Eshel Ben-Jacob

MicroRNA-based regulation of epithelial–hybrid–mesenchymal fate determination

Significance Epithelial–mesenchymal transitions play crucial roles in embryonic development, wound healing, and cancer metastasis. It is clearly of interest to quantitatively understand the core circuitry that takes input from the cell’s environment to perform this cell-fate decision. We devised a unique model of the microRNA (miR)-based coupled chimeric modules underlying this core circuit; this effort utilizes a unique theoretical framework for treating miR dynamics. We show that the miR-200/ZEB module functions as a ternary switch, allowing not only for the epithelial and mesenchymal phenotypes but also for a hybrid phenotype with mixed characteristics of collective cell migration, as seen in branching morphogenesis and wound closure. Our results provide valuable clues on how to control and reverse epithelial–hybrid–mesenchymal transitions. Forward and backward transitions between epithelial and mesenchymal phenotypes play crucial roles in embryonic development and tissue repair. Aberrantly regulated transitions are also a hallmark of cancer metastasis. The genetic network that regulates these transitions appears to allow for the existence of a hybrid phenotype (epithelial/mesenchymal). Hybrid cells are endowed with mixed epithelial and mesenchymal characteristics, enabling specialized capabilities such as collective cell migration. Cell-fate determination between the three phenotypes is in fact regulated by a circuit composed of two highly interconnected chimeric modules—the miR-34/SNAIL and the miR-200/ZEB mutual-inhibition feedback circuits. Here, we used detailed modeling of microRNA-based regulation to study this core unit. More specifically, we investigated the functions of the two isolated modules and subsequently of the combined unit when the two modules are integrated into the full regulatory circuit. We found that miR-200/ZEB forms a tristable circuit that acts as a ternary switch, driven by miR-34/SNAIL, that is a monostable module that acts as a noise-buffering integrator of internal and external signals. We propose to associate the three stable states—(1,0), (high miR-200)/(low ZEB); (0,1), (low miR-200)/(high ZEB); and (1/2,1/2), (medium miR-200)/(medium ZEB)—with the epithelial, mesenchymal, and hybrid phenotypes, respectively. Our (1/2,1/2) state hypothesis is consistent with recent experimental studies (e.g., ZEB expression measurements in collectively migrating cells) and explains the lack of observed mesenchymal-to-hybrid transitions in metastatic cells and in induced pluripotent stem cells. Testable predictions of dynamic gene expression during complete and partial transitions are presented.

Implications of the Hybrid Epithelial/Mesenchymal Phenotype in Metastasis

Transitions between epithelial and mesenchymal phenotypes – the epithelial to mesenchymal transition (EMT) and its reverse the mesenchymal to epithelial transition (MET)u2009– are hallmarks of cancer metastasis. While transitioning between the epithelial and mesenchymal phenotypes, cells can also attain a hybrid epithelial/mesenchymal (E/M) (i.e., partial or intermediate EMT) phenotype. Cells in this phenotype have mixed epithelial (e.g., adhesion) and mesenchymal (e.g., migration) properties, thereby allowing them to move collectively as clusters. If these clusters reach the bloodstream intact, they can give rise to clusters of circulating tumor cells (CTCs), as have often been seen experimentally. Here, we review the operating principles of the core regulatory network for EMT/MET that acts as a “three-way” switch giving rise to three distinct phenotypes – E, M and hybrid E/M – and present a theoretical framework that can elucidate the role of many other players in regulating epithelial plasticity. Furthermore, we highlight recent studies on partial EMT and its association with drug resistance and tumor-initiating potential; and discuss how cell–cell communication between cells in a partial EMT phenotype can enable the formation of clusters of CTCs. These clusters can be more apoptosis-resistant and have more tumor-initiating potential than singly moving CTCs with a wholly mesenchymal (complete EMT) phenotype. Also, more such clusters can be formed under inflammatory conditions that are often generated by various therapies. Finally, we discuss the multiple advantages that the partial EMT or hybrid E/M phenotype have as compared to a complete EMT phenotype and argue that these collectively migrating cells are the primary “bad actors” of metastasis.

Bacterial survival strategies suggest rethinking cancer cooperativity.

Despite decades of a much improved understanding of cancer biology, we are still baffled by questions regarding the deadliest traits of malignancy: metastatic colonization, dormancy and relapse, and the rapid evolution of multiple drug and immune resistance. New ideas are needed to resolve these critical issues. Relying on finding and demonstrating parallels between collective behavior capabilities of cancer cells and that of bacteria, we suggest communal behaviors of bacteria as a valuable model system for new perspectives and research directions. Understanding the ways in which bacteria thrive in competitive habitats and their cooperative strategies for surviving extreme stress can shed light on cooperativity in tumorigenesis and portray tumors as societies of smart communicating cells. This may translate into progress in fathoming cancer pathogenesis. We outline new experiments to test the cancer cooperativity hypothesis and reason that cancer may be outsmarted through its own social intelligence.

