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.

Epigenetic Regulation of EMT (Epithelial to Mesenchymal Transition) and Tumor Aggressiveness: A View on Paradoxical Roles of KDM6B and EZH2.

EMT (epithelial to mesenchymal transition) is a plastic phenomenon involved in metastasis formation. Its plasticity is conferred in a great part by its epigenetic regulation. It has been reported that the trimethylation of lysine 27 histone H3 (H3K27me3) was a master regulator of EMT through two antagonist enzymes that regulate this mark, the methyltransferase EZH2 (enhancer of zeste homolog 2) and the lysine demethylase KDM6B (lysine femethylase 6B). Here we report that EZH2 and KDM6B are overexpressed in numerous cancers and involved in the aggressive phenotype and EMT in various cell lines by regulating a specific subset of genes. The first paradoxical role of these enzymes is that they are antagonistic, but both involved in cancer aggressiveness and EMT. The second paradoxical role of EZH2 and KDM6B during EMT and cancer aggressiveness is that they are also inactivated or under-expressed in some cancer types and linked to epithelial phenotypes in other cancer cell lines. We also report that new cancer therapeutic strategies are targeting KDM6B and EZH2, but the specificity of these treatments may be increased by learning more about the mechanisms of action of these enzymes and their specific partners or target genes in different cancer types.

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.

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.

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.

Regulation of autophagy by protein post-translational modification.

Autophagy is a lysosome-mediated intracellular protein degradation process that involves about 38 autophagy-related genes as well as key signaling pathways that sense cellular metabolic and redox status, and has an important role in quality control of macromolecules and organelles. As with other major cellular pathways, autophagy proteins are subjected to regulatory post-translational modification. Phosphorylation is so far the most intensively studied post-translational modification in the autophagy process, followed by ubiquitination and acetylation. An interesting and new area is also now emerging, which appears to complement these more traditional mechanisms, and includes O-GlcNAcylation and redox regulation at thiol residues. Identification of the full spectrum of post-translational modifications of autophagy proteins, and determination of their impact on autophagy will be crucial for a better understanding of autophagy regulation, its deficits in diseases, and how to exploit this process for disease 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.

Mitophagy mechanisms and role in human diseases.

Mitophagy is a process of mitochondrial turnover through lysosomal mediated autophagy activities. This review will highlight recent studies that have identified mediators of mitophagy in response to starvation, loss of mitochondrial membrane potential or perturbation of mitochondrial integrity. Furthermore, we will review evidence of mitophagy dysfunction in various human diseases and discuss the potential for therapeutic interventions that target mitophagy processes.

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.

Over-expression of an inactive mutant cathepsin D increases endogenous alpha-synuclein and cathepsin B activity in SH-SY5Y cells.

Parkinson's disease is a neurodegenerative movement disorder. The histopathology of Parkinson's disease comprises proteinaceous inclusions known as Lewy bodies, which contains aggregated α-synuclein. Cathepsin D (CD) is a lysosomal protease previously demonstrated to cleave α-synuclein and decrease its toxicity in both cell lines and mouse brains in vivo. Here, we show that pharmacological inhibition of CD, or introduction of catalytically inactive mutant CD, resulted in decreased CD activity and increased cathepsin B activity, suggesting a possible compensatory response to inhibition of CD activity. However, this increased cathepsin B activity was not sufficient to maintain α-synuclein degradation, as evidenced by the accumulation of endogenous α-synuclein. Interestingly, the levels of LC3, LAMP1, and LAMP2, proteins involved in autophagy-lysosomal activities, as well as total lysosomal mass as assessed by LysoTracker flow cytometry, were unchanged. Neither autophagic flux nor proteasomal activities differs between cells over-expressing wild-type versus mutant CD. These observations point to a critical regulatory role for that endogenous CD activity in dopaminergic cells in α-synuclein homeostasis which cannot be compensated for by increased Cathepsin B. These data support the potential need to enhance CD function in order to attenuate α-synuclein accumulation as a therapeutic strategy against development of synucleinopathy.

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.

Dopamine and its metabolites in cathepsin D heterozygous mice before and after MPTP administration.

Cathepsin D (CD) is a lysosomal aspartyl protease which plays an important role in α-synuclein degradation, and neuronal survival. CD knockout mice die by post-natal day 25±1 due to intestinal necrosis. We analyzed the young adult male heterozygous mice, and found no behavior abnormalities in the heterozygous mice compared to wildtype littermates. LC3-II, p62, and α-synuclein levels are similar, while LAMP1 is higher in the striatum in CD heterozygous compared to wildtype mice. Interestingly, we found that dopamine and metabolites in the striatum and olfactory bulbs are at higher levels than wildtype littermates, while the DOPAC/DA and HVA/DA ratio remain similar between wildtype and CD heterozygous mice. In response to sub-chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration, dopamine, DOPAC, and HVA are depleted to similar levels in the striatum in both heterozygous and wildtype mice. Dopamine synthesizing enzyme tyrosine hydroxylase, metabolic enzyme monoamine oxidase, and catechol-O-methyltransferase (COMT) levels are similar in the striatum in wildtype and heterozygous mice. These studies provide valuable information regarding how lysosomal function may contribute to neurochemical homeostasis in animal models.

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.

GABARAPL1 (GEC1): original or copycat?

The GABARAPL1 (GABARAP-LIKE 1) gene was first described as an early estrogen-regulated gene that shares a high sequence homology with GABARAP and is thus a part of the GABARAP family. GABARAPL1, like GABARAP, interacts with the GABAA receptor and tubulin and promotes tubulin polymerization. The GABARAP family members (GABARAP, GABARAPL1 and GABARAPL2) and their close homologs (LC3 and Atg8) are not only involved in the transport of proteins or vesicles but are also implicated in various mechanisms such as autophagy, cell death, cell proliferation and tumor progression. However, despite these similarities, GABARAPL1 displays a complex regulation that is different from that of other GABARAP family members. Moreover, it presents a regulated tissue expression and is the most highly expressed gene among the family in the central nervous system. In this review article, we will outline the specific functions of this protein and also hypothesize about the roles that GABARAPL1 might have in several important biological processes such as cancer or neurodegenerative diseases.

GABARAPL1 (GEC1) associates with autophagic vesicles.

Gabarapl1 (gec1) was first described as an estrogen regulated gene which shares a high sequence homology with the gabarap gene. We previously demonstrated that GABARAPL1, like GABARAP, interacts with the GABAA receptor and tubulin and promotes tubulin polymerization. Previous work has demonstrated that the GABARAP family members (GABARAP, LC3, GATE-16 and Atg8) are not only involved in the transport of proteins or vesicles but are also implicated in various mechanisms such as autophagy, cell death, cell proliferation and tumor progression. We therefore asked whether GABARAPL1 might also play a role in autophagy. First, we showed that GABARAPL1 is cleaved at glycine 116, a residue which is conserved in other members of the family. We also demonstrated that GABARAPL1 is linked to phospholipids, delipidated by Atg4B, associated with intracellular membranes and accumulated in intracellular vesicles after inhibition of lysosomal activity. Finally, we showed that GABARAPL1 partially colocalizes with LC3 or Lysotracker green in intracellular vesicles. Taken together, our results demonstrate that GABARAPL1 associates with autophagic vesicles.