V-ATPase is a universal regulator of LC3-associated phagocytosis and non-canonical autophagy.

Non-canonical autophagy is a key cellular pathway in immunity, cancer, and neurodegeneration, characterized by conjugation of ATG8 to endolysosomal single membranes (CASM). CASM is activated by engulfment (endocytosis, phagocytosis), agonists (STING, TRPML1), and infection (influenza), dependent on K490 in the ATG16L1 WD40-domain. However, factors associated with non-canonical ATG16L1 recruitment and CASM induction remain unknown. Here, using pharmacological inhibitors, we investigate a role for V-ATPase during non-canonical autophagy. We report that increased V0-V1 engagement is associated with, and sufficient for, CASM activation. Upon V0-V1 binding, V-ATPase recruits ATG16L1, via K490, during LC3-associated phagocytosis (LAP), STING- and drug-induced CASM, indicating a common mechanism. Furthermore, during LAP, key molecular players, including NADPH oxidase/ROS, converge on V-ATPase. Finally, we show that LAP is sensitive to Salmonella SopF, which disrupts the V-ATPase-ATG16L1 axis and provide evidence that CASM contributes to the Salmonella host response. Together, these data identify V-ATPase as a universal regulator of CASM and indicate that SopF evolved in part to evade non-canonical autophagy.

Targeted protein degradation: from small molecules to complex organelles-a Keystone Symposia report.

Targeted protein degradation is critical for proper cellular function and development. Protein degradation pathways, such as the ubiquitin proteasomes system, autophagy, and endosome-lysosome pathway, must be tightly regulated to ensure proper elimination of misfolded and aggregated proteins and regulate changing protein levels during cellular differentiation, while ensuring that normal proteins remain unscathed. Protein degradation pathways have also garnered interest as a means to selectively eliminate target proteins that may be difficult to inhibit via other mechanisms. On June 7 and 8, 2021, several experts in protein degradation pathways met virtually for the Keystone eSymposium "Targeting protein degradation: from small molecules to complex organelles." The event brought together researchers working in different protein degradation pathways in an effort to begin to develop a holistic, integrated vision of protein degradation that incorporates all the major pathways to understand how changes in them can lead to disease pathology and, alternatively, how they can be leveraged for novel therapeutics.

GABARAP sequesters the FLCN-FNIP tumor suppressor complex to couple autophagy with lysosomal biogenesis.

Adaptive changes in lysosomal capacity are driven by the transcription factors TFEB and TFE3 in response to increased autophagic flux and endolysosomal stress, yet the molecular details of their activation are unclear. LC3 and GABARAP members of the ATG8 protein family are required for selective autophagy and sensing perturbation within the endolysosomal system. Here, we show that during the conjugation of ATG8 to single membranes (CASM), Parkin-dependent mitophagy, and Salmonella-induced xenophagy, the membrane conjugation of GABARAP, but not LC3, is required for activation of TFEB/TFE3 to control lysosomal capacity. GABARAP directly binds to a previously unidentified LC3-interacting motif (LIR) in the FLCN/FNIP tumor suppressor complex and mediates sequestration to GABARAP-conjugated membrane compartments. This disrupts FLCN/FNIP GAP function toward RagC/D, resulting in impaired substrate-specific mTOR-dependent phosphorylation of TFEB. Thus, the GABARAP-FLCN/FNIP-TFEB axis serves as a molecular sensor that coordinates lysosomal homeostasis with perturbations and cargo flux within the autophagy-lysosomal network.

An iron-dependent metabolic vulnerability underlies VPS34-dependence in RKO cancer cells.

