The ATG8 Family Proteins GABARAP and GABARAPL1 Target Antigen to Dendritic Cells to Prime CD4+ and CD8+ T Cells.

Vaccine therapy is a promising method of research to promote T cell immune response and to develop novel antitumor immunotherapy protocols. Accumulating evidence has shown that autophagy is involved in antigen processing and presentation to T cells. In this work, we investigated the potential role of GABARAP and GABARAPL1, two members of the autophagic ATG8 family proteins, as surrogate tumor antigen delivery vectors to prime antitumor T cells. We showed that bone marrow-derived dendritic cells, expressing the antigen OVALBUMIN (OVA) fused with GABARAP or GABARAPL1, were able to prime OVA-specific CD4+ T cells in vitro. Interestingly, the fusion proteins were also degraded by the proteasome pathway and the resulting peptides were presented by the MHC class I system. We then asked if the aforementioned fusion proteins could improve tumor cell immunogenicity and T cell priming. The B16-F10 melanoma was chosen as the tumor cell line to express the fusion proteins. B16-F10 cells that expressed the OVA-ATG8 fused proteins stimulated OVA-specific CD8+ T cells, but demonstrated no CD4+ T cell response. In the future, these constructions may be used in vaccination trials as potential candidates to control tumor growth.

EZH2 and KDM6B Expressions Are Associated with Specific Epigenetic Signatures during EMT in Non Small Cell Lung Carcinomas.

The role of Epigenetics in Epithelial Mesenchymal Transition (EMT) has recently emerged. Two epigenetic enzymes with paradoxical roles have previously been associated to EMT, EZH2 (Enhancer of Zeste 2 Polycomb Repressive Complex 2 (PRC2) Subunit), a lysine methyltranserase able to add the H3K27me3 mark, and the histone demethylase KDM6B (Lysine Demethylase 6B), which can remove the H3K27me3 mark. Nevertheless, it still remains unclear how these enzymes, with apparent opposite activities, could both promote EMT. In this study, we evaluated the function of these two enzymes using an EMT-inducible model, the lung cancer A549 cell line. ChIP-seq coupled with transcriptomic analysis showed that EZH2 and KDM6B were able to target and modulate the expression of different genes during EMT. Based on this analysis, we described INHBB, WTN5B, and ADAMTS6 as new EMT markers regulated by epigenetic modifications and directly implicated in EMT induction.

EMT is associated with an epigenetic signature of ECM remodeling genes.

Type III epithelial-mesenchymal transition (EMT) has been previously associated with increased cell migration, invasion, metastasis, and therefore cancer aggressiveness. This reversible process is associated with an important gene expression reprogramming mainly due to epigenetic plasticity. Nevertheless, most of the studies describing the central role of epigenetic modifications during EMT were performed in a single-cell model and using only one mode of EMT induction. In our study, we studied the overall modulations of gene expression and epigenetic modifications in four different EMT-induced cell models issued from different tissues and using different inducers of EMT. Pangenomic analysis (transcriptome and ChIP-sequencing) validated our hypothesis that gene expression reprogramming during EMT is largely regulated by epigenetic modifications of a wide range of genes. Indeed, our results confirmed that each EMT model is unique and can be associated with a specific transcriptome profile and epigenetic program. However, we could select some genes or pathways that are similarly regulated in the different models and that could therefore be used as a common signature of all EMT models and become new biomarkers of the EMT phenotype. As an example, we can cite the regulation of gene-coding proteins involved in the degradation of the extracellular matrix (ECM), which are highly induced in all EMT models. Based on our investigations and results, we identified ADAM19 as a new biomarker of in vitro and in vivo EMT and we validated this biological new marker in a cohort of non-small lung carcinomas.

ATG9A Is Overexpressed in Triple Negative Breast Cancer and Its In Vitro Extinction Leads to the Inhibition of Pro-Cancer Phenotypes.

