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.

High expression of QSOX1 reduces tumorogenesis, and is associated with a better outcome for breast cancer patients.

INTRODUCTION: The gene quiescin/sulfhydryl oxidase 1, QSOX1, encodes an enzyme directed to the secretory pathway and excreted into the extracellular space. QSOX1 participates in the folding and stability of proteins and thus could regulate the biological activity of its substrates in the secretory pathway and/or outside the cell. The involvement of QSOX1 in oncogenesis has been studied primarily in terms of its differential expression in systemic studies. QSOX1 is overexpressed in prostate cancers and in pancreatic adenocarcinoma. In contrast, QSOX1 gene expression is repressed in endothelial tumors. In the present study, we investigated the role of QSOX1 in breast cancer. METHODS: We analyzed QSOX1 mRNA expression in a cohort of 217 invasive ductal carcinomas of the breast. Moreover, we investigated QSOX1's potential role in regulating tumor growth and metastasis using cellular models in which we overexpressed or extinguished QSOX1 and xenograft experiments. RESULTS: We showed that the QSOX1 expression level is inversely correlated to the aggressiveness of breast tumors. Our results show that QSOX1 leads to a decrease in cell proliferation, clonogenic capacities and promotes adhesion to the extracellular matrix. QSOX1 also reduces the invasive potential of cells by reducing cell migration and decreases the activity of the matrix metalloproteinase, MMP-2, involved in these mechanisms. Moreover, in vivo experiments show that QSOX1 drastically reduces the tumor development. CONCLUSIONS: Together, these results suggest that QSOX1 could be posited as a new biomarker of good prognosis in breast cancer and demonstrate that QSOX1 inhibits human breast cancer tumorogenesis.

The functions of Atg8-family proteins in autophagy and cancer: linked or unrelated?

The Atg8-family proteins are subdivided into two subfamilies: the GABARAP and LC3 subfamilies. These proteins, which are major players of the autophagy pathway, present a conserved glycine in their C-terminus necessary for their association to the autophagosome membrane. This family of proteins present multiple roles from autophagy induction to autophagosome-lysosome fusion and have been described to play a role during cancer progression. Indeed, GABARAPs are described to be downregulated in cancers, and high expression has been linked to a good prognosis. Regarding LC3 s, their expression does not correlate to a particular tumor type or stage. The involvement of Atg8-family proteins during cancer, therefore, remains unclear, and it appears that their anti-tumor role may be associated with their implication in selective protein degradation by autophagy but might also be independent, in some cases, of their conjugation to autophagosomes. In this review, we will then focus on the involvement of GABARAP and LC3 subfamilies during autophagy and cancer and highlight the similarities but also the differences of action of each subfamily member.Abbreviations: AIM: Atg8-interacting motif; AMPK: adenosine monophosphate-associated protein kinase; ATG: autophagy-related; BECN1: beclin 1; BIRC6/BRUCE: baculoviral IAP repeat containing 6; BNIP3L/NIX: BCL2 interacting protein 3 like; GABARAP: GABA type A receptor-associated protein; GABARAPL1/2: GABA type A receptor associated protein like 1/2; GABRA/GABAA: gamma-aminobutyric acid type A receptor subunit; LAP: LC3-associated phagocytosis; LMNB1: lamin B1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; PI4K2A/PI4KIIα: phosphatidylinositol 4-kinase type 2 alpha; PLEKHM1: plecktrin homology and RUN domain containing M1; PtdIns3K-C1: class III phosphatidylinositol 3-kinase complex 1; SQSTM1: sequestosome 1; ULK1: unc51-like autophagy activating kinase 1.

GABARAPL1 Inhibits EMT Signaling through SMAD-Tageted Negative Feedback.

The pathway of selective autophagy, leading to a targeted elimination of specific intracellular components, is mediated by the ATG8 proteins, and has been previously suggested to be involved in the regulation of the Epithelial-mesenchymal transition (EMT) during cancer's etiology. However, the molecular factors and steps of selective autophagy occurring during EMT remain unclear. We therefore analyzed a cohort of lung adenocarcinoma tumors using transcriptome analysis and immunohistochemistry, and found that the expression of ATG8 genes is correlated with that of EMT-related genes, and that GABARAPL1 protein levels are increased in EMT+ tumors compared to EMT- ones. Similarly, the induction of EMT in the A549 lung adenocarcinoma cell line using TGF-ß/TNF-α led to a high increase in GABARAPL1 expression mediated by the EMT-related transcription factors of the SMAD family, whereas the other ATG8 genes were less modified. To determine the role of GABARAPL1 during EMT, we used the CRISPR/Cas9 technology in A549 and ACHN kidney adenocarcinoma cell lines to deplete GABARAPL1. We then observed that GABARAPL1 knockout induced EMT linked to a defect of GABARAPL1-mediated degradation of the SMAD proteins. These findings suggest that, during EMT, GABARAPL1 might intervene in an EMT-regulatory loop. Indeed, induction of EMT led to an increase in GABARAPL1 levels through the activation of the SMAD signaling pathway, and then GABARAPL1 induced the autophagy-selective degradation of SMAD proteins, leading to EMT inhibition.

