Germline BARD1 variants predispose to mesothelioma by impairing DNA repair and calcium signaling.

We report that ~1.8% of all mesothelioma patients and 4.9% of those younger than 55, carry rare germline variants of the BRCA1 associated RING domain 1 (BARD1) gene that were predicted to be damaging by computational analyses. We conducted functional assays, essential for accurate interpretation of missense variants, in primary fibroblasts that we established in tissue culture from a patient carrying the heterozygous BARD1V523A mutation. We found that these cells had genomic instability, reduced DNA repair, and impaired apoptosis. Investigating the underlying signaling pathways, we found that BARD1 forms a trimeric protein complex with p53 and SERCA2 that regulates calcium signaling and apoptosis. We validated these findings in BARD1-silenced primary human mesothelial cells exposed to asbestos. Our study elucidated mechanisms of BARD1 activity and revealed that heterozygous germline BARD1 mutations favor the development of mesothelioma and increase the susceptibility to asbestos carcinogenesis. These mesotheliomas are significantly less aggressive compared to mesotheliomas in asbestos workers.

RACER-m leverages structural features for sparse T cell specificity prediction.

Reliable prediction of T cell specificity against antigenic signatures is a formidable task, complicated by the immense diversity of T cell receptor and antigen sequence space and the resulting limited availability of training sets for inferential models. Recent modeling efforts have demonstrated the advantage of incorporating structural information to overcome the need for extensive training sequence data, yet disentangling the heterogeneous TCR-antigen interface to accurately predict MHC-allele-restricted TCR-peptide interactions has remained challenging. Here, we present RACER-m, a coarse-grained structural model leveraging key biophysical information from the diversity of publicly available TCR-antigen crystal structures. Explicit inclusion of structural content substantially reduces the required number of training examples and maintains reliable predictions of TCR-recognition specificity and sensitivity across diverse biological contexts. Our model capably identifies biophysically meaningful point-mutant peptides that affect binding affinity, distinguishing its ability in predicting TCR specificity of point-mutants from alternative sequence-based methods. Its application is broadly applicable to studies involving both closely related and structurally diverse TCR-peptide pairs.

BAP1 is a novel regulator of HIF-1α.

BAP1 is a powerful tumor suppressor gene characterized by haplo insufficiency. Individuals carrying germline BAP1 mutations often develop mesothelioma, an aggressive malignancy of the serosal layers covering the lungs, pericardium, and abdominal cavity. Intriguingly, mesotheliomas developing in carriers of germline BAP1 mutations are less aggressive, and these patients have significantly improved survival. We investigated the apparent paradox of a tumor suppressor gene that, when mutated, causes less aggressive mesotheliomas. We discovered that mesothelioma biopsies with biallelic BAP1 mutations showed loss of nuclear HIF-1α staining. We demonstrated that during hypoxia, BAP1 binds, deubiquitylates, and stabilizes HIF-1α, the master regulator of the hypoxia response and tumor cell invasion. Moreover, primary cells from individuals carrying germline BAP1 mutations and primary cells in which BAP1 was silenced using siRNA had reduced HIF-1α protein levels in hypoxia. Computational modeling and co-immunoprecipitation experiments revealed that mutations of BAP1 residues I675, F678, I679, and L691 -encompassing the C-terminal domain-nuclear localization signal- to A, abolished the interaction with HIF-1α. We found that BAP1 binds to the N-terminal region of HIF-1α, where HIF-1α binds DNA and dimerizes with HIF-1ß forming the heterodimeric transactivating complex HIF. Our data identify BAP1 as a key positive regulator of HIF-1α in hypoxia. We propose that the significant reduction of HIF-1α activity in mesothelioma cells carrying biallelic BAP1 mutations, accompanied by the significant reduction of HIF-1α activity in hypoxic tissues containing germline BAP1 mutations, contributes to the reduced aggressiveness and improved survival of mesotheliomas developing in carriers of germline BAP1 mutations.

Road-blocker HSP disease mutation disrupts pre-organization for ATP hydrolysis in kinesin through a second sphere control.

