other Rab family members participating in secretory autophagy are poorly understood. Herein, we identified 26 overlapped Rab proteins in purified autophagosomes of mouse pancreatic β‐cell “Min‐6” and human lung cancer cell “CL1‐5‐Q89L” with high secretory autophagy tendency by LC–MS/MS proteomics analysis. Six Rab proteins (Rab8a, Rab11b, Rab27a, Rab35, Rab37, and Rab7a) were detected in autophagosomes of four cell lines, associating them with autophagy‐related vesicle trafficking. We used CL1‐5‐Q89L cell line model to evaluate the levels of Rab proteins colocalization with autophagy LC3 proteins and presence in purified autophagosomes. We found five Rab proteins (Rab8a, Rab11b, Rab27a, Rab35, and Rab37) are highly expressed in the autophagosome compared to the normal control by immunoblotting under active secretion conditions. However, only Rab8a, Rab35, and Rab37 showing high colocalization with LC3 protein by cofocal microscopy. Despite the discrepancy between the image and immunoblotting analysis, our data sustains the speculation that Rab8a, Rab11b, Rab27a, Rab35, and Rab37 are possibly associated with the secretory autophagy machinery. In contrast, Rab7a shows low colocalization with LC3 puncta and low level in the autophagosome, suggesting it regulates different vesicle trafficking machineries. Our findings open a new direction toward exploring the role of Rab proteins in secretory autophagy‐related cargo exocytosis and identifying the cargoes and effectors regulated by specific Rab proteins.

Abstract Autophagy is a self‐recycling machinery to maintain cellular homeostasis by degrading harmful materials in the cell. Autophagy‐related gene 5 (Atg5) is required for autophagosome maturation. However, the role of Atg5 in tumorigenesis under autophagy deficient conditions remains unclear. This study focused on the autophagy‐independent role of Atg5 and the underlying mechanism in tumorigenesis. We demonstrated that knockout of autophagy‐related genes including Atg5, Atg7, Atg9, and p62 in mouse embryonic fibroblast (MEF) cells consistently decreased cell proliferation and motility, implying that autophagy is required to maintain diverse cellular functions. An Atg7 knockout MEF (Atg7−/− MEF) cell line representing deprivation of autophagy function was used to clarify the role of Atg5 transgene in tumorigenesis. We found that Atg5‐overexpressed Atg7−/‐MEF (clone A) showed increased cell proliferation, colony formation, and migration under autophagy deficient conditions. Accordingly, rescuing the autophagy deficiency of clone A by overexpression of Atg7 gene shifts the role of Atg5 from pro‐tumor to anti‐tumor status, indicating the dual role of Atg5 in tumorigenesis. Notably, the xenograft mouse model showed that clone A of Atg5‐overexpressed Atg7−/− MEF cells induced temporal tumor formation, but could not prolong further tumor growth. Finally, biomechanical analysis disclosed increased Wnt5a secretion and p‐JNK expression along with decreased β‐catenin expression. In summary, Atg5 functions as a tumor suppressor to protect the cell under normal conditions. In contrast, Atg5 shifts to a pro‐tumor status under autophagy deprivation conditions.

Abstract Formosanin C (FC) is a natural compound extracted from Paris formosana Hayata with anticancer activity. FC induces both autophagy and apoptosis in human lung cancer cells. FC‐induced depolarization of mitochondrial membrane potential (MMP) may trigger mitophagy. In this study, we clarified the effect of FC on autophagy, mitophagy, and the role of autophagy in FC‐related cell death and motility. We found FC caused the continuous increase of LC3 II (representing autophagosomes) from 24 to 72 h without degradation after treatment of lung and colon cancer cells, indicating that FC blocks autophagic progression. In addition, we confirmed that FC also induces early stage autophagic activity. Altogether, FC is not only an inducer but also a blocker of autophagy progression. Moreover, FC increased MMP accompanied by overexpression of COX IV (mitochondria marker) and phosphorylated Parkin (p‐Parkin, mitophagy marker) in lung cancer cells, but no colocalization of LC3 with COX IV or p‐Parkin was detected under confocal microscopy. Moreover, FC could not block CCCP (mitophagy inducer)‐induced mitophagy. These results imply that FC disrupts mitochondria dynamics in the treated cells, and the underlying mechanism deserves further exploration. Functional analysis reveals that FC suppresses cell proliferation and motility through apoptosis and EMT‐related pathway, respectively. In conclusion, FC acts as an inducer as well as a blocker of autophagy that results in cancer cell apoptosis and decreased motility. Our findings shed the light on the development of combined therapy with FC and clinical anticancer drugs for cancer treatment.

