Elda Grabocka: Stress-buffering comes in granules

Elda Grabocka investigates the role of stress granules in obesity and cancer.

When one thinks of high school, sharing hallways with students from 80 different countries is not the usual image that springs to mind. This was indeed Elda Grabocka’s experience. She grew up in Pogradec, a remote town in Albania—her parents, both physicians, were assigned to this location by the state. Elda won one of the two spots available for Albanian students in a national competition to attend the United World College of the Adriatic in Trieste, Italy, a high school focused on social change that brings together students from around the globe to promote intercultural understanding. Elda still remembers, with a smile on her face, the first glimpse at the laboratories as the senior students were working on their thesis projects: “That was exactly what I wanted to do!” She barely spoke English at the time and had to catch up to the level of her peers, but her perseverance and passion prevailed, and she obtained the International Baccalaureate Diploma (IBD). For the independent study of the IBD program, she submitted a research project in chemistry, which ended up being an important learning and life lesson: “That helped me understand that I was more suited to biology! In hindsight, it was great to have that experience so early; I certainly had no awareness then how essential failing and then learning from your failures is to science, but having a level of comfort with it from the beginning was probably a bonus.”Elda Grabocka. Photo courtesy of Chris Hamilton Photography.But science was not the only professional option Elda contemplated—her volunteering experience with relief organizations in various refugee camps made her consider a career in public health and humanitarian relief efforts. She finally sought a PhD degree in molecular pharmacology and structural biology in the laboratory of Phil Wedegaertner at Thomas Jefferson University. After studying heterotrimeric G-proteins and how the subcellular localization of their exchange factors regulates function, Elda felt the need to seek greener pastures. She went on to do a postdoc on one of longest-studied oncogenes, RAS—her choice wasn’t motivated by the field, but by the mentor, Dafna Bar-Sagi. Elda’s admiration for Dafna is notable when she speaks about her time at the New York University Langone Medical Center: “It’s remarkable how many novel aspects of RAS biology that have shaped and then re-shaped our thinking about this oncogene have come out of her lab; I felt there was a depth and breadth to her approach to scientific research that if I could learn, I’d be able to see more of the angles, so to speak, ask better questions; she has really expanded my mind in all those aspects.” Elda’s work focused on the interplay between the mutated forms of RAS and the wild-type isoforms, which she and others have shown is context dependent, with the wild-type isoforms acting as both tumor suppressors and tumor promoters (1). While still in Dafna’s laboratory, Elda pursued a more independent scientific interest: the role of stress granules in mutant KRAS cells. In 2016, Elda returned to her alma mater, joining the Department of Cancer Biology at the Sidney Kimmel Cancer Center at Jefferson as an assistant professor, with stress granules in cancer as the focus of her laboratory. We contacted her to learn more about her research journey.What interested you about stress granules and their connection with obesity and cancer?I became interested in stress granules and their potential role in cancer early in my postdoc. I read a review by Stephen Elledge’s group where they described the “stress phenotype” of cancer as an important player in tumorigenesis. I realized that cancer cells exist mostly in a state of stress—for example, mutated genes, like oncogenic RAS, are potent inducers of many types of cellular stresses. I was working on a RAS ubiquitination project, and one of the candidates for a RAS de-ubiquitinating enzyme we were looking at was implicated in stress granule formation. Little was known about stress granules at the time—they are induced by types of stresses associated with tumors (hypoxia, oxidative stress, osmotic pressure, proteotoxic stress, metabolic stress, etc.), so the question I asked was whether stress granules could function as a stress coping/adaptation mechanism in cancer. Indeed, I found that stress granules are prevalent in tissues from patients with pancreatic cancer and mouse models of pancreatic cancer. Remarkably, not all cancer cells are the same in their capacity to form stress granules—all cells will make stress granules under stress, but KRAS mutant cancer cells have a heightened ability to do so because signaling from mutant KRAS enhances the levels of a critical molecule to stress granule formation, 15-deoxy-prostaglandin J2 (2). This enhanced capacity to make stress granules, in turn, renders KRAS mutant cells more resistant to stress and more dependent on stress granules; inhibition of stress granules leads to increased cell death in KRAS mutant versus KRAS wild-type cancer cells.Immunofluorescence staining of pancreatic ductal adenocarcinoma tissue showing cancer cells in red, stress granules in green, and nuclei in blue. Image courtesy of the Grabocka laboratory.The work establishing this dependence was in vitro, so the primary goal when I started my laboratory was to determine their relevance in tumorigenesis, which led me to explore their connection to obesity and cancer for several reasons. First, obesity is a major predisposing factor for several cancers, including pancreatic and colon, which are prevalent KRAS-driven cancers for which treatment options are limited. Second, obesity is a complex pathology which likely impacts the pathobiology, the therapy response, and even the evolution of cancers that arise in this setting. Given that cell stress and inflammation