High Autophagic Flux Research Articles

ULK2 Is a Key Pro-Autophagy Protein That Contributes to the High Chemoresistance and Disease Relapse in FLT3-Mutated Acute Myeloid Leukemia.

We recently demonstrated that a small subset of cells in FLT3-mutated acute myeloid leukemia (AML) cell lines exhibit SORE6 reporter activity and cancer stem-like features including chemoresistance. To study why SORE6+ cells are more chemoresistant than SORE6- cells, we hypothesized that these cells carry higher autophagy, a mechanism linked to chemoresistance. We found that cytarabine (Ara-C) induced a substantially higher protein level of LC3B-II in SORE6+ compared to SORE6- cells. Similar observations were made using a fluorescence signal-based autophagy assay. Furthermore, chloroquine (an autophagy inhibitor) sensitized SORE6+ but not SORE6- cells to Ara-C. To decipher the molecular mechanisms underlying the high autophagic flux in SORE6+ cells, we employed an autophagy oligonucleotide array comparing gene expression between SORE6+ and SORE6- cells before and after Ara-C treatment. ULK2 was the most differentially expressed gene between the two cell subsets. To demonstrate the role of ULK2 in conferring higher chemoresistance in SORE6+ cells, we treated the two cell subsets with a ULK1/2 inhibitor, MRT68921. MRT68921 significantly sensitized SORE6+ but not SORE6- cells to Ara-C. Using our in vitro model for AML relapse, we found that regenerated AML cells contained higher ULK2 expression compared to pretreated cells. Importantly, inhibition of ULK2 using MRT68921 prevented in vitro AML relapse. Lastly, using pretreatment and relapsed AML patient bone marrow samples, we found that ULK2 expression was higher in relapsed AML. To conclude, our results supported the importance of autophagy in the relapse of FLT3-mutated AML and highlighted ULK2 in this context.

Exercise-promoted adiponectin secretion activates autolysosomes to protect the liver of ApoE-/- mice from a high-fat diet.

Fat is a "double-edged sword": while it is a necessary substance for the body, the long-term intake of excessive fat will cause obesity, with the liver subjected to lipotoxicity as it accumulates. It will then continue to deteriorate, eventually leading to liver failure, which is a negative impact of high-fat food intake. Research has shown that exercise can reverse the side effects of a chronic high-fat diet and help the body to mitigate the harmful effects of lipotoxicity. In our study, it was found that moderate-intensity cardio-training (MICT) and high-intensity interval exercise (HIIT) effectively protected the livers of high-fat diet (HFD) ApoE-/- mice against lipotoxicity. Previous results demonstrated that 12 weeks of HFD resulted in a significant elevation of CD36 in the livers of C57BL/6J mice, while knockdown of CD36 did not reduce the accumulation of fat in the liver. Therefore, we used ApoE-/- mice as experimental subjects. Although HFD caused the development of hyperlipidemia and atherosclerosis, it is interesting to note that, due to the knockdown of ApoE, the livers of ApoE-/- mice in the non-exercise group did not show significant lipid deposition; however, after 12 weeks of MICT and HIIT, the livers of ApoE-/- mice showed significant lipid deposition. After we analyzed the lipid metabolism in their livers, we found that this was caused by the promotion of transport of peripheral fat into the liver due to exercise. Moreover, 12 weeks of exercise effectively reduced atherosclerosis, and the livers of ApoE-/- mice in the exercise group were not damaged by lipotoxicity. The results showed that a 12-week exercise treatment activated AMPK in the livers of HFD ApoE-/- mice through the APN-AdipoR1 signaling pathway, improved hepatic lipid metabolism disorders, and promoted the nuclear translocation of TFEB to enhance autophagic-lysosomal lipid scavenging. After the peripheral lipid is input into the liver due to exercise, the energy generated through gluconeogenesis can be used to replenish the energy consumed by exercise and maintain the normal operation of various functions in the liver, based on which the high autophagic flux in the liver can be maintained and the lipid clearance rate can be enhanced to protect the liver from lipotoxicity.

Sushi domain-containing protein 4 binds to epithelial growth factor receptor and initiates autophagy in an EGFR phosphorylation independent manner

BackgroundSushi domain-containing protein 4 (SUSD4) is a recently discovered protein with unknown cellular functions. We previously revealed that SUSD4 can act as complement inhibitor and as a potential tumor suppressor.MethodsIn a syngeneic mouse model of breast cancer, tumors expressing SUSD4 had a smaller volume compared with the corresponding mock control tumors. Additionally, data from three different expression databases and online analysis tools confirm that for breast cancer patients, high mRNA expression of SUSD4 in the tumor tissue correlates with a better prognosis. In vitro experiments utilized triple-negative breast cancer cell lines (BT-20 and MDA-MB-468) stably expressing SUSD4. Moreover, we established a cell line based on BT-20 in which the gene for EGFR was knocked out with the CRISPR-Cas9 method.ResultsWe discovered that the Epithelial Growth Factor Receptor (EGFR) interacts with SUSD4. Furthermore, triple-negative breast cancer cell lines stably expressing SUSD4 had higher autophagic flux. The initiation of autophagy required the expression of EGFR but not phosphorylation of the receptor. Expression of SUSD4 in the breast cancer cells led to activation of the tumor suppressor LKB1 and consequently to the activation of AMPKα1. Finally, autophagy was initiated after stimulation of the ULK1, Atg14 and Beclin-1 axis in SUSD4 expressing cells.ConclusionsIn this study we provide novel insight into the molecular mechanism of action whereby SUSD4 acts as an EGFR inhibitor without affecting the phosphorylation of the receptor and may potentially influence the recycling of EGFR to the plasma membrane.

