Ruth Scherz-Shouval

Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4.

Autophagy is a major catabolic pathway by which eukaryotic cells degrade and recycle macromolecules and organelles. This pathway is activated under environmental stress conditions, during development and in various pathological situations. In this study, we describe the role of reactive oxygen species (ROS) as signaling molecules in starvation‐induced autophagy. We show that starvation stimulates formation of ROS, specifically H2O2. These oxidative conditions are essential for autophagy, as treatment with antioxidative agents abolished the formation of autophagosomes and the consequent degradation of proteins. Furthermore, we identify the cysteine protease HsAtg4 as a direct target for oxidation by H2O2, and specify a cysteine residue located near the HsAtg4 catalytic site as a critical for this regulation. Expression of this regulatory mutant prevented the formation of autophagosomes in cells, thus providing a molecular mechanism for redox regulation of the autophagic process.

Regulation of autophagy by ROS: physiology and pathology

Reactive oxygen species (ROS) are small and highly reactive molecules that can oxidize proteins, lipids and DNA. When tightly controlled, ROS serve as signaling molecules by modulating the activity of the oxidized targets. Accumulating data point to an essential role for ROS in the activation of autophagy. Be the outcome of autophagy survival or death and the initiation conditions starvation, pathogens or death receptors, ROS are invariably involved. The nature of this involvement, however, remains unclear. Moreover, although connections between ROS and autophagy are observed in diverse pathological conditions, the mode of activation of autophagy and its potential protective role remain incompletely understood. Notably, recent advances in the field of redox regulation of autophagy focus on the role of mitochondria as a source of ROS and on mitophagy as a means for clearance of ROS.

p53-dependent regulation of autophagy protein LC3 supports cancer cell survival under prolonged starvation

The p53 tumor suppressor is mutated in a high percentage of human tumors. However, many other tumors retain wild-type (wt) p53 expression, raising the intriguing possibility that they actually benefit from it. Recent studies imply a role for p53 in regulation of autophagy, a catabolic pathway by which eukaryotic cells degrade and recycle macromolecules and organelles, particularly under conditions of nutrient deprivation. Here, we show that, in many cell types, p53 confers increased survival in the face of chronic starvation. We implicate regulation of autophagy in this effect. In HCT116 human colorectal cancer cells exposed to prolonged nutrient deprivation, the endogenous wt p53 posttranscriptionally down-regulates LC3, a pivotal component of the autophagic machinery. This enables reduced, yet sustainable autophagic flux. Loss of p53 impairs autophagic flux and causes excessive LC3 accumulation upon starvation, culminating in apoptosis. Thus, p53 increases cell fitness by maintaining better autophagic homeostasis, adjusting the rate of autophagy to changing circumstances. We propose that some cancer cells retain wt p53 to benefit from the resultant increased fitness under limited nutrient supply.

Oxidation as a Post-Translational Modification that Regulates Autophagy

The toxicity associated with accumulation of reactive oxygen species (ROS) has led to the evolution of various defense strategies to overcome oxidative stress, including autophagy. This pathway is involved in the removal and degradation of damaged mitochondria and oxidized proteins. At low levels, however, ROS act as signal transducers in various intracellular pathways. In a recent study we described the role of ROS as signaling molecules in starvation-induced autophagy. We showed that starvation stimulates formation of ROS, specifically H2O2, in the mitochondria. Furthermore, we identified the cysteine protease HsAtg4 as a direct target for oxidation by H2O2, and specified a cysteine residue located near the HsAtg4 catalytic site as critical for this regulation. Here we focus on Atg4, the target of regulation, and discuss possible mechanisms for the regulation of this enzyme in the autophagic process. Addendum to: Reactive Oxygen Species Are Essential for Autophagy and Specifically Regulate the Activity of Atg4 R. Scherz-Shouval, E. Shvets, E. Fass, H. Shorer, L. Gil and Z. Elazar EMBO J 2007; doi: 10.1038/sj.emboj.7601623

Structure–Activity Relationships for Withanolides as Inducers of the Cellular Heat-Shock Response

To understand the relationship between the structure and the remarkably diverse bioactivities reported for withanolides, we obtained withaferin A (WA; 1) and 36 analogues (2-37) and compared their cytotoxicity to cytoprotective heat-shock-inducing activity (HSA). By analyzing structure-activity relationships for the series, we found that the ring A enone is essential for both bioactivities. Acetylation of 27-OH of 4-epi-WA (28) to 33 enhanced both activities, whereas introduction of β-OH to WA at C-12 (29) and C-15 (30) decreased both activities. Introduction of β-OAc to 4,27-diacetyl-WA (16) at C-15 (37) decreased HSA without affecting cytotoxicity, but at C-12 (36), it had minimal effect. Importantly, acetylation of 27-OH, yielding 15 from 1, 16 from 14, and 35 from 34, enhanced HSA without increasing cytotoxicity. Our findings demonstrate that the withanolide scaffold can be modified to enhance HSA selectively, thereby assisting development of natural product-inspired drugs to combat protein aggregation-associated diseases by stimulating cellular defense mechanisms.