are key features in obesity, this would make the ideal background to study the contribution of stress granules in tumorigenesis. I think this pre-existing stress [obesity] might necessitate the engagement of stress adaptive mechanisms from the early stages of tumorigenesis and may also lead to a high dependence on these processes.What are you currently working on, and what is up next for you?It’s a very exciting time to be working on stress granules! The field has grown significantly over the past 10 yr or so, especially with the renewed interest in phase separation. As organelles that form via phase separation when a cell is under stress, stress granules are perhaps one of the best examples of phase separation in vivo and a great platform to understand its relevance. The recent advances in defining the composition, as well as key molecular drivers and their functional domains in stress granule assembly, have been of great benefit. We are now better positioned to define the stress granule–specific functions in health and disease. Because stress granules are induced by various types of stresses, they could function as a pan-stress adaptation mechanism in cancer. This is a very appealing angle, as if we can solve how stress granules enable stress adaptation, which is a major focus of my laboratory, we could have better anti-cancer therapies.The composition of stress granules, comprising hundreds of proteins and mRNAs involved in several aspects of cell biology, prompted me to ask whether cytoprotection under stress is their main and/or only function. What other cellular processes stress granules regulate, whether these vary with the type of stress, and how such processes are integrated into the stress response of cancer cells are burning questions we are currently working on, as the answers will advance our understanding of the role of stress granules in cancer. The “chronic stress” of cancer is heterogenous in both spatial and temporal terms, as well as in the type of stress and intensity. I am also very curious to see if and how heterogeneity in stress stimuli impact the composition of stress granules and the processes they regulate, and how this may affect tumor evolution. Also, cancer cells are not the only cells in the tumor that make stress granules. As a matter of fact, we reported that KRAS mutant cells can stimulate stress granule formation in a paracrine manner. An ongoing project in the laboratory that I’m very excited about is focused on understanding the contribution of stress granules to the pro-tumorigenic microenvironment.What kind of approach do you bring to your work?My approach is very hypothesis and observation driven; the latter in the sense that it can often be that initial spark that inspires an idea, draws connections, and looks for context and meaning. I also find that sometimes the answer to my next question or the question I don’t know to ask yet is hidden right in front of my eyes, so paying careful attention to the data is key. It is also where objective and critical evaluation of experimental results starts. There’s one line that’s firmly ingrained in my mind from my postdoctoral training, which is “Science is self-correcting.” It’s a note of caution that if you don’t pay attention and see only what you want to see, it will still eventually prove you wrong, and you’d have wasted a lot of time in the process. So I try to minimize that waste as much as possible—unavoidable entirely, having a favorite hypothesis is part of the scientific thinking process, but crucial to remember to follow the data and not just convince yourself.What has been the biggest accomplishment in your career so far?I’m still quite early in my career to start listing accomplishments. I feel privileged to do the work I do; I essentially get funded to pursue ideas that I find interesting. So I have a hard time with this question because it has a hint of pride, and when you start adding pride to privilege, as a junior principal investigator especially, it gets a bit too self-serving. I hope that the work we are doing stands the test of time and leads to or helps lead to a meaningful impact on patients’ lives—that would be a great accomplishment.What has been the biggest challenge in your career so far?The past two years of COVID have certainly been a different reality, and a constantly shifting one at that. From a career perspective, so much of a scientific career happens at the bench: experiments happen at the bench, we train at the bench, animal work is long and requires multiple dedicated essential personnel and facilities, so inevitably, remote work, or shift work, limited occupancy, and the shortages we are now seeing in the supply chain have been a major challenge for everyone. I do think junior laboratories like mine experience that a bit harder. The bandwidth to absorb these challenges is much smaller if you’re just starting out, or if you’ve had a laboratory for a couple of years and are just ramping up. I must say though that it has made for stronger teamwork in the laboratory, and we’ve had to be really focused and efficient—so there’s an upside!Out for a paddle. Photo courtesy of Elda Grabocka.Any tips for a successful research career?Hard to say, because certainly it means different things to different people. The only tip I would give perhaps is to define what that means, what that success looks like for oneself, and be true to that. I expect how each one defines it also changes with time and experience, but I do think it’s very important to identify what success means as early as possible and let that be what you measure your efforts against. It’s easy to get distracted, overwhelmed, or even disheartened otherwise. My own definition is quite simple: success is doing what I love to do, working toward answering a meaningful scientific question, and enabling/supporting my trainees to reach their potential—keeping that in mind has been very important and helpful.     

