Research Center

Systems Analysis of Phenotypic Switch in Control of Cancer Invasion

Overview

Center Title

Systems Analysis of Phenotypic Switch in Control of Cancer Invasion

Center Website

http://levchenkolab.yale.edu/

Center Summary

Aerial view of Yale’s West Campus, which is where the Yale Systems Biology and Yale Cancer Institutes are located.

Over 90% of cancer related mortality is linked to invasive and metastatic spread of cancer cells from the primary tumor. In spite of the crucial importance of invasive cancer phenotype, we still have only fragmentary knowledge and understanding of the mechanisms leading to transition from proliferative to aggressive, migratory behavior of cancer cells, which we refer to as Proliferative-to-Aggressive phenotype switching. Increasing evidence suggests that this switch is a reflection of the inherent capacity of cancer cells to adopt both proliferative and migratory phenotypes, with the probability and rate of switching between these two phenotypes controlled by the cell genome, environmental conditions, and cell-cell interactions. The Cancer Systems Biology at Yale ([email protected]) will address the problem of regulation of invasive cancer spread and, more specifically, the Proliferative-to-Aggressive phenotypic switch. A combination of diverse expertise and innovative methods, ranging from synthetic biology, micro- and nano-fabrication technology, evolutionary biology, and mathematical analysis of intracellular molecular networks to the creation of novel CRISPR-based animal models and the design of novel kinase inhibitors that can lay the basis for novel therapeutic compounds will be used.

The Yale Systems Biology Institute features open laboratories (depicted here), dedicated support rooms, shared equipment rooms, and interactive office and community space.

[email protected] brings together researchers from seven Yale departments based at Yale schools of Arts and Sciences, Engineering and Applied Science, and Medicine and at Emory University, in close collaboration with Yale Cancer Institute, Yale Cancer Center, Yale skin cancer SPORE, and Yale Neurosurgery department. The work at [email protected] will be initially based on the tightly knit two Research Projects and two support shared resource Cores, first focused on the analysis of glioblastoma and melanoma cells, and normal cells of various species modeling invasive growth behavior and phenotypic switching. The work will be supported by the Administrative Core and the results disseminated through various mechanisms mediated by the Outreach and Education Core. The orthogonal and unconventional approaches, characteristic of the highly collaborative use of cutting edge, innovative approaches, will provide an opportunity to advance our understanding of the molecular networks controlling invasive, aggressive cancer spread and lead to new approaches to controlling and treating highly invasive and metastatic malignancies.

Investigators

Andre Levchenko, Ph.D.

Dr. Andre Levchenko is the Principal Investigator of [email protected] He is John C. Malone Professor of Biomedical Engineering at Yale and is also the Founding Director of Yale Systems Biology Institute (YSBI), whose major unifying activity will be centered on this Project. Dr. Levchenko has been the director of YSBI since Fall 2013, guiding it through a 2-fold expansion, consolidation in a new space, and establishment of vigorous research experience on a new campus. Dr. Levchenko is a systems biologist, bio-engineer, and biophysicist who has been among the active developers of Systems Biology as a discipline since its most recent emergence, and has made considerable impact on the integrative computational/experimental analysis of cellular signaling and cell-cell communication. His more recent efforts are in development of novel micro- and nano-fabricated devices for enhanced control of cellular micro-environments. His work on understanding of the innate immune response and mechanisms of cell migration has received wide acclaim (with the latter leading to his election as a Fellow of the American Physical Society earlier this year). His current focus is on understanding of the mechanisms of aggressive cell migration in diverse settings and in response to different cues. This research emphasis created the platform for unifying the efforts of multiple Yale researchers.

Mark Lemmon, Ph.D.

Dr. Mark Lemmon is a cellular and molecular biologist and pharmacologist, is Co-Director of the Yale Cancer Biology Institute, and David A. Sackler Professor of Pharmacology. The Lemmon laboratory has made significant contributions to understanding molecular mechanisms of cellular signaling by growth factor receptor tyrosine kinases – notably the EGF receptor – and through lipid second messengers, notably the phosphoinositides and other anionic lipids that bind pleckstrin homology (PH), FYVE, KA1, and other domains. The Lemmon lab made highly significant and high impact contributions to the structural understanding of both of these phenomena, and to understanding these signaling axes from structural/mechanistic perspectives – combining cellular, biochemical, structural, and (increasingly) organismal studies. Recently, a key goal of the laboratory has been to understand how mutational alterations of receptor tyrosine kinases, in particular the therapeutic targets EGFR and ALK, lead to aberrant signaling and cancer – and how this information can be used to impact clinical approaches (through direct collaboration with clinicians). The goal is to bring more biochemistry to personalized medicine in oncology. Another key aim of work of the Lemmon laboratory is to understand signaling systems and networks more broadly by studying the signaling consequences of molecular perturbations that are well defined (through the lab’s in vitro and structural work). This has been applied to studies of the role of the EGF receptor in Drosophila development, and is now being pursued in several cancer cell contexts.

