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The mechanism of the positive regulation of autophagy by mammalian target of rapamycin (mTOR) remains largely unknown. In the present study, we observed that inhibition of mTOR either genetically or pharmacologically suppressed basal and H2O2-induced autophagic processes concomitant with marked upregulation of apoptosis inducing factor (AIF) expression. In cells with mTOR knockdown, deprivation of AIF partially rescued both basal and induced autophagy. Importantly, we found that AIF interacted with either mTOR or Beclin1 and that AIF loss markedly enhanced the association of Beclin1 with VPS34, which is essential for autophagy initiation. In contrast, mTOR loss increased the binding of AIF to Beclin1, concomitantly decreasing the interaction between Beclin1 and VPS34. Collectively, the data presented here revealed a previously unnoticed autophagic regulatory pathway in which mTOR functioned as a positive regulator by directly interacting with AIF and autophagic proteins.

Akt is usually considered to be a negative regulator of both autophagy and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling. In the present study, we found that SC66, a pyridine-based allosteric Akt inhibitor, suppressed basal and H2 O2 -induced autophagy concurrent with decreased phosphorylation and activity of AMPK. SC66 treatment led to the formation of a high molecular weight (HMW) form of SQSTM1/p62 (p62), which is an autophagic substrate and is essential for selective autophagy. Moreover, we observed that SC66 inhibited the binding of p62 and microtubule-associated protein light chain 3 (LC3). The immunoprecipitation results revealed the interaction between p62 and epidermal growth factor receptor (EGFR), and knockdown of EGFR reversed SC66-mediated autophagy inhibition without affecting the phosphorylation of acetyl-CoA carboxylase (ACC), a well-known substrate of AMPK. SC66 increased the interaction between EGFR and Beclin 1 and markedly decreased the association of EGFR with VPS34, a critical protein for autophagy induction. Collectively, the data presented here indicate that EGFR-p62 pathway plays a critical role in Akt-mediated positive regulation of autophagy. This article is protected by copyright. All rights reserved.

Objective To construct a prediction model for more precise evaluation of prognosis which will allow personalized treatment recommendations for adjuvant therapy in patients following resection of esophageal squamous cell carcinoma (ESCC). Background Marked heterogeneity of patient prognosis and limited evidence regarding survival benefit of various adjuvant therapy regimens pose challenges in the clinical treatment of ESCC. Methods Based on comprehensive clinical data obtained from 4,129 consecutive patients with resected ESCC in a high-risk region in China, we identified predictors for overall survival (OS) through a two-phase selection based on Cox proportional hazard regression and minimization of Akaike information criterion. The model was internally validated using bootstrapping and externally validated in 1,815 patients from a non-high-risk region in China. Results The final model incorporates nine variables: age, sex, primary site, T stage, N stage, number of lymph nodes harvested, tumor size, adjuvant treatment, and hemoglobin level. A significant interaction was also observed between N stage and adjuvant treatment. N1+ stage patients were likely to benefit from addition of adjuvant therapy as opposed to surgery alone, but adjuvant therapy did not improve OS for N0 stage patients. The C-index of the model was 0.729 in the training cohort, 0.723 after bootstrapping, and 0.695 in the external validation cohort. This model outperformed the seventh edition American Joint Committee on Cancer staging system in prognostic prediction and risk stratification. Conclusion The prediction model constructed in this study may facilitate precise prediction of survival and inform decision-making about adjuvant therapy according to N stage.