Mitochondrial ROS Produced in Human Colon Carcinoma Associated with Cell Survival via Autophagy.

Simple SummaryHuman colon carcinoma remains one of the major causes of cancer-related death worldwide. Ubiquinol cytochrome c reductase binding protein (UQCRB) has been reported as a biomarker of colorectal cancer, but its role in tumor growth has not been clarified. CRC cells often exhibit high autophagic flux under nutrient deprivation or hypoxic condition and increased autophagy activation in cancer cells involving the recycling of cell components to facilitate survival in a tumor microenvironment. Here we show that UQCRB is overexpressed in HCT116 cells compared to CCD18co, normal colon fibroblast cells. Mechanistically, the increasing level of mitochondrial ROS (mROS) caused by UQCRB overexpression can release Ca2+ by the activation of the lysosomal transient receptor potential mucolipin 1 channels. This activation triggers transcription factor EB nuclear translocation and lysosome biogenesis leading to autophagy flux. Collectively, we identified that the increasing level of mROS by the overexpression of UQCRB in human colon carcinoma could link to autophagy for colorectal cancer survival. These results lead to a translational impact that a UQCRB inhibitor could be a potential anticancer agent for human colon carcinoma treatment.Human colon carcinomas, including HCT116 cells, often exhibit high autophagic flux under nutrient deprivation or hypoxic conditions. Mitochondrial ROS (mROS) is known as a ‘molecular switch’ for regulating the autophagic pathway, which is critical for directing cancer cell survival or death. In early tumorigenesis, autophagy plays important roles in maintaining cellular homeostasis and contributes to tumor growth. However, the relationships between mROS and the autophagic capacities of HCT116 cells are poorly understood. Ubiquinol cytochrome c reductase binding protein (UQCRB) has been reported as a biomarker of colorectal cancer, but its role in tumor growth has not been clarified. Here, we showed that UQCRB is overexpressed in HCT116 cells compared to CCD18co cells, a normal colon fibroblast cell line. Pharmacological inhibition of UQCRB reduced mROS levels, autophagic flux, and the growth of HCT116 tumors in a xenograft mouse model. We further investigated mutant UQCRB-overexpressing cell lines to identify functional links in UQCRB-mROS-autophagy. Notably, an increasing level of mROS caused by UQCRB overexpression released Ca2+ by the activation of lysosomal transient receptor potential mucolipin 1 (TRPML1) channels. This activation induced transcription factor EB (TFEB) nuclear translocation and lysosome biogenesis, leading to autophagy flux. Collectively, our study showed that increasing levels of mROS caused by the overexpression of UQCRB in human colon carcinoma HCT116 cells could be linked to autophagy for cell survival.

Metformin promotes autophagy activity and constrains HSV-1 replication in neuroblastoma cells

Problem consideredMetformin is an antihyperglycemic drug with effective clinical functions in type 2 diabetes patients. Recent evidence indicates that metformin can change natural cellular signaling to induce autophagy. Furthermore, infection could rise autophagy activity, but herpes simplex virus 1 (HSV-1) constructs, particularly virulence factors, constrain autophagy promotion, we used HSV-1 because of its ability to escape from autophagy by inhibiting beclin-1 formation, reversing PKR signaling and also induce mTOR activity. This study aimed to compare the extent of autophagic activity in metformin-treated cells infected with HSV-1 and determine the effect of metformin on HSV-1 replication. MethodsNeuroblastoma cells were cultured and then divided into the two separate groups of infected with HSV-1 and expose with metformin. Autophagy was evaluated via microtubule-associated protein 1 light chain 3 (LC3-II) monitoring by affix staining antibodies to this protein using flow cytometry. To evaluate viral load, HSV-1 was inoculated for 4 h post metformin treatment, and to evaluate viral load in the post-infection pathway, cells were inoculated with HSV-1. Then, after a 2-hour viral attachment time, metformin was administered to the cells. Viral nucleic acid was purified 48 h post-infection, and virus titer was calculated by real-time polymerase chain reaction (PCR). ResultsOur real-time PCR results indicated that HSV-1 viral load in pre-infection pathway reduced from 1.3 × 106 in positive control to 8 × 105 in cells exposed to metformin. Additionally, the TCID50/ml at 12 and 24 h after infection was 10 times lower than in HSV-1 positive control. However, after 48 h, HSV-1 titer in the metformin-treated group was equal with non-treated cells. For the post-infection pathway, real-time PCR results exhibited that HSV-1 titer was 2.3 × 106 in virus control cells compared to 1.8 × 106 in treated cells. That is, TCID50/ml in the medium of metformin-treated cells was 10 times lower in comparison with non-treated cells after 72 h. ConclusionThe study of the effect of metformin on neuroblastoma cells indicates high autophagic flux and lesser HSV-1 propagation in treated cells.

