Eran Schmukler

Beclin 1 self‐association is independent of autophagy induction by amino acid deprivation and rapamycin treatment

Autophagy, a process of self‐digestion of cellular constituents, regulates the balance between protein synthesis and protein degradation. Beclin 1 represents an important component of the autophagic machinery. It interacts with proteins that positively regulate autophagy, such as Vps34, UVRAG, and Ambra1, as well as with anti‐apoptotic proteins such as Bcl‐2 via its BH3‐like domain to negatively regulate autophagy. Thus, Beclin 1 interactions with several proteins may regulate autophagy. To identify novel Beclin 1 interacting proteins, we utilized a GST‐Beclin 1 fusion protein. Using mass spectroscopic analysis, we identified Beclin 1 as a protein that interacts with GST‐Beclin 1. Further examination by cross linking and co‐immunoprecipitation experiments confirmed that Beclin 1 self‐interacts and that the coiled coil and the N‐terminal region of Beclin 1 contribute to its oligomerization. Importantly, overexpression of vps34, UVRAG, or Bcl‐xL, had no effect on Beclin 1 self‐interaction. Moreover, this self‐interaction was independent of autophagy induction by amino acid deprivation or rapamycin treatment. These results suggest that full‐length Beclin 1 is a stable oligomer under various conditions. Such an oligomer may provide a platform for further protein–protein interactions. J. Cell. Biochem. 110: 1262–1271, 2010. Published 2010 Wiley‐Liss, Inc.

Impaired Autophagy in APOE4 Astrocytes

Alzheimers disease (AD) is the most prevalent form of dementia in elderly. Genetic studies revealed allelic segregation of the apolipoprotein E (ApoE) gene in sporadic AD and in families with higher risk of AD. The mechanisms underlying the pathological effects of ApoE4 are not yet entirely clear. Several studies indicate that autophagy, which plays an important role in degradation pathways of proteins, organelles and protein aggregates, may be impaired in AD. In the present study, we investigated the effects of ApoE4 versus the ApoE3 isoform on the process of autophagy in mouse-derived astrocytes. The results obtained reveal that under several autophagy-inducing conditions, astrocytes expressing ApoE4 exhibit lower autophagic flux compared to astrocytes expressing ApoE3. Using an in situ model, we examined the role of autophagy and the effects thereon of ApoE4 in the elimination of Aβ plaques from isolated brain sections of transgenic 5xFAD mice. This revealed that ApoE4 astrocytes eliminate Aβ plaques less effectively than the corresponding ApoE3 astrocytes. Additional experiments showed that the autophagy inducer, rapamycin, enhances Aβ plaque degradation by ApoE4 astrocytes whereas the autophagy inhibitor, chloroquine, blocks Aβ plaque degradation by ApoE3 astrocytes. Taken together, these findings show that ApoE4 impairs autophagy in astrocyte cultures and that this effect is associated with reduced capacity to clear Aβ plaques. This suggests that impaired autophagy may play a role in mediating the pathological effects of ApoE4 in AD.

DJ‐1 Deficiency Impairs Autophagy and Reduces Alpha‐synuclein Phagocytosis by Microglia

Parkinsons disease (PD) is a progressive neurodegenerative disorder, of which 1% of the hereditary cases are linked to mutations in DJ‐1, an oxidative stress sensor. The pathological hallmark of PD is intercellular inclusions termed Lewy Bodies, composed mainly of α‐Synuclein (α‐Syn) protein. Recent findings have shown that α‐Syn can be transmitted from cell to cell, suggesting an important role of microglia, as the main scavenger cells of the brain, in clearing α‐Syn. We previously reported that the knock down (KD) of DJ‐1 in microglia increased cells’ neurotoxicity to dopaminergic neurons. Here, we discovered that α‐Syn significantly induced elevated secretion of the proinflammatory cytokines IL‐6 and IL‐1β and a significant dose‐dependent elevation in the production of nitric oxide in DJ‐1 KD microglia, compared to control microglia. We further investigated the ability of DJ‐1 KD microglia to uptake and degrade soluble α‐Syn, and discovered that DJ‐1 KD reduces cell‐surface lipid raft expression in microglia and impairs their ability to uptake soluble α‐Syn. Autophagy is an important mechanism for degradation of intracellular proteins and organelles. We discovered that DJ‐1 KD microglia exhibit an impaired autophagy‐dependent degradation of p62 and LC3 proteins, and that manipulation of autophagy had less effect on α‐Syn uptake and clearance in DJ‐1 KD microglia, compared to control microglia. Further studies of the link between DJ‐1, α‐Syn uptake and autophagy may provide useful insights into the role of microglia in the etiology of the PD.