Extracellular matrix regulates the amount of the β-amyloid precursor protein and its amyloidogenic fragments

We have studied the influence of the extracellular matrix (ECM) on the amount of β-amyloid precursor protein (APP) and C-terminal amyloid-bearing fragments in 3T3 fibroblasts. After incubation with ECM components, the cellular APP content of 3T3 cells changed. Besides, different substrata including collagen, fibronectin, laminin, vitronectin, and heparin, determined changes in the amount of a C-terminal 22 kDa-fragment. The regulation of amyloidogenic fragments by the ECM was transient; in fact, when 3T3 cells were plated on tissue culture dishes coated with collagen or vitronectin, maximal levels of the 22 kDa fragment were observed 12 h after plating; in the presence of fibronectin, the maximum level of the amyloidogenic fragment was obtained 36 h after plating. These results indicate that the ECM modulates in a transient way the generation of APP-derived polypeptides containing the amyloid-β-peptide (Aβ). The ECM does not have a generalized effect on 3T3 fibroblasts, because no significant differences in cell attachment, growth rate, whole-cell polypeptide pattern, β₁ integrin and α-tubulin levels were observed on cells grown on various matrix proteins. Laminin, collagen, and heparin also influence the level of an amyloidogenic fragment of 35 kDa in Neuro 2A neuronal cells, without a significant change in the neuronal marker acetylcholinesterase. In this case, however, a long-lasting response to ECM molecules was observed. These observations provide evidence that ECM molecules influence APP biogenesis, including the generation of amyloidogenic fragments containing the Aβ peptide. Our studies might prove significant to understand the localized increment of β-amyloid deposition in selected areas of the brain of Alzheimer's patients. © 1996 Wiley-Liss, Inc.     

The auto-orientation in high magnetic fields of oxidized cytochrome b562 as source of constraints for solution structure determination

¹⁵N-¹H ¹J couplings were measured at 500 MHz and 800 MHz for ¹⁵N enriched oxidized cytochrome b ₅₆₂ from E. coli. The magnetic field dependence of 70 ¹J values, which could be measured without signal overlap, shows that there is a molecular magnetic anisotropy which provides partial molecular orientation in the magnetic field and, consequently, residual dipolar couplings (rdc). The rdc were used as further constraints to improve the existing structure [Arnesano et al. (1999) Biochemistry, 38, 8657–8670] with a protocol which uses the rhombic anisotropy [Banci et al. (1998) J. Am. Chem. Soc., 120, 12903–12909]. The overall large molecular magnetic anisotropy has been found to be determined by both the low spin iron (III) and the four helix bundle structure magnetic susceptibility anisotropy contributions.     