Autophagy Triggers Tamoxifen Resistance in Human Breast Cancer Cells by Preventing Drug-Induced Lysosomal Damage.

Simple SummaryEndocrine therapy with tamoxifen or other endocrine drugs represents the standard treatment for estrogen receptor-positive breast cancer. In spite of effectiveness of this therapy, onset of drug resistance worsens the prognosis of about 30% of patients. Autophagy has recently been proposed as a key player of drug resistance, but the underlying mechanisms are not completely understood. In this research, the authors investigate how autophagy triggers drug resistance in breast cancer cells. The results evidence that tamoxifen affects lysosome integrity, which suggests that this effect may contribute to the anticancer activity of this drug. Activation of autophagy and overexpression of iron-binding proteins synergize in protecting the lysosomal compartment, restraining drug effectiveness in breast cancer cells. According to these results, tamoxifen-resistant cells show an increased autophagic flux and overexpress iron-binding proteins. These findings indicate that screening for the level of iron-binding proteins may help to identify patients at risk for developing drug resistance.Endocrine resistance is a major complication during treatment of estrogen receptor-positive breast cancer. Although autophagy has recently gained increasing consideration among the causative factors, the link between autophagy and endocrine resistance remains elusive. Here, we investigate the autophagy-based mechanisms of tamoxifen resistance in MCF7 cells. Tamoxifen (Tam) triggers autophagy and affects the lysosomal compartment of MCF7 cells, such that activated autophagy supports disposal of tamoxifen-damaged lysosomes by lysophagy. MCF7 cells resistant to 5 µM tamoxifen (MCF7-TamR) have a higher autophagic flux and an enhanced resistance to Tam-induced lysosomal alterations compared to parental cells, which suggests a correlation between the two events. MCF7-TamR cells overexpress messenger RNAs (mRNAs) for metallothionein 2A and ferritin heavy chain, and they are re-sensitized to Tam by inhibition of autophagy. Overexpressing these proteins in parental MCF7 cells protects lysosomes from Tam-induced damage and preserves viability, while inhibiting autophagy abrogates lysosome protection. Consistently, we also demonstrate that other breast cancer cells that overexpress selected mRNAs encoding iron-binding proteins are less sensitive to Tam-induced lysosomal damage when autophagy is activated. Collectively, our data demonstrate that autophagy triggers Tam resistance in breast cancer cells by favoring the lysosomal relocation of overexpressed factors that restrain tamoxifen-induced lysosomal damage.

Combining Everolimus and Ku0063794 Promotes Apoptosis of Hepatocellular Carcinoma Cells via Reduced Autophagy Resulting from Diminished Expression of miR-4790-3p.

It is challenging to overcome the low response rate of everolimus in the treatment of patients with hepatocellular carcinoma (HCC). To overcome this challenge, we combined everolimus with Ku0063794, the inhibitor of mTORC1 and mTORC2, to achieve higher anticancer effects. However, the precise mechanism for the synergistic effects is not clearly understood yet. To achieve this aim, the miRNAs were selected that showed the most significant variation in expression according to the mono- and combination therapy of everolimus and Ku0063794. Subsequently, the roles of specific miRNAs were determined in the processes of the treatment modalities. Compared to individual monotherapies, the combination therapy significantly reduced viability, increased apoptosis, and reduced autophagy in HepG2 cells. The combination therapy led to significantly lower expression of miR-4790-3p and higher expression of zinc finger protein225 (ZNF225)—the predicted target of miR-4790-3p. The functional study of miR-4790-3p and ZNF225 revealed that regarding autophagy, miR-4790-3p promoted it, while ZNF225 inhibited it. In addition, regarding apoptosis, miR-4790-3p inhibited it, while ZNF225 promoted it. It was also found that HCC tissues were characterized by higher expression of miR-4790-3p and lower expression of ZNF225; HCC tissues were also characterized by higher autophagic flux. We, thus, conclude that the potentiated anticancer effect of the everolimus and Ku0063794 combination therapy is strongly associated with reduced autophagy resulting from diminished expression of miR-4790-3p, as well as higher expression of ZNF225.

Autophagy Plays a Role in the CUL4A-Related Poor Prognosis of Intrahepatic Cholangiocarcinoma.

