Abrogating GPT2 in triple-negative breast cancer inhibits tumor growth and promotes autophagy.

Uncontrolled proliferation and altered metabolic reprogramming are hallmarks of cancer. Active glycolysis and glutaminolysis are characteristic features of these hallmarks and required for tumorigenesis. A fine balance between cancer metabolism and autophagy is a prerequisite of homeostasis within cancer cells. Here we show that glutamate pyruvate transaminase 2 (GPT2), which serves as a pivot between glycolysis and glutaminolysis, is highly upregulated in aggressive breast cancers, particularly the triple-negative breast cancer subtype. Abrogation of this enzyme results in decreased tricarboxylic acid cycle intermediates, which promotes the rewiring of glucose carbon atoms and alterations in nutrient levels. Concordantly, loss of GPT2 results in an impairment of mechanistic target of rapamycin complex 1 activity as well as the induction of autophagy. Furthermore, in vivo xenograft studies have shown that autophagy induction correlates with decreased tumor growth and that markers of induced autophagy correlate with low GPT2 levels in patient samples. Taken together, these findings indicate that cancer cells have a close network between metabolic and nutrient sensing pathways necessary to sustain tumorigenesis and that aminotransferase reactions play an important role in maintaining this balance.

Increased autophagy blocks HER2-mediated breast tumorigenesis.

Allelic loss of the autophagy gene, beclin 1/BECN1, increases the risk of patients developing aggressive, including human epidermal growth factor receptor 2 (HER2)-positive, breast cancers; however, it is not known whether autophagy induction may be beneficial in preventing HER2-positive breast tumor growth. We explored the regulation of autophagy in breast cancer cells by HER2 in vitro and the effects of genetic and pharmacological strategies to increase autophagy on HER2-driven breast cancer growth in vivo. Our findings demonstrate that HER2 interacts with Beclin 1 in breast cancer cells and inhibits autophagy. Mice with increased basal autophagy due to a genetically engineered mutation in Becn1 are protected from HER2-driven mammary tumorigenesis, and HER2 fails to inhibit autophagy in primary cells derived from these mice. Moreover, treatment of mice with HER2-positive human breast cancer xenografts with the Tat-Beclin 1 autophagy-inducing peptide inhibits tumor growth as effectively as a clinically used HER2 tyrosine kinase inhibitor (TKI). This inhibition of tumor growth is associated with a robust induction of autophagy, a disruption of HER2/Beclin 1 binding, and a transcriptional signature in the tumors distinct from that observed with HER2 TKI treatment. Taken together, these findings indicate that the HER2-mediated inhibition of Beclin 1 and autophagy likely contributes to HER2-mediated tumorigenesis and that strategies to block HER2/Beclin 1 binding and/or increase autophagy may represent a new therapeutic approach for HER2-positive breast cancers.

Regulation of Beclin 1-Mediated Autophagy by Oncogenic Tyrosine Kinases.

Beclin 1 is a major regulator of autophagy, and it is a core component of the class III PI3K complexes. Beclin 1 is a highly conserved protein and its function is regulated in a number of ways, including post-translational modifications. Several studies indicate that receptor and non-receptor tyrosine kinases regulate autophagy activity in cancer, and some suggest the importance of Beclin 1 tyrosine phosphorylation in this process. Here we summarize the current knowledge of the mechanism whereby some oncogenic tyrosine kinases regulate autophagy through Beclin 1.

Regulation of TFEB and V-ATPases by mTORC1.

Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is an important, highly conserved, regulator of cell growth. Ancient among the signals that regulate mTORC1 are nutrients. Amino acids direct mTORC1 to the surface of the late endosome/lysosome, where mTORC1 becomes receptive to other inputs. However, the interplay between endosomes and mTORC1 is poorly understood. Here, we report the discovery of a network that links mTORC1 to a critical component of the late endosome/lysosome, the V-ATPase. In an unbiased screen, we found that mTORC1 regulated the expression of, among other lysosomal genes, the V-ATPases. mTORC1 regulates V-ATPase expression both in cells and in mice. V-ATPase regulation by mTORC1 involves a transcription factor translocated in renal cancer, TFEB. TFEB is required for the expression of a large subset of mTORC1 responsive genes. mTORC1 coordinately regulates TFEB phosphorylation and nuclear localization and in a manner dependent on both TFEB and V-ATPases, mTORC1 promotes endocytosis. These data uncover a regulatory network linking an oncogenic transcription factor that is a master regulator of lysosomal biogenesis, TFEB, to mTORC1 and endocytosis.

