Deubiquitylase PSMD14 inhibits autophagy to promote ovarian cancer progression via stabilization of LRPPRC

Autophagy is a key cellular process, which exists in many tumors and plays dual roles in tumor promotion and suppression. However, the role and mechanism of aberrant autophagy in ovarian cancer remains unclear. Ubiquitin-proteasome pathway is the most important pathway for specific protein degradation. Deubiquitinases (DUBs) have crucial roles in all the stages of tumorigenesis and progression. Herein, we explore the DUBs which contribute to aberrant autophagy in ovarian cancer. TCGA data analysis shows that the autophagy level is suppressed, and the selective autophagy receptor SQSTM1/p62 is abnormally high expressed in ovarian cancer. We screen and identify that the deubiquitinase PSMD14 negatively regulates autophagy level. Functional studies show that increased PSMD14 expression remarkably enhances ovarian cancer cells malignancy, whereas knockdown of PSMD14 has the opposite effect. Furthermore, in vivo assays show that knockdown of PSMD14 inhibits the growth, lung and abdominal metastasis of ovarian cancer. Mechanistically, PSMD14 directly interacts with LRPPRC and inhibits its ubiquitination, thereby inhibiting autophagy through LRPPRC/Beclin1-Bcl-2/SQSTM1 signaling pathway. Next, we demonstrate that PSMD14 is upregulated in ovarian cancer and high expression of PSMD14 positively correlates with LRPPRC. Taken together, we clarify the role of autophagy in regulating the ovarian cancer phenotype and provide insights into regulatory mechanism of autophagy in ovarian cancer.     

AMDE-1 Is a Dual Function Chemical for Autophagy Activation and Inhibition

Autophagy is the process by which cytosolic components and organelles are delivered to the lysosome for degradation. Autophagy plays important roles in cellular homeostasis and disease pathogenesis. Small chemical molecules that can modulate autophagy activity may have pharmacological value for treating diseases. Using a GFP-LC3-based high content screening assay we identified a novel chemical that is able to modulate autophagy at both initiation and degradation levels. This molecule, termed as Autophagy Modulator with Dual Effect-1 (AMDE-1), triggered autophagy in an Atg5-dependent manner, recruiting Atg16 to the pre-autophagosomal site and causing LC3 lipidation. AMDE-1 induced autophagy through the activation of AMPK, which inactivated mTORC1 and activated ULK1. AMDE-1did not affect MAP kinase, JNK or oxidative stress signaling for autophagy induction. Surprisingly, treatment with AMDE-1 resulted in impairment in autophagic flux and inhibition of long-lived protein degradation. This inhibition was correlated with a reduction in lysosomal degradation capacity but not with autophagosome-lysosome fusion. Further analysis indicated that AMDE-1 caused a reduction in lysosome acidity and lysosomal proteolytic activity, suggesting that it suppressed general lysosome function. AMDE-1 thus also impaired endocytosis-mediated EGF receptor degradation. The dual effects of AMDE-1 on autophagy induction and lysosomal degradation suggested that its net effect would likely lead to autophagic stress and lysosome dysfunction, and therefore cell death. Indeed, AMDE-1 triggered necroptosis and was preferentially cytotoxic to cancer cells. In conclusion, this study identified a new class of autophagy modulators with dual effects, which can be explored for potential uses in cancer therapy.     

Autophagy contributes to gefitinib-induced glioma cell growth inhibition

Epidermal growth factor receptor tyrosine kinase inhibitors, including gefitinib, have been evaluated in patients with malignant gliomas. However, the molecular mechanisms involved in gefitinib-mediated anticancer effects against glioma are incompletely understood. In the present study, the cytostatic potential of gefitinib was demonstrated by the inhibition of glioma cell growth, long-term clonogenic survival, and xenograft tumor growth. The cytostatic consequences were accompanied by autophagy, as evidenced by monodansylcadaverine staining of acidic vesicle formation, conversion of microtubule-associated protein-1 light chain 3-II (LC3-II), degradation of p62, punctate pattern of GFP-LC3, and conversion of GFP-LC3 to cleaved-GFP. Autophagy inhibitor 3-methyladenosine and chloroquine and genetic silencing of LC3 or Beclin 1 attenuated gefitinib-induced growth inhibition. Gefitinib-induced autophagy was not accompanied by the disruption of the Akt/mammalian target of rapamycin signaling. Instead, the activation of liver kinase-B1/AMP-activated protein kinase (AMPK) signaling correlated well with the induction of autophagy and growth inhibition caused by gefitinib. Silencing of AMPK suppressed gefitinib-induced autophagy and growth inhibition. The crucial role of AMPK activation in inducing glioma autophagy and growth inhibition was further supported by the actions of AMP mimetic AICAR. Gefitinib was shown to be capable of reducing the proliferation of glioma cells, presumably by autophagic mechanisms involving AMPK activation.     