Kinesin motor proteins perform several essential cellular functions powered by the adenosine triphosphate (ATP) hydrolysis reaction. Several single-point mutations in the kinesin motor protein KIF5A have been implicated to hereditary spastic paraplegia disease (HSP), a lethal neurodegenerative disease in humans. In earlier studies, we have shown that a series of HSP-related mutations can impair the kinesin's long-distance displacement or processivity by modulating the order-disorder transition of the linker connecting the heads to the coiled coil. On the other hand, the reduction of kinesin's ATP hydrolysis reaction rate by a distal asparagine-to-serine mutation is also known to cause HSP disease. However, the molecular mechanism of the ATP hydrolysis reaction in kinesin by this distal mutation is still not fully understood. Using classical molecular dynamics simulations combined with quantum mechanics/molecular mechanics calculations, the pre-organization geometry required for optimal hydrolysis in kinesin motor bound to α/ß-tubulin is determined. This optimal geometry has only a single salt-bridge (of the possible two) between Arg203-Glu236, putting a reactive water molecule at a perfect position for hydrolysis. Such geometry is also needed to create the appropriate configuration for proton translocation during ATP hydrolysis. The distal asparagine-to-serine mutation is found to disrupt this optimal geometry. Therefore, the current study along with our previous one demonstrates how two different effects on kinesin dynamics (processivity and ATP hydrolysis), caused by a different set of genotypes, can give rise to the same phenotype leading to HSP disease.

Decoding the coupled decision-making of the epithelial-mesenchymal transition and metabolic reprogramming in cancer.

Cancer metastasis relies on an orchestration of traits driven by different interacting functional modules, including metabolism and epithelial-mesenchymal transition (EMT). During metastasis, cancer cells can acquire a hybrid metabolic phenotype (W/O) by increasing oxidative phosphorylation without compromising glycolysis and they can acquire a hybrid epithelial/mesenchymal (E/M) phenotype by engaging EMT. Both the W/O and E/M states are associated with high metastatic potentials, and many regulatory links coupling metabolism and EMT have been identified. Here, we investigate the coupled decision-making networks of metabolism and EMT. Their crosstalk can exhibit synergistic or antagonistic effects on the acquisition and stability of different coupled metabolism-EMT states. Strikingly, the aggressive E/M-W/O state can be enabled and stabilized by the crosstalk irrespective of these hybrid states' availability in individual metabolism or EMT modules. Our work emphasizes the mutual activation between metabolism and EMT, providing an important step toward understanding the multifaceted nature of cancer metastasis.

Nrf2 Modulates the Hybrid Epithelial/Mesenchymal Phenotype and Notch Signaling During Collective Cancer Migration.

Hybrid epithelial/mesenchymal cells (E/M) are key players in aggressive cancer metastasis. It remains a challenge to understand how these cell states, which are mostly non-existent in healthy tissue, become stable phenotypes participating in collective cancer migration. The transcription factor Nrf2, which is associated with tumor progression and resistance to therapy, appears to be central to this process. Here, using a combination of immunocytochemistry, single cell biosensors, and computational modeling, we show that Nrf2 functions as a phenotypic stability factor for hybrid E/M cells by inhibiting a complete epithelial-mesenchymal transition (EMT) during collective cancer migration. We also demonstrate that Nrf2 and EMT signaling are spatially coordinated near the leading edge. In particular, computational analysis of an Nrf2-EMT-Notch network and experimental modulation of Nrf2 by pharmacological treatment or CRISPR/Cas9 gene editing reveal that Nrf2 stabilizes a hybrid E/M phenotype which is maximally observed in the interior region immediately behind the leading edge. We further demonstrate that the Nrf2-EMT-Notch network enhances Dll4 and Jagged1 expression at the leading edge, which correlates with the formation of leader cells and protruding tips. Altogether, our results provide direct evidence that Nrf2 acts as a phenotypic stability factor in restricting complete EMT and plays an important role in coordinating collective cancer migration.

A VDAC1-mediated NEET protein chain transfers [2Fe-2S] clusters between the mitochondria and the cytosol and impacts mitochondrial dynamics.

Mitochondrial inner NEET (MiNT) and the outer mitochondrial membrane (OMM) mitoNEET (mNT) proteins belong to the NEET protein family. This family plays a key role in mitochondrial labile iron and reactive oxygen species (ROS) homeostasis. NEET proteins contain labile [2Fe-2S] clusters which can be transferred to apo-acceptor proteins. In eukaryotes, the biogenesis of [2Fe-2S] clusters occurs within the mitochondria by the iron-sulfur cluster (ISC) system; the clusters are then transferred to [2Fe-2S] proteins within the mitochondria or exported to cytosolic proteins and the cytosolic iron-sulfur cluster assembly (CIA) system. The last step of export of the [2Fe-2S] is not yet fully characterized. Here we show that MiNT interacts with voltage-dependent anion channel 1 (VDAC1), a major OMM protein that connects the intermembrane space with the cytosol and participates in regulating the levels of different ions including mitochondrial labile iron (mLI). We further show that VDAC1 is mediating the interaction between MiNT and mNT, in which MiNT transfers its [2Fe-2S] clusters from inside the mitochondria to mNT that is facing the cytosol. This MiNT-VDAC1-mNT interaction is shown both experimentally and by computational calculations. Additionally, we show that modifying MiNT expression in breast cancer cells affects the dynamics of mitochondrial structure and morphology, mitochondrial function, and breast cancer tumor growth. Our findings reveal a pathway for the transfer of [2Fe-2S] clusters, which are assembled inside the mitochondria, to the cytosol.