Neuregulin Promotes Incomplete Autophagy of Prostate Cancer Cells That Is Independent of mTOR Pathway Inhibition

Background Growth factors activating the ErbB receptors have been described in prostate tumors. The androgen dependent prostate cancer cell line, LNCaP, expresses the ErbB-1, ErbB-2 and ErbB-3 receptor tyrosine kinases. Previously, it was demonstrated that NRG activates ErbB-2/ErbB-3 heterodimers to induce LNCaP cell death, whereas, EGF activates ErbB-1/ErbB-1 or ErbB-1/ErbB-2 dimers to induce cell growth and survival. It was also demonstrated that PI3K inhibitors repressed this cell death suggesting that in androgen deprived LNCaP cells, NRG activates a PI3K-dependent pathway associated with cell death. Methodology/Principal Findings In the present study we demonstrate that NRG induces autophagy in LNCaP cells, using LC3 as a marker. However, the autophagy induced by NRG may be incomplete since p62 levels elevate. We also demonstrated that NRG- induced autophagy is independent of mammalian target of rapamycin (mTOR) inhibition since NRG induces Akt and S6K activation. Interestingly, inhibition of reactive oxygen species (ROS) by N-acetylcysteine (NAC), inhibited NRG-induced autophagy and cell death. Our study also identified JNK and Beclin 1 as important components in NRG-induced autophagy and cell death. NRG induced elevation in JNK phosphorylation that was inhibited by NAC. Moreover, inhibitor of JNK inhibited NRG-induced autophagy and cell death. Also, in cells overexpressing Bcl-2 or cells expressing sh-RNA against Beclin 1, the effects of NRG, namely induction of autophagy and cell death, were inhibited. Conclusions/Significance Thus, in LNCaP cells, NRG-induces incomplete autophagy and cell death that depend on ROS levels. These effects of NRG are mediated by signaling pathway that activates JNK and Beclin 1, but is independent of mTOR inhibition.

Epizootic Hemorrhagic Disease Virus Induces and Benefits from Cell Stress, Autophagy, and Apoptosis

ABSTRACT The mode and timing of virally induced cell death hold the potential of regulating viral yield, viral transmission, and the severity of virally induced disease. Orbiviruses such as the epizootic hemorrhagic disease virus (EHDV) are nonenveloped and cytolytic. To date, the death of cells infected with EHDV, the signal transduction pathways involved in this process, and the consequence of their inhibition have yet to be characterized. Here, we report that the Ibaraki strain of EHDV2 (EHDV2-IBA) induces apoptosis, autophagy, a decrease in cellular protein synthesis, the activation of c-Jun N-terminal kinase (JNK), and the phosphorylation of the JNK substrate c-Jun. The production of infectious virions decreased upon inhibition of apoptosis with the pan-caspase inhibitor Q-VD-OPH (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone), upon inhibition of autophagy with 3-methyladenine or via the knockout of the autophagy regulator Atg5, or upon treatment of infected cells with the JNK inhibitor SP600125 or the cyclin-dependent kinase (CDK) inhibitor roscovitine, which also inhibited c-Jun phosphorylation. Moreover, Q-VD-OPH, SP600125, and roscovitine partially reduced EHDV2-IBA-induced cell death, and roscovitine diminished the induction of autophagy by EHDV2-IBA. Taken together, our results imply that EHDV induces and benefits from the activation of signaling pathways involved in cell stress and death.

Enhancing FTS (Salirasib) efficiency via combinatorial treatment

The Ras oncogene transmits signals, which regulate various cellular processes including cell motility, differentiation, growth and death. Since Ras signalling is abnormally activated in more than 30% of human cancers, Ras and its downstream signalling pathways are considered good targets for therapeutic interference. Ras is post‐translationally modified by the addition of a farnesyl group, which permits its attachment to the plasma membrane. Exploiting this knowledge, a synthetic Ras inhibitor, S‐trans, trans‐farnesylthiosalicylic acid (FTS; Salirasib), was developed. FTS resembles the farnesylcysteine group of Ras, and acts as an effective Ras antagonist. In the present review, the effect of FTS in combination with various other drugs, as tested in vitro and in vivo, and its therapeutic potential are discussed. As reviewed, FTS cooperates with diverse therapeutic agents, which significantly improves treatment outcome. Therefore, combinations of FTS with other agents have a potential to serve as anti‐cancer or anti‐inflammatory therapies.