Early detection and targeted treatments have led to a significant decrease in mortality linked to breast cancer (BC), however, important issues need to be addressed in the future. One of them will be to find new triple negative breast cancer (TNBC) therapeutic strategies, since none are currently efficiently targeting this subtype of BC. Since numerous studies have reported the possibility of targeting the autophagy pathway to treat or limit cancer progression, we analyzed the expression of six autophagy genes (ATG9A, ATG9B, BECLIN1, LC3B, NIX and P62/SQSTM1) in breast cancer tissue, and compared their expression with healthy adjacent tissue. In our study, we observed an increase in ATG9A mRNA expression in TNBC samples from our breast cancer cohort. We also showed that this increase of the transcript was confirmed at the protein level on paraffin-embedded tissues. To corroborate these in vivo data, we designed shRNA- and CRISPR/Cas9-driven inhibition of ATG9A expression in the triple negative breast cancer cell line MDA-MB-436, in order to determine its role in the regulation of cancer phenotypes. We found that ATG9A inhibition led to an inhibition of in vitro cancer features, suggesting that ATG9A can be considered as a new marker of TNBC and might be considered in the future as a target to develop new specific TNBC therapies.

Specific or not specific recruitment of DNMTs for DNA methylation, an epigenetic dilemma.

Our current view of DNA methylation processes is strongly moving: First, even if it was generally admitted that DNMT3A and DNMT3B are associated with de novo methylation and DNMT1 is associated with inheritance DNA methylation, these distinctions are now not so clear. Secondly, since one decade, many partners of DNMTs have been involved in both the regulation of DNA methylation activity and DNMT recruitment on DNA. The high diversity of interactions and the combination of these interactions let us to subclass the different DNMT-including complexes. For example, the DNMT3L/DNMT3A complex is mainly related to de novo DNA methylation in embryonic states, whereas the DNMT1/PCNA/UHRF1 complex is required for maintaining global DNA methylation following DNA replication. On the opposite to these unspecific DNA methylation machineries (no preferential DNA sequence), some recently identified DNMT-including complexes are recruited on specific DNA sequences. The coexistence of both types of DNA methylation (un/specific) suggests a close cooperation and an orchestration between these systems to maintain genome and epigenome integrities. Deregulation of these systems can lead to pathologic disorders.

GABARAPL1 tumor suppressive function is independent of its conjugation to autophagosomes in MCF-7 breast cancer cells.

The GABARAPL1 protein belongs to the ATG8 family whose members are involved in autophagy. Our laboratory previously demonstrated that GABARAPL1 associates with autophagic vesicles, regulates autophagic flux and acts as a tumor suppressor protein in breast cancer. In this study, we aimed to determine whether GABARAPL1 conjugation to autophagosomes is necessary for its tumor suppressive functions using the MCF-7 breast cancer cell line overexpressing GABARAPL1 or a G116A mutant, which is unable to be lipidated and associated to autophagosomes. We show that the G116A mutation impaired GABARAPL1 function in autophagosome/lysosome fusion and inhibited lysosome activity but did not alter MTOR and ULK1 activities or tumor growth in vivo. Our results demonstrate for the first time that GABARAPL1 plays different regulatory functions during early and late stages of autophagy, independently or not of its conjugation to autophagosomes, but its tumor suppressive function appeared to be independent of its conjugation to autophagic vesicles.

Carbon nanotubes as gene carriers: Focus on internalization pathways related to functionalization and properties.

Carbon nanotubes represent promising transporters for delivery of DNA and other biomolecules into living cells. Various methods of CNTs surface functionalization have been developed. These are essential to improve CNTs dispersibility and permit their interactions with biological structures that broaden their use in advanced biomedical applications. The present review discusses the different single walled carbon nanotubes and multiwalled carbon nanotubes functionalization methods, leading to the formation of optimized and functionalized-CNT complexes with DNA. F-CNTs are recognized as efficient and promising gene carriers. Emphasis is then placed on the processes used by f-CNTs/DNA complexes to cross cell membranes. Energy independent pathways and uptake mechanisms dependent on energy, such as endocytosis or phagocytosis, are reported by many studies, and if these mechanisms seem contradictory at first sight, a detailed review of the literature illustrates that they are rather complementary. Preferential use of one or the other depends on the DNA and CNTs chemical nature and physical parameters, experimental procedures and cell types. STATEMENT OF SIGNIFICANCE: Efficient non-viral gene delivery is desirable, yet challenging. CNTs appear as a promising solution to penetrate into cells and successfully deliver DNA. Moreover, the field of use of CNTs as gene carrier is large and is currently growing. This critical review summarizes the development and evaluation of CNTs as intracellular gene delivery system and provides an overview of functionalized CNTs/DNA cellular uptake mechanisms, depending on several parameters of CNTs/DNA complexes.