To evaluate the long-term and short-term outcomes of minimally invasive esophagectomy (MIE) compared with open esophagectomy (OE) in localized esophageal squamous cell carcinoma (ESCC) patients in real-world settings.MIE is an alternative to OE, despite the limited evidence regarding its effect on long-term survival.We recruited 5822 consecutive patients with resectable ESCC in two typical high-volume centers in southern and northern China, 1453 of whom underwent MIE. Propensity score-based overlap weighted regression adjusted for multifaceted confounding factors was used to compare outcomes in the MIE and OE groups.Five-year overall survival (OS) was 62.7% in the MIE group and 57.7% in the OE group. The overlap weighted Cox regression showed slightly better OS in the MIE group (HR 0.93, 95% CI: 0.82-1.06). Although duration of surgery was longer and treatment cost higher in the MIE group than in the OE group, the number of lymph nodes harvested was larger, the proportion of intraoperative blood transfusions lower, and postoperative complications less in the MIE group. 30-day (RR 0.77, 0.38-1.55) and 90-day (RR 0.79, 0.46-1.35) mortality were lower in the MIE group versus the OE group, although not statistically significant. These findings were consistent across different analytic approaches and subgroups, notably in the subset of ESCC patients with large tumors.MIE can be performed safely with OS comparable to OE for patients with localized ESCC, indicating MIE may be recommended as the primary surgical approach for resectable ESCC in health facilities with requisite technical capacity.

Gliocladicillin C (3) is a cytotoxic epipolythiodioxopiperazine (ETP) isolated from the Ophiocordyceps-associated fungus Clonostachys rogersoniana. Although the disulfides/polysulfides in ETPs are believed to account for their cytotoxicity, and 11′-deoxyverticillin A was demonstrated to induce apoptosis and autophagy, how they mediate apoptosis and autophagy remained unknown. Here, we revealed that 3 activated caspase-dependent apoptosis and autophagy in human tumor cells, while the prepared disulfide-cleavage product failed to induce reactive oxygen species production and PARP cleavage, but further enhanced the autophagic flux compared to 3. Gliocladicillin C and its derivative also increased the phosphorylation of AMP-activated protein kinase and stimulated autophagy by affecting the glycolytic pathway. These results demonstrated that the disulfides played an essential role in inducing apoptosis, but not autophagy.

Sunitinib (ST), a multitargeted receptor tyrosine kinase inhibitor, has been demonstrated to be effective for the treatment of renal carcinoma. It has been reported that ST is involved in the mediation of autophagy; however, its regulatory role in the autophagic process remains controversial. Furthermore, the mechanism by which activated AMP-activated protein kinase (AMPK) negatively regulates autophagy remains nearly unexplored. In the present study, we revealed that ST inhibited AMPK activity and regulated autophagy in a cell type- and dose-dependent manner. In a number of cell lines, ST was demonstrated to inhibit H2O2-induced autophagy and the phosphorylation of acetyl-CoA carboxylase (ACC), whereas alone it could block the autophagic flux concurrent with increased expression of p62. An immunoprecipitation assay revealed that LC3 directly interacted with p62, whereas ST increased punctate LC3 staining, which was well colocalized with p62. Taken together, we reveal a previously unnoticed pathway for ST to regulate the autophagic process, and p62, although often utilized as a substrate in autophagy, plays a critical role in regulating the inhibition of ST in both basal and induced autophagy.

Two new diterpenoids, hypoxyterpoids A (1) and B (2), and four new isocoumarin derivatives, hypoxymarins A–D (4–7), together, with seven known metabolites (3 and 8–13) were obtained from the crude extract of the mangrove-derived fungus Hypoxylon sp. The structures of the new compounds were elucidated on the basis of 1- and 2-dimensional (1D/2D) nuclear magnetic resonance (NMR) spectroscopic and mass spectrometric analysis. The absolute configurations of compounds 1, 2, 4, 5, and 7 were determined by comparison of experimental and calculated electronic circular dichroism (ECD) spectra, and the absolute configurations of C-4′ in 6 and C-9 in 7 were determined by [Rh2(OCOCF3)4]-induced ECD spectra. Compound 1 showed moderate α-glucosidase inhibitory activities with IC50 values of 741.5 ± 2.83 μM. Compounds 6 and 11 exhibited DPPH scavenging activities with IC50 values of 15.36 ± 0.24 and 3.69 ± 0.07 μM, respectively.