Involvement of LMA1 and GATE-16 family members in intracellular membrane dynamics

Intracellular membrane fusion is conserved from yeast to man as well as among different intracellular trafficking pathways. This process can be generally divided into several well-defined biochemical reactions. First, an early recognition (or tethering) takes place between donor and acceptor membranes, mediated by ypt/rab GTPases and complexes of tethering factors. Subsequently, a closer association between the two membranes is achieved by a docking process, which involves tight association between membrane proteins termed SNAREs. The formation of such a trans-SNARE complex leads to the final membrane fusion, resulting in an accumulation of cis-SNARE complexes on the acceptor membrane. Thus, multiple rounds of transport and delivery of the donor SNARE back to its original membrane require dissociation of the SNARE complexes. SNARE dissociation, termed priming, is mediated by the AAA ATPase, N-ethylmaleimide-sensitive factor (NSF) and its partner, soluble NSF attachment protein (SNAP), in a reaction that requires ATP hydrolysis. In the present review we focus on LMA1 and GATE-16, two low-molecular-weight proteins, which assist in priming SNARE molecules in the vacuole in yeast and the Golgi complex in mammals, respectively. LMA1 and GATE-16 are suggested to keep the dissociated cis-SNAREs apart from each other, allowing multiple fusion processes to take place. GATE-16 belongs to a novel family of ubiquitin-like proteins conserved from yeast to man. We discuss here the involvement of this family in multiple intracellular trafficking pathways.

Monitoring Starvation-Induced Reactive Oxygen Species Formation

Reactive oxygen species (ROS) are potentially harmful to cells because of their ability to oxidize cell constituents such as DNA, proteins, and lipids. However, at low levels, and under tight control, this feature makes them excellent modifiers in a variety of signal transduction pathways, including autophagy. Autophagy was traditionally associated with oxidative stress, acting in the degradation of oxidized proteins and organelles. Recently, a signaling role was suggested for ROS in the regulation of autophagy, leading, under different circumstances, either to survival or to death. To study the effects of ROS on this pathway, one must determine the localization, intensity, kinetics, and essentiality of the oxidative signal in autophagy. Moreover, once characterized, detection and manipulation of ROS formation could be used to monitor and control autophagic activity. In this chapter we discuss methods to examine ROS in the context of autophagy.

Abstract PR07: Mechanisms of stromal reprogramming mediated by heat shock factor 1

For tumors to form, progress and metastasize, they must recruit and reprogram normal cells in their microenvironment into a pro-tumorigenic stroma. Yet little is known about the pathways leading to stromal reprogramming. We hypothesized that such reprogramming would not be mediated by classic oncogenes, since the stroma is relatively genetically stable. Instead we propose that cancer cells hijack normal cytoprotective stress responses, and subvert them to enable stromal reprogramming. We found that Heat-shock factor 1 (HSF1), master regulator of the heat-shock response, plays a crucial role in this process. Across a broad range of human cancers, HSF1 is activated in cancer-associated fibroblasts (CAFs). In early stage breast and lung cancer, high stromal HSF1 activation is strongly associated with poor patient outcome. In fibroblasts co-cultured with cancer cells, HSF1 drives a transcriptional program that supports cancer phenotypes. This program is profoundly different from the transcriptional program it drives in cancer cells or in heat-shocked cells. Here we characterize mechanisms leading to activation of this unique stromal program and describe the contribution of cancer cells to this process. We then explore the stromal HSF1 program in patients. We apply computational and experimental approaches to define independent cancer and stromal HSF1 signatures and highlight the prognostic value of these signatures in human breast cancer. Billions of years of evolution through changing environments led organisms to develop an arsenal of cytoprotective pathways to promote their survival under stressful conditions. Our work provides insights into the ways by which tumors co-opt these normal biological networks to support their survival in the stressful tumor microenvironment. This abstract is also presented as Poster B40. Citation Format: Ruth Scherz-Shouval, Marc L. Mendillo, Giorgio Gaglia, Irit Ben-Aharon, Andrew H. Beck, Luke Whitesell, Susan Lindquist. Mechanisms of stromal reprogramming mediated by heat shock factor 1. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR07.

