Inhibition Of Lysosomal Degradation Research Articles

Lysosomal degradation of PD-L1 is associated with immune-related adverse events during anti-PD-L1 immunotherapy in NSCLC patients.

Background: Immune checkpoint inhibitors (ICIs) can induce immune-related adverse events (irAEs). Liquid biomarkers to predict irAE occurrence are urgently needed. We previously developed an ELISA system to specifically detect soluble PD-L1 (sPD-L1) with PD-1-binding capacity (bsPD-L1). Here, we investigated the relationship between sPD-L1 and bsPD-L1 in gastric cancer (GC) and non-small cell lung cancer (NSCLC) treated with PD-1/PD-L1 blockade and their association with irAEs. Methods: We examined sPD-L1, bsPD-L1, matrix metalloproteinases (MMPs), and proinflammatory cytokine levels by ELISA in plasma samples from 117 GC patients prior to surgery and 72 NSCLC patients prior to and at 2 months after ICI treatment (anti-PD-1, n = 48; anti-PD-L1, n = 24). In mice treated with anti-PD-1/PD-L1 antibodies (Abs), sPD-L1 levels and localization of Abs were examined by ELISA and immunohistochemistry, respectively. Results:sPD-L1 was detected with higher frequency in GC patients than in NSCLC patients, whereas bsPD-L1 was detected with similar frequencies in GC and NSCLC patients. sPD-L1 levels were correlated with IL-1α, IL-1β, TNF-α, and IL-6 levels, while bsPD-L1 levels were correlated with MMP13, MMP3, and IFN-γ levels. In NSCLC patients, anti-PD-L1, but not anti-PD-1, treatment increased sPD-L1, which was associated with irAE development, but not with clinical outcomes. In mice, trafficking of anti-PD-L1 Abs to lysosomes in F4/80+ macrophages resulted in sPD-L1 production, which was suppressed by treatment with lysosomal degradation inhibitor chloroquine and macrophage depletion. Conclusion: Anti-PD-L1-mediated lysosomal degradation induces sPD-L1 production, which can serve as an indicator to predict irAE development during anti-PD-L1 treatment.

KIF16B Mediates Anterograde Transport and Modulates Lysosomal Degradation of the HIV-1 Envelope Glycoprotein.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) is incorporated into virions at the site of particle assembly on the plasma membrane (PM). The route taken by Env to reach the site of assembly and particle incorporation remains incompletely understood. Following initial delivery to the PM through the secretory pathway, Env is rapidly endocytosed, suggesting that recycling is required for particle incorporation. Endosomes marked by the small GTPase Rab14 have been previously shown to play a role in Env trafficking. Here, we examined the role of KIF16B, the molecular motor protein that directs outward movement of Rab14-dependent cargo, in Env trafficking. Env colocalized extensively with KIF16B+ endosomes at the cellular periphery, while expression of a motor-deficient mutant of KIF16B redistributed Env to a perinuclear location. The half-life of Env labeled at the cell surface was markedly reduced in the absence of KIF16B, while a normal half-life was restored through inhibition of lysosomal degradation. In the absence of KIF16B, Env expression on the surface of cells was reduced, leading to a reduction in Env incorporation into particles and a corresponding reduction in particle infectivity. HIV-1 replication in KIF16B knockout cells was substantially reduced compared to that in wild-type cells. These results indicated that KIF16B regulates an outward sorting step involved in Env trafficking, thereby limiting lysosomal degradation and enhancing particle incorporation. IMPORTANCE The HIV-1 envelope glycoprotein is an essential component of HIV-1 particles. The cellular pathways that contribute to incorporation of envelope into particles are not fully understood. Here, we have identified KIF16B, a motor protein that directs movement from internal compartments toward the plasma membrane, as a host factor that prevents envelope degradation and enhances particle incorporation. This is the first host motor protein identified that contributes to HIV-1 envelope incorporation and replication.

Puerarin Prevents Cadmium-Induced Neuronal Injury by Alleviating Autophagic Dysfunction in Rat Cerebral Cortical Neurons.

Autophagic dysfunction is one of the main mechanisms of cadmium (Cd)-induced neurotoxicity. Puerarin (Pue) is a natural antioxidant extracted from the medicinal and edible homologous plant Pueraria lobata. Studies have shown that Pue has neuroprotective effects in a variety of brain injuries, including Cd-induced neuronal injury. However, the role of Pue in the regulation of autophagy to alleviate Cd-induced injury in rat cerebral cortical neurons remains unclear. This study aimed to elucidate the protective mechanism of Pue in alleviating Cd-induced injury in rat cerebral cortical neurons by targeting autophagy. Our results showed that Pue alleviated Cd-induced injury in rat cerebral cortical neurons in vitro and in vivo. Pue activates autophagy and alleviates Cd-induced autophagic blockade in rat cerebral cortical neurons. Further studies have shown that Pue alleviates the Cd-induced inhibition of autophagosome-lysosome fusion, as well as the inhibition of lysosomal degradation. The specific mechanism is related to Pue alleviating the inhibition of Cd on the expression levels of the key proteins Rab7, VPS41, and SNAP29, which regulate autophagosome-lysosome fusion, as well as the lysosome-related proteins LAMP2, CTSB, and CTSD. In summary, these results indicate that Pue alleviates Cd-induced autophagic dysfunction in rat cerebral cortical neurons by alleviating autophagosome-lysosome fusion dysfunction and lysosomal degradation dysfunction, thereby alleviating Cd-induced neuronal injury.