Both Raptor and Rictor are the key components in the complexes of mammalian target of rapamycin (mTOR), which play a vital role in mediating autophagy. Unlike mTOR, the regulatory role of either Raptor or Rictor in the regulation of autophagic process is relatively less explored. In present study, we found that rasfonin, which isolated from Talaromyces sp. 3656-A1 and was a fungal natural product, activated both caspase-dependent apoptosis and autophagy in ACHN, a renal carcinoma cell line. Knockdown of Raptor decreased both rasfonin-induced autophagic flux and PARP-1 cleavage, and in contrast, Rictor silencing increased apoptosis concomitantly enhancing rasfonin-induced autophagy. Unexpectedly, API-2, which was widely used as an inhibitor of Akt, promoted rasfonin-dependent autophagy in Raptor-depleted but not Rictor-deprived cells. Collectively, these results demonstrated that Raptor and Rictor could play a distinctly regulatory role in rasfonin-enhanced autophagy and apoptosis.

Nuclear-localized mitochondria were discovered over sixty years ago1; however, the function of these organelles in the initiation of nuclear formation and development remains unknown. Here, we showed that mitochondria fragmented into dense particles to initiate and develop a nucleus, and multiple nuclei were separately and simultaneously formed by fragmented mitochondria in a single cell. The combination of nuclei individually constructed by the mitochondrial assembly of dense particles for nuclear transition partitioned the cytoplasm to form an intranuclear inclusion (INC), whose formation was not related to herniation or invagination of the cytoplasm. During nuclear conversion of itself and neighbouring organelles, the mitochondrion was incorporated into the nucleus to become a nuclear mitochondrion. Knockdown of microtubule-associated protein light chain 3 (LC3), a key autophagic protein, increased free micronuclei by delaying nuclear fusion and enhancing the mitochondria-to-micronuclei transition.

The function of nuclear-localized mitochondria remains unknown. We found that mitochondria assembled dense particles to fragment and disperse into the particles, which reassembled to initiate nuclear formation and development. Individually-formed nuclei in one single cell were joined together by mitochondrial fragmentation concurrently partitioning cytoplasm to form an intranuclear inclusion (INC), whose creation was not related to herniation or invagination of the cytoplasm. Along with the nuclear transition of a mitochondrion and its neighboring counterparts, the organelle included itself in the nucleus to become nuclear mitochondrion through peripherally assembling of dense particles. New medium reversed the nuclear formation of the organelles to recovery and re-establishment via the return of the particles, which consisted of dense microvesicles (MIVs).

Chloropupukeananin (RN56-6) and Pestalofone C (RN56-49), isolated from the culture of the plant endophytic fungus Pestalotiopsis fici, have been shown cytotoxic, anti-HIV, and antimicrobial activities. However, the underlying mechanism of their regulatory roles in autophagy remains unknown. In the present study, we revealed that both compounds increased the formation of autophagosome and enhanced autophagic flux. While RN56-6 upregulated the expression of HK2, one of the key rate-limiting enzymes of glycolysis, the inhibition of glycolysis chemically attenuated RN56-6-induced autophagy. On the contrary, RN56-49 downregulated the expression of HK2, while the suppression of glycolysis promoted RN56-49-dependent autophagic flux. Moreover, the knockdown of AMPKβ1, a scaffolding subunit of AMPK, decreased autophagy induced by these two compounds. Collectively, these findings revealed that RN56-6 and RN56-49 regulated autophagic process through AMPK and glycolytic pathway.