BAP1 forms a trimer with HMGB1 and HDAC1 that modulates gene × environment interaction with asbestos.

Carriers of heterozygous germline BAP1 mutations (BAP1+/-) are affected by the "BAP1 cancer syndrome." Although they can develop almost any cancer type, they are unusually susceptible to asbestos carcinogenesis and mesothelioma. Here we investigate why among all carcinogens, BAP1 mutations cooperate with asbestos. Asbestos carcinogenesis and mesothelioma have been linked to a chronic inflammatory process promoted by the extracellular release of the high-mobility group box 1 protein (HMGB1). We report that BAP1+/- cells secrete increased amounts of HMGB1, and that BAP1+/- carriers have detectable serum levels of acetylated HMGB1 that further increase when they develop mesothelioma. We linked these findings to our discovery that BAP1 forms a trimeric protein complex with HMGB1 and with histone deacetylase 1 (HDAC1) that modulates HMGB1 acetylation and its release. Reduced BAP1 levels caused increased ubiquitylation and degradation of HDAC1, leading to increased acetylation of HMGB1 and its active secretion that in turn promoted mesothelial cell transformation.

Decoding leader cells in collective cancer invasion.

Collective cancer invasion with leader-follower organization is increasingly recognized as a predominant mechanism in the metastatic cascade. Leader cells support cancer invasion by creating invasion tracks, sensing environmental cues and coordinating with follower cells biochemically and biomechanically. With the latest developments in experimental and computational models and analysis techniques, the range of specific traits and features of leader cells reported in the literature is rapidly expanding. Yet, despite their importance, there is no consensus on how leader cells arise or their essential characteristics. In this Perspective, we propose a framework for defining the essential aspects of leader cells and provide a unifying perspective on the varying cellular and molecular programmes that are adopted by each leader cell subtype to accomplish their functions. This Perspective can lead to more effective strategies to interdict a major contributor to metastatic capability.

Design and proof of concept for targeted phage-based COVID-19 vaccination strategies with a streamlined cold-free supply chain.

Development of effective vaccines against coronavirus disease 2019 (COVID-19) is a global imperative. Rapid immunization of the entire human population against a widespread, continually evolving, and highly pathogenic virus is an unprecedented challenge, and different vaccine approaches are being pursued. Engineered filamentous bacteriophage (phage) particles have unique potential in vaccine development due to their inherent immunogenicity, genetic plasticity, stability, cost-effectiveness for large-scale production, and proven safety profile in humans. Herein we report the development and initial evaluation of two targeted phage-based vaccination approaches against SARS-CoV-2: dual ligand peptide-targeted phage and adeno-associated virus/phage (AAVP) particles. For peptide-targeted phage, we performed structure-guided antigen design to select six solvent-exposed epitopes of the SARS-CoV-2 spike (S) protein. One of these epitopes displayed on the major capsid protein pVIII of phage induced a specific and sustained humoral response when injected in mice. These phage were further engineered to simultaneously display the peptide CAKSMGDIVC on the minor capsid protein pIII to enable their transport from the lung epithelium into the systemic circulation. Aerosolization of these "dual-display" phage into the lungs of mice generated a systemic and specific antibody response. In the second approach, targeted AAVP particles were engineered to deliver the entire S protein gene under the control of a constitutive CMV promoter. This induced tissue-specific transgene expression, stimulating a systemic S protein-specific antibody response in mice. With these proof-of-concept preclinical experiments, we show that both targeted phage- and AAVP-based particles serve as robust yet versatile platforms that can promptly yield COVID-19 vaccine prototypes for translational development.

Exploring Energy Landscapes of Intrinsically Disordered Proteins: Insights into Functional Mechanisms.