Nanovectorization of DNA Through Cells Using Protamine Complexation.

Carbon nanotubes (CNT) are currently used as a promising family of nanovectors able to deliver different types of therapeutic molecules. Several applications dealing with CNT used as drug nanocarriers have been developed since their ability to penetrate into the cells has been proved. CNT can thus load several active molecules to various cells. In this paper, we will use molecular dynamic simulation to describe theoretically the potential of CNT to transport and deliver DNA through the formation of protamine-DNA-CNT complex.

The autophagy GABARAPL1 gene is epigenetically regulated in breast cancer models.

BACKGROUND: The GABARAP family members (GABARAP, GABARAPL1/GEC1 and GABARAPL2 /GATE-16) are involved in the intracellular transport of receptors and the autophagy pathway. We previously reported that GABARAPL1 expression was frequently downregulated in cancer cells while a high GABARAPL1 expression is a good prognosis marker for patients with lymph node-positive breast cancer. METHODS: In this study, we asked using qRT-PCR, western blotting and epigenetic quantification whether the expression of the GABARAP family was regulated in breast cancer by epigenetic modifications. RESULTS: Our data demonstrated that a specific decrease of GABARAPL1 expression in breast cancers was associated with both DNA methylation and histone deacetylation and that CREB-1 recruitment on GABARAPL1 promoter was required for GABARAPL1 expression. CONCLUSIONS: Our work strongly suggests that epigenetic inhibitors and CREB-1 modulators may be used in the future to regulate autophagy in breast cancer cells.

Proximity Ligation In situ Assay is a Powerful Tool to Monitor Specific ATG Protein Interactions following Autophagy Induction.

Macroautophagy is a highly regulated intracellular degradation process which has been extensively studied over the last decade. This pathway has been initially described as a non selective process inducing the degradation of parts of the cytoplasm as well as organelles at random. Nevertheless, over the last few years, new research highlighted the existence of a more selective autophagy pathway specifically recruiting some organelles or aggregates to the autophagosomes in order to induce their degradation. These selective autophagy pathways such as aggrephagy, mitophagy, pexophagy or xenophagy, involve the intervention of a cargo, the material to be degraded, cargo adapters, the molecules allowing the recruitment of the cargo to the autophagosome, and the proteins of the ATG8 family which link the cargo adapters to the autophagosome. One of the main questions which now remain is to develop new techniques and protocols able to discriminate between these different types of induced autophagy. In our work, we studied the possibility to use the P-LISA technique, which has been recently developed to study endogenous in vivo protein interactions, as a new technique to characterize the ATG proteins specifically involved in bulk or selective autophagy. In this manuscript, we indeed demonstrate that this technique allows the study of endogenous ATG protein interactions in cells following autophagy induction, but more interestingly that this technique might be used to characterize the ATG proteins involved in selective autophagy.

Induction of oxiapoptophagy on 158N murine oligodendrocytes treated by 7-ketocholesterol-, 7ß-hydroxycholesterol-, or 24(S)-hydroxycholesterol: Protective effects of α-tocopherol and docosahexaenoic acid (DHA; C22:6 n-3).