Stability of the hybrid epithelial/mesenchymal phenotype

Epithelial-to-Mesenchymal Transition (EMT) and its reverse – Mesenchymal to Epithelial Transition (MET) – are hallmarks of cellular plasticity during embryonic development and cancer metastasis. During EMT, epithelial cells lose cell-cell adhesion and gain migratory and invasive traits either partially or completely, leading to a hybrid epithelial/mesenchymal (hybrid E/M) or a mesenchymal phenotype respectively. Mesenchymal cells move individually, but hybrid E/M cells migrate collectively as observed during gastrulation, wound healing, and the formation of tumor clusters detected as Circulating Tumor Cells (CTCs). Typically, the hybrid E/M phenotype has largely been tacitly assumed to be transient and ‘metastable’. Here, we identify certain ‘phenotypic stability factors’ (PSFs) such as GRHL2 that couple to the core EMT decision-making circuit (miR-200/ZEB) and stabilize hybrid E/M phenotype. Further, we show that H1975 lung cancer cells can display a stable hybrid E/M phenotype and migrate collectively, a behavior that is impaired by knockdown of GRHL2 and another previously identified PSF - OVOL. In addition, our computational model predicts that GRHL2 can also associate hybrid E/M phenotype with high tumor-initiating potential, a prediction strengthened by the observation that the higher levels of these PSFs may be predictive of poor patient outcome. Finally, based on these specific examples, we deduce certain network motifs that can stabilize the hybrid E/M phenotype. Our results suggest that partial EMT, i.e. a hybrid E/M phenotype, need not be ‘metastable’, and strengthen the emerging notion that partial EMT, but not necessarily a complete EMT, is associated with aggressive tumor progression.

Hyperbaric Oxygen Induces Late Neuroplasticity in Post Stroke Patients - Randomized, Prospective Trial

Background Recovery after stroke correlates with non-active (stunned) brain regions, which may persist for years. The current study aimed to evaluate whether increasing the level of dissolved oxygen by Hyperbaric Oxygen Therapy (HBOT) could activate neuroplasticity in patients with chronic neurologic deficiencies due to stroke. Methods and Findings A prospective, randomized, controlled trial including 74 patients (15 were excluded). All participants suffered a stroke 6–36 months prior to inclusion and had at least one motor dysfunction. After inclusion, patients were randomly assigned to treated or cross groups. Brain activity was assessed by SPECT imaging; neurologic functions were evaluated by NIHSS, ADL, and life quality. Patients in the treated group were evaluated twice: at baseline and after 40 HBOT sessions. Patients in the cross group were evaluated three times: at baseline, after a 2-month control period of no treatment, and after subsequent 2-months of 40 HBOT sessions. HBOT protocol: Two months of 40 sessions (5 days/week), 90 minutes each, 100% oxygen at 2 ATA. We found that the neurological functions and life quality of all patients in both groups were significantly improved following the HBOT sessions while no improvement was found during the control period of the patients in the cross group. Results of SPECT imaging were well correlated with clinical improvement. Elevated brain activity was detected mostly in regions of live cells (as confirmed by CT) with low activity (based on SPECT) – regions of noticeable discrepancy between anatomy and physiology. Conclusions The results indicate that HBOT can lead to significant neurological improvements in post stroke patients even at chronic late stages. The observed clinical improvements imply that neuroplasticity can still be activated long after damage onset in regions where there is a brain SPECT/CT (anatomy/physiology) mismatch. Trial Registration ClinicalTrials.gov NCT00715897

Towards elucidating the connection between epithelial–mesenchymal transitions and stemness

Epithelial cells undergoing epithelial-to-mesenchymal transitions have often been shown to behave as cancer stem cells, but the precise molecular connection remains elusive. At the genetic level, stemness is governed by LIN28/let-7 double inhibition switch, whereas EMT/MET is controlled by miR-200/ZEB double inhibition circuit and LIN28 is inhibited by miR-200, coupling the two modules. Here, using a specially devised theoretical framework to investigate the dynamics of the LIN28/let-7 system, we show that it can operate as a three-way switch (between low, high and intermediate LIN28 levels termed the D, U and hybrid D/U states) similar to the three-way operation of the miR-200/ZEB circuit that allows for the existence of a hybrid epithelial/mesenchymal (E/M) phenotype. We find significant correspondence between the existence of the three states of the two circuits: E–D, M–U and E/M–D/U. Incorporating the activation of OCT4 by LIN28, we find that the hybrid E/M phenotype has high likelihood (when compared with either the E or M states) to gain stemness. Combining the LIN28/let-7 regulation by NF-κB and c-MYC, we find that NF-κB, but not c-MYC, elevates the likelihood of E/M phenotype to gain stemness. Our results are consistent with emerging concept that partial EMT can lead to stemness.