Determination of the autophagic flux in murine and human peripheral blood mononuclear cells.

The autophagy lysosomal system (ALS) is crucial for cellular homeostasis, contributing to maintain whole body health and alterations are associated with diseases like cancer or cardiovascular diseases. For determining the autophagic flux, inhibition of lysosomal degradation is mandatory, highly complicating autophagy measurement in vivo. To overcome this, herein blood cells were used as they are easy and routinely to isolate. Within this study we provide detailed protocols for determination of the autophagic flux in peripheral blood mononuclear cells (PBMCs) isolated from human and, to our knowledge the first time, also from murine whole blood, extensively discussing advantages and disadvantages of both methods. Isolation of PBMCs was performed using density gradient centrifugation. To minimize changes on the autophagic flux through experimental conditions, cells were directly treated with concanamycin A (ConA) for 2h at 37°C in their serum or for murine cells in serum filled up with NaCl. ConA treatment decreased lysosomal cathepsins activity and increased Sequestosome 1 (SQSTM1) protein and LC3A/B-II:LC3A/B-I ratio in murine PBMCs, while transcription factor EB was not altered yet. Aging further enhanced ConA-associated increase in SQSTM1 protein in murine PBMCs but not in cardiomyocytes, indicating tissue-specific differences in autophagic flux. In human PBMCs, ConA treatment also decreased lysosomal activity and increased LC3A/B-II protein levels, demonstrating successful autophagic flux detection in human subjects. In summary, both protocols are suitable to determine the autophagic flux in murine and human samples and may facilitate a better mechanistic understanding of altered autophagy in aging and disease models and to further develop novel treatment strategies.

Neu1 deficiency increases the susceptibility of zebrafish to Edwardsiella piscicida infection via lysosomal dysfunction

Neu1 is a lysosomal glycosidase that catalyzes the removal of sialic acids from glycoconjugates. Although Neu1 sialidase is highly conserved among vertebrates, the role of fish Neu1 is not fully understood because of its unique aquatic living situation. Compared to land animals, fish have a higher chance of bacterial infection, and to understand the role of fish Neu1, the susceptibility of Neu1 knockout zebrafish (Neu1-KO) was evaluated using Edwardsiella piscicida, a fish pathogen. Neu1-KO larvae showed high susceptibility to E. piscicida, despite the activation of macrophages, and presented increased lysosomal signals induced by the accumulation of Sia α2-3 linked oligosaccharides. The accumulation coincided with the signal of the macrophage marker, suggesting that the dysfunction of lysosomes in macrophages would result in a high susceptibility of Neu1-KO to E. piscicida. Chloroquine, an inhibitor of lysosomal degradation, induced high mortality of wild type zebrafish with E. piscicida infection accompanied by increased lysosomal accumulation, similar to Neu1-KO zebrafish. This study revealed that Neu1 sialidase plays a crucial role in the lysosomal degradation of macrophages with a bacterial infection.

Alteration in Rab11-mediated endocytic trafficking of LDL receptor contributes to angiotensin II-induced cholesterol accumulation and injury in podocytes.

ObjectivesExposure of podocytes to angiotensin II (Ang II) enhances the abundance of the cell surface glycoprotein, low‐density lipoprotein receptor (LDLR) and promotes significant changes in the cellular cholesterol content. Recent investigation provides evidence that the small GTPase Rab11 is involved in the regulation of LDLR, but the exact mechanisms remain unknown. In this study, the role of Rab11 in post‐transcriptional regulation of LDLR was evaluated to investigate potential mechanisms of podocyte cholesterol dysregulation in chronic kidney disease.Materials and MethodsCholesterol content, LDLR and Rab11 expression were assessed in podocytes from Ang II‐infused mice. In vitro, the intracellular localization of LDLR was detected under different conditions. Rab11 expression was modulated and we then explored the effect of anti‐lipid cytotoxicity by detecting LDLR expression and trafficking, cholesterol content and apoptosis in podocytes.ResultsCholesterol accumulation, upregulated expression of LDLR and Rab11 were discovered in podocytes from Ang II‐infused mice. Ang II enhanced the co‐precipitation of LDLR with Rab11 and accelerated the endocytic recycling of LDLR to the plasma membrane. Additionally, silencing Rab11 promoted lysosomal degradation of LDLR and alleviated Ang II‐induced cholesterol accumulation and apoptosis in podocytes. Conversely, overexpression of Rab11 or inhibition of lysosomal degradation up‐regulated the abundance of LDLR and aggravated podocyte cholesterol deposition.ConclusionsRab11 triggers the endocytic trafficking and recycling of LDLR; overactivation of this pathway contributes to Ang II‐induced podocyte cholesterol accumulation and injury.