VPS34 is a key regulator of endomembrane dynamics and cargo trafficking, and is essential in cultured cell lines and in mice. To better characterize the role of VPS34 in cell growth, we performed unbiased cell line profiling studies with the selective VPS34 inhibitor PIK-III and identified RKO as a VPS34-dependent cellular model. Pooled CRISPR screen in the presence of PIK-III revealed endolysosomal genes as genetic suppressors. Dissecting VPS34-dependent alterations with transcriptional profiling, we found the induction of hypoxia response and cholesterol biosynthesis as key signatures. Mechanistically, acute VPS34 inhibition enhanced lysosomal degradation of transferrin and low-density lipoprotein receptors leading to impaired iron and cholesterol uptake. Excess soluble iron, but not cholesterol, was sufficient to partially rescue the effects of VPS34 inhibition on mitochondrial respiration and cell growth, indicating that iron limitation is the primary driver of VPS34-dependency in RKO cells. Loss of RAB7A, an endolysosomal marker and top suppressor in our genetic screen, blocked transferrin receptor degradation, restored iron homeostasis and reversed the growth defect as well as metabolic alterations due to VPS34 inhibition. Altogether, our findings suggest that impaired iron mobilization via the VPS34-RAB7A axis drive VPS34-dependence in certain cancer cells.

TMEM41B is a novel regulator of autophagy and lipid mobilization.

Autophagy maintains cellular homeostasis by targeting damaged organelles, pathogens, or misfolded protein aggregates for lysosomal degradation. The autophagic process is initiated by the formation of autophagosomes, which can selectively enclose cargo via autophagy cargo receptors. A machinery of well-characterized autophagy-related proteins orchestrates the biogenesis of autophagosomes; however, the origin of the required membranes is incompletely understood. Here, we have applied sensitized pooled CRISPR screens and identify the uncharacterized transmembrane protein TMEM41B as a novel regulator of autophagy. In the absence of TMEM41B, autophagosome biogenesis is stalled, LC3 accumulates at WIPI2- and DFCP1-positive isolation membranes, and lysosomal flux of autophagy cargo receptors and intracellular bacteria is impaired. In addition to defective autophagy, TMEM41B knockout cells display significantly enlarged lipid droplets and reduced mobilization and ß-oxidation of fatty acids. Immunostaining and interaction proteomics data suggest that TMEM41B localizes to the endoplasmic reticulum (ER). Taken together, we propose that TMEM41B is a novel ER-localized regulator of autophagosome biogenesis and lipid mobilization.

Autophagy-Independent Lysosomal Targeting Regulated by ULK1/2-FIP200 and ATG9.

Iron is vital for many homeostatic processes, and its liberation from ferritin nanocages occurs in the lysosome. Studies indicate that ferritin and its binding partner nuclear receptor coactivator-4 (NCOA4) are targeted to lysosomes by a form of selective autophagy. By using genome-scale functional screening, we identify an alternative lysosomal transport pathway for ferritin that requires FIP200, ATG9A, VPS34, and TAX1BP1 but lacks involvement of the ATG8 lipidation machinery that constitutes classical macroautophagy. TAX1BP1 binds directly to NCOA4 and is required for lysosomal trafficking of ferritin under basal and iron-depleted conditions. Under basal conditions ULK1/2-FIP200 controls ferritin turnover, but its deletion leads to TAX1BP1-dependent activation of TBK1 that regulates redistribution of ATG9A to the Golgi enabling continued trafficking of ferritin. Cells expressing an amyotrophic lateral sclerosis (ALS)-associated TBK1 allele are incapable of degrading ferritin suggesting a molecular mechanism that explains the presence of iron deposits in patient brain biopsies.

An AMPK-independent signaling pathway downstream of the LKB1 tumor suppressor controls Snail1 and metastatic potential.

The serine/threonine kinase LKB1 is a tumor suppressor whose loss is associated with increased metastatic potential. In an effort to define biochemical signatures of metastasis associated with LKB1 loss, we discovered that the epithelial-to-mesenchymal transition transcription factor Snail1 was uniquely upregulated upon LKB1 deficiency across cell types. The ability of LKB1 to suppress Snail1 levels was independent of AMPK but required the related kinases MARK1 and MARK4. In a screen for substrates of these kinases involved in Snail regulation, we identified the scaffolding protein DIXDC1. Similar to loss of LKB1, DIXDC1 depletion results in upregulation of Snail1 in a FAK-dependent manner, leading to increased cell invasion. MARK1 phosphorylation of DIXDC1 is required for its localization to focal adhesions and ability to suppress metastasis in mice. DIXDC1 is frequently downregulated in human cancers, which correlates with poor survival. This study defines an AMPK-independent phosphorylation cascade essential for LKB1-dependent control of metastatic behavior.

