Showing papers on "MAP1LC3B published in 2013"

IFNB1/interferon-β-induced autophagy in MCF-7 breast cancer cells counteracts its proapoptotic function

Abstract: IFNB1/interferon (IFN)-β belongs to the type I IFNs and exerts potent antiproliferative, proapoptotic, antiangiogenic and immunemodulatory functions. Despite the beneficial effects of IFNB1 in experimental breast cancers, clinical translation has been disappointing, possibly due to induction of survival pathways leading to treatment resistance. Defects in autophagy, a conserved cellular degradation pathway, are implicated in numerous cancer diseases. Autophagy is induced in response to cancer therapies and can contribute to treatment resistance. While the type II IFN, IFNG, which in many aspects differs significantly from type I IFNs, can induce autophagy, no such function for any type I IFN has been reported. We show here that IFNB1 induces autophagy in MCF-7, MDAMB231 and SKBR3 breast cancer cells by measuring the turnover of two autophagic markers, MAP1LC3B/LC3 and SQSTM1/p62. The induction of autophagy in MCF-7 cells occurred upstream of the negative regulator of autophagy MTORC1, and autophagosome formation was dependent on the known core autophagy molecule ATG7 and the IFNB1 signaling molecule STAT1. Using siRNA-mediated silencing of several core autophagy molecules and STAT1, we provide evidence that IFNB1 mediates its antiproliferative effects independent of autophagy, while the proapoptotic function of IFNB1 was strongly enhanced in the absence of autophagy. This suggests that autophagy induced by IFNB1 promoted survival, which might contribute to tumor resistance against IFNB1 treatment. It may therefore be clinically relevant to reconcile a role for IFNB1 in the treatment of breast cancer with concomitant inhibition of autophagy.

Progesterone receptor membrane component 1/Sigma-2 receptor associates with MAP1LC3B and promotes autophagy

Abstract: Autophagy resembles a recycling process in which proteins, organelles, or regions of the cytoplasm are enveloped and degraded. We have found that two of the central autophagy proteins, MAP1LC3 (microtubule-associated protein 1 light chain 3, also described as LC3) and UVRAG (UV radiation resistance associated/UV radiation associated gene), complex with PGRMC1/S2R (progesterone receptor membrane component 1, also known as sigma-2 receptor). PGRMC1 is a cytochrome that is induced in cancer and is essential for tumor formation, invasion, and metastasis. Autophagy contributes to the turnover of long-lived and/or ubiquitinated proteins and the clearance of damaged organelles, and we have shown that PGRMC1 promotes both processes. Inhibition of PGRMC1 by RNAi or small molecule inhibitors causes autophagy substrates to increase and aberrant mitochondria to accumulate. We propose that this disruption of autophagy upon PGRMC1 inhibition increases AMPK activation, elevating the levels of TSC1 (tuberous sclerosis complex) and TSC2 and inactivating MTOR and RPS6KB/p70S6K, causing cleaved MAP1LC3B levels to increase. Thus, PGRMC1 binds to key components of the autophagy machinery and is required for the degradative activity of autophagy.

LITAF, a BCL6 target gene, regulates autophagy in mature B-cell lymphomas

Abstract: We have previously reported that LITAF is silenced by promoter hypermethylation in germinal centre-derived B-cell lymphomas, but beyond these data the regulation and function of lipopolysaccharide-induced tumour necrosis factor (TNF) factor (LITAF) in B cells are unknown. Gene expression and immunohistochemical studies revealed that LITAF and BCL6 show opposite expression in tonsil B-cell subpopulations and B-cell lymphomas, suggesting that BCL6 may regulate LITAF expression. Accordingly, BCL6 silencing increased LITAF expression, and chromatin immunoprecipitation and luciferase reporter assays demonstrated a direct transcriptional repression of LITAF by BCL6. Gain- and loss-of-function experiments in different B-cell lymphoma cell lines revealed that, in contrast to its function in monocytes, LITAF does not induce lipopolysaccharide-mediated TNF secretion in B cells. However, gene expression microarrays defined a LITAF-related transcriptional signature containing genes regulating autophagy, including MAP1LC3B (LC3B). In addition, immunofluorescence analysis co-localized LITAF with autophagosomes, further suggesting a possible role in autophagy modulation. Accordingly, ectopic LITAF expression in B-cell lymphoma cells enhanced autophagy responses to starvation, which were impaired upon LITAF silencing. Our results indicate that the BCL6-mediated transcriptional repression of LITAF may inhibit autophagy in B cells during the germinal centre reaction, and suggest that the constitutive repression of autophagy responses in BCL6-driven lymphomas may contribute to lymphomagenesis.