Decreased turnover of the CNS myelin protein Opalin in a mouse model of hereditary spastic paraplegia 35.

Spastic paraplegia 35 (SPG35) (OMIM: 612319) or fatty acid hydroxylase-associated neurodegeneration (FAHN) is caused by deficiency of fatty acid 2-hydroxylase (FA2H). This enzyme synthesizes sphingolipids containing 2-hydroxylated fatty acids, which are particularly abundant in myelin. Fa2h-deficient (Fa2h-/-) mice develop symptoms reminiscent of the human disease and therefore serve as animal model of SPG35. In order to understand further the pathogenesis of SPG35, we compared the proteome of purified CNS myelin isolated from wild type and Fa2h-/- mice at different time points of disease progression using tandem mass tag labeling. Data analysis with a focus on myelin membrane proteins revealed a significant increase of the oligodendrocytic myelin paranodal and inner loop protein (Opalin) in Fa2h-/- mice, whereas the concentration of other major myelin proteins was not significantly changed. Western blot analysis revealed an almost 6-fold increase of Opalin in myelin of Fa2h-/- mice aged 21-23months. A concurrent unaltered Opalin gene expression suggested a decreased turnover of the Opalin protein in Fa2h-/- mice. Supporting this hypothesis, Opalin protein half-life was reduced significantly when expressed in CHO cells synthesizing 2-hydroxylated sulfatide, compared to cells synthesizing only non-hydroxylated sulfatide. Degradation of Opalin was inhibited by inhibitors of lysosomal degradation but unaffected by proteasome inhibitors. Taken together, these results reveal a new function of 2-hydroxylated sphingolipids namely affecting the turnover of a myelin membrane protein. This may play a role in the pathogenesis of SPG35.

Monoamine oxidase A deletion leads to autophagy inhibition and impairment in cardiomyocyte differentiation from hiPSCs

Reactive oxygen species (ROS) and autophagy play a major role in cardiac pathophysiology, including cardiomyocyte differentiation. Yet, the exact source of ROS controlling the process of differentiation remains unknown. Within mitochondria, monoamine oxidases A and B (MAO-A and -B) are one of the major sources of ROS and play an important role in cardiac tissue homeostasis. Nevertheless, their involvement in cardiomyocyte differentiation has not been investigated yet. Here, we assessed whether ablation of MAO-A, the isoform expressed in human induced pluripotent stem cells (hiPSCs), could affect cardiomyocyte differentiation. MAO-A deletion negatively affected contractile properties of hiPSC-derived cardiomyocytes, resulting in higher frequency and lower amplitude of calcium transients. These features were accompanied by sarcomere disorganization and a reduction in the α- to β-myosin heavy chain ratio in MAO-A−/− cells, similarly to what occurs in failing hearts. GATA4, one of the key genes involved in cardiac remodeling, was significantly upregulated in these cells. To understand the underlying mechanisms, we investigated whether MAO-A deletion affected macroautophagy, one of the key processes required for cardiomyocyte differentiation. Macroautophagy levels were reduced in MAO-A−/− hiPSCs both at baseline and upon starvation or rapamycin administration, as evidenced by reduced accumulation of LC3-II in the presence of inhibitors of lysosomal degradation. The inhibitory effect on macroautophagy is likely due to the reduction of AMPK phosphorylation levels observed in MAO-A−/− hiPSCs. Taken together, these results suggest that MAO-A is required for the regulation of macroautophagy in hiPSCs and, consequently, for the process of differentiation and maturation of cardiac myocytes.