Growth factor receptor bound protein 2 (Grb2) is an adaptor protein critical for signal transduction and endocytosis, but its role in DNA damage response (DDR) remains unknown. Here, we report that either knockdown of Grb2 or overexpression of the mutated Grb2 promotes micronuclei formation in response to oxidative stress. Furthermore, Grb2 was demonstrated to interact with phosphatase and tensin homologue (PTEN; a tumor suppressor essential for nuclear stability), and the loss of Grb2 reduced the nuclear-localized PTEN, which was further decreased upon stimulation with hydrogen peroxide (H2O2). Overexpression of the T398A-mutated, nuclear-localized PTEN reduced micronuclei frequency in the cells deficient of functional Grb2 via rescuing the H2O2-dependent expression of Rad51, a protein essential for the homologous recombination (HR) repair process. Moreover, depletion of Grb2 markedly decreased the expression of Rad51 and its interaction with PTEN. Notably, Rad51 showed a preference to immunoprecipation with the T398A-PTEN mutant, and silencing of Rad51 alone accumulated micronuclei concurring with decreased expression of both Grb2 and PTEN. Our findings indicate that Grb2 interacts with PTEN and Rad51 to regulate genomic stability in DDR by mediating the nuclear translocation of PTEN to affect the expression of Rad51.

Aims To investigate the role and underlying mechanism of 4E-BP1 and S6K1 in regulating autophagy and hepatitis B virus (HBV) replication. Main methods The mRNA relative expression of HBx and its DNA level were detected by real-time PCR. The relative levels of hepatitis B surface antigen (HBsAg) were measured by enzyme-linked immunosorbent assay (ELISA). HBx DNA level of HepG2 cells transfected with pcDNA3.1(+)-HBV1.3 plasmids were detected by Southern blot. Moreover, we determined autophagy through electron microscopy, confocal microscopy and Western blot. Key findings Rapamycin promoted autophagy and the X protein synthesis concomitantly with elevation in Akt phosphorylation and Beclin1 expression. Either Beclin1 or Akt depletion suppresses the Rapa-enhanced HBV replication, whereas mTOR silencing inhibited HBV replication concurring with a decreased in both S6K1 and 4E-BP1 phosphorylation. Unexpectedly, Akt inhibitor suppressed Rapa-dependent autophagic flux and increased the level of p62/SQSTM1. While S6K1 ablation impaired autophagy and decreased X protein expression, 4E-BP1 silencing slightly influenced autophagy and increased X protein level. Significance The underlying mechanism of 4E-BP1 and S6K1, two main downstream effectors of mTOR, in mediating HBV replication and HBV-induced autophagy remains largely unknown. Here, we propose that Akt is required for both HBV replication and Rapa-induced autophagy, and 4E-BP1 and S6K1 play a distinct role in the virus replication and autophagic process.

Deficiency of autophagy has been linked to increase in nuclear instability, but the role of autophagy in regulating the formation and elimination of micronuclei, a diagnostic marker for genomic instability, is limited in mammalian cells. Utilizing immunostaining and subcellular fractionation, we found that either LC3-II or the phosphorylated Ulk1 localized in nuclei, and immunoprecipitation results showed that both LC3 and Unc-51-like kinase 1 (Ulk1) interacted with γ-H2AX, a marker for the DNA double-strand breaks (DSB). Sunitinib, a multi-targeted receptor tyrosine kinase inhibitor, was found to enhance the autophagic flux concurring with increase in the frequency of micronuclei accrued upon inhibition of autophagy, and similar results were also obtained in the rasfonin-treated cells. Moreover, the punctate LC3 staining colocalized with micronuclei. Unexpectedly, deprivation of SQSTM1/p62 alone accumulated micronuclei, which was not further increased upon challenge with ST. Rad51 is a protein central to repairing DSB by homologous recombination and treatment with ST or rasfonin decreased its expression. In several cell lines, p62 appeared in the immunoprecipites of Rad51, whereas LC3, Ulk1 and p62 interacted with PARP-1, another protein involved in DNA repair and genomic stability. In addition, knockdown of either Rad51 or PARP-1 completely inhibited the ST-induced autophagic flux. Taken together, the data presented here demonstrated that both LC3-II and the phosphorylated Ulk1 localized in nuclei and interacted with the proteins essential for nuclear stability, thereby revealing a more intimate relationship between autophagy and genomic stability.