Megan M. Young

Autophagosomal Membrane Serves as Platform for Intracellular Death-inducing Signaling Complex (iDISC)-mediated Caspase-8 Activation and Apoptosis

Background: It remains a matter of debate whether autophagy contributes to apoptosis. Results: Atg5 and p62 are required for an intracellular death-inducing signaling complex (iDISC) formation on autophagosomal membranes for caspase-8 self-processing. Conclusion: Autophagosome serves as a platform for the intracellular activation of caspase-8. Significance: Induction of iDISC formation may shift cytoprotective autophagy to apoptosis for more effective cancer therapies. Autophagy and apoptosis are two evolutionarily conserved processes that regulate cell fate in response to cytotoxic stress. However, the functional relationship between these two processes remains far from clear. Here, we demonstrate an autophagy-dependent mechanism of caspase-8 activation and initiation of the apoptotic cascade in response to SKI-I, a pan-sphingosine kinase inhibitor, and bortezomib, a proteasome inhibitor. Autophagy is induced concomitantly with caspase-8 activation, which is responsible for initiation of the caspase cascade and the mitochondrial amplification loop that is required for full execution of apoptosis. Inhibition of autophagosome formation by depletion of Atg5 or Atg3 results in a marked suppression of caspase-8 activation and apoptosis. Although caspase-8 self-association depends on p62/SQSTM1, its self-processing requires the autophagosomal membrane. Caspase-8 forms a complex with Atg5 and colocalizes with LC3 and p62. Moreover, FADD, an adaptor protein for caspase-8 activation, associates with Atg5 on Atg16L- and LC3-positive autophagosomal membranes and loss of FADD suppresses cell death. Taken together, these results indicate that the autophagosomal membrane serves as a platform for an intracellular death-inducing signaling complex (iDISC) that recruits self-associated caspase-8 to initiate the caspase-8/-3 cascade.

Sphingolipids: regulators of crosstalk between apoptosis and autophagy

Apoptosis and autophagy are two evolutionarily conserved processes that maintain homeostasis during stress. Although the two pathways utilize fundamentally distinct machinery, apoptosis and autophagy are highly interconnected and share many key regulators. The crosstalk between apoptosis and autophagy is complex, as autophagy can function to promote cell survival or cell death under various cellular conditions. The molecular mechanisms of crosstalk are beginning to be elucidated and have critical implications for the treatment of various diseases, such as cancer. Sphingolipids are a class of bioactive lipids that mediate many key cellular processes, including apoptosis and autophagy. By targeting several of the shared regulators, sphingolipid metabolites differentially regulate the induction of apoptosis and autophagy. Importantly, individual sphingolipid species appear to “switch” autophagy toward cell survival (e.g., sphingosine-1-phosphate) or cell death (e.g., ceramide, gangliosides). This review assesses the current understanding of sphingolipid-induced apoptosis and autophagy to address how sphingolipids mediate the “switch” between the cell survival and cell death. As sphingolipid metabolism is frequently dysregulated in cancer, sphingolipid-modulating agents, or sphingomimetics, have emerged as a novel chemotherapeutic strategy. Ultimately, a greater understanding of sphingolipid-mediated crosstalk between apoptosis and autophagy may be critical for enhancing the chemotherapeutic efficacy of these agents.

Bif-1 haploinsufficiency promotes chromosomal instability and accelerates Myc-driven lymphomagenesis via suppression of mitophagy

Malignant transformation by oncogenes requires additional genetic/epigenetic changes to overcome enhanced susceptibility to apoptosis. In the present study, we report that Bif-1 (Sh3glb1), a gene encoding a membrane curvature–driving endophilin protein, is a haploinsufficient tumor suppressor that plays a key role in the prevention of chromosomal instability and suppresses the acquisition of apoptosis resistance during Myc-driven lymphomagenesis. Although a large portion of Bif-1–deficient mice harboring an Eμ-Myc transgene displayed embryonic lethality, allelic loss of Bif-1 dramatically accelerated the onset of Myc-induced lymphoma. At the premalignant stage, hemizygous deletion of Bif-1 resulted in an increase in mitochondrial mass, accumulation of DNA damage, and up-regulation of the antiapoptotic protein Mcl-1. Consistently, allelic loss of Bif-1 suppressed the activation of caspase-3 in Myc-induced lymphoma cells. Moreover, we found that Bif-1 is indispensable for the autophagy-dependent clearance of damaged mitochondria (mitophagy), because loss of Bif-1 resulted in the accumulation of endoplasmic reticulum–associated immature autophagosomes and suppressed the maturation of autophagosomes. The results of the present study indicate that Bif-1 haploinsufficiency attenuates mitophagy and results in the promotion of chromosomal instability, which enables tumor cells to efficiently bypass the oncogenic/metabolic pressures for apoptosis. .