Hif-1 - альтернативные сигнальные механизмы активации и формирования толерантности к гипоксии/ишемии

Альтернативные сигнальные механизмы активации транскрипционного фактора HIF-1 принципиально можно подразделить на 3 типа: связанные с ингибированием пролилгидроксилаз и ограничением деградации HIF-1α; связанные с индукцией HIF-1α; связанные с активацией HIF-1α. Особый практический интерес представляет активация фактора HIF-1 во время гиперкапнии, которая часто сопутствует гипоксии. Пермиссивная гиперкапния вызывает повышение концентрации HIF-1α в ткани головного мозга и потенцирует активацию HIF-1α, что выражается в повышении содержания HIF-1α в гиппокампальных нейронах СА1 региона после гиперкапнически-гипоксического воздействия. При изолированном применении гипоксии аналогичный эффект отсутствует. Перспективным для разработки методов повышения толерантности органов и тканей к гипоксии/ишемии является комбинирование средств, повышающих активность HIF-1, с респираторными гиперкапнически-гипоксическими тренировками и препаратами, влияющими на другие адаптогенные механизмы. Along with the classical oxygen-dependent mechanism of activation of the transcription factor HIF-1, there are alternative signaling mechanisms of this process. These mechanisms can be fundamentally divided into the following types: associated with the inhibition of prolyl hydroxylases and limiting the degradation of HIF-1α (iron chelators, NO, succinate, inhibitors of lysosomal degradation); associated with the induction of HIF-1α (TNFα, IL-1, angiotensin); associated with the activation of HIF-1α (MAPK, PI3K). Of particular practical interest is the activation of HIF-1 factor when exposed to CO2 during hypercapnia, which is often associated with hypoxia. Permissive hypercapnia causes an increase in the concentration of HIF-1α in brain tissue at an equivalent level of hypoxia, and also potentiates the effectiveness of hypoxia in relation to HIF-1α activation, which is expressed in an increase in the content of HIF-1α in hippocampal neurons of the CA1 region after hypercapnic-hypoxic exposure, on the absence of a similar effect with the isolated use of hypoxia. Promising for the development of methods to increase the tolerance of organs and tissues to hypoxia / ischemia is a combination of agents that increase the activity of HIF-1, with respiratory hypercapnic-hypoxic training and drugs that affect other adaptogenic mechanisms (A1-adenosine receptors, mitoK+ATP-channels, opioid receptors).

A protective role of autophagy in Pb-induced developmental neurotoxicity in zebrafish

Lead (Pb) is one of the most toxic heavy metals and has aroused widespread concern as it can cause severe impairments in the developing nervous system. Autophagy has been proposed as an injury factor in Pb-induced neurotoxicity. In this study, we used zebrafish embryo as a model, measured the general toxic effects of Pb, and investigated the effect of Pb exposure on autophagy, and its role in Pb-induced developmental neurotoxicity. Zebrafish embryos were exposed to Pb at concentrations of 0, 0.1, 1 or 10 μM until 4 days post-fertilization. Our data showed that exposure to 10 μM Pb significantly reduced survival rates and impaired locomotor activity. Uptake of Pb was enhanced as the concentration and duration of exposure increased. Inhibition of lysosomal degradation with bafilomycin A1 treatment abolished the suppression of Lc3-II protein expression by Pb. Furthermore, autophagosome formation was inhibited by Pb in the brain. In addition, mRNA expression of beclin1, one of the critical genes in autophagy, were decreased in Pb exposure groups at 72 h post-fertilization. Whole-mount in situ hybridization assay showed that beclin1 gene expression in the brain was reduced by Pb. Rapamycin, an autophagy inducer, partly resolved developmental neurotoxicity induced by Pb exposure. Our results suggest that autophagy plays a protective role in the developmental neurotoxicity of Pb in zebrafish embryos and larvae.

Rapid degradation of progressive ankylosis protein (ANKH) in craniometaphyseal dysplasia

Mutations in the progressive ankylosis protein (NP_473368, human ANKH) cause craniometaphyseal dysplasia (CMD), characterized by progressive thickening of craniofacial bones and widened metaphyses in long bones. The pathogenesis of CMD remains largely unknown, and treatment for CMD is limited to surgical intervention. We have reported that knock-in mice (AnkKI/KI) carrying a F377del mutation in ANK (NM_020332, mouse ANK) replicate many features of CMD. Interestingly, ablation of the Ank gene in AnkKO/KO mice also leads to several CMD-like phenotypes. Mutations causing CMD led to decreased steady-state levels of ANK/ANKH protein due to rapid degradation. While wild type (wt) ANK was mostly associated with plasma membranes, endoplasmic reticulum (ER), Golgi apparatus and lysosomes, CMD-linked mutant ANK was aberrantly localized in cytoplasm. Inhibitors of proteasomal degradation significantly restored levels of overexpressed mutant ANK, whereas endogenous CMD-mutant ANK/ANKH levels were more strongly increased by inhibitors of lysosomal degradation. However, these inhibitors do not correct the mislocalization of mutant ANK. Co-expressing wt and CMD-mutant ANK in cells showed that CMD-mutant ANK does not negatively affect wt ANK expression and localization, and vice versa. In conclusion, our finding that CMD mutant ANK/ANKH protein is short-lived and mislocalized in cells may be part of the CMD pathogenesis.