Storage globulins in Gnetopsida. 2. A serotaxonomic study of legumin-like proteins

Abstract

Legumin-like proteins from Ephedra distachya, Ephedra foeminea, Gnetum gnemon, Gnetum montanum and Welwitschia mirabilis and four Angiosperms (Magnolia grandiflora, Pisum sativum, Glycine max and Cucurbita moschata) were used in a serological investigation. The immunological tests demonstrate the heterogeneity of Gnetopsida and indicate discriminative correlations of Welwitschia, Gnetum and Ephedra to the examined Angiosperms.     

Influence of fatty acid‐free diet on mammary tumor development and growth rate in HER‐2/neu transgenic mice

Numerous investigations have found a relationship between higher risk of cancer and increased intake of fats, while results of clinical studies of fat reduction and breast cancer recurrence have been mixed. A diet completely free of fats cannot be easily administered to humans, but experimental studies in mice can be done to determine whether this extreme condition influences tumor development. Here, we examined the effects of a FA‐free diet on mammary tumor development and growth rate in female FVB‐neu proto‐oncogene transgenic mice that develop spontaneous multifocal mammary tumors after a long latency period. Mice were fed a fatty acid‐free diet beginning at 112, 35, and 30 days of age. In all these experiments, tumor appearance was delayed, tumor incidence was reduced and the mean number of palpable mammary tumors per mouse was lower, as compared to standard diet‐fed mice. By contrast, tumor growth rate was unaffected in mice fed the fatty acid‐free diet. Plasma of mice fed the fatty acid‐free diet revealed significantly higher contents of oleic, palmitoleic and 20:3ω9 acids and lower contents of linoleic and palmitic acids. In conclusion, these findings indicate that a FA‐free diet reduces tumor incidence and latency but not tumor growth rate, suggesting that a reduction in dietary FAs in humans may have a protective effect on tumorigenesis but not on tumors once they appear. J. Cell. Physiol. 228: 242–249, 2013. © 2012 Wiley Periodicals, Inc.     

Task Performance of Midden Workers of Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae)

Material brought to midden piles of leaf-cutting ants is considered to be hazardous. It is therefore expected that midden workers should not re-enter the colony, to reduce pathogen transmission. Here, we examined whether the midden workers of Atta sexdens rubropilosa remain confined to the waste compartment and, if not, whether they could perform many different behaviors in the absence of specialized nestmates. Eleven subcolonies received either midden workers or non-midden workers in addition to pupae and 50 mL of fungus garden. Survival, fungal and brood care, foraging and waste manipulation were observed daily until subcolonies death. Subcolonies maintained by midden workers died earlier: average survival times were 13.92 and 22.66 days for midden and non-midden workers, respectively. Midden workers cared for the brood and foraged as non-midden workers did but they were not as efficient in caring for the garden. Activities related to waste manipulation were more frequently performed by midden workers. These results show that midden workers are not a behaviorally rigid caste and retain many labor capabilities being able to leave the waste compartment and perform internal activities in the absence of specialized internal workers.     

Visually inexperienced chicks exhibit spontaneous preference for biological motion patterns

When only a small number of points of light attached to the torso and limbs of a moving organism are visible, the animation correctly conveys the animal's activity. Here we report that newly hatched chicks, reared and hatched in darkness, at their first exposure to point-light animation sequences, exhibit a spontaneous preference to approach biological motion patterns. Intriguingly, this predisposition is not specific for the motion of a hen, but extends to the pattern of motion of other vertebrates, even to that of a potential predator such as a cat. The predisposition seems to reflect the existence of a mechanism in the brain aimed at orienting the young animal towards objects that move semi-rigidly (as vertebrate animals do), thus facilitating learning, i.e., through imprinting, about their more specific features of motion.