Abstract 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.

Abstract Background Esophageal squamous cell carcinoma (ESCC) is widely prevalent in Taiwan, and high metastatic spread of ESCC leads to poor survival rate. Fibronectin (FN) assembly on the cell membrane may induce ESCC mobility. MicroRNAs (MiRNAs) are abundant in and participate in tumorigenesis in many cancers. However, the role of MiRNA in FN assembly-related ESCC mobility remains unexplored. Methods We divided ESCC CE81T cells into high-FN assembly (CE81FN+) and low-FN assembly (CE81FN−) groups by flow cytometry. MiRNA microarray analysis identified miR-146a expression as the most down-regulated miRNA in comparison of CE81FN+ and CE81FN− cells. Results Cell proliferation and migration were decreased when CE81FN+ cells overexpressed transgenic miR-146a compared to the parental cells, indicating an inverse correlation between low miR-146a expression and high proliferation as well as motility of FN assembly ESCC cells. Furthermore, vimentin is the target gene of miR-146a involved in ESCC tumorigenesis. MiR-146a suppressed cell proliferation, migration and invasion of CE81FN+ cells through the inhibition of vimentin expression, as confirmed by real-time PCR, Western blotting and Transwell™ assay. Analysis of one hundred and thirty-six paired ESCC patient specimens revealed that low miR-146a and high vimentin levels were frequently detected in tumor, and that the former was associated with late tumor stages (III and IV). Notably, either low miR-146a expression or high vimentin level was significantly associated with poor overall survival rate among ESCC patients. Conclusions This is the first report to link FN assembly in the cell membrane with miR-146a, vimentin and ESCC tumorigenesis both in vitro and in ESCC patients.

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.

Background: In our preliminary screening, expression of miR-338-5p was found to be higher in primary colorectal cancer (CRC) with metastasis. The autophagy related gene- phosphatidylinositol 3-kinase, catalytic subunit type 3 (PIK3C3) appeared to be targeted by miR-338-5p. Here, we provide solid evidence in support of PIK3C3 involved in miR-338-5p related metastasis of CRC in vitro and in vivo. Methods: The potential clinical relevance of miR-338-5p and its target gene was analysed on benign colorectal polyps and primary CRCs by QPCR. Mouse spleen xenograft experiment was performed to examine the importance of miR-338-5p for metastasis. Findings: PIK3C3 was one of target genes of miR-338-5p. In primary CRCs, expression of miR-338-5p is positively related to tumour staging, distant metastasis and poor patient survival. Patients with higher ratios of miR-338-5p/PIK3C3 also had significantly poor overall survival, supporting their significance in the progression of CRC. Over-expression of miR-338-5p promotes CRC metastasis to the liver and lung in vivo, in which PIK3C3 was down-regulated in the metastatic tumours. In contrast, overexpression of PIK3C3 in miR-338-5p stable cells inhibited the growth of metastatic tumours. Both migration and invasion of CRC in vitro induced by miR-338-5p are mediated by suppression of PIK3C3. Using forward and reverse approaches, autophagy was proved to involve in CRC migration and invasion induced by miR-338-5p. Interpretation: MiR-338-5p induces migration, invasion and metastasis of CRC in part through PIK3C3-related autophagy pathway. The miR-338-5p/PIK3C3 ratio may become a prognostic biomarker for CRC patients. Fund: NCKU Hospital, Taiwan, Ministry of Science and Technology, Taiwan. Keywords: miR-338-5p, PIK3C3, Autophagy and colorectal cancer

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.