Autophagy in filamentous fungi

Autophagy is a ubiquitous, non-selective degradation process in eukaryotic cells that is conserved from yeast to man. Autophagy research has increased significantly in the last ten years, as autophagy has been connected with cancer, neurodegenerative disease and various human developmental processes. Autophagy also appears to play an important role in filamentous fungi, impacting growth, morphology and development. In this review, an autophagy model developed for the yeast Saccharomyces cerevisiae is used as an intellectual framework to discuss autophagy in filamentous fungi. Studies imply that, similar to yeast, fungal autophagy is characterized by the presence of autophagosomes and controlled by Tor kinase. In addition, fungal autophagy is apparently involved in protection against cell death and has significant effects on cellular growth and development. However, the only putative autophagy proteins characterized in filamentous fungi are Atg1 and Atg8. We discuss various strategies used to study and monitor fungal autophagy as well as the possible relationship between autophagy, physiology, and morphological development.     

Resveratrol attenuates vascular endothelial inflammation by inducing autophagy through the cAMP signaling pathway

Inflammation participates centrally in all stages of atherosclerosis (AS), which begins with inflammatory changes in the endothelium, characterized by expression of the adhesion molecules. Resveratrol (RSV) is a naturally occurring phytoalexin that can attenuate endothelial inflammation; however, the exact mechanisms have not been thoroughly elucidated. Autophagy refers to the normal process of cell degradation of proteins and organelles, and is protective against certain inflammatory injuries. Thus, we intended to determine the role of autophagy in the antiinflammatory effects of RSV in human umbilical vein endothelial cells (HUVECs). We found that RSV pretreatment reduced tumor necrosis factor α (TNF/TNFα)-induced inflammation and increased MAP1LC3B2 (microtubule-associated protein 1 light chain 3 β 2) expression and SQSTM1/p62 (sequestosome 1) degradation in a concentration-dependent manner. A bafilomycin A₁ (BafA1) challenge resulted in further accumulation of MAP1LC3B2 in HUVECs. Furthermore, autophagy inhibitors 3-methyladenine (3-MA), chloroquine as well as ATG5 and BECN1 siRNA significantly attenuated RSV-induced autophagy, which, subsequently, suppressed the downregulation of RSV-induced inflammatory factors expression. RSV also increased cAMP (cyclic adenosine monophosphate) content, the expression of PRKA (protein kinase A) and SIRT1 (sirtuin 1), as well as the activity of AMPK (AMP-activated protein kinase). RSV-induced autophagy in HUVECs was abolished in the presence of inhibitors of ADCY (adenylyl cyclase, KH7), PRKA (H-89), AMPK (compound C), or SIRT1 (nicotinamide and EX-527), as well as ADCY, PRKA, AMPK, and SIRT1 siRNA transfection, indicating that the effects of RSV on autophagy induction were dependent on cAMP, PRKA, AMPK and SIRT1. In conclusion, RSV attenuates endothelial inflammation by inducing autophagy, and the autophagy in part was mediated through the activation of the cAMP-PRKA-AMPK-SIRT1 signaling pathway.     

Regulation of autophagy by transforming growth factor-β (TGF-β) signaling

Transforming growth factor-β (TGF-β) has broad impacts on an array of diverse cellular functions including cell growth, differentiation, adhesion, migration, and apoptosis. Perturbations of the TGF-β signaling pathways are involved in progression of various tumors. Autophagy is a pivotal response of normal and cancer cells to environmental stresses and is induced by various stimuli. Otherwise, autophagy has an intrinsic function in tumor suppression. Recently, we demonstrated that TGF-β induces autophagy in hepatocellular carcinoma cells and mammary carcinoma cells. Autophagy activation by TGF-β is mediated through the Smad and JNK pathways. We show that siRNA-mediated knockdown of autophagy genes suppresses the growth inhibitory function of TGF-β and that autophagy activation potentiates TGF-β-mediated induction of proapoptotic genes, Bim and Bmf, in hepatoma cells. In this context, the autophagy pathway might contribute to the growth inhibitory effect of TGF-β, in conjunction with other anti-proliferative pathways downstream of TGF-β signaling. The context and manner by which the TGF-β signaling pathway regulates autophagy have implications for a better understanding of pathological and bidirectional roles of TGF-β signaling pathways in tumorigenesis.     

