LC3 Cleavage Research Articles

Mutation of the elongin C binding domain of human respiratory syncytial virus non-structural protein 1 (NS1) results in degradation of NS1 and attenuation of the virus

BackgroundHuman respiratory syncytial virus (RSV) is an important cause of lower respiratory tract disease in the paediatic population, immunocompromised individuals and the elderly worldwide. However, despite global efforts over the past several decades there are no commercially available vaccines. RSV encodes 2 non-structural proteins, NS1 and NS2, that are type I interferon antagonists. RSV restricts type I interferon signaling and the expression of antiviral genes by degrading STAT2. It has been proposed that NS1 binds to elongin C to form a ubiquitin ligase (E3) complex that targets STAT2 for ubiquitination and proteosomal degradation.ResultsHere, we have engineered a live recombinant RSV in which the 3 consensus amino acids of the NS1 elongin C binding domain have been replaced with alanine (NS1F-ELCmut). Mutation of this region of NS1 resulted in attenuation of RSV replication in A549 cells to levels similar to that observed when the NS1 gene is completely deleted (ΔNS1). This mutation also resulted in moderate attenuation in Vero cells. Attenuation was correlated to intracellular degradation of the mutated NS1 protein. Time course analysis showed that mutant NS1 protein accumulated in cytoplasmic bodies that contained the lysosomal marker LAMP1. However lack of cleavage of LC3 suggested that autophagy was not involved. Induction of IFN-β mRNA expression also was observed in association with the degradation of NS1 protein and attenuation of viral growth.ConclusionsThese results indicate that the elongin C binding region of NS1 is crucial for survival of the protein and that disruption of this region results in the degradation of NS1 and restriction of RSV replication.

Abstract 849: Autophagic cell death response to benzyl isothiocyanate exposure in human breast cancer cells is mediated by FoxO1

Abstract Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables (e.g., gardencress), is empowered to prevent chemically-induced cancers as well as oncogene-driven spontaneous mammary cancer development in rodent models. Furthermore, BITC treatment effectively inhibits growth of cultured as well as xenografted human breast cancer cells. However, the mechanisms underlying cancer chemopreventive responses to BITC are not fully understood. We now demonstrate, for the first time, that BITC causes FoxO1-mediated autophagic cell death in human breast cancer cells. Exposure of MDA-MB-231 (an estrogen-independent cell line with mutant p53) and MCF-7 (an estrogen-receptor positive cell line with wild-type p53) human breast cancer cells to BITC resulted in autophagy characterized by appearance of double-membrane structures judged by transmission electron microscopy, formation of acidic vesicular organelles evidenced by microscopy following staining with lysosomotropic agent acridine orange, cleavage of LC3 evidenced by immunoblotting, and punctuate localization of LC3. Interestingly, a spontaneously immortalized and non-tumorigenic human mammary epithelial cell line (MCF-10A) was resistant to autophagy induction by BITC. BITC-mediated growth inhibition and apoptotic cell death resulting from 6 hour treatment was significantly attenuated in the presence of autophagy inhibitor 3-methyladenineand (3-MA; a phosphatidylinositol 3-phosphate kinase inhibitor) and bafilomycin A1 (an inhibitor of vacuolar type H+-ATPase). On the other hand, growth inhibition and apoptotic DNA fragmentation resulting from 24 hour exposure to BITC was not prevented by either 3-MA or bafilomycin A. Furthermore, inhibition of MDA-MB-231 xenograft growth in female athymic mice by BITC administration (2.5 and 7.5 µmol BITC/mouse by i.p. injection) correlated with cleavage of LC3. BITC-induced autophagy was accompanied by induction of FoxO1. Moreover, RNA interference of FoxO1 conferred significant protection against BITC-mediated growth inhibition and autophagy. In conclusion, these results indicate that autophagy is an early event in growth inhibition by BITC against human breast cancer cells, which culminates into apoptotic cell death following longer exposures (e.g., 24 hours). This investigation was supported by the USPHS grant RO1 CA129347-04, awarded by the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 849. doi:10.1158/1538-7445.AM2011-849

Autophagy as a therapeutic target in cancer treatment.