Günter Wagner, Ph.D.

Dr. Günter Wagner is an evolutionary biologist studying the evolution of the mammalian female reproductive tract, and other aspects of the evolution of development. He is Alison Richard Professor of Ecology and Evolutionary Biology and a core faculty member in the Yale Systems Biology Institute. His laboratory focuses on the evolution of gene regulation and the evolutionary origin of the uterine decidual cell, a cell type critical for the implantation of the fetus and the maintenance of pregnancy. One of the main functions of the decidual cell is to regulate the invasion of the fetal trophoblast and thus the regulation of invasive behavior. Last year Dr. Wagner, together with a student Alaric D’Souza, published a paper that documented a correlation between the (secondary) evolution of non-invasive placentation and a lower rate of malignancy in certain forms of cancer. This observation motivates his participation [email protected], where he will investigate the mechanisms by which endometrial stromal cells oppose the invasion of the fetus and how, in bovines and related species, this may also have conveyed resistance to melanoma malignancy.

Murat Acar, Ph.D.

Dr. Murat Acar is an Assistant Professor in Molecular, Cellular and Developmental Biology and in Physics, and is a core faculty member of the Yale Systems Biology Institute. He is a systems biologist who combines experimental and computational tools in his research by using the budding yeast as a model organism. Trained as a physicist, Dr. Acar is interested in understanding and solving the complexity in the organization of natural gene networks by using a reductionist approach. Since even the most complex gene networks can be broken down to modular pieces operating as locally-embedded network motifs in the larger gene networks, Dr. Acar’s research aims to characterize the function and evolution of these frequently-occurring modular network motifs. He has many years of hands-on experience in yeast genetics, cell biology, and computational modeling. Dr. Acar will lead the research efforts aiming at characterizing the evolution of phenotypic switching rates in the yeast galactose network during cellular adaptation to fluctuating environments.

Jesse Rinehart, Ph.D.

Dr. Jesse Rinehart is an Assistant Professor in the Department of Cellular & Molecular Physiology at the Yale University School of Medicine and also a core faculty member in the Yale Systems Biology Institute. He is a physiologist and proteomics expert. Dr. Rinehart studied protein synthesis and the evolution of the genetic code during his graduate work and completed his postdoctoral training focused on protein phosphorylation in physiological systems. Dr. Rinehart’s research aims to understand and “decode” complex signaling networks in physiological systems. Researchers in the Rinehart laboratory use an innovative combination of quantitative phosphoproteomics and synthetic biology to achieve their aims. They are now applying this technology to understand the properties of phosphoserine in human kinases and singling networks. Dr. Rinehart’s team will deploy these enabling technologies to advance our understanding of how kinases control aggressive cancers and specifically to identify small molecule kinase inhibitors to target human cancers.

Farren Isaacs, Ph.D.

Dr. Isaacs is an Associate Professor in the Department of Molecular, Cellular and Developmental Biology and a core faculty member in the Systems Biology Institute at Yale. He is a synthetic biologist focused on developing foundational genomic and cellular engineering technologies to understand and engineer biological systems. His lab integrates engineering and evolution through the construction of genes, networks and genomes alongside quantitative models to gain a better understanding of biological systems. In turn, they utilize these insights to program and evolve organisms with new biological function. A particular emphasis is the development of genome engineering technologies to construct ‘Genomically Recoded Organisms’ that possess an alternate genetic code. In the context of [email protected], re-coded organisms improve properties for incorporating nonstandard amino acids into proteins, including the site-specific incorporation of phosphoserine to activate human kinases in engineered bacteria. Dr. Isaacs’ work focuses on incorporating phosphorylated amino acids to enable the production of active kinases implicated in the tumorigenesis of glioblastomas. These efforts will be extended in three key areas: (1) to recapitulate phosphorylation-based mammalian signaling in recoded bacteria, (2) to elucidate biomolecular interactions of kinase functions, and (3) will be leveraged to identify novel inhibitors of kinase activity.