Intrinsically disordered proteins (IDPs) lack a rigid three-dimensional structure and populate a polymorphic ensemble of conformations. Because of the lack of a reference conformation, their energy landscape representation in terms of reaction coordinates presents a daunting challenge. Here, our newly developed energy landscape visualization method (ELViM), a reaction coordinate-free approach, shows its prime application to explore frustrated energy landscapes of an intrinsically disordered protein, prostate-associated gene 4 (PAGE4). PAGE4 is a transcriptional coactivator that potentiates the oncogene c-Jun. Two kinases, namely, HIPK1 and CLK2, phosphorylate PAGE4, generating variants phosphorylated at different serine/threonine residues (HIPK1-PAGE4 and CLK2-PAGE4, respectively) with opposing functions. While HIPK1-PAGE4 predominantly phosphorylates Thr51 and potentiates c-Jun, CLK2-PAGE4 hyperphosphorylates PAGE4 and attenuates transactivation. To understand the underlying mechanisms of conformational diversity among different phosphoforms, we have analyzed their atomistic trajectories simulated using AWSEM forcefield, and the energy landscapes were elucidated using ELViM. This method allows us to identify and compare the population distributions of different conformational ensembles of PAGE4 phosphoforms using the same effective phase space. The results reveal a predominant conformational ensemble with an extended C-terminal segment of WT PAGE4, which exposes a functional residue Thr51, implying its potential of undertaking a fly-casting mechanism while binding to its cognate partner. In contrast, for HIPK1-PAGE4, a compact conformational ensemble enhances its population sequestering phosphorylated-Thr51. This clearly explains the experimentally observed weaker affinity of HIPK1-PAGE4 for c-Jun. ELViM appears as a powerful tool, especially to analyze the highly frustrated energy landscape representation of IDPs where appropriate reaction coordinates are hard to apprehend.

Towards decoding the coupled decision-making of metabolism and epithelial-to-mesenchymal transition in cancer.

Cancer cells have the plasticity to adjust their metabolic phenotypes for survival and metastasis. A developmental programme known as epithelial-to-mesenchymal transition (EMT) plays a critical role during metastasis, promoting the loss of polarity and cell-cell adhesion and the acquisition of motile, stem-cell characteristics. Cells undergoing EMT or the reverse mesenchymal-to-epithelial transition (MET) are often associated with metabolic changes, as the change in phenotype often correlates with a different balance of proliferation versus energy-intensive migration. Extensive crosstalk occurs between metabolism and EMT, but how this crosstalk leads to coordinated physiological changes is still uncertain. The elusive connection between metabolism and EMT compromises the efficacy of metabolic therapies targeting metastasis. In this review, we aim to clarify the causation between metabolism and EMT on the basis of experimental studies, and propose integrated theoretical-experimental efforts to better understand the coupled decision-making of metabolism and EMT.

Design and proof-of-concept for targeted phage-based COVID-19 vaccination strategies with a streamlined cold-free supply chain.

Development of effective vaccines against Coronavirus Disease 2019 (COVID-19) is a global imperative. Rapid immunization of the world human population against a widespread, continually evolving, and highly pathogenic virus is an unprecedented challenge, and many different vaccine approaches are being pursued to meet this task. Engineered filamentous bacteriophage (phage) have unique potential in vaccine development due to their inherent immunogenicity, genetic plasticity, stability, cost-effectiveness for large-scale production, and proven safety profile in humans. Herein we report the design, development, and initial evaluation of targeted phage-based vaccination approaches against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) by using dual ligand peptide-targeted phage and adeno-associated virus/phage (AAVP) particles. Towards a unique phage- and AAVP-based dual-display candidate approach, we first performed structure-guided antigen design to select six solvent-exposed epitopes of the SARS-CoV-2 spike (S) protein for display on the recombinant major capsid coat protein pVIII. Targeted phage particles carrying one of these epitopes induced a strong and specific humoral response. In an initial experimental approach, when these targeted phage particles were further genetically engineered to simultaneously display a ligand peptide (CAKSMGDIVC) on the minor capsid protein pIII, which enables receptor-mediated transport of phage particles from the lung epithelium into the systemic circulation (termed "dual-display"), they enhanced a systemic and specific spike (S) protein-specific antibody response upon aerosolization into the lungs of mice. In a second line of investigation, we engineered targeted AAVP particles to deliver the entire S protein gene under the control of a constitutive cytomegalovirus (CMV) promoter, which induced tissue-specific transgene expression stimulating a systemic S protein-specific antibody response. As proof-of-concept preclinical experiments, we show that targeted phage- and AAVP-based particles serve as robust yet versatile enabling platforms for ligand-directed immunization and promptly yield COVID-19 vaccine prototypes for further translational development. SIGNIFICANCE: The ongoing COVID-19 global pandemic has accounted for over 2.5 million deaths and an unprecedented impact on the health of mankind worldwide. Over the past several months, while a few COVID-19 vaccines have received Emergency Use Authorization and are currently being administered to the entire human population, the demand for prompt global immunization has created enormous logistical challenges--including but not limited to supply, access, and distribution--that justify and reinforce the research for additional strategic alternatives. Phage are viruses that only infect bacteria and have been safely administered to humans as antibiotics for decades. As experimental proof-of-concept, we demonstrated that aerosol pulmonary vaccination with lung-targeted phage particles that display short epitopes of the S protein on the capsid as well as preclinical vaccination with targeted AAVP particles carrying the S protein gene elicit a systemic and specific immune response against SARS-CoV-2 in immunocompetent mice. Given that targeted phage- and AAVP-based viral particles are sturdy yet simple to genetically engineer, cost-effective for rapid large-scale production in clinical grade, and relatively stable at room temperature, such unique attributes might perhaps become additional tools towards COVID-19 vaccine design and development for immediate and future unmet needs.