TPS343 Background: Autophagy is a catabolic cellular process whereby cytoplasm, proteins and organelles are sequestered in double- membrane vesicles, the autophagosomes, and are delivered to and degraded in lysosomes. Autophagy is induced under stress, such as nutrient deprivation. In tumors, autophagy is activated in hypoxic areas and in response to anticancer drugs, providing a survival mechanism that counteracts endogenous and treatment-induced metabolic stress. As such, autophagy prolongs tumor cell survival and is a treatment resistance mechanism. Preclinical studies showed that autophagy inhibition concurrently with chemotherapy preferentially sensitized tumor cells to treatment by depriving them of a critical prosurvival function. Thus, autophagy inhibition in combination with chemotherapy is expected to increase therapeutic efficacy in cancer treatment. Methods: Several phase I/II studies involving autophagy modulation by hydroxychloroquine (HCQ) in combination with chemotherapy for the treatment of different cancers, including breast, colon, prostate and NSCLC, are currently accruing patients at the Cancer Institute of New Jersey. As an example, for patients with metastatic breast cancer who have received no more than 3 prior regimens, we are conducting a phase I/II study of ixabepilone given every 3 weeks with daily HCQ. The phase I portion will determine the MTD. The endpoints for phase II are to assess antitumor activity and time to progression, determine change in CTCs upon treatment, evaluate pharmacodynamic markers for autophagy detection in tumor specimens and characterize the effects of drug treatment on autophagy in PBMCs as a surrogate tissue. The autophagy potential of breast tumors prior to treatment is evaluated by IHC for the autophagy regulators Beclin 1 and LC3, for p62, which accumulates upon autophagy inhibition, and for PTEN and phospho-Akt, as both PTEN loss and Akt activation suppress autophagy. Ixabepilone is given alone on Day 1 of Cycle 1 (HCQ is started on Day 3), and PBMCs are examined for autophagy induction by monitoring Beclin 1, p62 and LC3 levels, as well as LC3 cleavage, by WB at 24 and 48 hours. The same assays are performed in Cycle 2, when both drugs are started on Day 1. No significant financial relationships to disclose.

BCL2 and CASP8 regulation by NF‐κB differentially affect mitochondrial function and cell fate in antiestrogen‐sensitive and ‐resistant breast cancer cells

Resistance to endocrine therapies remains a major problem in the management of estrogen receptor-alpha (ER)-positive breast cancer. We show that inhibition of NF-kappaB (p65/RELA), either by overexpression of a mutant IkappaB (IkappaBSR) or a small-molecule inhibitor of NF-kappaB (parthenolide; IC(50)=500 nM in tamoxifen-resistant cells), synergistically restores sensitivity to 4-hydroxytamoxifen (4HT) in resistant MCF7/RR and MCF7/LCC9 cells and further sensitizes MCF-7 and MCF7/LCC1 control cells to 4HT. These effects are independent of changes in either cell cycle distribution or in the level of autophagy measured by inhibition of p62/SQSTM1 expression and cleavage of LC3. NF-kappaB inhibition restores the ability of 4HT to decrease BCL2 expression, increase mitochondrial membrane permeability, and induce a caspase-dependent apoptotic cell death in resistant cells. Each of these effects is reversed by a caspase 8 (CASP8)-specific inhibitor that blocks enzyme-substrate binding. Thus, increased activation of NF-kappaB can alter sensitivity to tamoxifen by modulating CASP8 activity, with consequent effects on BCL2 expression, mitochondrial function, and apoptosis. These data provide significant new insights into how molecular signaling affects antiestrogen responsiveness and strongly suggest that a combination of parthenolide and tamoxifen may offer a novel therapeutic approach to the management of some ER-positive breast cancers.