Michael Murrell, Ph.D.

Dr. Michael Murrell, an Assistant Professor in the Department of Biomedical Engineering and a core faculty member in the Systems Biology Institute at Yale, is a biophysicist whose primary expertise is in mechanical force production in cell biology. His laboratory develops approaches to understanding mechanical force generation by two distinct but complementary approaches. First, he measures the mechanical stresses produced by living cells subject to genetic and pharmacological perturbation. In doing so, he identifies how the dynamics and composition of the cell cytoskeleton and cell membrane influence the generation and transmission of mechanical stresses to the extracellular matrix. In parallel, he designs and engineers artificial cells in vitro, using purified proteins and lipids to reconstruct the cell cytoskeleton and membrane, for the goal of reproducing force production in a simplified system. As there are few components, and no biochemical regulation, this allows him to isolate the role of individual components, whose abundance and activity can be easily manipulated. To this end, Dr. Murrell has developed precise control of physical variables that can influence the generation and transmission of mechanical stresses, including cell volume, F-actin organization, membrane tension, fluid pressure and adhesion. From this, his lab learns basic physical principles regarding how cells produce mechanical force, which may otherwise be obfuscated by the overlapping biochemical and genetic regulation that exits in living cells. Thus, their simplified systems can inform us as to the basic physical relationships that underlie mechanical force production in cells, as well as provide potential targets for genetic and pharmacological perturbations. Dr. Murrell will seek to identify the role of NKCC1 in mediating mechanical force production in invasive glioblastoma and melanoma cells, by exploring its role in coupling to the cytoskeleton, as well as modulating fluid flow, hydrostatic pressure, and the volume of the cell.

Rong Fan, Ph.D.

Dr. Rong Fan is an Associate Professor of Biomedical Engineering and a microfabrication and microfluidics expert at Yale University. He was trained as an analytical and materials chemist before becoming a trainee of the NCI-founded Nano Systems Biology Cancer Center at Caltech, where he developed a high-density antibody barcode chip for multiplexed protein biomarker detection in microliters of blood or even single cells. His research interest is focused on the development of microfluidic systems and bioMEMS technology for multiparameter analysis of single cells and cell-cell paracrine signaling. His laboratory developed a high-throughput microchip technology for single cell, 42-plex cytokine profiling that was utilized to quantify the deep functional heterogeneity in human immune cells in response to pathogenic stimulation and in hematologic cancer cells. It was also used to quantify paracrine communication between cancer and immune cells. His laboratory is also working on the development of other microfluidic devices for single-cell functional genomics analysis tools (e.g., methylomics, transcriptomics, etc). He is the recipient of the Howard Temin Pathway to Independence award (K99/R00) from the National Cancer Institute, the NSF Early Stage Faculty Career Development (CAREER) Award and the Packard Fellowship for Science and Engineering. In this project, Dr. Fan provides support in microdevice design and single-cell proteomic assay for investigating cell-cell interaction and offers training in microfluidics techniques to researchers in the Center and local community for outreach activities.

Sidi Chen, Ph.D.

Dr. Sidi Chen is a geneticist, who is an Assistant Professor of Genetics and a core faculty member of the Systems Biology at Yale. His current research focuses on cancer systems biology, in particular in vivo CRISPR/Cas9-mediated cancer modeling and genetic screening. He has led studies on the essential function of new genes in animal development and tumorigenesis, microRNA regulation of tumor hypoxia and angiogenesis, in vivo modeling of lung cancer and liver cancer using gene editing, and genome-wide screens for metastasis regulators. Dr. Chen will lead the studies aimed at validation of the role of molecular components predicted by Research Projects to play key roles in promoting or suppressing invasive cancer spread.

Lynne Regan, Ph.D.