Autophagy is involved in endogenous and NVP-AUY922-induced KIT degradation in gastrointestinal stromal tumors

Abstract: Gastrointestinal stromal tumor (GIST) is a prototype of mutant KIT oncogene-driven tumor. Prolonged tyrosine kinase inhibitor (TKI) treatment may result in a resistant phenotype through acquired secondary KIT mutation. Heat shock protein 90 (HSP90AA1) is a chaperone protein responsible for protein maturation and stability, and KIT is a known client protein of HSP90AA1. Inhibition of HSP90AA1 has been shown to destabilize KIT protein by enhancing its degradation via the proteasome-dependent pathway. In this study, we demonstrated that NVP-AUY922 (AUY922), a new class of HSP90AA1 inhibitor, is effective in inhibiting the growth of GIST cells expressing mutant KIT protein, the imatinib-sensitive GIST882 and imatinib-resistant GIST48 cells. The growth inhibition was accompanied with a sustained reduction of both total and phosphorylated KIT proteins and the induction of apoptosis in both cell lines. Surprisingly, AUY922-induced KIT reduction could be partially reversed by pharmacological inhibition of either autophagy or proteasome degradation pathway. The blockade of autophagy alone led to the accumulation of the KIT protein, highlighting the role of autophagy in endogenous KIT turnover. The involvement of autophagy in endogenous and AUY922-induced KIT protein turnover was further confirmed by the colocalization of KIT with MAP1LC3B-, acridine orange- or SQSTM1-labeled autophagosome, and by the accumulation of KIT in GIST cells by silencing either BECN1 or ATG5 to disrupt autophagosome activity. Therefore, the results not only highlight the potential application of AUY922 for the treatment of KIT-expressing GISTs, but also provide the first evidence for the involvement of autophagy in endogenous and HSP90AA1 inhibitor-induced KIT degradation.

The Immune System GTPase GIMAP6 Interacts with the Atg8 Homologue GABARAPL2 and Is Recruited to Autophagosomes

Abstract: The GIMAPs (GTPases of the immunity-associated proteins) are a family of small GTPases expressed prominently in the immune systems of mammals and other vertebrates. In mammals, studies of mutant or genetically-modified rodents have indicated important roles for the GIMAP GTPases in the development and survival of lymphocytes. No clear picture has yet emerged, however, of the molecular mechanisms by which they perform their function(s). Using biotin tag-affinity purification we identified a major, and highly specific, interaction between the human cytosolic family member GIMAP6 and GABARAPL2, one of the mammalian homologues of the yeast autophagy protein Atg8. Chemical cross-linking studies performed on Jurkat T cells, which express both GIMAP6 and GABARAPL2 endogenously, indicated that the two proteins in these cells readily associate with one another in the cytosol under normal conditions. The GIMAP6-GABARAPL2 interaction was disrupted by deletion of the last 10 amino acids of GIMAP6. The N-terminal region of GIMAP6, however, which includes a putative Atg8-family interacting motif, was not required. Over-expression of GIMAP6 resulted in increased levels of endogenous GABARAPL2 in cells. After culture of cells in starvation medium, GIMAP6 was found to localise in punctate structures with both GABARAPL2 and the autophagosomal marker MAP1LC3B, indicating that GIMAP6 re-locates to autophagosomes on starvation. Consistent with this finding, we have demonstrated that starvation of Jurkat T cells results in the degradation of GIMAP6. Whilst these findings raise the possibility that the GIMAPs play roles in the regulation of autophagy, we have been unable to demonstrate an effect of GIMAP6 over-expression on autophagic flux.