Autophagy is associated with a robust specific transcriptional signature in breast cancer subtypes.

Previous works have described that autophagy could be associated to both pro- and anti-cancer properties according to numerous factors, such as the gene considered, the step of autophagy involved or the cancer model used. These data might be explained by the fact that some autophagy-related genes may be involved in other cellular processes and therefore differently regulated according to the type or the grade of the tumor. Indeed, using different approaches of transcriptome analysis in breast cancers, and further confirmation using digital PCR, we identified a specific signature of autophagy gene expression associated to Luminal A or Triple Negative Breast Cancers (TNBC). Moreover, we confirmed that ATG5, an autophagy gene specifically expressed in TNBC, favored cell migration, whereas BECN1, an autophagy gene specifically associated with ER-positive breast cancers, induced opposite effects. We also showed that overall inhibition of autophagy promoted cell migration suggesting that the role of individual ATG genes in cancer phenotypes was not strictly dependent of their function during autophagy. Finally, our work led to the identification of TXNIP1 as a potential biomarker associated to autophagy induction in breast cancers. This gene could become an essential tool to quantify autophagy levels in fixed biopsies, sort tumors according to their autophagy levels and determine the best therapeutic treatment.

Fertility defects revealing germline biallelic nonsense NBN mutations.

Biallelic mutations in the NBN/NBS1 gene are the cause of Nijmegen breakage syndrome (NBS), a severe pediatric disease characterized by dysmorphy with a bird-like face, microcephaly, growth retardation, immune deficiency, and proneness to cancer. We here report two adult siblings that are compound heterozygotes for two previously unreported NBN nonsense mutations. These patients presented with the unique clinical symptom of fertility defects. Contrasting with the absence of any developmental abnormality, biological analyses revealed defects similar to those observed in NBS patients, including chromosomal instability, cellular hyperradiosensitivity and checkpoint defects as measured by radioresistant DNA synthesis (RDS). NBN mutations should thus be considered a new cause of infertility, and should be searched for if associated with the biological abnormalities of NBS.

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.

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.

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.

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.

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 (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.

Oxidative stress promotes myofibroblast differentiation and tumour spreading.

JunD regulates genes involved in antioxidant defence. We took advantage of the chronic oxidative stress resulting from junD deletion to examine the role of reactive oxygen species (ROS) in tumour development. In a model of mammary carcinogenesis, junD inactivation increased tumour incidence and revealed an associated reactive stroma. junD-inactivation in the stroma was sufficient to shorten tumour-free survival rate and enhance metastatic spread. ROS promoted conversion of fibroblasts into highly migrating myofibroblasts through accumulation of the hypoxia-inducible factor (HIF)-1alpha transcription factor and the CXCL12 chemokine. Accordingly, treatment with an antioxidant reduced the levels of HIF and CXCL12 and numerous myofibroblast features. CXCL12 accumulated in the stroma of HER2-human breast adenocarcinomas. Moreover, HER2 tumours exhibited a high proportion of myofibroblasts, which was significantly correlated to nodal metastases. Interestingly, this subset of tumours exhibited a significant nuclear exclusion of JunD and revealed an associated oxido-reduction signature, further demonstrating the relevance of our findings in human cancers. Collectively, our data uncover a new mechanism by which oxidative stress increases the migratory properties of stromal fibroblasts, which in turn potentiate tumour dissemination.

The TCL1A oncoprotein interacts directly with the NF-kappaB inhibitor IkappaB.