Angiogenesis inhibition using an oncolytic herpes simplex virus expressing endostatin in a murine lung cancer model.

Herpes-mediated viral oncolysis alone is not sufficient to completely eradicate tumors. In this study we used a replication conditional, endostatin-expressing herpes simplex virus-1 mutant (HSV-Endo) in a murine lung cancer model. We hypothesized that the anti-angiogenic action of endostatin would improve upon the oncolytic effect of HSV-1. HSV-Endo was evaluated in a pulmonary metastases and orthotopic flank model, where there was significantly less tumor burden and reduced microvessel density compared to a control virus. Endostatin expression appears to improve the anti-tumor effect of HSV-1 in a lung cancer model.

Mouse model of carbon tetrachloride induced liver fibrosis: Histopathological changes and expression of CD133 and epidermal growth factor.

BACKGROUND: In the setting of chronic liver injury in humans, epidermal growth factor (EGF) and EGF receptor (EGFR) are up-regulated and have been proposed to have vital roles in both liver regeneration and development of hepatocellular carcinoma (HCC). Chronic liver injury also leads to hepatic stellate cell (HSC) differentiation and a novel subpopulation of HSCs which express CD133 and exhibit properties of progenitor cells has been described in rats. The carbon tetrachloride (CCl4)-induced mouse model has been historically relied upon to study liver injury and regeneration. We exposed mice to CCl4 to assess whether EGF and CD133+ HSCs are up-regulated in chronically injured liver. METHODS: CCl4 in olive oil was administered to strain A/J mice three times per week by oral gavage. RESULTS: Multiple well-differentiated HCCs were found in all livers after 15 weeks of CCl4 treatment. Notably, HCCs developed within the setting of fibrosis and not cirrhosis. CD133 was dramatically up-regulated after CCl4 treatment, and increased expression of desmin and glial fibrillary acidic protein, representative markers of HSCs, was also observed. EGF expression significantly decreased, contrary to observations in humans, whereas the expression of amphiregulin, another EGFR ligand, was significantly increased. CONCLUSIONS: Species-specific differences exist with respect to the histopathological and molecular pathogenesis of chronic liver disease. CCl4-induced chronic liver injury in A/J mice has important differences compared to human cirrhosis leading to HCC.

A functional epidermal growth factor (EGF) polymorphism, EGF serum levels, and esophageal adenocarcinoma risk and outcome.

PURPOSE: The epidermal growth factor (EGF) pathway is important in esophageal adenocarcinoma (EAC) tumorigenesis. We hypothesized that the EGF A61G homozygous variant genotype (GG) is (a) both a risk and poor prognostic factor for EAC and (b) associated with higher EGF serum levels in individuals with gastroesophageal reflux disease (GERD). EXPERIMENTAL DESIGN: Using unconditional logistic regression, we compared EGF A61G in 312 EAC cases and 447 GERD-free controls, adjusting for age, gender, smoking history, and healthy adult body mass index. Using the method of Kaplan and Meier, log-rank tests, and Cox proportional hazard models, we correlated EGF A61G with overall and failure-free survival in the EAC cases. Serum EGF levels and EGF genotype (G/G versus others) were correlated in 144 GERD patients using Wilcoxon rank sum tests. RESULTS: The EGF A61G G/G genotype conferred increased EAC risk, with an adjusted odds ratio of 1.81 (95% confidence interval, 1.2-2.7), and was even higher in the subgroup of EAC patients with concurrent Barrett's esophagus (adjusted odds ratio, 2.18; 95% confidence interval, 1.3-3.7). However, EGF A61G was not associated with a more aggressive phenotype or prognosis in EAC patients. Higher serum EGF levels were found in GERD patients carrying G/G compared with A/A or A/G (P = 0.03, Wilcoxon rank sum test). CONCLUSION: The EGF A61G G/G genotype is associated with a near 2-fold greater risk of EAC. The G/G allele was also associated with higher EGF levels in tumor-free patients with GERD. EGF genotyping can potentially identify high-risk patients with GERD and Barrett's metaplasia who might benefit from increased surveillance.

Epithelial-to-mesenchymal transition and integrin-linked kinase mediate sensitivity to epidermal growth factor receptor inhibition in human hepatoma cells.

