A Bregman-proximal point algorithm for robust non-negative matrix factorization with possible missing values and outliers - application to gene expression analysis.

BACKGROUND: Non-Negative Matrix factorization has become an essential tool for feature extraction in a wide spectrum of applications. In the present work, our objective is to extend the applicability of the method to the case of missing and/or corrupted data due to outliers. RESULTS: An essential property for missing data imputation and detection of outliers is that the uncorrupted data matrix is low rank, i.e. has only a small number of degrees of freedom. We devise a new version of the Bregman proximal idea which preserves nonnegativity and mix it with the Augmented Lagrangian approach for simultaneous reconstruction of the features of interest and detection of the outliers using a sparsity promoting ℓ 1 penality. CONCLUSIONS: An application to the analysis of gene expression data of patients with bladder cancer is finally proposed.

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.

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.

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.

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.

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.

Specific regional distribution of gec1 mRNAs in adult rat central nervous system.

GEC1 protein shares high identity with GABARAP (GABA(A) Receptor-Associated Protein), interacts with tubulin and GABA(A) receptors and is potentially involved in intracellular transport processes. Recently, using quantitative real time PCR, we have reported the gec1 mRNA expression in different rat brain areas. In the present study, we investigated the cell types expressing gec1 in rat brain. Sense and anti-sense gec1 RNA probes, corresponding to the 3'-untranslated region, were generated. In northern blotting experiments, the anti-sense probe revealed only the 1.75 kb gec1 mRNAs. On the other hand, in immunohistochemistry experiments, GEC1 polyclonal antibodies did not discriminate between GEC1 and GABARAP proteins. Therefore, we used digoxigenin-labeled RNA probes for in situ hybridization (ISH) experiments to map the gec1 expression. Using the anti-sense probe, we detected the gec1 mRNAs specifically in neurons throughout the rostrocaudal extent of the brain as well as in the spinal cord. Although a majority of neurons expressed the gec1 mRNAs, different intensities of labeling were observed depending on the areas: the strongest labeling was observed in the isocortex, hippocampus, basal telencephalon, some thalamic and most of hypothalamic nuclei, cerebellum, and numerous brainstem nuclei. Furthermore, the gec1 mRNAs were intensely expressed in neurons involved in somatomotor and neuroendocrine functions and weakly expressed in sensory and reticular structures. These results corroborate the putative role of the GEC1 protein in the trafficking of receptor GABA(A).

Involvement of sulfhydryl oxidase QSOX1 in the protection of cells against oxidative stress-induced apoptosis.

The QSOX1 protein, belonging to a new class of FAD-linked Quiescin/Sulfhydryl oxidase, catalyzes disulfide bond formation. To give new insight into the biological function of QSOX1, we studied its involvement in oxidative stress-induced apoptosis and cell recovery of PC12 cells. By real time RT-PCR and flow cytometric analysis, we show that the QSOX1 mRNA and protein levels increased late after the beginning of oxidative treatment and were sustained for 72 h. These levels were still high when the PC12 cells were not dying but had resumed proliferation. The kinetics of QSOX1 expression suggest a more protective effect of QSOX1 rather than an involvement of this protein in apoptosis. Human breast cancer MCF-7 cell lines overexpressing the guinea pig QSOX1 protein submitted to the same treatments appeared less sensitive to cell death than the MCF-7 control cells. The protective effect is partly due to a preservation of the mitochondrial polarization generally lost after an oxidative stress. These results strengthen our hypothesis of a protective role of QSOX1 against apoptosis.

Effect of 7-O-beta-D-glucopyranosylchrysin and its aglycone chrysin isolated from Podocytisus caramanicus on estrogen receptor alpha transcriptional activity.

A screening of Greek Fabaceae extracts identified the methanolic extract of Podocytisus caramanicus Boiss. & Heldr. as having proliferative activity on human breast cancer cells (MCF-7). Using transient transfection experiments, we have first used three compounds described for their estrogen-like properties, E (2), genistein (Gen) and biochanin A (Bch), as controls to evaluate our cellular model. Secondly, we have demonstrated that the 7- O-beta- D-glucopyranosylchrysin (Glc-chr), the most abundant flavone of the extract, and its aglycone chrysin were able to increase estrogen receptor alpha transcriptional activity in MCF-7 cells. We have also shown that the estrogenic activity of Glc-chr could be completely suppressed by the pure estrogen antagonist ICI 182,780 suggesting that the effect of Glc-chr is mediated by ERalpha.

Identification and expression of a new splicing variant of FAD-sulfhydryl oxidase in adult rat brain.

Flavoproteins of the quiescin/sulfhydryl oxidase (QSOX) family catalyze oxidation of peptide and protein thiols to disulfides with the reduction of oxygen to hydrogen peroxide. We report here the molecular cloning of a new putative sulfhydryl oxidase cDNA, rQSOX-L (GenBank Accession no ), from adult rat brain and its expression studied by RT-PCR, Northern and Western blots in rat tissues. DNA-sequencing demonstrated the existence of two cDNAs in rat cortex, corresponding to a long transcript (rQSOX-L) and a short transcript (rQSOX-S) which differed by 851 nucleotides due to alternative splicing. The new transcript, rQSOX-L (3356 nucleotides), was specifically expressed in brain, hypophysis, heart, testis and seminal vesicle. The distribution of this variant is not homogeneous in the different tissues studied and suggests a complex gene regulation. The full-length rQSOX-L cDNA has an open reading frame of 2250-bp encoding a protein of 750 amino acids that contains a signal peptide sequence, a protein-disulfide-isomerase-type thioredoxin and ERV1-ALR domains and a long form specific C-terminal extension. The rQSOX-L protein is highly homologous to members of the sulfhydryl oxidase/Quiescin family and contains particularly two potential sites for N-glycosylation. This protein isoform was specifically detected in rat brain tissues in opposition to the low molecular form that was ubiquitous. Matrix-assisted laser desorption/ionization time of flight mass spectrometry analysis of the immunoprecipitate tryptic fragments allowed the identification of rQSOX-L protein.