GRB2 is a BECN1 interacting protein that regulates autophagy.

GRB2 is an adaptor protein of HER2 (and several other tyrosine kinases), which we identified as a novel BECN1 (Beclin 1) interacting partner. GRB2 co-immunoprecipitated with BECN1 in several breast cancer cell lines and regulates autophagy through a mechanism involving the modulation of the class III PI3Kinase VPS34 activity. In ovo studies in a CAM (Chicken Chorioallantoic Membrane) model indicated that GRB2 knockdown, as well as overexpression of GRB2 loss-of-function mutants (Y52A and S86A-R88A) compromised tumor growth. These differences in tumor growth correlated with differential autophagy activity, indicating that autophagy effects might be related to the effects on tumorigenesis. Our data highlight a novel function of GRB2 as a BECN1 binding protein and a regulator of autophagy.

TROP2 expression and SN38 antitumor activity in malignant pleural mesothelioma cells provide a rationale for antibody-drug conjugate therapy.

OBJECTIVES: Malignant pleural mesothelioma (MPM) is an aggressive cancer which at large is not amenable to curative surgery. Despite the recent approval of immune checkpoint inhibitor therapy, the response rates and survival following systemic therapy is still limited. Sacituzumab govitecan is an antibody-drug conjugate targeting the topoisomerase I inhibitor SN38 to trophoblast cell-surface antigen 2 (TROP-2)-positive cells. Here we have explored the therapeutic potential of sacituzumab govitecan in MPM models. MATERIALS AND METHODS: TROP2 expression was analyzed in a panel of two well established and 15 pleural effusion derived novel lines by RT-QPCR and immunoblotting, TROP2 membrane-localization was studied by flow cytometry and immunohistochemistry. Cultured mesothelial cells and pneumothorax pleura served as controls. The sensitivity of MPM cell lines to irinotecan and SN38 was studied using cell viability, cell cycle, apoptosis and DNA damage assays. Drug sensitivity of cell lines was correlated with RNA expression of DNA repair genes. Drug sensitivity was defined as an IC50 below 5 nM in the cell viability assay. RESULTS: TROP2 expression was detected at RNA and protein level in 6 of the 17 MPM cell lines, but not in in cultured mesothelial control cells or in the mesothelial layer of the pleura. TROP2 was detectable on the cell membrane in 5 MPM lines and was present in the nucleus in 6 cell models. Ten of 17 MPM cell lines showed sensitivity to SN38 treatment, among those 4 expressed TROP2. High AURKA RNA expression and high proliferation rate correlated with sensitivity to SN38-induced cell death, DNA damage response, cell cycle arrest and cell death. Sacituzumab govitecan treatment effectively induced cell cycle arrest and cell death in TROP2-positive MPM cells. CONCLUSION: TROP2 expression and sensitivity to SN38 in MPM cell lines support biomarker-selected clinical exploration of sacituzumab govitecan in patients with MPM.

Studying Autophagy In Vivo in the Mammary Gland and in Xenograft Samples.

Autophagy is a dynamic process that can be monitored in multiple ways, both in vitro and in vivo. Studies in mice are a widely used tool to understand multiple diseases and conditions where autophagy plays a role, and therefore autophagic flux measurement in tissues of rodent models are of utmost importance. Here, we present some assays successfully used in determining the autophagy status in the mice mammary gland as well as in xenografts.

A Dual HiBiT-GFP-LC3 Lentiviral Reporter for Autophagy Flux Assessment.

Autophagy is an intracellular degradation process that maintains the cellular homeostasis and it is regulated in multiple ways, both in health and disease. Assessment of autophagic flux in cells is an important approach for understanding the function of autophagy in biological contexts. Here, we describe a new tool for the qualitative and quantitative determination of autophagic flux using a dual lentiviral reporter system that generates a fusion HiBiT-GFP-LC3B protein suitable for generating stable cell lines.