In demyelinating or non-demyelinating neurodegenerative diseases, increased levels of 7-ketocholesterol (7KC), 7ß-hydroxycholesterol (7ß-OHC) and 24(S)-hydroxycholesterol (24S-OHC) can be observed in brain lesions. In 158N murine oligodendrocytes, 7KC triggers a complex mode of cell death defined as oxiapoptophagy, involving simultaneous oxidative stress, apoptosis and autophagy. In these cells, 7KC as well as 7ß-OHC and 24S-OHC induce a decrease of cell proliferation evaluated by phase contrast microscopy, an alteration of mitochondrial activity quantified with the MTT test, an overproduction of reactive oxygen species revealed by staining with dihydroethidium and dihydrorhodamine 123, caspase-3 activation, PARP degradation, reduced expression of Bcl-2, and condensation and/or fragmentation of the nuclei which are typical criteria of oxidative stress and apoptosis. Moreover, 7KC, 7ß-OHC and 24S-OHC promote conversion of microtubule-associated protein light chain 3 (LC3-I) to LC3-II which is a characteristic of autophagy. Consequently, 7ß-OHC and 24S-OHC, similarly to 7KC, can be considered as potent inducers of oxiapoptophagy. Furthermore, the different cytotoxic effects associated with 7KC, 7ß-OHC and 24S-OHC-induced oxiapoptophagy are attenuated by vitamin E (VitE, α-tocopherol) and DHA which enhances VitE protective effects. In 158N murine oligodendrocytes, our data support the concept that oxiapoptophagy, which can be inhibited by VitE and DHA, could be a particular mode of cell death elicited by cytotoxic oxysterols.

Interplay between ROS and autophagy in cancer cells, from tumor initiation to cancer therapy.

Cancer formation is a complex and highly regulated multi-step process which is highly dependent of its environment, from the tissue to the patient. This complexity implies the development of specific treatments adapted to each type of tumor. The initial step of cancer formation requires the transformation of a healthy cell to a cancer cell, a process regulated by multiple intracellular and extracellular stimuli. The further steps, from the anarchic proliferation of cancer cells to form a primary tumor to the migration of cancer cells to distant organs to form metastasis, are also highly dependent of the tumor environment but of intracellular molecules and pathways as well. In this review, we will focus on the regulatory role of reactive oxygen species (ROS) and autophagy levels during the course of cancer development, from cellular transformation to the formation of metastasis. These data will allow us to discuss the potential of this molecule or pathway as putative future therapeutic targets.

Use of epigenetic modulators as a powerful adjuvant for breast cancer therapies.

Breast cancer (BC) is one of the five most frequent cancers in the world. Despite earlier diagnosis and development of specific treatments, mortality has only declined of about 30 % during the past two decades. Two of the main reasons are the emergence of drug resistance and the absence of specific therapy for triple negative breast cancers (TNBC), which are characterized by a poor prognosis due to high proliferation rate. Therefore, the future goal of the fight against BC will be to find new therapeutic approaches to overcome drug resistances and cure TNBC. Recent research on gene expression profiles linked to the different types of BC cells have led to consider the use of epigenetic modulators to modulate the expression of genes deregulated in cancer. The preliminary encouraging results have demonstrated a positive effect of DNA Methyl Transferase (DNMT) and Histone DeAcetylase (HDAC) inhibitors on different types of BC, as well as drug-resistant cells, with low side effects. In this review, we will describe the different epigenetic modulators currently used or investigated in BC therapy research in vitro as well as preclinical and clinical trials, and promising compounds, which might be used in future BC therapies.

The role of GABARAPL1/GEC1 in autophagic flux and mitochondrial quality control in MDA-MB-436 breast cancer cells.

GABARAPL1/GEC1 is an early estrogen-induced gene which encodes a protein highly conserved from C. elegans to humans. Overexpressed GABARAPL1 interacts with GABAA or kappa opioid receptors, associates with autophagic vesicles, and inhibits breast cancer cell proliferation. However, the function of endogenous GABARAPL1 has not been extensively studied. We hypothesized that GABARAPL1 is required for maintaining normal autophagic flux, and plays an important role in regulating cellular bioenergetics and metabolism. To test this hypothesis, we knocked down GABARAPL1 expression in the breast cancer MDA-MB-436 cell line by shRNA. Decreased expression of GABARAPL1 activated procancer responses of the MDA-MB-436 cells including increased proliferation, colony formation, and invasion. In addition, cells with decreased expression of GABARAPL1 exhibited attenuated autophagic flux and a decreased number of lysosomes. Moreover, decreased GABARAPL1 expression led to cellular bioenergetic changes including increased basal oxygen consumption rate, increased intracellular ATP, increased total glutathione, and an accumulation of damaged mitochondria. Taken together, our results demonstrate that GABARAPL1 plays an important role in cell proliferation, invasion, and autophagic flux, as well as in mitochondrial homeostasis and cellular metabolic programs.