Autophagy protects intestinal epithelial Cells against Deoxynivalenol toxicity by alleviating oxidative stress via IKK signaling pathway

Autophagy is an intracellular process of homeostatic degradation that promotes cell survival under various stressors. Deoxynivalenol (DON), a fungal toxin, often causes diarrhea and disturbs the homeostasis of the intestinal system. To investigate the function of intestinal autophagy in response to DON and associated mechanisms, we firstly knocked out ATG5 (autophagy-related gene 5) in porcine intestinal epithelial cells (IPEC-J2) using CRISPR-Cas9 technology. When treated with DON, autophagy was induced in IPEC-J2 cells but not in IPEC-J2.Atg5ko cells. The deficiency in autophagy increased DON-induced apoptosis in IPEC-J2.atg5ko cells, in part, through the generation of reactive oxygen species (ROS). The cellular stress response can be restored in IPEC-J2.atg5ko cells by overexpressing proteins involved in protein folding. Interestingly, we found that autophagy deficiency downregulated the expression of endoplasmic reticulum folding proteins BiP and PDI when IPEC-J2.atg5ko cells were treated with DON. In addition, we investigated the molecular mechanism of autophagy involved in the IKK, AMPK, and mTOR signaling pathway and found that Bay-117082 and Compound C, specific inhibitors for IKK and AMPK, respectively, inhibited the induction of autophagy. Taken together, our results suggest that autophagy is pivotal for protection against DON in pig intestinal cells.     

Folliculin Regulates Ampk-Dependent Autophagy and Metabolic Stress Survival

Dysregulation of AMPK signaling has been implicated in many human diseases, which emphasizes the importance of characterizing AMPK regulators. The tumor suppressor FLCN, responsible for the Birt-Hogg Dubé renal neoplasia syndrome (BHD), is an AMPK-binding partner but the genetic and functional links between FLCN and AMPK have not been established. Strikingly, the majority of naturally occurring FLCN mutations predisposing to BHD are predicted to produce truncated proteins unable to bind AMPK, pointing to the critical role of this interaction in the tumor suppression mechanism. Here, we demonstrate that FLCN is an evolutionarily conserved negative regulator of AMPK. Using Caenorhabditis elegans and mammalian cells, we show that loss of FLCN results in constitutive activation of AMPK which induces autophagy, inhibits apoptosis, improves cellular bioenergetics, and confers resistance to energy-depleting stresses including oxidative stress, heat, anoxia, and serum deprivation. We further show that AMPK activation conferred by FLCN loss is independent of the cellular energy state suggesting that FLCN controls the AMPK energy sensing ability. Together, our data suggest that FLCN is an evolutionarily conserved regulator of AMPK signaling that may act as a tumor suppressor by negatively regulating AMPK function.     

Autophagy Negatively Regulates Early Axon Growth in Cortical Neurons

Neurite growth requires neurite extension and retraction, which are associated with protein degradation. Autophagy is a conserved bulk degradation pathway that regulates several cellular processes. However, little is known about autophagic regulation during early neurite growth. In this study, we investigated whether autophagy was involved in early neurite growth and how it regulated neurite growth in primary cortical neurons. Components of autophagy were expressed and autophagy was activated during early neurite growth. Interestingly, inhibition of autophagy by atg7 small interfering RNA (siRNA) caused elongation of axons, while activation of autophagy by rapamycin suppressed axon growth. Surprisingly, inhibition of autophagy reduced the protein level of RhoA. Moreover, expression of RhoA suppressed axon overelongation mediated by autophagy inhibition, whereas inhibition of the RhoA signaling pathway by Y-27632 recovered rapamycin-mediated suppression of axon growth. Interestingly, hnRNP-Q1, which negatively regulates RhoA, accumulated in autophagy-deficient neurons, while its protein level was reduced by autophagy activation. Overall, our study suggests that autophagy negatively regulates axon extension via the RhoA-ROCK pathway by regulating hnRNP-Q1 in primary cortical neurons. Therefore, autophagy might serve as a fine-tuning mechanism to regulate early axon extension.     