Isopeptide and ester bond ubiquitination both regulate degradation of the human dopamine receptor 4

How an optimal level of human dopamine D4 receptor (hD4R) is maintained in synaptic membranes is not known. We show here that hD4R is ubiquitinated in primary neurons. We go on to show that ubiquitin is attached to hD4R through isopeptide and ester bonds. When lysine (Lys) residues of the hD4R are substituted with arginine (Arg) residues, cellular hD4R protein levels increase. A synergistic effect on hD4R levels is noted when cytoplasmic serine (Ser) and threonine (Thr) residues are mutated. Chloroquine, an inhibitor of lysosomal degradation, did not have an effect on hD4R protein levels. However, treatment with bortezomib, an inhibitor of the 20S proteasome, caused a dose-dependent increase in hD4R protein levels. The effect of bortezomib was attenuated in the receptor variants that lacked Lys or Ser/Thr residues, and the hD4R mutant that lacked 17 cytoplasmic Lys, Ser, and Thr residues was nearly insensitive to bortezomib treatment. We conclude that both isopeptide and ester bond ubiquitination regulate proteasomal degradation of hD4R.

Chaperone-mediated autophagy compensates for impaired macroautophagy in the cirrhotic liver to promote hepatocellular carcinoma.

Macroautophagy and chaperone-mediated autophagy (CMA) represent two major lysosomal degradation processes and often compensate for one another to facilitate cell survival. The aim of this study was to determine whether these autophagy pathways could compensate for one another to promote HCC cell survival in the cirrhotic liver. Analysis of normal liver tissue showed no expression of glypican-3 or p62 proteins, suggesting that macroautophagy is the major contributor to autophagic flux under non-pathological conditions. Of 46 cirrhotic livers with HCC examined, 39 (84%) of HCCs showed increased expression of p62, and 36 (78%) showed increased expression of glypican-3, while adjacent non-tumorous hepatocytes were negative for expression of p62 and glypican-3, similar to normal liver tissue. These results suggest that macroautophagy flux is impaired in HCC. Furthermore, more than 95% of HCCs showed altered expression of LAMP-2A compared to the surrounding non-tumorous cirrhotic liver, consistent with induction of CMA in HCC. Elevated expression of glucose-regulated protein 78 (GRP78) and heat shock cognate protein (Hsc70) were detected in 100% of HCC and adjacent non-tumorous cirrhotic livers, suggesting that unresolved ER-stress is associated with HCC risk in liver cirrhosis. Interestingly, inhibition of lysosomal degradation using hydroxychloroquine (HCQ) induced expression of the tumor suppressor p53, promoted apoptosis, and inhibited HCC growth, whereas activation of autophagy using an mTOR inhibitor (Torin1) promoted HCC growth. Results of this study suggest that induction of CMA compensates for the impairment of macroautophagy to promote HCC survival in the cirrhotic liver.

Oxidant-induced autophagy and ferritin degradation contribute to epithelial-mesenchymal transition through lysosomal iron.

PurposeTransforming growth factor (TGF)-β1 triggers epithelial–mesenchymal transition (EMT) through autophagy, which is partly driven by reactive oxygen species (ROS). The aim of this study was to determine whether leaking lysosomes and enhanced degradation of H-ferritin could be involved in EMT and whether it could be possible to prevent EMT by iron chelation targeting of the lysosome.Materials and methodsEMT, H-ferritin, and autophagy were evaluated in TGF-β1-stimulated A549 human lung epithelial cells cultured in vitro using Western blotting, with the additional morphological assessment of EMT. By using immunofluorescence and flow cytometry, lysosomes and ROS were assessed by acridine orange and 6-carboxy-2′,7′-dichlorodihydrofluorescein acetate assays, respectively.ResultsTGF-β1-stimulated cells demonstrated a loss of H-ferritin, which was prevented by the antioxidant N-acetyl-L-cysteine (NAC) and inhibitors of lysosomal degradation. TGF-β1 stimulation generated ROS and autophagosome formation and led to EMT, which was further promoted by the additional ROS-generating cytokine, tumor necrosis factor-α. Lysosomes of TGF-β1-stimulated cells were sensitized to oxidants but also completely protected by lysosomal loading with dextran-bound deferoxamine (DFO). Autophagy and EMT were prevented by NAC, DFO, and inhibitors of autophagy and lysosomal degradation.ConclusionThe findings of this study support the role of enhanced autophagic degradation of H-ferritin as a mechanism for increasing the vulnerability of lysosomes to iron-driven oxidant injury that triggers further autophagy during EMT. This study proposes that lysosomal leakage is a novel pathway of TGF-β1-induced EMT that may be prevented by iron-chelating drugs that target the lysosome.

Angiotensin II reduces the surface abundance of KV 1.5 channels in arterial myocytes to stimulate vasoconstriction.