Dr. Lynne Regan is a biophysicist and biochemist. She is Professor of Molecular Biophysics and Biochemistry and of Chemistry and is an active member of the Yale Cancer Center. Her research investigates novel strategies by which to inhibit Hsp90 as a route to a new class of anti-cancer agents. She is also studying the multifaceted roles of heat shock factor-1 (HSF1), the mechanisms of HSF1 activation, and the role of HSF1 in cancer. She is Director of the Raymond and Beverley Sackler Institute for Biological, Physical and Engineering Sciences (RBSI), Director of the Integrated Graduate Program in Physical and Engineering Biology (IGPPEB) and co-Director of the NSF REU Site: Convergence of Research at the Interface of the Biological, Physical, and Engineering Sciences. The IGPPEB brings students with backgrounds in the physical and biological sciences into educational and research programs that apply quantitative physical approaches to key questions in biology. Students entering through the IGPPEB program will be well prepared to perform research in any [email protected] laboratory. The RBSI supports multiple activities, for researchers at all levels, to enhance interdisciplinary research at Yale. Institute activities and events are synergistic with [email protected] plans and Dr. Regan’s involvement with both will enhance this connection. The NSF-REU site that Regan co-directs has proven to be an effective route by which to encourage undergraduates from under-represented and disadvantaged groups to embark upon STEM careers. 75% of students who have participated are now enrolled in Ph.D. or M.D./Ph.D. programs.

Adam Marcus, Ph.D.

Dr. Adam Marcus, an Associate Professor of Hematology and Medical Oncology at Emory University School of Medicine, is a cancer biologist who focuses on understanding how cancer cells invade using a combination of molecular and imaging-based approaches. He has numerous publications studying the mechanisms of cancer cell invasion and metastasis that incorporates clinical and pre-clinical studies. His laboratory has developed a new image-guided genomics approach to dissect tumor cell heterogeneity in the context of cancer cell invasion. This work has revealed a symbiotic mechanism for cancer cell invasion and has led to generation of novel cell lines to probe the biology of cell:cell communication. This work will be used to generate a portion of the data related [email protected] research. Dr. Marcus serves as the Director of the Emory Integrated Cellular Imaging Core, which houses 17 microscopes throughout Emory’s campus and is part of the NCI-Designated Winship Cancer Institute. Dr. Marcus also serves as the Director of Graduate Studies for the Cancer Biology Ph.D. program at Emory, and founded the K-12 STEM outreach organization Students for Science. Dr. Marcus will enable the development and use of the SAGA methodology to explore the homotypic cell-cell communication.

Kshitiz Gupta, Ph.D.

Dr. Kshitiz Gupta is a bioengineer who is an Associate Research Scientist in the Department of Biomedical Engineering at Yale University. Dr. Gupta has broad expertise in mechanobiology, and studying intercellular communication. He has a B.Tech. in Computer Science and a Ph.D. in Biomedical Engineering, and served as the founder and Chief Scientific Officer of CardiacMimetics, a startup company based on detecting cardiotoxic drugs during drug development. Dr. Gupta is developing techniques to study cell-cell interactions, including platforms to measure collective cell invasion, and cellular intercommunication.

Anatoly Kiyatkin, Ph.D.

Dr. Anatoly Kiyatkin is a Research Scientist in the Department of Pharmacology and Cancer Biology Institute at the Yale University School of Medicine. He is a systems biologist working in the Lemmon lab on the projects that are focused on the analysis of the dynamics of receptor tyrosine kinase-mediated cell signaling networks with a goal to understand design principles of regulatory network structures that are crucial for network function and cell fate decisions. To reconstruct signaling routes from receptors at the plasma membrane to the activation of MAPK and immediate early genes he uses systematic perturbations and measures activation patterns of signaling proteins. This analysis will help to determine network vulnerabilities in cancerous cells and target them with molecular therapeutics.

Maria Apostolidi, Ph.D.

Dr. Apostolidi is a postdoctoral fellows in the Rinehart lab. Her project will aim to decode the molecular mechanisms of a novel class of kinases thought to control the migration of aggressive cancer cells. She will utilize novel synthetic biology platform technologies developed in the Yale Center to develop new therapeutic strategies designed to arrest cancer cell migration. Dr. Apostolidi trained with Prof. Constantinos Stathopoulos at the University of Patras School of Medicine in the Department of Biochemistry. Her PhD studies focused on the role of aminoacyl-tRNA synthesis in the regulation of ribosomal and exo-ribosomal protein synthesis in pathogens.

Projects

Project 1: Analysis of Cell Autonomous Mechanisms of Phenotypic Plasticity in Invasive Cell Spread

The use of nano-fabricated patterns to mimic in vivo migration of glioblastoma and melanoma cells. Cell shapes and migration patterns on the “tissue-mimetic surfaces” is similar to that found in vivo and inside collagen/laminin gels for patient derived tumor cells (GBM612; MRI of tumor shown). This surface allows faster cells to separate from slower, more proliferative ones, in the process of directed cell migration (see RACE, Project 1). The cell population can be modified, for example by initial transfection with an shRNA library, such that sequential RACE assays can be performed to observe gradual enrichment of specific shRNAs in faster vs. slower cells, which can then be further analyzed biochemically.