MCP-1 causes cardiomyoblast death via autophagy resulting from ER stress caused by oxidative stress generated by inducing a novel zinc-finger protein, MCPIP

MCP-1 (monocyte chemotactic protein-1) plays a critical role in the development of heart failure that is known to involve apoptosis. How MCP-1 contributes to cell death involved in the development of heart disease is not understood. In the present study we show that MCP-1 causes death in cardiac myoblasts, H9c2 cells, by inducing oxidative stress which causes ER stress leading to autophagy via a novel zinc-finger protein, MCPIP (MCP-1-induced protein). MCPIP expression caused cell death, and knockdown of MCPIP attenuated MCP-1-induced cell death. It caused induction of iNOS (inducible NO synthase), translocation of the NADPH oxidase subunit phox47 from the cytoplasm to the membrane, production of ROS (reactive oxygen species), and induction of ER (endoplasmic reticulum) stress markers HSP40 (heat-shock protein 40), PDI (protein disulfide-isomerase), GRP78 (guanine-nucleotide-releasing protein 78) and IRE1alpha (inositol-requiring enzyme 1alpha). It also caused autophagy, as indicated by beclin-1 induction, cleavage of LC3 (microtubule-associated protein 1 light chain 3) and autophagolysosome formation, and apoptosis, as indicated by caspase 3 activation and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) assay. Inhibitors of oxidative stress, including CeO2 nanoparticles, inhibited ROS formation, ER stress, autophagy and cell death. Specific inhibitors of ER stress inhibited autophagy and cell death as did knockdown of the ER stress signalling protein IRE1. Knockdown of beclin-1 and autophagy inhibitors prevented cell death. This cell death involved caspase 2 and caspase 12, as specific inhibitors of these caspases prevented MCPIP-induced cell death. Microarray analysis showed that MCPIP expression caused induction of a variety of genes known to be involved in cell death. MCPIP caused activation of JNK (c-Jun N-terminal kinase) and p38 and induction of p53 and PUMA (p53 up-regulated modulator of apoptosis). Taken together, these results suggest that MCPIP induces ROS/RNS (reactive nitrogen species) production that causes ER stress which leads to autophagy and apoptosis through caspase 2/12 and IRE1alpha-JNK/p38-p53-PUMA pathway. These results provide the first molecular insights into the mechanism by which elevated MCP-1 levels associated with chronic inflammation may contribute to the development of heart failure.

Co-Inhibition of BCL-W and BCL2 Restores Antiestrogen Sensitivity through BECN1 and Promotes an Autophagy-Associated Necrosis

BCL2 family members affect cell fate decisions in breast cancer but the role of BCL-W (BCL2L2) is unknown. We now show the integrated roles of the antiapoptotic BCL-W and BCL2 in affecting responsiveness to the antiestrogen ICI 182,780 (ICI; Fulvestrant Faslodex), using both molecular (siRNA; shRNA) and pharmacologic (YC137) approaches in three breast cancer variants; MCF-7/LCC1 (ICI sensitive), MCF-7/LCC9 (ICI resistant), and LY2 (ICI resistant). YC137 inhibits BCL-W and BCL2 and restores ICI sensitivity in resistant cells. Co-inhibition of BCL-W and BCL2 is both necessary and sufficient to restore sensitivity to ICI, and explains mechanistically the action of YC137. These data implicate functional cooperation and/or redundancy in signaling between BCL-W and BCL2, and suggest that broad BCL2 family member inhibitors will have greater therapeutic value than targeting only individual proteins. Whereas ICI sensitive MCF-7/LCC1 cells undergo increased apoptosis in response to ICI following BCL-W±BCL2 co-inhibition, the consequent resensitization of resistant MCF-7/LCC9 and LY2 cells reflects increases in autophagy (LC3 cleavage; p62/SQSTM1 expression) and necrosis but not apoptosis or cell cycle arrest. Thus, de novo sensitive cells and resensitized resistant cells die through different mechanisms. Following BCL-W+BCL2 co-inhibition, suppression of functional autophagy by 3-methyladenine or BECN1 shRNA reduces ICI-induced necrosis but restores the ability of resistant cells to die through apoptosis. These data demonstrate the plasticity of cell fate mechanisms in breast cancer cells in the context of antiestrogen responsiveness. Restoration of ICI sensitivity in resistant cells appears to occur through an increase in autophagy-associated necrosis. BCL-W, BCL2, and BECN1 integrate important functions in determining antiestrogen responsiveness, and the presence of functional autophagy may influence the balance between apoptosis and necrosis.