Several different voltage-dependent K+ (KV ) channel isoforms are expressed in arterial smooth muscle cells (myocytes). Vasoconstrictors inhibit KV currents, but the isoform selectivity and mechanisms involved are unclear. We show that angiotensin II (Ang II), a vasoconstrictor, stimulates degradation of KV 1.5, but not KV 2.1, channels through a protein kinase C- and lysosome-dependent mechanism, reducing abundance at the surface of mesenteric artery myocytes. The AngII-induced decrease in cell surface KV 1.5 channels reduces whole-cell KV 1.5 currents and attenuates KV 1.5 function in pressurized arteries. We describe a mechanism by which AngII stimulates protein kinase C-dependent KV 1.5 channel degradation, reducing the abundance of functional channels at the myocyte surface. Smooth muscle cells (myocytes) of resistance-size arteries express several different voltage-dependent K+ (KV ) channels, including KV 1.5 and KV 2.1, which regulate contractility. Myocyte KV currents are inhibited by vasoconstrictors, including angiotensinII (AngII), but the mechanisms involved are unclear. Here, we tested the hypothesis that AngII inhibits KV currents by reducing the plasma membrane abundance of KV channels in myocytes. AngiotensinII (applied for 2h) reduced surface and total KV 1.5 protein in rat mesenteric arteries. In contrast, AngII did not alter total or surface KV 2.1, or KV 1.5 or KV 2.1 cellular distribution, measured as the percentage of total protein at the surface. Bisindolylmaleimide (BIM; a protein kinaseC blocker), a protein kinaseC inhibitory peptide or bafilomycinA (a lysosomal degradation inhibitor) each blocked the AngII-induced decrease in total and surface KV 1.5. Immunofluorescence also suggested that AngII reduced surface KV 1.5 protein in isolated myocytes; an effect inhibited by BIM. Arteries were exposed to AngII or AngII plus BIM (for 2h), after which these agents were removed and contractility measurements performed or myocytes isolated for patch-clamp electrophysiology. AngiotensinII reduced both whole-cell KV currents and currents inhibited by Psora-4, a KV 1.5 channel blocker. AngiotensinII also reduced vasoconstriction stimulated by Psora-4 or 4-aminopyridine, another KV channel inhibitor. These data indicate that AngII activates protein kinaseC, which stimulates KV 1.5 channel degradation, leading to a decrease in surface KV 1.5, a reduction in whole-cell KV 1.5 currents and a loss of functional KV 1.5 channels in myocytes of pressurized arteries.

Hydroxychloroquine potentiates carfilzomib toxicity towards myeloma cells.

Cells degrade proteins either by proteasomes that clinically are targeted by for example bortezomib or carfilzomib, or by formation of autophagosomes and lysosomal degradation that can be inhibited by hydroxychloroquine (HCQ). Multiple myeloma is unique among cancers because proteasomal inhibition has good clinical effects. However, some multiple myeloma patients display intrinsic resistance to the treatment and most patients acquire resistance over time. We hypothesized that simultaneous targeting both arms of protein degradation could be a way to improve treatment of multiple myeloma. Here we tested the combined effects of the lysosomal inhibitor HCQ and clinically relevant proteasome inhibitors on myeloma cell lines and primary cells. Carfilzomib and bortezomib both induced immunoglobulin-containing aggregates in myeloma cells. HCQ significantly potentiated the effect of carfilzomib in both cell lines and in primary myeloma cells. In contrast, HCQ had little or no effects on the toxicity of bortezomib. Furthermore, cells adapted to tolerate high levels of carfilzomib could be re-sensitized to the drug by co-treatment with HCQ. Thus, we show that inhibition of lysosomal degradation can overcome carfilzomib resistance, suggesting that the role of autophagy in myeloma cells is dependent on type of proteasome inhibitor. In conclusion, attempts should be made to combine HCQ with carfilzomib in the treatment of multiple myeloma.

Abstract 4656: ER stress, unfolded protein response, and autophagy are pro-survival responses to fenretinide + safingol treatment in GBM cells