The Bif-1-Dynamin 2 membrane fission machinery regulates Atg9-containing vesicle generation at the Rab11-positive reservoirs

Atg9 is a multispanning transmembrane protein that is required for autophagosome formation. During autophagy, vesicles containing Atg9 are generated through an unknown mechanism and delivered to the autophagosome formation sites. We have previously reported that Atg9-containing membranes undergo continuous tubulation and fission during nutrient starvation in a manner dependent on the curvature-inducing protein Bif-1/Sh3glb1. Here, we identify Dynamin 2 (DNM2) as a Bif-1-interacting protein that mediates the fission of Atg9-containing membranes during autophagy. The interaction of Bif-1 and DNM2 is enhanced upon nutrient starvation, and Bif-1 and DNM2 cooperatively induce the generation of Atg9-containing vesicles. Inhibition of the GTPase activity of DNM2 results in the accumulation of Atg9-positive tubular structures that originate from a Rab11-positive reservoir. Although Atg9 seems to be constitutively trafficked to the reservoir regardless of Bif-1 expression, membrane tubulation from the Atg9 reservoir is dependent on Bif-1 and is strongly induced upon nutrient starvation. These findings suggest that the generation of Atg9 vesicles from a Rab11-positive reservoir is tightly controlled by the Bif-1-DNM2 membrane fission machinery in response to cellular demand for autophagy.

Inhibition of NADPH oxidase by glucosylceramide confers chemoresistance

The bioactive sphingolipid ceramide induces oxidative stress by disrupting mitochondrial function and stimulating NADPH oxidase (NOX) activity, both implicated in cell death mechanisms. Many anticancer chemotherapeutics (anthracyclines, Vinca alkaloids, paclitaxel, and fenretinide), as well as physiological stimuli such as tumor necrosis factor α (TNFα), stimulate ceramide accumulation and increase oxidative stress in malignant cells. Consequently, ceramide metabolism in malignant cells and, in particular the up-regulation of glucosylceramide synthase (GCS), has gained considerable interest in contributing to chemoresistance. We hypothesized that increases in GCS activity and thus glucosylceramide, the product of GCS activity, represents an important resistance mechanism in glioblastoma. In our study, we determined that increased GCS activity effectively blocked reactive oxygen species formation by NOX. We further showed, in both glioblastoma and neuroblastoma cells that glucosylceramide directly interfered with NOX assembly, hence delineating a direct resistance mechanism. Collectively, our findings indicated that pharmacological or molecular targeting of GCS, using non-toxic nanoliposome delivery systems, successfully augmented NOX activity, and improved the efficacy of known chemotherapeutic agents.

Altered Sphingolipid Metabolism in Patients with Metastatic Pancreatic Cancer

Although numerous genetic mutations and amplifications have been identified in pancreatic cancer, much of the molecular pathogenesis of the disease remains undefined. While proteomic and transcriptomic analyses have been utilized to probe and characterize pancreatic tumors, lipidomic analyses have not been applied to identify perturbations in pancreatic cancer patient samples. Thus, we utilized a mass spectrometry-based lipidomic approach, focused towards the sphingolipid class of lipids, to quantify changes in human pancreatic cancer tumor and plasma specimens. Subgroup analysis revealed that patients with positive lymph node metastasis have a markedly higher level of ceramide species (C16:0 and C24:1) in their tumor specimens compared to pancreatic cancer patients without nodal disease or to patients with pancreatitis. Also of interest, ceramide metabolites, including phosphorylated (sphingosine- and sphinganine-1-phosphate) and glycosylated (cerebroside) species were elevated in the plasma, but not the pancreas, of pancreatic cancer patients with nodal disease. Analysis of plasma level of cytokine and growth factors revealed that IL-6, IL-8, CCL11 (eotaxin), EGF and IP10 (interferon inducible protein 10, CXCL10) were elevated in patients with positive lymph nodes metastasis, but that only IP10 and EGF directly correlated with several sphingolipid changes. Taken together, these data indicate that sphingolipid metabolism is altered in human pancreatic cancer and associated with advanced disease. Assessing plasma and/or tissue sphingolipids could potentially risk stratify patients in the clinical setting.

Sphingosine Kinase 1 Cooperates with Autophagy to Maintain Endocytic Membrane Trafficking

Sphingosine kinase 1 (Sphk1) associates with early endocytic membranes during endocytosis; however, the role of sphingosine or sphingosine-1-phosphate as the critical metabolite in endocytic trafficking has not been established. Here, we demonstrate that the recruitment of Sphk1 to sphingosine-enriched endocytic vesicles and the generation of sphingosine-1-phosphate facilitate membrane trafficking along the endosomal pathway. Exogenous sphingosine and sphingosine-based Sphk1 inhibitors induce the Sphk1-dependent fusion of endosomal membranes to accumulate enlarged late endosomes and amphisomes enriched in sphingolipids. Interestingly, Sphk1 also appears to facilitate endosomal fusion independent of its catalytic activity, given that catalytically inactive Sphk1G82D is recruited to endocytic membranes by sphingosine or sphingosine-based Sphk1 inhibitor and promotes membrane fusion. Furthermore, we reveal that the clearance of enlarged endosomes is dependent on the activity of ceramide synthase, lysosomal biogenesis, and the restoration of autophagic flux. Collectively, these studies uncover intersecting roles for Sphk1, sphingosine, and autophagic machinery in endocytic membrane trafficking.