Rapid Assessment of T-Cell Receptor Specificity of the Immune Repertoire.

Accurate assessment of TCR-antigen specificity at the whole immune repertoire level lies at the heart of improved cancer immunotherapy, but predictive models capable of high-throughput assessment of TCR-peptide pairs are lacking. Recent advances in deep sequencing and crystallography have enriched the data available for studying TCR-p-MHC systems. Here, we introduce a pairwise energy model, RACER, for rapid assessment of TCR-peptide affinity at the immune repertoire level. RACER applies supervised machine learning to efficiently and accurately resolve strong TCR-peptide binding pairs from weak ones. The trained parameters further enable a physical interpretation of interacting patterns encoded in each specific TCR-p-MHC system. When applied to simulate thymic selection of an MHC-restricted T-cell repertoire, RACER accurately estimates recognition rates for tumor-associated neoantigens and foreign peptides, thus demonstrating its utility in helping address the large computational challenge of reliably identifying the properties of tumor antigen-specific T-cells at the level of an individual patient's immune repertoire.

A Scalable Computational Approach for Simulating Complexes of Multiple Chromosomes.

Significant efforts have been recently made to obtain the three-dimensional (3D) structure of the genome with the goal of understanding how structures may affect gene regulation and expression. Chromosome conformational capture techniques such as Hi-C, have been key in uncovering the quantitative information needed to determine chromatin organization. Complementing these experimental tools, co-polymers theoretical methods are necessary to determine the ensemble of three-dimensional structures associated to the experimental data provided by Hi-C maps. Going beyond just structural information, these theoretical advances also start to provide an understanding of the underlying mechanisms governing genome assembly and function. Recent theoretical work, however, has been focused on single chromosome structures, missing the fact that, in the full nucleus, interactions between chromosomes play a central role in their organization. To overcome this limitation, MiChroM (Minimal Chromatin Model) has been modified to become capable of performing these multi-chromosome simulations. It has been upgraded into a fast and scalable software version, which is able to perform chromosome simulations using GPUs via OpenMM Python API, called Open-MiChroM. To validate the efficiency of this new version, analyses for GM12878 individual autosomes were performed and compared to earlier studies. This validation was followed by multi-chain simulations including the four largest human chromosomes (C1-C4). These simulations demonstrated the full power of this new approach. Comparison to Hi-C data shows that these multiple chromosome interactions are essential for a more accurate agreement with experimental results. Without any changes to the original MiChroM potential, it is now possible to predict experimentally observed inter-chromosome contacts. This scalability of Open-MiChroM allow for more audacious investigations, looking at interactions of multiple chains as well as moving towards higher resolution chromosomes models.

Exploring chromosomal structural heterogeneity across multiple cell lines.

Using computer simulations, we generate cell-specific 3D chromosomal structures and compare them to recently published chromatin structures obtained through microscopy. We demonstrate using machine learning and polymer physics simulations that epigenetic information can be used to predict the structural ensembles of multiple human cell lines. Theory predicts that chromosome structures are fluid and can only be described by an ensemble, which is consistent with the observation that chromosomes exhibit no unique fold. Nevertheless, our analysis of both structures from simulation and microscopy reveals that short segments of chromatin make two-state transitions between closed conformations and open dumbbell conformations. Finally, we study the conformational changes associated with the switching of genomic compartments observed in human cell lines. The formation of genomic compartments resembles hydrophobic collapse in protein folding, with the aggregation of denser and predominantly inactive chromatin driving the positioning of active chromatin toward the surface of individual chromosomal territories.