CUL4A regulate the termination of autophagy in a physical process. However, the relationship between CUL4A and autophagy in cancer is unclear. We retrospectively investigated 99 intrahepatic cholangiocarcinoma (iCCA) cases. Whole sections were used for immunohistochemical analysis for p62, and LC3B expression. Q-score was defined as the sum of the labeling intensity and proportion. The cut-off point for immunoreactivity was set. CUL4A was overexpressed in cell lines and autophagy reflux was compared after manipulation. The iCCA cases with CUL4A overexpression had significantly higher prevalence of intact activated autophagy (42.4 vs. 15.2%; p = 0.003), which was significantly associated with advance tumor stage (34.1% vs. 15.4%; p = 0.032), less extensive necrosis (8.3 vs. 49.3%; p < 0.001), and shortened disease-free survival (mean, 19.6 vs. 65.5 months, p = 0.015). In vitro, iCCA cells with CUL4A overexpression significantly increased LC3II level as compared to the cells under basal condition. Although both cell types showed intact autophagy with increased LC3II expression after bafilomycin A1 treatment, the accumulation of LC3II was higher in CUL4A-overexpressing cells. CUL4A overexpression increased the proliferation of cells as compared with control cells. After treatment with bafilomycin A1, proliferation was inhibited in both cell types, but the effects were more prominent in the cells overexpressing CUL4A. CUL4A promotes autophagy, and exhibits significantly higher autophagic flux which affects the proliferation of iCCA cells; these effects correlated with advance tumor stage and poor prognosis. Thus, targeting autophagy may be potentially therapeutic in iCCA.

Virtual screening and repurposing of FDA-approved drugs from ZINC database to identify potential autophagy inhibitors exploiting autophagy related 4A cysteine peptidase as a target: potential as novel anti-cancer molecule.

Cancer cells utilize extensive autophagy in effort to adapt to high metabolic stress. This indicates that impairing the high autophagic flux might be an attractive target for cancer therapy. Autophagy related gene 4A (ATG4A) is a key player for autophagy and its inhibition may help in tumor clearance. The present study aims to screen candidate drugs from FDA-approved drugs, a subset of Zinc database, to identify potential ATG4A inhibitors that may have anti-cancer activity. Computer aided drug design approach was applied for the study using the virtual screening tools Raccoon and MGLTools-1.5.6. We have identified the drug Lumacaftor as a potent inhibitor of ATG4A on the basis of computational approaches viz. molecular docking, molecular dynamics simulation and MM/PBSA method. The drug is likely to be a potent regimen candidate to be used as an anti-cancer molecule. However, this potent inhibitor against ATG4A as anti-cancer molecule needs further investigation and validation. Communicated by Ramaswamy H. Sarma

Thermogenic Activation Downregulates High Mitophagy Rate in Human Masked and Mature Beige Adipocytes.

Thermogenic brown and beige adipocytes oxidize metabolic substrates producing heat, mainly by the mitochondrial uncoupling protein UCP1, and can thus counteract obesity. Masked beige adipocytes possess white adipocyte-like morphology, but can be made thermogenic by adrenergic stimuli. We investigated the regulation of mitophagy upon thermogenic activation of human masked and mature beige adipocytes. Human primary abdominal subcutaneous adipose-derived stromal cells (hASCs) and Simpson–Golabi–Behmel syndrome (SGBS) preadipocytes were differentiated to white and beige adipocytes, then their cAMP-induced thermogenic potential was assessed by detecting increased expressions of UCP1, mitochondrial DNA content and respiratory chain complex subunits. cAMP increased the thermogenic potential of white adipocytes similarly to beige ones, indicating the presence of a masked beige population. In unstimulated conditions, a high autophagic flux and mitophagy rates (demonstrated by LC3 punctae and TOM20 co-immunostaining) were observed in white adipocytes, while these were lower in beige adipocytes. Silencing and gene expression experiments showed that the ongoing mitophagy was Parkin-independent. cAMP treatment led to the downregulation of mitophagy through PKA in both types of adipocytes, resulting in more fragmented mitochondria and increased UCP1 levels. Our data indicates that mitophagy is repressed upon encountering a short-term adrenergic stimulus, as a fast regulatory mechanism to provide high mitochondrial content for thermogenesis.

The alarmin HMGB1 in cell cytoplasm can modulate the apoptosis to autophagy switch of pancreatic beta cells under stress

Abstract High mobility group box 1 (HMGB1) is an infamous alarmin which is known to be harmful to pancreatic beta cells and associated with diabetes mellitus pathogenesis and pancreatic islet graft failure. However, recent studies have indicated that cytosolic HMGB1 could function as a modulator to relieve cells from apoptotic stress by inducing autophagy. Particularly, pancreatic beta cells have been well-known to demonstrate the apoptosis to autophagy switch when exposed to hypoxia or lipoxiticy. In this study, we have investigated the beta cells under hypoxic and lipotoxic stress with CCK8 assay and flow cytometry while utilizing a small molecule inhibitor of HMGB1 which can suppress the cytosolic accumulation of HMGB1. The results demonstrated that the cytoplasmic HMGB1 blockade decreased the viability of mouse beta cell line MIN6 and primary islets. Moreover, the cytoplasmic HMGB1 blockade decreased the autophagic flux and increased the ratio of apoptotic MIN6 cells. Also, it was noteworthy that the MIN6 cells have shown high autophagic flux even in basal state. We believe this study linked the role of cytoplasmic HMGB1 in apoptosis to autophagy switch and the importance of autophagy in beta cells under apoptotic stress. We expect this study to open up a novel field of HMGB1 utilization within diabetes- and islet transplantation-related therapeutics, where the current field is mainly focused on the exhaustive blockade of HMGB1.