QSOX1 inhibits autophagic flux in breast cancer cells.

The QSOX1 protein (Quiescin Sulfhydryl oxidase 1) catalyzes the formation of disulfide bonds and is involved in the folding and stability of proteins. More recently, QSOX1 has been associated with tumorigenesis and protection against cellular stress. It has been demonstrated in our laboratory that QSOX1 reduces proliferation, migration and invasion of breast cancer cells in vitro and reduces tumor growth in vivo. In addition, QSOX1 expression has been shown to be induced by oxidative or ER stress and to prevent cell death linked to these stressors. Given the function of QSOX1 in these two processes, which have been previously linked to autophagy, we wondered whether QSOX1 might be regulated by autophagy inducers and play a role in this catabolic process. To answer this question, we used in vitro models of breast cancer cells in which QSOX1 was overexpressed (MCF-7) or extinguished (MDA-MB-231). We first showed that QSOX1 expression is induced following amino acid starvation and maintains cellular homeostasis. Our results also indicated that QSOX1 inhibits autophagy through the inhibition of autophagosome/lysosome fusion. Moreover, we demonstrated that inhibitors of autophagy mimic the effect of QSOX1 on cell invasion, suggesting that its role in this process is linked to the autophagy pathway. Previously published data demonstrated that extinction of QSOX1 promotes tumor growth in NOG mice. In this study, we further demonstrated that QSOX1 null tumors present lower levels of the p62 protein. Altogether, our results demonstrate for the first time a role of QSOX1 in autophagy in breast cancer cells and tumors.

Induction of oxiapoptophagy, a mixed mode of cell death associated with oxidative stress, apoptosis and autophagy, on 7-ketocholesterol-treated 158N murine oligodendrocytes: impairment by α-tocopherol.

7-Ketocholesterol (7KC) has been suggested to induce a complex mode of cell death on monocytic cells: oxiapoptophagy (OXIdation, APOPTOsis, and autoPHAGY) (Monier et al. (2003) [12]). The aim of the present study, realized on 158N murine oligodendrocytes, was to bring new evidence on this mixed form of cell death. On 158N cells, 7KC induces an overproduction of reactive oxygen species (ROS) revealed by dihydroethidium staining, a loss of transmembrane mitochondrial potential measured with DiOC6(3), caspase-3 activation, and condensation and/or fragmentation of the nuclei which are typical criteria of oxidative stress and apoptosis. Moreover, 7KC enhances cytoplamic membrane permeability to propidium iodide, and induces acidic vesicular organelle formation evaluated with acridine orange. In addition, 7KC promotes conversion of microtubule-associated protein light chain 3 (LC3-I) to LC3-II which is characteristic of autophagy. These different side effects were impaired by α-tocopherol. Altogether, our data demonstrate that oxiapoptophagy including ROS overproduction, apoptosis and autophagy could be a particular type of cell death activated by 7KC which can be inhibited by α-tocopherol.

Specific distribution of the autophagic protein GABARAPL1/GEC1 in the developing and adult mouse brain and identification of neuronal populations expressing GABARAPL1/GEC1.

Macroautophagy is a highly conserved cellular degradation process, regulated by autophagy-related (atg) factors, in which a double membrane autophagosome engulfs cytoplasmic components to target them for degradation. In yeast, the Atg8 protein is indispensable for autophagosome formation. In mammals, this is complicated by the presence of six Atg8 homologues grouped into the GABARAP and MAP1LC3 subfamilies. Although these proteins share a high similarity, their transcript expression, regulation and protein interactions differ, suggesting they may display individual properties and specific functions. GABARAPL1/GEC1 is a member of the GABARAP subfamily and its mRNA is the most highly expressed Atg8 homologue in the central nervous system. Consequently, we performed an in depth study of GABARAPL1 distribution in the developing and adult murine brain. Our results show that GABARAPL1 brain expression is visible as early as embryonic day 11 and progressively increases to a maximum level in the adult. Immunohistochemical staining was detected in both fibers and immature neurons in embryos but was restrained to neurons in adult tissue. By E17, intense punctate-like structures were visible and these accumulated in cortical primary neurons treated with the autophagosome/lysosome fusion inhibitor Bafilomycin A1 (Baf A1), suggesting that they represent autophagosomes. Finally, GABARAPL1 expression was particularly intense in motoneurons in the embryo and in neurons involved in somatomotor and neuroendocrine functions in the adult, particularly in the substantia nigra pars compacta, a region affected in Parkinson's disease. Our study of cerebral GABARAPL1 protein expression provides insight into its role in the development and homeostasis of the mouse brain.