Abstract Background Arsenic is a widely distributed metalloid compound that has biphasic effects on cultured cells. In large doses, arsenic can be toxic enough to trigger cell death. In smaller amounts, non-toxic doses may promote cell proliferation and induces carcinogenesis. Aberration of chromosome is frequently detected in epithelial cells and lymphocytes of individuals from arsenic contaminated areas. Overexpression of Aurora-A, a mitotic kinase, results in chromosomal instability and cell transformation. We have reported that low concentration (≦1 μM) of arsenic treatment increases Aurora-A expression in immortalized bladder urothelial E7 cells. However, how arsenic induces carcinogenesis through Aurora-A activation remaining unclear. Methods Bromodeoxyuridine (BrdU) staining, MTT assay, and flow cytometry assay were conducted to determine cell proliferation. Messenger RNA and protein expression levels of Aurora-A were detected by reverse transcriptional-PCR and Western blotting, respectively. Centrosome of cells was observed by immunofluorescent staining. The transcription factor of Aurora-A was investigated by promoter activity, chromosome immunoprecipitation (ChIP), and small interfering RNA (shRNA) assays. Mouse model was utilized to confirm the relationship between arsenic and Aurora-A. Results We reveal that low dosage of arsenic treatment increased cell proliferation is associated with accumulated cell population at S phase. We also detected increased Aurora-A expression at mRNA and protein levels in immortalized bladder urothelial E7 cells exposed to low doses of arsenic. Arsenic-treated cells displayed increased multiple centrosome which is resulted from overexpressed Aurora-A. Furthermore, the transcription factor, E2F1, is responsible for Aurora-A overexpression after arsenic treatment. We further disclosed that Aurora-A expression and cell proliferation were increased in bladder and uterus tissues of the BALB/c mice after long-term arsenic (1 mg/L) exposure for 2 months. Conclusion We reveal that low dose of arsenic induced cell proliferation is through Aurora-A overexpression, which is transcriptionally regulated by E2F1 both in vitro and in vivo. Our findings disclose a new possibility that arsenic at low concentration activates Aurora-A to induce carcinogenesis.

Analysis of various public databases revealed that HRAS gene mutation frequency and mRNA expression are higher in bladder urothelial carcinoma. Further analysis revealed the roles of oncogenic HRAS, autophagy, and cell senescence signaling in bladder cancer cells sensitized to the anticancer drug cisplatin using the phytochemical pterostilbene. A T24 cell line with the oncogenic HRAS was chosen for further experiments. Indeed, coadministration of pterostilbene increased stronger cytotoxicity on T24 cells compared to HRAS wild-type E7 cells, which was paralleled by neither elevated apoptosis nor induced cell cycle arrest, but rather a marked elevation of autophagy and cell senescence in T24 cells. Pterostilbene-induced autophagy in T24 cells was paralleled by inhibition of class I PI3K/mTOR/p70S6K as well as activation of MEK/ERK (a RAS target) and class III PI3K pathways. Pterostilbene-induced cell senescence on T24 cells was paralleled by increased pan-RAS and decreased phospho-RB expression. Coadministration of PI3K class III inhibitor 3-methyladenine or MEK inhibitor U0126 suppressed pterostilbene-induced autophagy and reversed pterostilbene-enhanced cytotoxicity, but did not affect pterostilbene-elevated cell senescence in T24 cells. Animal study data confirmed that pterostilbene enhanced cytotoxicity of cisplatin plus gemcitabine. These results suggest a therapeutic application of pterostilbene in cisplatin-resistant bladder cancer with oncogenic HRAS.