Rapamycin does not reproduce the alterations to Caco‐2 transepithelial electrical resistance caused by the knockdown of Raptor

Intestinal homeostasis is achieved through well‐coordinated interactions between the mucus layer, the epithelial layer and the immune cells of the lamina propria. Owing to its proximity with the luminal content, the intestinal epithelium is exposed to insults of chemical, physical or dysbiotic nature. These insults may perturb the epithelium permeability and trigger a localized immune response, which if not controlled may progress to damage the epithelium. Although the mechanistic target of rapamycin complex 1 (mTORC1) is deactivated in differentiated enterocytes, mTORC1 becomes active under repeated insult or injury to the epithelium and participates in epithelial cell repair. However, many aspects of mTORC1 re‐activation remain to be elucidated. To investigate the effects of graded levels of mTORC1 activity on epithelial barrier function, we knocked down (&gt;90% down) mTORC1 signaling‐associated proteins, Raptor and TSC2, to create three Caco‐2 cell lines in which, after full differentiation in Transwell®, mTORC1 activity was either normal (shScramble cells), low (shRaptor cells, mTORC1 activity 50% lower than shScramble cells) or high (shTSC2 cells, mTORC1 activity 80% higher than shScramble cells). When compared with shScramble cells, shRaptor cells had higher abundance of p‐(Thr172)AMPK (+46%, P&lt;0.05), lower abundance of p‐(Ser757)ULK‐1 (−50%, P&gt;0.05), higher autophagic flux (+180%, P&lt;0.05), and lower expression of pore‐forming tight junction protein Claudin‐2 (−65%, P&lt;0.05). In contrast, shTSC2 cells had higher levels of p‐(Ser757)ULK‐1 (+100%, P&lt;0.05) and Claudin‐2 (+250%, P&lt;0.05) compared with shScramble cells. These mTORC1‐related changes were accompanied by significant alteration to the Caco‐2 transepithelial electrical resistance (TEER), which was 180% higher in shRaptor cells vs. shScramble cells, and conversely 60% lower in shTSC2 cells vs. shScramble cells. Although rapamycin suppressed mTORC1 activity in these cells, rapamycin caused a modest 34% (P&lt;0.05) increase of TEER in shScramble cells, suggesting that factors other than mTORC1 ablation were responsible for the high TEER of shRaptor cells. The use of chloroquine (an inhibitor of autophagosome‐lysosome fusion) showed that autophagy alone could not explain the strong electrical resistance of shRaptor cells. We are currently investigating whether TSC2, whose expression increases as Caco‐2 cells differentiate concomitantly with mTORC1 silencing, might regulate paracellular permeability and tight junction assembly in the epithelium.Support or Funding InformationUSDA‐NIFA

P23 Co‐chaperone Protects Aryl Hydrocarbon Receptor (AHR) from the Autophagy‐Mediated Degradation

The aryl hydrocarbon receptor (AHR) is first discovered as a transcription factor in response to toxic compounds such as dioxins. It also plays important roles in organ development, immune responses, metabolisms and carcinogenesis. Without ligand activation, AHR resides in the cytosol and is accompanied by Hsp90, p23 and XAP2. AHR is degraded via the 26S proteasome after ligand activation. However, it is still unclear how basal AHR protein levels are regulated. We previously discovered that AHR protein levels are lower in p23 stable knockdown (p23KD) cell lines when there is no ligand treatment. Here we report that treatment of proteasome inhibitor MG132 could not restore AHR in p23KD HeLa cells while the autophagy inhibitor chloroquine increased AHR proteins levels to 2.5 to 3‐fold in p23KD HeLa cells and 2‐fold in WT HeLa cells. Similar results were observed when cells were treated with other autophagy inhibitors (bafilomycin A1 and 3MA). There was higher autophagic flux in p23KD HeLa cells than in WT HeLa cells. Results from the co‐immunoprecipitation experiment and proximity ligation assay showed interaction between AHR and LC3B (LC3B‐II), which is an important protein in macroautophagy. These results support that the AHR protein levels are regulated by autophagy and p23 can protect AHR from the autophagy‐mediated degradation. Additional data will be presented to address the mechanisms of basal AHR protein degradation via autophagy.Support or Funding InformationNIH R15ES023104

Rapamycin and chloroquine insensitivity reveal mTORC1‐independent regulation of epithelial permeability in differentiated intestinal Caco‐2 cells