Abstract C132: Targeting heat shock factor 1 improves the antitumor efficiency of hyperthermia.

The heat-shock response is a powerful transcriptional program which acts genome-wide, not only to restore normal protein folding through the induction of heat shock proteins (HSP), but to re-shape global cellular pathways controlling survival, growth and metabolism. In mammals, this response is regulated primarily by the Heat Shock Factor 1 (HSF1) transcription factor. We have previously shown that HSF1 plays a fundamental role in tumorigenesis, by promoting the survival and malignance of tumor cells, both in tissue culture and in mouse models of cancer [1]. HSF1 exerts its role by activating a unique transcriptional program in the cancer cells [2]. Indeed, increased HSF1 levels, as well as activation of its transcriptional signature, are associated with reduced survival in breast, lung and colon cancer patients [3]. Cancer cells are exquisitely dependent on HSF1 for survival. Exposure to additional stress, such as heat, further increases their dependency on HSF1. Recently we described how translation is linked to HSF1 activation using a derivative of the natural compound rocaglamide [4]. We found that this drug-like inhibitor of translation-initiation inhibits HSF1 and leads to tumor regression in hematopoietic malignancies. Here we combine this compound, or genetic inhibition of HSF1 expression, with focal heat therapy delivered via gold nano rods. We find that inhibiting HSF1 in solid tumors increases the efficiency of hyperthermia as an anticancer treatment. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C132. Citation Format: Ruth Scherz-Shouval, Alexander F. Bagley, Luke Whitesell, Sangeeta N. Bhatia, Susan Lindquist. Targeting heat shock factor 1 improves the antitumor efficiency of hyperthermia. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C132.

Abstract A23: Cell autonomous and nonautonomous activities of heat shock factor 1 support tumor initiation, progression, and metastasis

The heat-shock response is a powerful transcriptional program which acts genome-wide, not only to restore the normal protein folding through the induction of heat shock proteins (HSP), but to re-shape global cellular pathways controlling survival, growth and metabolism. In mammals, this response is regulated primarily by the Heat Shock Factor 1 (HSF1) transcription factor. We have previously shown that HSF1 plays a fundamental role in tumorigenesis, by promoting the survival and malignance of tumor cells, both in tissue culture and in mouse models of cancer [1]. Recently we demonstrated that HSF1 exerts its role by activating a unique transcriptional program in the cancer cells, that is distinct from the one activated during heat shock [2]. In breast cancer and several other types of carcinoma, we found that high HSF1 protein levels and activation of the HSF1-dependent transcriptional program are associated with poor clinical outcome [3]. Here we show that HSF1 is activated not only in the tumor cells, but also in the stromal cells infiltrating the tumor. Examining human patient samples, we find immunohistochemical evidence for activation of HSF1 in the stroma. Using mouse xenograft models and in vitro co-culture we show that HSF1 in the stroma supports tumor cell growth. Finally, expression profiling and analysis of the DNA binding pattern of HSF1 in tumors and in cell culture indicates that stromal HSF1 supports tumorigenesis by activating a unique, stroma-specific transcriptional program. Taken together, our data suggests that HSF1 acts in the cancer cells and in the stroma to activate distinct, yet complimentary transcriptional programs that will facilitate tumor initiation, progression and metastasis. References 1. Dai, C., et al., Heat shock factor 1 is a powerful multifaceted modifier of carcinogenesis. Cell, 2007. 130(6): p. 1005-18. 2. Mendillo, M.L., et al., HSF1 drives a transcriptional program distinct from heat shock to support highly malignant human cancers. Cell, 2012. 150(3): p. 549-62. 3. Santagata, S., et al., High levels of nuclear heat-shock factor 1 (HSF1) are associated with poor prognosis in breast cancer. Proc Natl Acad Sci U S A, 2011. 108(45): p. 18378-83. Citation Format: Ruth Scherz-Shouval, Sandro Santagata, Martina Koeva, Luke Whitesell, Susan Lindquist. Cell autonomous and nonautonomous activities of heat shock factor 1 support tumor initiation, progression, and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A23.