Nintedanib and Dasatinib Treatments Induce Protective Autophagy as a Potential Resistance Mechanism in MPM Cells.

Malignant pleural mesothelioma (MPM) is a rare type of cancer with a grim prognosis. So far, no targetable oncogenic mutation was identified in MPM and biomarkers with predictive value toward drug sensitivity or resistance are also lacking. Nintedanib (BIBF1120) is a small-molecule tyrosine kinase inhibitor that showed promising efficacy preclinically and in phase II trial in MPM as an angiogenesis inhibitor combined with chemotherapy. However, the extended phase III trial failed. In this study, we investigated the effect of nintedanib on one of its targets, the SRC kinase, in two commercial and six novel MPM cell lines. Surprisingly, nintedanib treatment did not inhibit SRC activation in MPM cells and even increased phosphorylation of SRC in several cell lines. Combination treatment with the SRC inhibitor dasatinib could reverse this effect in all cell lines, however, the cellular response was dependent on the drug sensitivity of the cells. In 2 cell lines, with high sensitivity to both nintedanib and dasatinib, the drug combination had no synergistic effect but cell death was initiated. In 2 cell lines insensitive to nintedanib combination treatment reduced cell viability synergisticaly without cell death. In contrast, in these cells both treatments increased the autophagic flux assessed by degradation of the autophagy substrate p62 and increased presence of LC3B-II, increased number of GFP-LC3 puncta and decreased readings of the HiBiT-LC3 reporter. Additionaly, autophagy was synergistically promoted by the combined treatment. At the transcriptional level, analysis of lysosomal biogenesis regulator Transcription Factor EB (TFEB) showed that in all cell lines treated with nintedanib and to a lesser extent, with dasatinib, it became dephosphorylated and accumulated in the nucleus. Interestingly, the expression of certain known TFEB target genes implicated in autophagy or lysosomal biogenesis were significantly modified only in 1 cell line. Finally, we showed that autophagy induction in our MPM cell lines panel by nintedanib and dasatinib is independent of the AKT/mTOR and the ERK pathways. Our study reveals that autophagy can serve as a cytoprotective mechanism following nintedanib or dasatinib treatments in MPM cells.

Beth Levine's Legacy: From the Discovery of BECN1 to Therapies. A Mentees' Perspective.

With great sadness, the scientific community received the news of the loss of Beth Levine on 15 June 2020. Dr. Levine was a pioneer in the autophagy field and work in her lab led not only to a better understanding of the molecular mechanisms regulating the pathway, but also its implications in multiple physiological and pathological conditions, including its role in development, host defense, tumorigenesis, aging or metabolism. This review does not aim to provide a comprehensive view of autophagy, but rather an outline of some of the discoveries made by the group of Beth Levine, from the perspective of some of her own mentees, hoping to honor her legacy in science.

A BAP1 synonymous mutation results in exon skipping, loss of function and worse patient prognosis.

Synonymous mutations are generally disregarded by genomic analyses because they are considered non-pathogenic. We identified and characterized a somatic synonymous mutation in the epigenetic modifier and tumor suppressor BAP1, resulting in exon skipping and complete protein inactivation. This radically altered the prognosis of a clear-cell renal cell carcinoma patient from The Cancer Genome Atlas (TCGA) with a PBRM1 mutation (a predictor biomarker for positive responses to immune checkpoint inhibitors) from good (an estimated overall survival of 117 months) to a very bad prognosis (an estimated overall survival of 31 months), emphasizing the importance of scrutinizing synonymous mutations near acceptor splice sites of cancer genes for accurate precision medicine.

Metabolism of cancer cells commonly responds to irradiation by a transient early mitochondrial shutdown.