TECPR2: a new autophagy link for neurodegeneration.

Abstract: Autophagy dysfunction has been implicated in a group of progressive neurodegenerative diseases, and has been reported to play a major role in the pathogenesis of these disorders. We have recently reported a recessive mutation in TECPR2, an autophagy-implicated WD repeat-containing protein, in five individuals with a novel form of monogenic hereditary spastic paraparesis (HSP). We found that diseased skin fibroblasts had a decreased accumulation of the autophagy-initiation protein MAP1LC3B/LC3B, and an attenuated delivery of both LC3B and the cargo-recruiting protein SQSTM1/p62 to the lysosome where they are subject to degradation. The discovered TECPR2 mutation reveals for the first time a role for aberrant autophagy in a major class of Mendelian neurodegenerative diseases, and suggests mechanisms by which impaired autophagy may impinge on a broader scope of neurodegeneration.

Arginine68 is an essential residue for the C-terminal cleavage of human Atg8 family proteins.

Abstract: Autophagy is a conserved cellular process that degrades and recycles cytoplasmic components via a lysosomal pathway. The phosphatidylethanolamine (PE)-conjugation of the Atg8 protein plays an important role in the yeast autophagy process. In humans, six Atg8 homologs, including MAP1LC3A, MAP1LC3B, MAP1LC3C (refer to LC3A, LC3B, and LC3C hereafter), GABARAP, GABARAPL1, and GABARAPL2 have been reported. All of them can be conjugated to PE through a ubiquitin-like conjugation system, and be located to autophagosomes. In this study, we found 3 new alternative splicing isoforms in LC3B, GABARAP, and GABARAPL1, (designated as LC3B-a, GABARAP-a and GABARAPL1-a, respectively). None of them can go through the PE-conjugation process and be located to autophagosomes. Interestingly, compared with LC3B, LC3B-a has a single amino acid (Arg68) deletion due to the NAGNAG alternative splicing in intron 3. Through structural simulations, we found that the C-terminal tail of LC3B-a is less mobile than that of LC3B, thus affecting its C-terminal cleavage by human ATG4 family proteins. Furthermore, we found that Arg68 is an essential residue facilitating the interaction between human Atg8 family proteins and ATG4B by forming a salt bridge with Asp171 of ATG4B. Depletion of this salt bridge reduces autophagosomes formation and autophagic flux under both normal and nutrition starvation conditions. These results suggest Arg68 is an essential residue for the C-terminal cleavage of Atg8 family proteins during the autophagy process.

Harnessing autophagy for cell fate control gene therapy

Abstract: We hypothesized that rapamycin, through induction of autophagy and promotion of an antiapoptotic phenotype, would permit lentiviral (LV)-based transgene delivery to human T-Rapa cells, which are being tested in phase II clinical trials in the setting of allogeneic hematopoietic cell transplantation. Manufactured T-Rapa cells were exposed to supernatant enriched for a LV vector encoding a fusion protein consisting of truncated CD19 (for cell surface marking) and DTYMK/TMPKΔ, which provides “cell-fate control” due to its ability to phosphorylate (activate) AZT prodrug. LV-transduction in rapamycin-treated T-Rapa cells: (1) resulted in mitochondrial autophagy and a resultant antiapoptotic phenotype, which was reversed by the autophagy inhibitor 3-MA; (2) yielded changes in MAP1LC3B and SQSTM1 expression, which were reversed by 3-MA; and (3) increased T-Rapa cell expression of the CD19-DTYMKΔ fusion protein, despite their reduced proliferative status. Importantly, although the transgene-expressing T-Rapa cells expressed an antiapoptotic phenotype, they were highly susceptible to cell death via AZT exposure both in vitro and in vivo (in a human-into-mouse xenogeneic transplantation model). Therefore, rapamycin induction of T cell autophagy can be used for gene therapy applications, including the CD19-DTYMKΔ cell-fate control axis to improve the safety of T cell immuno-gene therapy.