The MEK/ERK pathway is essential for maintenance of cytoprotective autophagy in <Emphasis Type="Italic">E1A</Emphasis>+<Emphasis Type="Italic">cHA</Emphasis>-<Emphasis Type="Italic">RAS</Emphasis> transformants after exposure to radiation

Autophagy is a conserved process of protein and organelle degradation that serves to maintain cell viability. Autophagy is frequently induced in response to stress or to exposure to DNA-damaging agents or retinoids, as well as to starvation and deficiency of growth factors. In this work, autophagy induced in E1A+cHA-RAS transformed cells in response to X-ray radiation was studied, with a focus on the role of the MEK/ERK signaling pathway in the regulation of radiation-induced autophagy. It was found that inhibition of the MEK/ERK pathway diminished cell viability and altered the sequence of events in radiation-induced autophagy. In particular, it caused aberrations in its final stages, leading to cytoplasmic accumulation of the p62/SQSTM1 adaptor protein in autophagic cavities of unclear origin. Thus, the MEK/ERK pathway activity is essential for the induction and maintenance of autophagy, increasing the viability of exposed cells in response to radiation.     

A critical role for autophagy in pancreatic cancer

Autophagy is a regulated catabolic process that leads to the lysosomal degradation of damaged proteins, organelles and other macromolecules, with subsequent recycling of bioenergetic intermediates. The role of autophagy in cancer is undoubtedly complex and likely dependent on tumor type and on the cellular and developmental context. While it has been well demonstrated that autophagy may function as a tumor suppressor, there is mounting evidence that autophagy may have pro-tumorigenic roles, e.g., promoting therapeutic resistance as well as survival under stresses such as hypoxia and nutrient deprivation. These two, seemingly disparate functions can be reconciled by a possible temporal role of autophagy during tumor development, initially suppressing tumor initiation yet supporting tumor growth at later stages.     

受体酪氨酸激酶对自噬的调控及其研究进展*

自噬是真核生物进化上保守的溶酶体降解的生物学过程,在维护细胞内的稳态、消除有害组分等方面起到了重要作用。受体酪氨酸激酶家族(receptor tyrosine kinase,RTKs)是一类激酶蛋白,在正常细胞和癌症细胞的运动和侵袭中起着重要作用。RTKs蛋白既能促进自噬,也能抑制自噬。研究显示,RTKs能够在肿瘤和相关疾病中发挥自噬作用,比如表皮生长因子受体(epidermal growth factor receptor,EGFR)可以抑制自噬,从而促进肿瘤生长、增殖;还能通过RTK/Ras/ERK信号通路诱导自噬,进而参与诸如细胞免疫反应之类的相关疾病。主要综述了RTKs对自噬的调控作用和相关研究成果,为靶点靶向疗法的理论依据提供了基础。     

Depletion of NBR1 in urothelial carcinoma cells enhances rapamycin-induced apoptosis through impaired autophagy and mitochondrial dysfunction

Rapamycin is well-recognized in the clinical therapeutic intervention for patients with cancer by specifically targeting mammalian target of rapamycin (mTOR) kinase. Rapamycin regulates general autophagy to clear damaged cells. Previously, we identified increased expression of messenger RNA levels of NBR1 (the neighbor of BRCA1 gene; autophagy cargo receptor) in human urothelial cancer (URCa) cells, which were not exhibited in response to rapamycin treatment for cell growth inhibition. Autophagy plays an important role in cellular physiology and offers protection against chemotherapeutic agents as an adaptive response required for maintaining cellular energy. Here, we hypothesized that loss of NBR1 sensitizes human URCa cells to growth inhibition induced by rapamycin treatment, leading to interruption of protective autophagic activation. Also, the potential role of mitochondria in regulating autophagy was tested to clarify the mechanism by which rapamycin induces apoptosis in NBR1-knockdown URCa cells. NBR1-knockdown URCa cells exhibited enhanced sensitivity to rapamycin associated with the suppression of autophagosomal elongation and mitochondrial defects. Loss of NBR1 expression altered the cellular responses to rapamycin treatment, resulting in impaired ATP homeostasis and an increase in reactive oxygen species (ROS). Although rapamycin treatment-induced autophagy by adenosine monophosphate-activated protein kinase (AMPK) phosphorylation in NBR1-knockdown cells, it did not process the conjugated form of LC3B-II after activation by unc-51 like autophagy-activating kinase 1 (ULK1). NBR1-knockdown URCa cells exhibited rather profound mitochondrial dysfunctions in response to rapamycin treatment as evidenced by Δψm collapse, ATP depletion, ROS accumulation, and apoptosis activation. Therefore, our findings provide a rationale for rapamycin treatment of NBR1-knockdown human urothelial cancer through the regulation of autophagy and mitochondrial dysfunction by regulating the AMPK/mTOR signaling pathway, indicating that NBR1 can be a potential therapeutic target of human urothelial cancer.     