Intestinal barrier integrity dictates the intestinal homeostasis by stringently regulating the permeability of the luminal contents into the intestinal tissue of the human body. The protective role of a well‐formed epithelial barrier is substantiated by incidences of increased mucosal inflammation upon epithelial barrier disruption. The pathophysiology of many serious intestinal disorders such as inflammatory bowel disease, necrotizing enterocolitis, and celiac disease is associated with increased permeability of the intestinal wall to luminal contents. In order to identify new therapeutic strategies against these disorders, it is critical to better understand the underpinning molecular mechanisms regulating epithelial barrier permeability. The role of the mTORC1 (mechanistic target of rapamycin complex 1) signaling in the regulation of intestinal barrier function and permeability remains to be fully elucidated. To that end, we genetically manipulated human epithelial intestinal Caco‐2 cells to generate three stable cell lines with baseline (shScramble), low (shRaptor) or high (shTSC2) mTORC1 activity. The three cell lines maintained their intended mTORC1 activity upon differentiation on Transwell® inserts, i.e., shRaptor cells exhibited 50% lower mTORC1 activity vs. shScramble cells, while shTSC2 cells exhibited 80% higher mTORC1 activity vs. shScramble cells. Transepithelial electrical resistance (TEER), a measure of epithelial permeability was inversely proportional to mTORC1 activity (r2= 0.98, P&lt;0.05). shRaptor cells had higher expression of p‐(Thr172)AMPK (+46%, P&lt;0.05), lower abundance of p‐(Ser757)ULK‐1 (−50%, P&gt;0.05), higher autophagic flux (+180%, P&lt;0.05), and lower expression of pore‐forming tight junction protein Claudin‐2 (−65%, P&lt;0.05) compared with shScramble cells. In contrast, shTSC2 cells had higher levels of p‐(Ser757)ULK‐1 (+100%, P&lt;0.05) and Claudin‐2 (+250%, P&lt;0.05) compared with shScramble cells. We treated shScramble cells with rapamycin in an attempt to pharmacologically reproduce the effects of Raptor knockdown on epithelial permeability and found that rapamycin modestly but statistically increased TEER (+34%, P&lt;0.05) and decreased Claudin2 (−36%, P&lt;0.05). The role of autophagy in rapamycin‐mediated increase of TEER was dismissed on the basis that chloroquine (inhibitor of autophagosome‐lysosome fusion) did not significantly reduce TEER in shScramble cells. Our results suggest that although mTORC1 regulates epithelial permeability during the early stages of Caco‐2 cell differentiation, TEER is not substantially affected by rapamycin and chloroquine in fully differentiated cells.Support or Funding InformationSupported by USDA‐NIFA

Metabolic effects of RUBCN/Rubicon deficiency in kidney proximal tubular epithelial cells

ABSTRACT Macroautophagy/autophagy is a lysosomal degradation system which plays a protective role against kidney injury. RUBCN/Rubicon (RUN domain and cysteine-rich domain containing, Beclin 1-interacting protein) inhibits the fusion of autophagosomes and lysosomes. However, its physiological role in kidney proximal tubular epithelial cells (PTECs) remains uncertain. In the current study, we analyzed the phenotype of newly generated PTEC-specific rubcn-deficient (KO) mice. Additionally, we investigated the role of RUBCN in lipid metabolism using isolated rubcn-deficient PTECs. Although KO mice exhibited sustained high autophagic flux in PTECs, they were not protected from acute ischemic kidney injury. Unexpectedly, KO mice exhibited hallmark features of metabolic syndrome accompanied by expanded lysosomes containing multi-layered phospholipids in PTECs. RUBCN deficiency in cultured PTECs promoted the mobilization of phospholipids from cellular membranes to lysosomes via enhanced autophagy. Treatment of KO PTECs with oleic acid accelerated fatty acids transfer to mitochondria. Furthermore, KO PTECs promoted massive triglyceride accumulation in hepatocytes (BNL-CL2 cells) co-cultured in transwell, suggesting accelerated fatty acids efflux from the PTECs contributes to the metabolic syndrome in KO mice. This study shows that sustained high autophagic flux by RUBCN deficiency in PTECs leads to metabolic syndrome concomitantly with an accelerated mobilization of phospholipids from cellular membranes to lysosomes. Abbreviations: ABC: ATP binding cassette; ACADM: acyl-CoA dehydrogenase medium chain; ACTB: actin, beta; ATG: autophagy related; AUC: area under the curve; Baf: bafilomycin A1; BAT: brown adipose tissue; BODIPY: boron-dipyrromethene; BSA: bovine serum albumin; BW: body weight; CAT: chloramphenicol acetyltransferase; CM: complete medium; CPT1A: carnitine palmitoyltransferase 1a, liver; CQ: chloroquine; CTRL: control; EGFP: enhanced green fluorescent protein; CTSD: cathepsin D; EAT: epididymal adipose tissue; EGFR: epidermal growth factor receptor; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; FA: fatty acid; FBS: fetal bovine serum; GTT: glucose tolerance test; HE: hematoxylin and eosin; HFD: high-fat diet; I/R: ischemia-reperfusion; ITT: insulin tolerance test; KAP: kidney androgen regulated protein; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LD: lipid droplet; LRP2: low density lipoprotein receptor related protein 2; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MAT: mesenteric adipose tissue; MS: mass spectrometry; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; NDRG1: N-myc downstream regulated 1; NDUFB5: NADH:ubiquinone oxidoreductase subunit B5; NEFA: non-esterified fatty acid; OA: oleic acid; OCT: optimal cutting temperature; ORO: Oil Red O; PAS: Periodic-acid Schiff; PFA: paraformaldehyde; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PPARA: peroxisome proliferator activated receptor alpha; PPARGC1A: PPARG coactivator 1 alpha; PTEC: proximal tubular epithelial cell; RAB7A: RAB7A, member RAS oncogene family; RPS6: ribosomal protein S6; RPS6KB1: ribosomal protein S6 kinase B1; RT: reverse transcription; RUBCN: rubicon autophagy regulator; SAT: subcutaneous adipose tissue; SFC: supercritical fluid chromatography; SQSTM1: sequestosome 1; SREBF1: sterol regulatory element binding transcription factor 1; SV-40: simian virus-40; TFEB: transcription factor EB; TG: triglyceride; TS: tissue specific; TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling; UN: urea nitrogen; UQCRB: ubiquinol-cytochrome c reductase binding protein; UVRAG: UV radiation resistance associated; VPS: vacuolar protein sorting; WAT: white adipose tissue.