Hepatocellular carcinoma (HCC) is associated with a poor prognosis due to late diagnoses and a lack of effective treatment options. Epidermal growth factor receptor (EGFR)-targeted therapies have been effective in other cancers. However, erlotinib and cetuximab have shown only modest efficacy in clinical trials of HCC. We examined epithelial-to-mesenchymal transition (EMT) as a determinant of sensitivity of HCC to EGFR inhibitors. A panel of 12 human hepatoma cell lines were classified as epithelial or mesenchymal based on their expression of E-cadherin and vimentin. The resulting classification correlated with a previous microarray analysis of human hepatoma cell lines whereby the mesenchymal cell lines were shown to have increased expression of genes involved in metastasis and invasion. Sensitivity to erlotinib, gefitinib, and cetuximab was assessed and the epithelial cell lines were found to be significantly more susceptible to all three agents. Analysis of the EGFR pathway showed that EMT status was independent of EGFR expression or downstream extracellular signal-regulated kinase activation and only the epithelial cell lines expressed ErbB3. Interestingly, mesenchymal cells resistant to EGFR inhibitors had increased AKT and signal transducer and activator of transcription-3 activation through elevated expression of integrin-linked kinase (ILK). Mesenchymal cell lines were therefore experimentally transformed with kinase-inactive ILK (KI-ILK) with a resulting decrease in ILK activity and activation of AKT. KI-ILK transformants showed increased sensitivity to EGFR inhibitors both in vitro and in an in vivo xenograft model. These data suggest that EMT predicts HCC sensitivity to EGFR-targeted therapies and that ILK is a novel target to overcome HCC resistance to EGFR inhibition.

Epidermal growth factor gene functional polymorphism and the risk of hepatocellular carcinoma in patients with cirrhosis.

CONTEXT: Overexpression of epidermal growth factor (EGF) in the liver induces transformation to hepatocellular carcinoma in animal models. Polymorphisms in the EGF gene modulate EGF levels. OBJECTIVE: To assess the relationship among human EGF gene single-nucleotide polymorphism, EGF expression, and risk of hepatocellular carcinoma. DESIGN, SETTING, AND PARTICIPANTS: Molecular mechanisms linking the 61*G allele polymorphism to EGF expression were examined in human hepatocellular carcinoma cell lines and human liver tissue. A case-control study involving 207 patients with cirrhosis was conducted at the Massachusetts General Hospital (1999-2006) and a validation case-control study involving 121 patients with cirrhosis was conducted at Hôpital Paul Brousse (1993-2006). Restriction fragment-length polymorphism was used to determine the EGF gene polymorphism genotype. Logistic regression analysis was used to assess the association between the EGF polymorphism and hepatocellular carcinoma risk. MAIN OUTCOME MEASURES: Mechanisms by which the EGF gene polymorphism modulates EGF levels and associations among EGF gene polymorphism, EGF levels, and hepatocellular carcinoma. RESULTS: Transcripts from the EGF 61*G allele exhibited more than a 2-fold longer half-life than those from the 61*A allele, and EGF secretion was 2.3-fold higher in G/G hepatocellular carcinoma cell lines than A/A cell lines. Serum EGF levels were 1.8-fold higher in G/G patients than A/A patients, and liver EGF levels were 2.4-fold higher in G/G patients than A/A patients. Among the 207 patients with cirrhosis in the Massachusetts study population, 59 also had hepatocellular carcinoma. Analysis of the distribution of allelic frequencies revealed that there was a 4-fold odds of hepatocellular carcinoma in G/G patients compared with A/A patients in the Massachusetts study population (odds ratio, 4.0; 95% confidence interval [CI], 1.6-9.6; P = .002). Logistic regression analysis demonstrated that the number of copies of G was significantly associated with hepatocellular carcinoma after adjusting for age, sex, race, etiology, and severity of cirrhosis (G/G or A/G vs A/A; hazard ratio, 3.49; 95% CI, 1.29-9.44; P = .01). The significant association was validated in the French patients with alcoholic cirrhosis and hepatocellular carcinoma. CONCLUSION: The EGF gene polymorphism genotype is associated with risk for development of hepatocellular carcinoma in liver cirrhosis through modulation of EGF levels.