The goal of this project is to obtain a better quantitative understanding of the complex regulation and characteristic of the invasive phenotypic state. The analysis in the project is aimed at the cell autonomous view of regulation, without explicit emphasis of cell-cell interactions. We will combine a variety of tools and approaches, including combining experimentation and mathematical modeling.

We are able to separate slow moving, proliferative cells from fast moving, migratory cells (and enrich each phenotype) using a “phenotypic filter” through an assay called Rapid Analysis of Cell migration Enhancement (RACE). This assay employs nano-fabricated surfaces that result in rapid and highly oriented cell migration. Preliminary results using RACE not only suggest that the assay can help identify molecular mechanisms of the Proliferative-to-Aggressive phenotypic switch but have also led us to develop a preliminary mathematical model describing how specific kinases, thought to be activated by the same growth factors, can be differentially active in different cell sub-populations. In addition, using synthetic biology techniques, we have created a new platform for generating novel kinase inhibitors that can be used to, for the first time, target kinases considered to be “difficult” to target due to their orphan status and unclear regulation mechanisms. When possible, the mechanistic significance in cancer invasion and spreading of newly identified and targeted kinases will be examined using biophysical approaches that can determine whether and how cytoskeletally-mediated processes leading to cell migration have been triggered.

 

Project 2: Analysis of Non-Cell-Autonomous (Cell Communication-Dependent) Mechanisms of Phenotypic Plasticity in Invasive Cell Spread

Non-cell autonomous interactions, including both those between cancer cells of different phenotypes (homotypic) and between cancer and stromal cells (heterotypic), will be studied in Project 2. Cancer cells are depicted in shades of red and purple, and stromal cells in shades of white.

Preliminary data from the RACE assay (see Project 1) combined with additional evidence suggests that both cancer cell-cancer cell and cancer cell-stroma interactions are critically important for controlling the Proliferative-to-Aggressive phenotypic switch and the ensuing invasive cancer spread. Therefore, in Project 2, our goal is to identify and analyze novel targets as possible regulators of invasive cancer phenotype, particularly ones involved in cell-cell interactions. The key novelty in this project is in the realization that invasive cancer migration has many similarities with normal, invasive processes occurring in developmental or physiological processes, such as wound healing or development of the placental tissue.

We will examine, in a species-dependent fashion, whether stromal cells exert differential resistance to or cooperation with the invasive cancer spread. A focus will be on the differential expression of specific chemo-attractants and chemo-repellents that are frequently associated with angigogenesis or neuronal migration. We will study the mechanisms regulating communication and putative interaction between cells adopting a more proliferative phenotype with cells adopting a more migratory phenotype. We will use novel techniques and approaches in Project 2 to perturb and validate possible novel targets as regulators of invasive cancer phenotype. Analysis of such targets will be conducted in collaboration with Project 1, since many of the techniques used in these projects are compatible and mutually enriching.

Cores

Core 1: Microfabrication Core

Core 1 will offer two key microfabricated platforms and assays to enable two different measurements. First, to measure at the scale of a single cancer cell, the migration and invasiveness of melanoma and glioblastoma cell populations in response to combinations of external cues. This will enable the RACE assay, analysis of the cell migration in different types of channels, and a new assay aimed at investigation of heterotypic cell communication. Second, to measure a panel of paracrine signals mediating heterotypic cell-cell communication that drive non-autonomous cancer progression.

Core 2: Animal Core

Core 2 will focus on the validation of the molecular targets and mechanisms involved in phenotypic switching between proliferative and invasive cancer phenotypes. This will be achieved using novel CRISPR-based models of cancer invasion and metastasis. This will allow us to explore the progression of disease following genetic perturbation of molecules of interest identified in the RACE assay, for example, in the analysis of homo- and hetero-typic cell interactions. Viral libraries generated by Core 2 will be used to perform in vivo high throughput analysis, aimed at assaying multiple genetic perturbations, ultimately mimicking the RACE assay in vivo. Both animal models and virus libraries will be of immense utility to researchers both at Yale and elsewhere, representing a valuable resource within the Consortium.