Abstract The cytotoxicity of the synthetic retinoid, fenretinide (4-HPR), is associated with increased dihydroceramide and reactive oxygen species (ROS) levels and enhanced by co-treatment with safingol (S), an L-threo-dihydroceramide precursor. The ER stress response displays dichotomic effects wherein mild short-term stressors activate responses to neutralize or adapt to stress, but severe or long-lasting stressors activate cell death cascades. Here, we report that ER stress and autophagy are pro-survival responses in glioblastoma multiforme (GBM) cells to unfolded proteins-related cytotoxicity induced by 4-HPR+S in vitro. In T98G and A172 cells, 4-HPR+S induced mixed cell death associated with time-dependent increase of TUNEL-positivity (+12-36 h) and cleavage of caspase-3 (+12-36 h), but pan- caspase inhibitor, boc-D-fmk, did not rescue cell death. Cytotoxicity (+6-24 h) was associated with minimal mitochondrial depolarization or dual-staining of Annexin V/PI. Assays for known non-apoptotic mechanisms were negative. There was time-dependent (+6-24 h) increase of GRP78 protein levels, a key regulator of ER stress response; ER stress sensors, PERK, IRE1α, and ATF6, were activated (+6-24 h); levels of pro-apoptotic transcription factor, CHOP, increased (+6-24 h). Cytotoxicity was increased by PERK pathway inhibitors, salubrinal (+24-72 h, p≤0.05), GSK 2606414 (+24-72 h, p≤0.05), and GSK 2656157 (+48-72 h, p≤0.05), indicating that PERK activation was pro-survival. Treatment increased levels of CHIP, a key co-chaperone targeting unfolded proteins for degradation (+6-24 h). There was strong activation of the ubiquitin-proteasome system (UPS) and autophagy preceding or concurrent with the accumulation of poly-ubiquitinated proteins (+12-24 h). Autophagy was evidenced by increased conversion of LC3B-I to LC3B-II (+6-24 h). LC3B-II levels were increased by lysosomal degradation inhibitor, bafilomycin A1, indicating increased autophagic flux. Cytosolic levels of HDAC6, a coordinator of protein turnover via UPS and autophagy through trafficking of protein aggregates into pre-lysosomal structures (aggresomes) prior to autophagosome engulfment, increased (+6-24 h). siRNA knockdown of CHIP enhanced cytotoxicity (+24-72 h, p≤0.05). siRNA ablation of autophagy (ATG7, BECN1)(+24-36 h, p≤0.05), or pharmacological disruption by mefloquine, increased cytotoxicity (+24-72 h, p≤0.05), indicating that autophagy was pro-survival. Cytotoxicity was increased by siRNA knockdown of HDAC6 (+24-72 h, p≤0.05) and selective HDAC6 inhibitor, ACY1215, (+24-72 h, p≤0.05). Together, these results support that ER stress/unfolded protein response (UPR) is pro-survival in GBM cells exposed to 4-HPR+S. This suggests that co-agents that reduce ER stress response, UPR, or autophagy processes may further sensitize GBM cells to 4-HPR+S. Citation Format: Nikhil M. Vad, Dong Wang, Barry J. Maurer. ER stress, unfolded protein response, and autophagy are pro-survival responses to fenretinide + safingol treatment in GBM cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4656.

Pentoxifylline modulates the AKT/mTOR/AMPK pathway: potential role as an adjunct in cancer chemotherapy

Targeting specific pathways involved in the growth and death of cells, such as AKT (signal transduction pathway promoting growth), mTOR (intracellular signaling pathway regulating cell cycle), and AMPK (coordination of growth, autophagy, and metabolism), is an established, effective strategy to inhibit cancer cell growth. Previously, we have shown that pentoxifylline (PTX), a clinically‐utilized non‐specific phosphodiesterase inhibitor, can inhibit growth and induce G1 arrest in prostate cancer cells. In the present studies, we examined the mechanism of PTX induced growth inhibition via modulation of growth/metabolic pathways, and the potential use of PTX as an adjunct in cancer chemotherapy. In prostate adenocarcinoma cells (LNCaP), we show PTX modulates the AKT pathway, as evidenced by the decreased phosphorylation of AKT. Downstream modulation of the mTOR pathway is indicated by decreased phosphorylation of mTOR, ribosomal S6K, and 4EBP1, and total cyclin D1. P‐mTOR levels return to baseline by 24h, while p‐S6K, p‐4EBP1, and cyclin D1 levels are suppressed for at least 48h, indicating a sustained decrease in the activity of cell growth pathway proteins. PTX also modulates the AMPK pathway, as revealed by modulation of downstream p‐RAPTOR, p‐ACC and p‐ULK. Both the AKT and AMPK pathways regulate the activation of cellular autophagy, an intracellular degradation system and determinant of cell survival. Treatment of LNCaP cells with PTX alone, and in combination with bafilomycin, a lysosomal degradation inhibitor, increased LC3‐II expression, indicating an induction of autophagic flux. Overall, PTX modulates both AKT and AMPK pathways, decreasing cell growth and increasing autophagy. Moreover, LNCaP cells treated with PTX were more susceptible to docetaxel (DOC; first‐line chemotherapy) induced cytotoxicity than DOC alone. Interestingly, the PTX/DOC combination decreased the expression of LC3 relative to PTX alone, suggesting activation of an alternate cell death pathway. Indeed, assessment of caspase cleavage, pBcl‐2, and annexin staining revealed a synergistic effect of PTX/DOC treatment on the induction of apoptosis. In summary, the findings provide a mechanistic basis for the potential use of PTX as an adjunct in the chemotherapy of prostate cancer. The ability of PTX to potentiate the effects of DOC on tumor growth inhibition in a mouse xenograft model is currently being assessed.Support or Funding InformationASPET SURF, P30ES006694, T32ES006694

TRIM5α Degradation via Autophagy Is Not Required for Retroviral Restriction.