Autophagy plays diverse roles in Ras-related tumorigenesis. H-rasval12 induces autophagy through multiple signaling pathways including Raf-1/ERK pathway, and various ERK downstream molecules of autophagy have been reported. In this study, Bcl-2/adenovirus E1B 19-kDa–interacting protein 3 (BNIP3) is identified as a downstream transducer of the Ras/Raf/ERK signaling pathway to induce autophagy. BNIP3 was upregulated by H-rasval12 at the transcriptional level to compete with Beclin 1 for binding with Bcl-2. H-rasval12–induced autophagy suppresses cell proliferation demonstrated both in vitro and in vivo by expression of ectopic BNIP3, Atg5, or interference RNA of BNIP3 (siBNIP3) and Atg5 (shAtg5) using mouse NIH3T3 and embryo fibroblast cells. H-rasval12 induces different autophagic responses depending on the duration of Ras overexpression. After a short time (48 hours) of Ras overexpression, autophagy inhibits cell proliferation. In contrast, a longer time (2 weeks) of Ras overexpression, cell proliferation was enhanced by autophagy. Furthermore, overexpression of mutant Ras, BNIP3, and LC3-II was detected in bladder cancer T24 cells and the tumor parts of 75% of bladder cancer specimens indicating a positive correlation between autophagy and tumorigenesis. Taken together, our mouse model demonstrates a balance between BNIP3-mediated autophagy and H-rasval12–induced tumor formation and reveals that H-rasval12 induces autophagy in a BNIP3-dependent manner, and the threshold of autophagy plays a decisive role in H-rasval12–induced tumorigenesis. Our findings combined with others’ reports suggest a new therapeutic strategy against Ras-related tumorigenesis by negative or positive regulation of autophagic activity, which is determined by the level of autophagy and tumor progression stages.

Background: Mutant Ras plays multiple functions in tumorigenesis including tumor formation and metastasis. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a metastasis inhibitor gene, suppresses matrix metalloproteinase (MMP) activity in the metastatic cascade. Clarifying the relationship between Ras and RECK and understanding the underlying molecular mechanism may lead to the development of better treatment for Ras-related tumors. Methods: Suppression subtractive hybridization PCR (SSH PCR) was conducted to identify Ha-rasval12 up-regulated genes in bladder cancer cells. Stable cell lines of human breast cancer (MCF-7-ras) and mouse NIH3T3 fibroblasts (7-4) harboring the inducible Ha-rasval12 oncogene, which could be induced by isopropylthio-β-D-galactoside (IPTG), were used to clarify the relationship between Ras and the up-regulated genes. Chromatin immunoprecipitation (ChIP) assay, DNA affinity precipitation assay (DAPA) and RECK reporter gene assay were utilized to confirm the complex formation and binding with promoters. Results: Retinoblastoma binding protein-7 (RbAp46) was identified and confirmed as a Ha-rasval12 up-regulated gene. RbAp46 could bind with histone deacetylase (HDAC1) and Sp1, followed by binding to RECK promoter at the Sp1 site resulting in repression of RECK expression. High expression of Ras protein accompanied with high RbAp46 and low RECK expression were detected in 75% (3/4) of the clinical bladder cancer tumor tissues compared to the adjacent normal parts. Ras induced RbAp46 expression increases invasion of the bladder cancer T24 cells and MMP-9 activity was increased, which was confirmed by specific lentiviral shRNAs inhibitors against Ras and RbAp46. Similarly, knockdown of RbAp46 expression in the stable NIH3T3 cells "7-4" by shRNA decreased Ras-related lung metastasis using a xenograft nude mice model. Conclusions: We confirmed that RbAp46 is a Ha-rasval12 up-regulated gene and binds with HDAC1 and Sp1. Furthermore, RbAp46 binds to the RECK promoter at the Sp1 site via recruitment by Sp1. RECK is subsequently activated, leading to increased MMP9 activity, which may lead to increased metastasis in vivo. Our findings of Ras upregulation of RbAp46 may lead to revealing a novel mechanism of Ras-related tumor cell metastasis. [ABSTRACT FROM AUTHOR]

Approximately 350 million people worldwide are chronically infected by hepatitis B virus (HBV). HBV causes severe liver diseases including cirrhosis and hepatocellular carcinoma (HCC). In about 25% of affected patients, HBV infection proceeds to HCC. Therefore, the mechanisms by which HBV affects the host cell to promote viral replication and its pathogenesis have been the subject of intensive research efforts. Emerging evidence indicates that both autophagy and microRNAs (miRNAs) are involved in HBV replication and HBV-related hepatocarcinogenesis. In this review, we summarize how HBV induces autophagy, the role of autophagy in HBV infection, and HBV-related tumorigenesis. We further discuss the emerging roles of miRNAs in HBV infection and how HBV affects miRNAs biogenesis. The accumulating knowledge pertaining to autophagy and miRNAs in HBV replication and its pathogenesis may lead to the development of novel strategies against HBV infection and HBV-related HCC tumorigenesis.