Gene network signaling in hormone responsiveness modifies apoptosis and autophagy in breast cancer cells

Resistance to endocrine therapies, whether de novo or acquired, remains a major limitation in the ability to cure many tumors that express detectable levels of the estrogen receptor alpha protein (ER). While several resistance phenotypes have been described, endocrine unresponsiveness in the context of therapy-induced tumor growth appears to be the most prevalent. The signaling that regulates endocrine resistant phenotypes is poorly understood but it involves a complex signaling network with a topology that includes redundant and degenerative features. To be relevant to clinical outcomes, the most pertinent features of this network are those that ultimately affect the endocrine-regulated components of the cell fate and cell proliferation machineries. We show that autophagy, as supported by the endocrine regulation of monodansylcadaverine staining, increased LC3 cleavage, and reduced expression of p62/SQSTM1, plays an important role in breast cancer cells responding to endocrine therapy. We further show that the cell fate machinery includes both apoptotic and autophagic functions that are potentially regulated through integrated signaling that flows through key members of the BCL2 gene family and beclin-1 (BECN1). This signaling links cellular functions in mitochondria and endoplasmic reticulum, the latter as a consequence of induction of the unfolded protein response. We have taken a seed-gene approach to begin extracting critical nodes and edges that represent central signaling events in the endocrine regulation of apoptosis and autophagy. Three seed nodes were identified from global gene or protein expression analyses and supported by subsequent functional studies that established their abilities to affect cell fate. The seed nodes of nuclear factor kappa B (NFκB), interferon regulatory factor-1 (IRF1), and X-box binding protein-1 (XBP1) are linked by directional edges that support signal flow through a preliminary network that is grown to include key regulators of their individual function: NEMO/IKKγ, nucleophosmin and ER respectively. Signaling proceeds through BCL2 gene family members and BECN1 ultimately to regulate cell fate.

PEA-15 Induces Autophagy in Human Ovarian Cancer Cells and Is Associated with Prolonged Overall Survival

Phospho-enriched protein in astrocytes (PEA-15) is a 15-kDa phosphoprotein that slows cell proliferation by binding to and sequestering extracellular signal-regulated kinase (ERK) in the cytoplasm, thereby inhibiting ERK-dependent transcription and proliferation. In previous studies of E1A human gene therapy for ovarian cancer, we discovered that PEA-15 induced the antitumor effect of E1A by sequestering activated ERK in the cytoplasm of cancer cells. Here, we investigated the role of PEA-15 in ovarian cancer tumorigenesis, the expression levels of PEA-15 in human ovarian cancer, and whether PEA-15 expression correlated with overall survival in women with ovarian cancer. We overexpressed PEA-15 in low-PEA-15-expressing cells and knocked down PEA-15 in high-PEA-15-expressing cells and analyzed the effects on proliferation, anchorage-independent growth, and cell cycle progression. We then assessed PEA-15 expression in an annotated tissue microarray of tumor samples from 395 women with primary epithelial ovarian cancer and tested whether PEA-15 expression was linked with overall survival. PEA-15 expression inhibited proliferation, and cell cycle analysis did not reveal apoptosis but did reveal autophagy, which was confirmed by an increase in LC3 cleavage. Inhibition of the ERK1/2 pathway decreased PEA-15-induced autophagy. These findings suggest that the antitumor activity of PEA-15 is mediated, in part, by the induction of autophagy involving activation of the ERK1/2 pathway. Multivariable analyses indicated that the women with high-PEA-15-expressing tumors survived longer than those with low-PEA-15-expressing tumors (hazard ratio, 1.973; P = 0.0167). Our findings indicate that PEA-15 expression is an important prognostic marker in ovarian cancer.