TRIM5α is an interferon-inducible retroviral restriction factor that prevents infection by inducing the abortive disassembly of capsid cores recognized by its C-terminal PRY/SPRY domain. The mechanism by which TRIM5α mediates the disassembly of viral cores is poorly understood. Previous studies demonstrated that proteasome inhibitors abrogate the ability of TRIM5α to induce premature core disassembly and prevent reverse transcription; however, viral infection is still inhibited, indicating that the proteasome is partially involved in the restriction process. Alternatively, we and others have observed that TRIM5α associates with proteins involved in autophagic degradation pathways, and one recent study found that autophagic degradation is required for the restriction of retroviruses by TRIM5α. Here, we show that TRIM5α is basally degraded via autophagy in the absence of restriction-sensitive virus. We observe that the autophagy markers LC3b and lysosome-associated membrane protein 2A (LAMP2A) localize to a subset of TRIM5α cytoplasmic bodies, and inhibition of lysosomal degradation with bafilomycin A1 increases this association. To test the requirement for macroautophagy in restriction, we examined the ability of TRIM5α to restrict retroviral infection in cells depleted of the autophagic mediators ATG5, Beclin1, and p62. In all cases, restriction of retroviruses by human TRIM5α, rhesus macaque TRIM5α, and owl monkey TRIM-Cyp remained potent in cells depleted of these autophagic effectors by small interfering RNA (siRNA) knockdown or clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome editing. Collectively, these results are consistent with observations that the turnover of TRIM5α proteins is sensitive to autophagy inhibition; however, the data presented here do not support observations that the inhibition of autophagy abrogates retroviral restriction by TRIM5 proteins. Restriction factors are a class of proteins that inhibit viral replication. Following fusion of a retrovirus with a host cell membrane, the retroviral capsid is released into the cytoplasm of the target cell. TRIM5α inhibits retroviral infection by promoting the abortive disassembly of incoming retroviral capsid cores; as a result, the retroviral genome is unable to traffic to the nucleus, and the viral life cycle is extinguished. In the process of restriction, TRIM5α itself is degraded by the proteasome. However, in the present study, we have shown that in the absence of a restriction-sensitive virus, TRIM5α is degraded by both proteasomal and autophagic degradation pathways. Notably, we observed that restriction of retroviruses by TRIM5α does not require autophagic machinery. These data indicate that the effector functions of TRIM5α can be separated from its degradation and may have further implications for understanding the mechanisms of other TRIM family members.

Abstract 15099: Suppression of Autophagic Flux by Sequestration of p62 Promotes RIP1-mediated Necroptosis in Cardiomyocytes

Purpose: Necroptosis, a programmed form of necrosis, has been implicated in various pathological conditions including heart failure. Here we examined whether and how receptor-interacting protein kinase (RIP) 1-dependent necroptosis is functionally linked with autophagy in cardiomyocytes. Methods and results: In H9c2 cells, the necroptotic pathway was activated by TNF-α (TNF, 50 ng/ml) alone or by co-incubation of TNF with 20 mM z-VAD-fmk (zVAD), a pan-caspase inhibitor. TNF increased LDH activity in the culture medium from 26.6±4.3% of total LDH activity in the vehicle-treated control to 39.1±4.8%, suggesting induction of necrosis. Addition of zVAD to TNF (TNF/zVAD) further increased the LDH activity to 70.6±2.7%. The augmented cell death by TNF/zVAD was completely reversed by necrostatin-1 (39.4±4.0%), a RIP1 inhibitor, and was attenuated by rapamycin (10 nM), a promoter of autophagy (49.4±2.0%). Immunoblot analyses showed that TNF/zVAD significantly increased the level of LC3-II. The accumulation of LC3-II protein was not further increased by bafilomycin A1 (100 nM), an inhibitor of lysosomal degradation, indicating that accumulation of LC3-II by TNF/zVAD reflected suppression of autophagic flux. Immunoprecipitation experiments revealed that TNF/zVAD reduced interaction between LC3-II and p62, an autophagy adaptor protein, and conversely increased interaction between RIP1 and p62. The changes in p62 interactosomes by TNF/zVAD were reversed by pretreatment with rapamycin. On the other hand, TNF/zVAD-induced cell death when p62 was knocked down was still sensitive to necrostatin-1, indicating that a p62-independent pathway is also involved in RIP1-mediated necroptosis. Conclusions: Suppression of autophagic flux by p62 sequesteration from p62-LC3-II complexes and its recruitment to RIP1 promotes RIP1-dependent necroptosis by TNF/zVAD.