Nucleolin and ErbB2 inhibition reduces tumorigenicity of ErbB2-positive breast cancer

ErbB2, a member of the ErbB family of receptor tyrosine kinases, is an essential player in the cell’s growth and proliferation signaling pathways. Amplification or overexpression of ErbB2 is observed in ∼30% of breast cancer patients, and often drives cellular transformation and cancer development. Recently, we have shown that ErbB2 interacts with the nuclear-cytoplasmic shuttling protein nucleolin, an interaction which enhances cell transformation in vitro, and increases mortality risk and disease progression rate in human breast cancer patients. Given these results, and since acquired resistance to anti-ErbB2-targeted therapy is a major obstacle in treatment of breast cancer, we have examined the therapeutic potential of targeting the ErbB2–nucleolin complex. The effect of the nucleolin-specific inhibitor GroA (AS1411) on ErbB2-positive breast cancer was tested in vivo, in a mouse xenograft model for breast cancer; as well as in vitro, alone and in combination with the ErbB2 kinase-inhibitor tyrphostin AG-825. Here, we show that in vivo treatment of ErbB2-positive breast tumor xenografts with GroA reduces tumor size and leads to decreased ErbB2-mediated signaling. Moreover, we found that co-treatment of breast cancer cell lines with GroA and the ErbB2 kinase-inhibitor tyrphostin AG-825 enhances the anti-cancer effects exerted by GroA alone in terms of cell viability, mortality, migration, and invasiveness. We, therefore, suggest a novel therapeutic approach, consisting of combined inhibition of ErbB2 and nucleolin, which has the potential to improve breast cancer treatment efficacy.

The Interplay Between Apolipoprotein E4 and the Autophagic–Endocytic–Lysosomal Axis

Since its discovery as a genetic risk factor for Alzheimer’s disease, the APOE4 allele has been linked to the majority of the pathological findings associated with the disease progression. These include abnormalities of the endocytic, autophagic, and lysosomal machineries, which begin at the most early stages of Alzheimer’s disease development. Considering that these three vesicular systems share common features and, in fact, comprise an interconnected cargo-trafficking and degradation network, some of the effects of APOE4 are interrelated, while others are system-specific. In turn, APOE4-driven impairments of endocytosis, autophagy, and lysosomal activity influence various aspects of Alzheimer’s disease pathology, ranging from Aβ generation and clearance to neuronal loss and cognitive deficits. This review discusses the detrimental effects of APOE4 on the endocytic–autophagic–lysosomal axis in the context of Alzheimer’s disease, as well as the various mechanisms underlying them.

Continuous treatment with FTS confers resistance to apoptosis and affects autophagy.

High percentage of human cancers involves alteration or mutation in Ras proteins, including the most aggressive malignancies, such as lung, colon and pancreatic cancers. FTS (Salirasib) is a farnesylcysteine mimetic, which acts as a functional Ras inhibitor, and was shown to exert anti-tumorigenic effects in vitro and in vivo. Previously, we have demonstrated that short-term treatment with FTS also induces protective autophagy in several cancer cell lines. Drug resistance is frequently observed in cancer cells exposed to prolonged treatment, and is considered a major cause for therapy inefficiency. Therefore, in the present study, we examined the effect of a prolonged treatment with FTS on drug resistance of HCT-116 human colon cancer cells, and the involvement of autophagy in this process. We found that cells grown in the presence of FTS for 6 months have become resistant to FTS-induced cell growth inhibition and cell death. Furthermore, we discovered that the resistant cells exhibit altered autophagy, reduced apoptosis and changes in Ras-related signaling pathways following treatment with FTS. Moreover we found that while FTS induces an apoptosis-related cleavage of p62, the FTS-resistant cells were more resistant to apoptosis and p62 cleavage.

Chapter 3 – Inhibition of ErbB Receptors and Autophagy in Cancer Therapy

Autophagy is a process that regulates the balance between synthesis of proteins and organelles and their degradation. Thus, autophagy can be viewed as a cell survival mechanism, which also maintain cell homeostasis. Unregulated or defective autophagy is linked to various pathologic conditions, including neurodegenerative diseases and cancer. Members of the ErbB family of transmembrane receptor tyrosine kinases are involved in progression of several types of human cancers. Amplification, overexpression, and activating mutations of ErbBs are associated with reduced survival. Therefore, ErbB directed therapies are often used to treat cancer patients with amplified ErbB activation. In this chapter, we discuss the link between autophagy and ErbB receptor tyrosine kinases, with special emphasis on drugs targeting these oncogenes and their impact on autophagy.