Cancer bioenergetics fuel processes necessary to maintain viability and growth under stress conditions. We hypothesized that cancer metabolism supports the repair of radiation-induced DNA double-stranded breaks (DSBs). We combined the systematic collection of metabolic and radiobiological data from a panel of irradiated cancer cell lines with mathematical modeling and identified a common metabolic response with impact on the DSB repair kinetics, including a mitochondrial shutdown followed by compensatory glycolysis and resumption of mitochondrial function. Combining ionizing radiation (IR) with inhibitors of the compensatory glycolysis or mitochondrial respiratory chain slowed mitochondrial recovery and DNA repair kinetics, offering an opportunity for therapeutic intervention. Mathematical modeling allowed us to generate new hypotheses on general and individual mechanisms of the radiation response with relevance to DNA repair and on metabolic vulnerabilities induced by cancer radiotherapy. These discoveries will guide future mechanistic studies for the discovery of metabolic targets for overcoming intrinsic or therapy-induced radioresistance.

Broad-Spectrum HDAC Inhibitors Promote Autophagy through FOXO Transcription Factors in Neuroblastoma.

Depending on context and tumor stage, deregulation of autophagy can either suppress tumorigenesis or promote chemoresistance and tumor survival. Histone deacetylases (HDACs) can modulate autophagy; however, the exact mechanisms are not fully understood. Here, we analyze the effects of the broad-spectrum HDAC inhibitors (HDACi) panobinostat and vorinostat on the transcriptional regulation of autophagy with respect to autophagy transcription factor activity (Transcription factor EB-TFEB, forkhead boxO-FOXO) and autophagic flux in neuroblastoma cells. In combination with the late-stage autophagic flux inhibitor bafilomycin A1, HDACis increase the number of autophagic vesicles, indicating an increase in autophagic flux. Both HDACi induce nuclear translocation of the transcription factors FOXO1 and FOXO3a, but not TFEB and promote the expression of pro-autophagic FOXO1/3a target genes. Moreover, FOXO1/3a knockdown experiments impaired HDACi treatment mediated expression of autophagy related genes. Combination of panobinostat with the lysosomal inhibitor chloroquine, which blocks autophagic flux, enhances neuroblastoma cell death in culture and hampers tumor growth in vivo in a neuroblastoma zebrafish xenograft model. In conclusion, our results indicate that pan-HDACi treatment induces autophagy in neuroblastoma at a transcriptional level. Combining HDACis with autophagy modulating drugs suppresses tumor growth of high-risk neuroblastoma cells. These experimental data provide novel insights for optimization of treatment strategies in neuroblastoma.

Structural analysis and functional implications of the negative mTORC1 regulator REDD1.

REDD1 is a conserved stress-response protein that regulates mTORC1, a critical regulator of cell growth and proliferation that is implicated in cancer. REDD1 is induced by hypoxia, and REDD1 overexpression is sufficient to inhibit mTORC1. mTORC1 is regulated by the small GTPase Rheb, which in turn is regulated by the GTPase-activating protein complex, TSC1/TSC2. REDD1 induced-mTORC1 inhibition requires the TSC1/TSC2 complex, and REDD1 has been proposed to act by directly binding to and sequestering 14-3-3 proteins away from TSC2 leading to TSC2-dependent inhibition of mTORC1. Structure/function analyses have led us to identify two segments in REDD1 that are essential for function, which act in an interdependent manner. We have determined a crystal structure of REDD1 at 2.0 A resolution, which shows that these two segments fold together to form an intact domain with a novel fold. This domain is characterized by an alpha/beta sandwich consisting of two antiparallel alpha-helices and a mixed beta-sheet encompassing an uncommon psi-loop motif. Structure-based docking and functional analyses suggest that REDD1 does not directly bind to 14-3-3 proteins. Sequence conservation mapping to the surface of the structure and mutagenesis studies demarcated a hotspot likely to interact with effector proteins that is essential for REDD1-mediated mTORC1 inhibition.

TFEB-mediated lysosomal biogenesis and lysosomal drug sequestration confer resistance to MEK inhibition in pancreatic cancer.