Sh3glb1/Bif-1 and mitophagy: acquisition of apoptosis resistance during Myc-driven lymphomagenesis.

Evasion of apoptosis, which enables cells to survive and proliferate under metabolic stress, is one of the hallmarks of cancer. We have recently reported that SH3GLB1/Bif-1 functions as a haploinsufficient tumor suppressor to prevent the acquisition of apoptosis resistance and malignant transformation during Myc-driven lymphomagenesis. SH3GLB1 is a membrane curvature-inducing protein that interacts with BECN1 though UVRAG and regulates the post-Golgi trafficking of membrane-integrated ATG9A for autophagy. At the premalignant stage, allelic loss of Sh3glb1 enhances Myc-induced chromosomal instability and results in the upregulation of anti-apoptotic proteins, including MCL1 and BCL2L1. Notably, we found that Sh3glb1 haploinsufficiency increases mitochondrial mass in overproliferated prelymphomatous Eμ-Myc cells. Moreover, loss of Sh3glb1 suppresses autophagy-dependent mitochondrial clearance (mitophagy) in PARK2/Parkin-expressing mouse embryonic fibroblasts (MEFs) treated with the mitochondrial uncoupler CCCP. Interestingly, PARK2-expressing Sh3glb1-deficient cells accumulate ER-associated immature autophagosome-like structures after treatment with CCCP. Taken together, we propose a model of mitophagy in which SH3GLB1 together with the class III phosphatidylinositol 3-kinase complex II (PIK3C3CII) (PIK3R4-PIK3C3-BECN1-UVRAG) regulates the trafficking of ATG9A-containing Golgi-derived membranes (A9+GDMs) to damaged mitochondria for autophagosome formation to counteract oncogene-driven tumorigenesis.

Atg2A/B deficiency switches cytoprotective autophagy to non-canonical caspase-8 activation and apoptosis

Autophagosomal membranes are emerging as platforms for various cell survival and death signaling networks beyond autophagy. While autophagy-dependent cell death has been reported in response to a variety of stimuli, the underlying molecular mechanisms remain far from clear. Here, we demonstrate that inhibition of autophagosome completion by Atg2A/B deletion accumulates immature autophagosomal membranes that promote non-canonical caspase-8 activation in response to nutrient starvation via an intracellular death-inducing signaling complex (iDISC). Importantly, iDISC-induced caspase-8 dimerization and activation occurs on accumulated autophagosomal membranes and requires the LC3 conjugation machinery but is independent from the extrinsic pathway of apoptosis. Moreover, we have identified NF-κB signaling and c-FLIP as negative regulators of iDISC-mediated caspase-8 activation and apoptosis. Collectively, these findings reveal autophagosomal membrane completion as a novel target to switch cytoprotective autophagy to apoptosis.

Insulin signaling in retinal neurons is regulated within cholesterol-enriched membrane microdomains

Neuronal cell death is an early pathological feature of diabetic retinopathy. We showed previously that insulin receptor signaling is diminished in retinas of animal models of diabetes and that downstream Akt signaling is involved in insulin-mediated retinal neuronal survival. Therefore, further understanding of the mechanisms by which retinal insulin receptor signaling is regulated could have therapeutic implications for neuronal cell death in diabetes. Here, we investigate the role of cholesterol-enriched membrane microdomains to regulate PKC-mediated inhibition of Akt-dependent insulin signaling in R28 retinal neurons. We demonstrate that PKC activation with either a phorbol ester or exogenous application of diacylglycerides impairs insulin-induced Akt activation, whereas PKC inhibition augments insulin-induced Akt activation. To investigate the mechanism by which PKC impairs insulin-stimulated Akt activity, we assessed various upstream mediators of Akt signaling. PKC activation did not alter the tyrosine phosphorylation of the insulin receptor or IRS-2. Additionally, PKC activation did not impair phosphatidylinositol 3-kinase activity, phosphoinositide-dependent kinase phosphorylation, lipid phosphatase (PTEN), or protein phosphatase 2A activities. Thus, we next investigated a biophysical mechanism by which insulin signaling could be disrupted and found that disruption of lipid microdomains via cholesterol depletion blocks insulin-induced Akt activation and reduces insulin receptor tyrosine phosphorylation. We also demonstrated that insulin localizes phosphorylated Akt to lipid microdomains and that PMA reduces phosphorylated Akt. In addition, PMA localizes and recruits PKC isotypes to these cholesterol-enriched microdomains. Taken together, these results demonstrate that both insulin-stimulated Akt signaling and PKC-induced inhibition of Akt signaling depend on cholesterol-enriched membrane microdomains, thus suggesting a putative biophysical mechanism underlying insulin resistance in diabetic retinopathy.