Epigenetic regulation of estrogen signaling in breast cancer.

Estrogen signaling is mediated by ERα and ERß in hormone dependent, breast cancer (BC). Over the last decade the implication of epigenetic pathways in BC tumorigenesis has emerged: cancer-related epigenetic modifications are implicated in both gene expression regulation, and chromosomal instability. In this review, the epigenetic-mediated estrogen signaling, controlling both ER level and ER-targeted gene expression in BC, are discussed: (1) ER silencing is frequently observed in BC and is often associated with epigenetic regulations while chemical epigenetic modulators restore ER expression and increase response to treatment;(2) ER-targeted gene expression is tightly regulated by co-recruitment of ER and both coactivators/corepressors including HATs, HDACs, HMTs, Dnmts and Polycomb proteins.

Identification of HSP90 as a new GABARAPL1 (GEC1)-interacting protein.

GABARAPL1 belongs to the small family of GABARAP proteins (including GABARAP, GABARAPL1 and GABARAPL2/GATE-16), one of the two subfamilies of the yeast Atg8 orthologue. GABARAPL1 is involved in the intracellular transport of receptors, via an interaction with tubulin and GABA(A) or kappa opioid receptors, and also participates in autophagy and cell proliferation. In the present study, we identify the HSP90 protein as a novel interaction partner for GABARAPL1 using GST pull-down, mass spectrometry and coimmunoprecipitation experiments. GABARAPL1 and HSP90 partially colocalize in MCF-7 breast cancer cells overexpressed Dsred-GABARAPL1 and in rat brain. Moreover, treatment of MCF-7 cells overexpressed FLAG-GABARAPL1-6HIS with the HSP90 inhibitor 17-AAG promotes the GABARAPL1 degradation, a process that is blocked by proteasome inhibitors such as MG132, bortezomib and lactacystin. Accordingly, we demonstrate that HSP90 interacts and protects GABARAPL1 from its degradation by the proteasome.

GABARAPL1 antibodies: target one protein, get one free!

Atg8 is a yeast protein involved in the autophagic process and in particular in the elongation of autophagosomes. In mammals, several orthologs have been identified and are classed into two subfamilies: the LC3 subfamily and the GABARAP subfamily, referred to simply as the LC3 or GABARAP families. GABARAPL1 (GABARAP-like protein 1), one of the proteins belonging to the GABARAP (GABA(A) receptor-associated protein) family, is highly expressed in the central nervous system and implicated in processes such as receptor and vesicle transport as well as autophagy. The proteins that make up the GABARAP family demonstrate conservation of their amino acid sequences and protein structures. In humans, GABARAPL1 shares 86% identity with GABARAP and 61% with GABARAPL2 (GATE-16). The identification of the individual proteins is thus very limited when working in vivo due to a lack of unique peptide sequences from which specific antibodies can be developed. Actually, and to our knowledge, there are no available antibodies on the market that are entirely specific to GABARAPL1 and the same may be true of the anti-GABARAP antibodies. In this study, we sought to examine the specificity of three antibodies targeted against different peptide sequences within GABARAPL1: CHEM-CENT (an antibody raised against a short peptide sequence within the center of the protein), PTG-NTER (an antibody raised against the N-terminus of the protein) and PTG-FL (an antibody raised against the full-length protein). The results described in this article demonstrate the importance of testing antibody specificity under the conditions for which it will be used experimentally, a caution that should be taken when studying the expression of the GABARAP family proteins.