Colorectal cancer cells respond differentially to autophagy inhibition in vivo

Autophagy has both tumor-promoting and -suppressing effects in cancer, including colorectal cancer (CRC), with transformed cells often exhibiting high autophagic flux. In established tumors, autophagy inhibition can lead to opposite responses resulting in either tumor cell death or hyperproliferation. The functional mechanisms underlying these differences are poorly understood. The present study aimed to investigate the relationship between the autophagic capacities of CRC cells and their sensitivities to autophagy inhibition. All studied CRC cell lines showed high basal autophagic flux. However, only HCT116 and Caco-2/15 cells displayed regulated autophagic flux upon starvation. Knockdown of ATG5 (which disrupts autophagosome elongation) or RAB21 (which decreases autophagosome/lysosome fusion) had little effect on CRC cell proliferation in vitro. Nonetheless, inhibition of autophagy in vivo had a substantial cell line-dependent impact on tumor growth, with some cells displaying decreased (HCT116 and Caco-2/15) or increased (SW480 and LoVo) proliferation. RNA sequencing and Western blot analyses in hyperproliferative SW480 tumors revealed that the mTORC2 and AKT pathways were hyperactivated following autophagy impairment. Inhibition of either mTOR or AKT activities rescued the observed hyperproliferation in autophagy-inhibited SW480 and reduced tumor growth. These results highlight that autophagy inhibition can lead, in specific cellular contexts, to compensatory mechanisms promoting tumor growth.

Estrogen enhances human osteoblast survival and function via promotion of autophagy

Estrogen increases bone formation by promoting mineralization and prolonging the lifespan of osteoblasts. To understand the underlying molecular mechanism/s, we identified estrogen-regulated proteins at different stages of human osteoblast differentiation using differential proteomics approach. Among the identified proteins, we observed that estrogen upregulated RAB3GAP1 on day 1 and 5 of differentiation. RAB3GAP1 is critically involved in the process of autophagy, a eukaryotic degradative pathway essential for cell survival. We, therefore, investigated the effect of estrogen on autophagy in differentiating human osteoblasts and their precursors, the mesenchymal stem cells (MSCs). MSCs exhibited high autophagic flux which declined during osteoblast differentiation, resulting in high basal apoptosis in osteoblasts. Estrogen reduced apoptosis in differentiating osteoblasts by promoting autophagy, thus contributing towards their longer lifespan. Further, MSCs were resistant against starvation-induced apoptosis, whereas, differentiating osteoblasts showed significant susceptibility towards it. Estrogen, in addition to promoting mineralization, protected differentiating osteoblasts from starvation-induced apoptosis by increasing autophagic flux. Autophagic flux in RAB3GAP1 knockdown osteoblasts appeared diminished, and showed increased apoptosis even in nutrient-rich conditions, and exhibited significantly impaired mineralization. However, irrespective of the presence of estrogen, starvation further enhanced apoptosis in these cells. Furthermore, estrogen failed to promote mineralization in these osteoblasts. Our study illustrates that autophagy is essential for human osteoblast survival and mineralization, and osteoblasts are susceptible to apoptosis due to reduced autophagy during differentiation. Estrogen, via upregulation of RAB3GAP1, promotes autophagy in osteoblasts during differentiation thereby increasing their survival and mineralization capacity. Our study demonstrates the positive role of autophagy in bone homeostasis.

Bovine papillomavirus E5 oncoprotein expression and its association with an interactor network in aggresome-autophagy pathway

E5 protein, the major oncoprotein of bovine Deltapapillomavirus (BPV), was found to be expressed in 18 of 21 examined urothelial cancers of cattle. E5 oncoprotein was found to interact with p62 which was degraded through the autophagosome-lysosome pathway as well as LC3-II and appeared to be involved in the phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α). Autophagy was morphologically documented by transmission electron microscope (TEM) through the detection of double-membrane autophagosomes and autolysosomes. Overexpression of Bag3 known to mediate selective autophagy was also demonstrated. Furthermore, Bag3 and BPV E5 oncoprotein were seen to co-localize with dynein and 14-3-3γ, which suggested that Bag3 could be involved in inducing the retrograde transport of BPV E5 along microtubules to aggresomes, perinuclear sites with high autophagic flux. Electron dense perinuclear structures consistent with aggresomes were also documented by TEM in urothelial cancer cells. Finally, Bag3 was found to also interact with synaptopodin 2 (Synpo2), which would seem to contribute to cargo degradation as it has been shown to facilitate autophagosome formation. This study provides mechanistic insights into the potential role(s) of autophagy in BPV disease, which can help to develop future treatment and control measures for BPV infection. Activation of autophagy correlates positively with BPV infection and may play a role in biological behavior of bladder cancer as urothelial carcinomas of cattle are known to be characterized by a relatively low rate of metastasis.