The T cell leukaemia/lymphoma 1A (TCL1A) oncoprotein plays key roles in several B and T cell malignancies. Lacking enzymatic activity, TCL1A's transforming action was linked to its capacity to co-activate the protein kinase AKT via binding to its pleckstrin homology (PH) domain. However, perturbation of AKT signalling alone was recently shown insufficient to explain TCL1A oncogenesis, suggesting that TCL1A has additional cellular partners. Searching for such additional targets, we found that TCL1A binds specifically and directly to the ankyrin domain of IkappaB, the inhibitor of the NF-kappaB transcription factors. Through binding assays and a structural analysis by small angle X-ray scattering, we show that TCL1A and IkappaB interact in yeast-two-hybrid systems, when transiently overexpressed in 293 cells, and as recombinant proteins in vitro. We further establish that the association between TCL1A and IkappaB is compatible with AKT binding to TCL1A, but incompatible with IkappaB binding to NF-kappaB. By interfering with the inhibition of NF-kappaB by IkappaB, TCL1A may increase the concentration of free NF-kappaB molecules sufficiently to trigger expression of anti-apoptotic genes. Thus our data suggest an additional route by which TCL1A might cause cancer.

Haploinsufficiency of CDKN1B contributes to leukemogenesis in T-cell prolymphocytic leukemia.

T-cell prolymphocytic leukemia (T-PLL) is consistently associated with inactivation of the ATM gene and chromosomal re-arrangements leading to an overexpression of MTCP1/TCL1 oncoproteins. These alterations are present at the earliest stage of malignant transformation, suggesting that additional events are required for overt malignancy. In this study, we pursued the investigation of the 12p13 deletion, previously shown to occur in approximately half of T-PLLs. We refined the minimal region of deletion by single nucleotide and microsatellite polymorphism allelotyping. We defined a 216-kb region containing the CDKN1B gene that encodes the cyclin-dependent kinase inhibitory protein p27(KIP1). Sequencing this gene in 47 T-PLL patient samples revealed a nonsense mutation in one case without 12p13 deletion. The absence of biallelic inactivation of CDKN1B for most patients suggested a haploinsufficiency mechanism for tumor suppression, which was investigated in an animal model of the disease. In a Cdkn1b(+/-) background, MTCP1 transgenics had consistent and multiple emergences of preleukemic clones not observed in control cohorts. The second Cdkn1b allele was maintained and expressed in these preleukemic clones. Altogether, these data strongly implicate CDKN1B haploinsufficiency in the pathogenesis of T-PLL.

The TCL1 oncoprotein inhibits activation-induced cell death by impairing PKCtheta and ERK pathways.

The TCL1/MTCP1 oncogenes were identified on the basis of their involvement in T-cell prolymphocytic leukemia (T-PLL). TCL1 and MTCP1 proteins directly interact with AKT and modulate the AKT signal-transduction pathway, but the relevance of this mechanism in leukemogenesis remains unclear. We investigate the biologic functions of TCL1 in the T-cell lineage using various cell lines, and primary malignant and normal lymphocytes. In the Jurkat cell line, expression of TCL1 had no effect in unstimulated cells, whereas it abrogated activation-induced cell death (AICD). These cellular effects were concomitant with a major inhibition by TCL1 of PKCtheta and ERK pathways. Secondly, the TCL1-driven T-cell leukemia cell line SUP-T11 was shown to have impaired PKCtheta and ERK phosphorylation upon stimulation, which were restored by TCL1 inhibition using RNA interference. Finally, defects in these pathways were also observed in primary malignant (T-PLL) and transduced normal T lymphocytes expressing TCL1. Altogether, our data demonstrated that TCL1 inhibits AICD in T cells by blocking PKCtheta and ERK activation, upon cellular activation.

Cyclin D3 is a cofactor of retinoic acid receptors, modulating their activity in the presence of cellular retinoic acid-binding protein II.

Ligand-induced transcription activation of retinoic acid (RA) target genes by nuclear receptors (retinoic acid (RAR) and retinoid X (RXR) receptors) depends on the recruitment of coactivators. We have previously demonstrated that the small 15-kDa cellular RA-binding protein II (CRABPII) is a coactivator present in the RA-dependent nuclear complex. As identifying cell-specific partners of CRABPII might help to understand the novel control of RA signaling, we performed a yeast two-hybrid screen of a hematopoietic HL-60 cDNA library using human CRABPII as bait and have subsequently identified human cyclin D3 as a partner of CRABPII. Cyclin D3 interacted with CRABPII in a ligand-independent manner and equally bound RAR alpha, but not RXR alpha, and only in the presence of RA. We further show that cyclin D3 positively modulated RA-mediated transcription through CRABPII. Therefore, cyclin D3 may be part of a ternary complex with CRABPII and RAR. Finally, we show that cyclin D3 expression paralleled HL-60 differentiation and arrest of cell growth. These findings led us to speculate that control of cell proliferation during induction of differentiation may directly involve, at the transcriptional level, nuclear receptors, coactivators, and proteins of the cell cycle in a cell- and nuclear receptor-specific manner.