Computational quest for understanding the role of astrocyte signaling in synaptic transmission and plasticity

The complexity of the signaling network that underlies astrocyte-synapse interactions may seem discouraging when tackled from a theoretical perspective. Computational modeling is challenged by the fact that many details remain hitherto unknown and conventional approaches to describe synaptic function are unsuitable to explain experimental observations when astrocytic signaling is taken into account. Supported by experimental evidence is the possibility that astrocytes perform genuine information processing by means of their calcium signaling and are players in the physiological setting of the basal tone of synaptic transmission. Here we consider the plausibility of this scenario from a theoretical perspective, focusing on the modulation of synaptic release probability by the astrocyte and its implications on synaptic plasticity. The analysis of the signaling pathways underlying such modulation refines our notion of tripartite synapse and has profound implications on our understanding of brain function.

Immunoproteasome deficiency is a feature of non-small cell lung cancer with a mesenchymal phenotype and is associated with a poor outcome

Significance The success rate of therapeutic trials that target tumor antigens is quite limited. We demonstrate for the first time to our knowledge that lung cancer cells that have undergone epithelial-to-mesenchymal transition lose immunoproteasome expression, resulting in markedly reduced antigen presentation. Reduced expression of the immunoproteasome was associated with and can predict poor outcome in non-small cell lung carcinoma (NSCLC) patients. Induction of the immunoproteasome with IFNγ or 5-aza-2′-deoxycytidine (5-aza-dC) treatment can overcome this immune escape mechanism of mesenchymal cells by restoring functional HLA class I-bound peptides. These findings have substantial relevance for development of effective strategies to target tumor cells with inherent resistance to T cell-mediated immunotherapy. The immunoproteasome plays a key role in generation of HLA peptides for T cell-mediated immunity. Integrative genomic and proteomic analysis of non-small cell lung carcinoma (NSCLC) cell lines revealed significantly reduced expression of immunoproteasome components and their regulators associated with epithelial to mesenchymal transition. Low expression of immunoproteasome subunits in early stage NSCLC patients was associated with recurrence and metastasis. Depleted repertoire of HLA class I-bound peptides in mesenchymal cells deficient in immunoproteasome components was restored with either IFNγ or 5-aza-2′-deoxycytidine (5-aza-dC) treatment. Our findings point to a mechanism of immune evasion of cells with a mesenchymal phenotype and suggest a strategy to overcome immune evasion through induction of the immunoproteasome to increase the cellular repertoire of HLA class I-bound peptides.

Coupling the modules of EMT and stemness: A tunable ‘stemness window’ model

Metastasis of carcinoma involves migration of tumor cells to distant organs and initiate secondary tumors. Migration requires a complete or partial Epithelial-to-Mesenchymal Transition (EMT), and tumor-initiation requires cells possessing stemness. Epithelial cells (E) undergoing a complete EMT to become mesenchymal (M) have been suggested to be more likely to possess stemness. However, recent studies suggest that stemness can also be associated with cells undergoing a partial EMT (hybrid E/M phenotype). Therefore, the correlation between EMT and stemness remains elusive. Here, using a theoretical framework that couples the core EMT and stemness modules (miR-200/ZEB and LIN28/let-7), we demonstrate that the positioning of ‘stemness window’ on the ‘EMT axis’ need not be universal; rather it can be fine-tuned. Particularly, we present OVOL as an example of a modulating factor that, due to its coupling with miR-200/ZEB/LIN28/let-7 circuit, fine-tunes the EMT-stemness interplay. Coupling OVOL can inhibit the stemness likelihood of M and elevate that of the hybrid E/M (partial EMT) phenotype, thereby pulling the ‘stemness window’ away from the M end of ‘EMT axis’. Our results unify various apparently contradictory experimental findings regarding the interconnection between EMT and stemness, corroborate the emerging notion that partial EMT associates with stemness, and offer new testable predictions.

Polarity mechanisms such as contact inhibition of locomotion regulate persistent rotational motion of mammalian cells on micropatterns

Significance During the growth of an embryo or the spreading of a tumor, cells may travel collectively. We study a computational model of a simple example of collective migration: two cells confined to a square adhesive pattern. In this confinement, some cell types rotate, whereas others do not. We model these crawling cells, the forces between them, and several possible ways that the cells could choose what direction they will crawl—their “polarity mechanism.” We show that the cell polarity mechanism can control whether the pairs of cells rotate or remain fixed. This suggests that we can learn about how large groups of cells choose their direction by studying the rotation of pairs. Pairs of endothelial cells on adhesive micropatterns rotate persistently, but pairs of fibroblasts do not; coherent rotation is present in normal mammary acini and kidney cells but absent in cancerous cells. Why? To answer this question, we develop a computational model of pairs of mammalian cells on adhesive micropatterns using a phase field method and study the conditions under which persistent rotational motion (PRM) emerges. Our model couples the shape of the cell, the cell’s internal chemical polarity, and interactions between cells such as volume exclusion and adhesion. We show that PRM can emerge from this minimal model and that the cell-cell interface may be influenced by the nucleus. We study the effect of various cell polarity mechanisms on rotational motion, including contact inhibition of locomotion, neighbor alignment, and velocity alignment, where cells align their polarity to their velocity. These polarity mechanisms strongly regulate PRM: Small differences in polarity mechanisms can create significant differences in collective rotation. We argue that the existence or absence of rotation under confinement may lead to insight into the cell’s methods for coordinating collective cell motility.