MiR4673 improves fitness profile of neoplastic cells by induction of autophagy

Therapeutic resistance of neoplasms is mainly attributed to gradual evolution of mutational profile1. Here, we demonstrate a microRNA-mediated mechanism that effectively improves fitness of SKBR3 mammary carcinoma cells by cytoplasmic reprogramming. The reprogramming is triggered by endogenous miR4673 transcribed from notch-1 locus. The miRNA downregulates cdk-18, a cyclin-dependent kinase that regulates M-G1 transition in cycling cells2,3. Suppression of cdk-18 triggers mitophagy and autophagy. Due to high autophagic flux, oestrogen receptor-1+/progesterone receptor+/p53+ (Esr1+/Pr+/p53+) SKBR3 cells are coerced into an Esr1−/Prlow/p53−profile. Increased mitophagy in combination with proteasomal degradation of p53 transiently arrests the cycling cells at G0 and enhances radio-resistance of the SKBR3 population. These findings highlight the impact on cancer therapy of non-encoded neoplastic resistance, arising as a consequence of miRNA-mediated autophagic reprogramming that uncouples phenotype and genotype.

Abstract 1431: Autophagic degradation of the endoplasmic reticulum leads to reduced phospholipid biosynthesis in mutant isocitrate dehydrogenase 1 gliomas

Abstract Introduction: Cancer cells up-regulate phospholipid biosynthesis in order to meet the demands of uncontrolled cell proliferation. Unusually, mutant isocitrate dehydrogenase 1 (IDHmut) gliomas down-regulate levels of the phospholipid precursors phosphocholine and phosphoethanolamine relative to wild-type IDH1 (IDHwt) gliomas. The goal of this study was therefore to investigate whether the phospholipids phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdE) are also down-regulated in IDHmut gliomas. Methods: Immortalized normal human astrocytes and U87 cells expressing IDHwt or IDHmut enzyme were studied. Steady-state phospholipid levels were measured by 31P-magnetic resonance spectroscopy (MRS) of cell extracts. De novo phospholipid biosynthesis was quantified by 13C-MRS of cells labeled with 56μM [1,2-13C]-choline and [1,2-13C]-ethanolamine. ER area was measured by STORM imaging of calnexin fluorescence. Results: Steady-state PtdCho and PtdE levels and de novo phospholipid biosynthesis were significantly reduced in IDHmut cells relative to IDHwt. Concomitantly, the activities of CTP:PC cytidylyltransferase (CCT) and CTP:PE cytidylyltransferase (ECT), the rate-limiting enzymes for phospholipid biosynthesis, were significantly reduced in IDHmut cells. CCT and ECT are localized to the endoplasmic reticulum (ER), the site of phospholipid biosynthesis. We therefore measured ER area by super-resolution STORM imaging and found a significant reduction in IDHmut cells relative to IDHwt while cell size remained unchanged. Autophagy of the ER (ER-phagy) can regulate ER area and we found that IDHmut cells showed increased expression of FAM134b, the ER-phagy receptor and higher autophagic flux. These results suggest that IDHmut cells undergo ER-phagy, leading to reduced phospholipid biosynthesis. Silencing FAM134b or the autophagy proteins Atg5 and Atg7 restored ER area and phospholipid biosynthesis and abrogated clonogenicity of IDHmut cells, linking ER-phagy to reduced phospholipid biosynthesis and pointing to a therapeutic opportunity. In the absence of specific ER-phagy inhibitors, we used the late-stage autophagy inhibitor chloroquine (CQ) to test proof-of-principle and found that CQ significantly inhibited orthotopic IDHmut glioma growth in vivo. Importantly, we observed ER-phagy and down-regulation of phospholipid levels in IDHmut glioma patient biopsies, highlighting the translational validity of our findings. Conclusions: We find that down-regulation of phospholipid biosynthesis is a unique metabolic outcome of ER-phagy in IDHmut gliomas. While autophagy has been linked to glucose and glutamine metabolism, this study is the first to link autophagy to phospholipid biosynthesis. Importantly, our study identifies a rationale for the development of novel therapies targeting ER-phagy in IDHmut gliomas. Citation Format: Pavithra Viswanath, Russell O. Pieper, Joanna J. Phillips, Sabrina M. Ronen. Autophagic degradation of the endoplasmic reticulum leads to reduced phospholipid biosynthesis in mutant isocitrate dehydrogenase 1 gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1431.