Oncogenic KRAS mutations are encountered in more than 90% of pancreatic ductal adenocarcinomas. MEK inhibition has failed to procure any clinical benefits in mutant RAS-driven cancers including pancreatic ductal adenocarcinoma (PDAC). To identify potential resistance mechanisms underlying MEK inhibitor (MEKi) resistance in PDAC, we investigated lysosomal drug accumulation in PDAC models both in vitro and in vivo. Mouse PDAC models and human PDAC cell lines as well as human PDAC xenografts treated with the MEK inhibitor trametinib or refametinib led to an enhanced expression of lysosomal markers and enrichment of lysosomal gene sets. A time-dependent, increase in lysosomal content was observed upon MEK inhibition. Strikingly, there was a strong activation of lysosomal biogenesis in cell lines of the classical compared to the basal-like molecular subtype. Increase in lysosomal content was associated with nuclear translocation of the Transcription Factor EB (TFEB) and upregulation of TFEB target genes. siRNA-mediated depletion of TFEB led to a decreased lysosomal biogenesis upon MEK inhibition and potentiated sensitivity. Using LC-MS, we show accumulation of MEKi in the lysosomes of treated cells. Therefore, MEK inhibition triggers lysosomal biogenesis and subsequent drug sequestration. Combined targeting of MEK and lysosomal function may improve sensitivity to MEK inhibition in PDAC.

The Role of Beclin 1-Dependent Autophagy in Cancer.

Autophagy (self-eating) is an intracellular degradation process used by cells to keep a "clean house"; as it degrades abnormal or damaged proteins and organelles, it helps to fight infections and also provides energy in times of fasting or exercising. Autophagy also plays a role in cancer, although its precise function in each cancer type is still obscure, and whether autophagy plays a protecting (through the clearing of damaged organelles and protein aggregates and preventing DNA damage) or a promoting (by fueling the already stablished tumor) role in cancer remains to be fully characterized. Beclin 1, the mammalian ortholog of yeast Atg6/Vps30, is an essential autophagy protein and has been shown to play a role in tumor suppression. Here, an update of the tumorigenesis regulation by Beclin 1-dependent autophagy is provided.

Multistep regulation of TFEB by MTORC1.

The master regulator of lysosome biogenesis, TFEB, is regulated by MTORC1 through phosphorylation at S211, and a S211A mutation increases nuclear localization. However, TFEBS211A localizes diffusely in both cytoplasm and nucleus and, as we show, retains regulation by MTORC1. Here, we report that endogenous TFEB is phosphorylated at S122 in an MTORC1-dependent manner, that S122 is phosphorylated in vitro by recombinant MTOR, and that S122 is important for TFEB regulation by MTORC1. Specifically, nuclear localization following MTORC1 inhibition is blocked by a S122D mutation (despite S211 dephosphorylation). Furthermore, such a mutation inhibits lysosomal biogenesis induced by Torin1. These data reveal a novel mechanism of TFEB regulation by MTORC1 essential for lysosomal biogenesis.

Molecular cloning and characterization of gilthead sea bream (Sparus aurata) growth hormone receptor (GHR). Assessment of alternative splicing.

The full-length growth hormone receptor (GHR) of gilthead sea bream (Sparus aurata) was cloned and sequenced by RT-PCR and rapid amplification of 5'and 3'ends. The open reading frame codes for a mature 609 amino acid protein with a hydrophobic transmembrane region and all the characteristic motifs of GHRs. Sequence analysis revealed a 96 and 76% of amino acid identity with black sea bream (Acanthopagrus schlegeli) and turbot (Scophthalmus maximus) GHRs, respectively, but this amino acid identity decreases up to 52% for goldfish (Carassius auratus) GHR. By means of real-time PCR assays, concurrent changes in the hepatic expression of GHRs and insulin-like growth factor-I (IGF-I) was evidenced. Moreover, their regulation occurred in conjunction with the summer spurt of growth rates and circulating levels of GH and IGF-I. Search of alternative splicing was carried out exhaustively for gilthead sea bream GHR, but Northern blot and 3' RACE failed to demonstrate the occurrence of short alternative messengers. Besides, RT-PCR screening did not reveal deletions or insertions that could lead to alternative reading frames. In agreement with this, cross-linking assays only evidenced two protein bands that match well with the size of glycosylated and non-glycosylated forms of the full-length GHR. If so, it appears that alternative splicing at the 3'end does not occur in gilthead sea bream, although different messengers for truncated or longer GHR variants already exist in turbot and black sea bream, respectively. The physiological relevance of this finding remains unclear, but perhaps it points out large inter-species differences in the heterogeneity of the GHR population.