Abstract B98: Chloroquin converts molecular iodine induced autophagy in MDAMB231 to apoptotic cell death: Implications for overcoming chemotherapeutic resistance

Abstract B98 Autophagic mechanism is known to provide survival advantage and chemo-resistant in various cancers. Molecular iodine (I2) has been shown to cause apoptotic cell death independent of estrogen receptor and p53 status in breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-453, ZR-75-1, T-47D). In MCF-7 cells apoptotic cell death has been shown to be independent of caspase activity and is mediated through AIF. Non-evident apoptosis in MDA-MB-231 in response to I2 treatment led us to investigate its cell death mechanism in detail. Electron microscopic observations and immunofluorescence performed in molecular iodine treated MDA-MB-231 cells showed increase in acidic vacuoles, autophagosome formation and subsequent autolysosome formation confirmed the autophagy. Blocking of various stages of I2 induced autophagy by PI3 kinase inhibitors (LY294002 and 3-MA), bafilomycin (lysosomal fusion inhibitor) resulted in enhanced cell death indicating that autophagy provides survival advantage to MDA-MB-231 cells. The evidence of autophagy is further supported by enhanced expression of Beclin, decreased Bcl-2, and increased LC-3 cleavage seen on western blot. Recent evidence that anti-malarial drug chloroquin inhibits chemotherapeutic regimen induced autophagy leading to increased cell death in CNS lymphoma is promising. To know whether the chloroquin mediated increased therapeutic efficacy is drug/system specific or is more generalized phenomenon we investigated its effect in I2 treated MDA-MB-231 cells. Co-treatment with I2 and Chloroquin resulted in increased cytotoxicity, accumulation of sub-G1 cell fraction, nuclear fragmentation with enhanced caspase-9 activation and decreased pro-caspase-3 indicates that chloroquin redirects the cells undergoing autophagy under the influence of molecular iodine to dominant apoptotic mechanism of cell death. In vivo efficiency of molecular iodine and chloroquine combined treatment was assessed in mammary tumor implanted ICRC mice. Mammary tumors were implanted in 18-20 mice from spontaneous mammary tumors harboring mice. Volumes were measured by NMR spectroscopy. Once tumor volume reached 0.5 - 0.7 cm3, mice were divided in three groups of six mice each of control, I2 treatment and I2 plus chloroquin co-treatment (n=6). Tumor volume in untreated mice showed a significant increase over a period of 6 weeks, no increase in iodine treated group and a significant decreased on combination treatment protocol The evidence provided so far indicates that the chloroquin probably increases the therapeutic efficacy of drug regimen in a ubiquitous manner. This switch over ability from autophagy to apoptosis provided by chloroquin makes it a potential adjuvant to overcome the survival advantage conferred by molecular Iodine to MDA-MB-231 cells. Supported by Department of Science & Technology, Government of India, Grant No SR/SO/HS/017/2003 Citation Information: Cancer Prev Res 2008;1(7 Suppl):B98.

Resveratrol inhibits glucose metabolism in human ovarian cancer cells

Objectives. Resveratrol is a phytoalexin found in grapes that inhibits the in vitro growth of multiple tumor cell types. We showed previously that resveratrol induces autophagic cell death in ovarian cancer cells. Because autophagy is typically an adaptive response to nutrient starvation, we hypothesized that autophagy would also be triggered when ovarian cancer cells are nutrient deprived and that resveratrol could in fact be acting by inducing a starvation-like signaling response. Methods. Ovarian cancer cells were incubated with normal media, media containing resveratrol, glucose free media, or media lacking amino acids. Growth inhibition was determined using the sulforhodamine assay. Cells were evaluated for autophagocytosis by analyzing cleavage of LC3. Glucose uptake, lactate production, and activation of glycolytic regulators pAkt and pmTOR were analyzed following resveratrol treatment. Results. We show here that epithelial ovarian cancer cells are highly sensitive to glucose-deprivation-induced cell death and like resveratrol, glucose deprivation induces caspase-independent cell death with hallmarks of autophagy. Consistent with the hypothesis that resveratrol treatment results in biochemical conditions that mirror a nutrient deprived state, we found that resveratrol dramatically reduces glucose uptake and lactate production. Moreover, resveratrol reduces the levels of phosphorylated Akt and mTOR, two signals that increase glucose uptake and the rate limiting steps in glycolysis. Conclusions. Our findings are consistent with the hypothesis that resveratrol-induced changes in glucose utilization comprise the mechanism that underlies resveratrol-induced autophagocytosis in ovarian cancer. Inhibition of glycolysis in ovarian cancer with resveratrol or other compounds may be effective therapy for ovarian cancer.