A critical role for autophagy in pancreatic cancer

Autophagy is a regulated catabolic process that leads to the lysosomal degradation of damaged proteins, organelles and other macromolecules, with subsequent recycling of bioenergetic intermediates. The role of autophagy in cancer is undoubtedly complex and likely dependent on tumor type and on the cellular and developmental context. While it has been well demonstrated that autophagy may function as a tumor suppressor, there is mounting evidence that autophagy may have pro-tumorigenic roles, e.g., promoting therapeutic resistance as well as survival under stresses such as hypoxia and nutrient deprivation. These two, seemingly disparate functions can be reconciled by a possible temporal role of autophagy during tumor development, initially suppressing tumor initiation yet supporting tumor growth at later stages.     

Phospholipase D-mediated autophagic regulation is a potential target for cancer therapy

Autophagy is a catabolic process in which cell components are degraded to maintain cellular homeostasis by nutrient limitations. Defects of autophagy are involved in numerous diseases, including cancer. Here, we demonstrate a new role of phospholipase D (PLD) as a regulator of autophagy. PLD inhibition enhances autophagic flux via ATG1 (ULK1), ATG5 and ATG7, which are essential autophagy gene products critical for autophagosome formation. Moreover, PLD suppresses autophagy by differentially modulating phosphorylation of ULK1 mediated by mTOR and adenosine monophosphate-activated protein kinase (AMPK), and by suppressing the interaction of Beclin 1 with vacuolar-sorting protein 34 (Vps34), indicating that PLD coordinates major players of the autophagic pathway, AMPK-mTOR-ULK1 and Vps34/Beclin 1. Ultimately, PLD inhibition significantly sensitized in vitro and in vivo cancer regression via genetic and pharmacological inhibition of autophagy, providing rationale for a new therapeutic approach to enhancing the anticancer efficacy of PLD inhibition. Collectively, we show a novel role for PLD in the molecular machinery regulating autophagy.     

Defective Autophagy and mTORC1 Signaling in Myotubularin Null Mice

Autophagy is a vesicular trafficking pathway that regulates the degradation of aggregated proteins and damaged organelles. Initiation of autophagy requires several multiprotein signaling complexes, such as the ULK1 kinase complex and the Vps34 lipid kinase complex, which generates phosphatidylinositol 3-phosphate [PtdIns(3)P] on the forming autophagosomal membrane. Alterations in autophagy have been reported for various diseases, including myopathies. Here we show that skeletal muscle autophagy is compromised in mice deficient in the X-linked myotubular myopathy (XLMTM)-associated PtdIns(3)P phosphatase myotubularin (MTM1). Mtm1-deficient muscle displays several cellular abnormalities, including a profound increase in ubiquitin aggregates and abnormal mitochondria. Further, we show that Mtm1 deficiency is accompanied by activation of mTORC1 signaling, which persists even following starvation. In vivo pharmacological inhibition of mTOR is sufficient to normalize aberrant autophagy and improve muscle phenotypes in Mtm1 null mice. These results suggest that aberrant mTORC1 signaling and impaired autophagy are consequences of the loss of Mtm1 and may play a primary role in disease pathogenesis.     

Inhibition of autophagy impairs tumor cell invasion in an organotypic model

Autophagy is a membrane-trafficking process that delivers cytoplasmic constituents to lysosomes for degradation. It contributes to energy and organelle homeostasis and the preservation of proteome and genome integrity. Although a role in cancer is unquestionable, there are conflicting reports that autophagy can be both oncogenic and tumor suppressive, perhaps indicating that autophagy has different roles at different stages of tumor development. In this report, we address the role of autophagy in a critical stage of cancer progression—tumor cell invasion. Using a glioma cell line containing an inducible shRNA that targets the essential autophagy gene Atg12, we show that autophagy inhibition does not affect cell viability, proliferation or migration but significantly reduces cellular invasion in a 3D organotypic model. These data indicate that autophagy may play a critical role in the benign to malignant transition that is also central to the initiation of metastasis.