Specific distribution of gabarap, gec1/gabarap Like 1, gate16/gabarap Like 2, lc3 messenger RNAs in rat brain areas by quantitative real-time PCR.

GABARAP and GEC1/GABARAPL1 interact with tubulin and GABA(A) receptor and belong to a new protein family. This family includes GATE 16 and LC3, potentially involved in intracellular transport processes. In this study, we combined brain dissection and quantitative real-time reverse transcription polymerase chain reaction to study discriminatively gabarap, gec1/gabarapL1, gate16/gabarapL2, lc3 mRNA distribution in multiple rat brain areas.

Analysis of the guinea-pig estrogen-regulated gec1/GABARAPL1 gene promoter and identification of a functional ERE in the first exon.

The gec1/GABARAPL1 (GABA(A)-receptor-associated protein like-1) gene has been identified as an early estrogen-regulated gene in guinea-pig cultured endometrial glandular epithelial cells (GEC). Guinea-pig and human gec1/GABARAPL1 proteins share 87% identity with GABARAP, which acts as a protein linker between microtubules and the GABA(A) receptor. To investigate the molecular mechanisms regulating gec1/GABARAPL1 gene expression, the 1.5-kbp region upstream of the translation initiation codon of the guinea-pig gec1/GABARAPL1 gene was cloned. A 300-bp fragment encompassing a pyrimidine-rich initiator element (INR) and the transcription start site (+1) was sufficient to initiate transcription. Transfection and gel shift experiments showed that a sequence located at +36/+50 in the first exon permitted induction of expression of this gene by estradiol acting via ERalpha. This sequence (GGGTCAACGTGACGT) differs only by one base pair from the consensus estrogen response element ERE (GGGTCAACGTGACCT). It can be concluded that the ERE located in the first exon encoding the 5'-untranslated region is sufficient for E2 activation of gec1/GABARAPL1 transcription.

Olfactory epithelium destruction by ZnSO4 modified sulfhydryl oxidase expression in mice.

Experimental destruction of olfactory neurons stimulates proliferation and differentiation of local neural precursors and is used as a model to study in vivo mechanisms for degeneration and regeneration of the nervous system. Quiescin-sulfhydryl oxidases (QSOX) have a potential role in the control of the cell cycle or growth regulation and have recently been described in the central nervous system. In mice, we show an expression of QSOX in olfactory mucosa. Northern- and western-blot analysis show that the destruction of olfactory epithelium is associated with a reversible reduction in QSOX expression. Interestingly, QSOX is not localized in olfactory neurons (ON) but in cells of the lamina propria, suggesting that olfactory epithelium destruction may act as a signal of down-regulation of QSOX expression.

GEC1, a protein related to GABARAP, interacts with tubulin and GABA(A) receptor.

We have previously identified in uterine cells a novel estrogen-regulated gene called gec1. GEC1 presents 87% identity with GABARAP which, so far, was the only protein found to associate with tubulin and GABA(A) receptor. We demonstrated then that GEC1 interacts in vitro with tubulin and GABA(A) receptor, and promotes tubulin assembly and microtubule bundling. Since all polyclonal antibodies failed in discrimination of both proteins GEC1 and GABARAP, a GEC1-GFP fusion protein was used to specifically localize GEC1. GEC1-GFP was distributed over the cytoplasm in perinuclear vesicles with a scattered pattern. Overall, our data show that GEC1 could be a new member of the GABARAP family involved in the transport of GABA(A) receptor.

Onset of direct 17-beta estradiol effects on proliferation and c-fos expression during oncogenesis of endometrial glandular epithelial cells.

In normal endometrial glandular epithelial cells (GEC), 17beta-estradiol (E2) enhances proliferation and c-fos expression only in the presence of growth factors. On the contrary, growth factors are not required for the E2 effects in cancerous cells. Thus, a repression of E2 action could exist in normal cells and be turned off in cancerous cells, allowing a direct estrogen-dependent proliferation. To verify this hypothesis, we established immortalized and transformed cell models, then investigated alterations of E2 effects during oncogenesis. SV40 large T-antigen was used to generate immortalized GEC model (IGEC). After observation of telomerase reactivation, IGEC model was transfected by activated c-Ha-ras to obtain transformed cell lines (TGEC1 and TGEC2). The phenotypic, morphological, and genetic characteristics of these models were determined before studying the E2 effects. In IGEC, the E2 action on proliferation and c-fos expression required the presence of growth factors, as observed in GECs. In TGECs, this action arose in the absence of growth factors. After IGEC transformation, the activation of ras pathway would substitute the priming events required for the release of repression in GEC and IGEC and thus permit direct E2 effects. Our cell models are particularly suitable to investigate alterations of gene regulation by E2 during oncogenesis.