Drug-Tolerant Idling Melanoma Cells Exhibit Theory-Predicted Metabolic Low-Low Phenotype.

Cancer cells adjust their metabolic profiles to evade treatment. Metabolic adaptation is complex and hence better understood by an integrated theoretical-experimental approach. Using a minimal kinetic model, we predicted a previously undescribed Low/Low (L/L) phenotype, characterized by low oxidative phosphorylation (OXPHOS) and low glycolysis. Here, we report that L/L metabolism is observed in BRAF-mutated melanoma cells that enter a drug-tolerant "idling state" upon long-term MAPK inhibition (MAPKi). Consistently, using publicly available RNA-sequencing data of both cell lines and patient samples, we show that melanoma cells decrease their glycolysis and/or OXPHOS activity upon MAPKi and converge toward the L/L phenotype. L/L metabolism is unfavorable for tumor growth, yet supports successful cell division at ~50% rate. Thus, L/L drug-tolerant idling cells are a reservoir for accumulating mutations responsible for relapse, and it should be considered as a target subpopulation for improving MAPKi outcomes in melanoma treatment.

Redox-dependent gating of VDAC by mitoNEET.

MitoNEET is an outer mitochondrial membrane protein essential for sensing and regulation of iron and reactive oxygen species (ROS) homeostasis. It is a key player in multiple human maladies including diabetes, cancer, neurodegeneration, and Parkinson's diseases. In healthy cells, mitoNEET receives its clusters from the mitochondrion and transfers them to acceptor proteins in a process that could be altered by drugs or during illness. Here, we report that mitoNEET regulates the outer-mitochondrial membrane (OMM) protein voltage-dependent anion channel 1 (VDAC1). VDAC1 is a crucial player in the cross talk between the mitochondria and the cytosol. VDAC proteins function to regulate metabolites, ions, ROS, and fatty acid transport, as well as function as a "governator" sentry for the transport of metabolites and ions between the cytosol and the mitochondria. We find that the redox-sensitive [2Fe-2S] cluster protein mitoNEET gates VDAC1 when mitoNEET is oxidized. Addition of the VDAC inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) prevents both mitoNEET binding in vitro and mitoNEET-dependent mitochondrial iron accumulation in situ. We find that the DIDS inhibitor does not alter the redox state of MitoNEET. Taken together, our data indicate that mitoNEET regulates VDAC in a redox-dependent manner in cells, closing the pore and likely disrupting VDAC's flow of metabolites.

NRF2 activates a partial epithelial-mesenchymal transition and is maximally present in a hybrid epithelial/mesenchymal phenotype.

The epithelial-mesenchymal transition (EMT) is a key process implicated in cancer metastasis and therapy resistance. Recent studies have emphasized that cells can undergo partial EMT to attain a hybrid epithelial/mesenchymal (E/M) phenotype - a cornerstone of tumour aggressiveness and poor prognosis. These cells can have enhanced tumour-initiation potential as compared to purely epithelial or mesenchymal ones and can integrate the properties of cell-cell adhesion and motility that facilitates collective cell migration leading to clusters of circulating tumour cells (CTCs) - the prevalent mode of metastasis. Thus, identifying the molecular players that can enable cells to maintain a hybrid E/M phenotype is crucial to curb the metastatic load. Using an integrated computational-experimental approach, we show that the transcription factor NRF2 can prevent a complete EMT and instead stabilize a hybrid E/M phenotype. Knockdown of NRF2 in hybrid E/M non-small cell lung cancer cells H1975 and bladder cancer cells RT4 destabilized a hybrid E/M phenotype and compromised the ability to collectively migrate to close a wound in vitro. Notably, while NRF2 knockout simultaneously downregulated E-cadherin and ZEB-1, overexpression of NRF2 enriched for a hybrid E/M phenotype by simultaneously upregulating both E-cadherin and ZEB-1 in individual RT4 cells. Further, we predict that NRF2 is maximally expressed in hybrid E/M phenotype(s) and demonstrate that this biphasic dynamic arises from the interconnections among NRF2 and the EMT regulatory circuit. Finally, clinical records from multiple datasets suggest a correlation between a hybrid E/M phenotype, high levels of NRF2 and its targets and poor survival, further strengthening the emerging notion that hybrid E/M phenotype(s) may occupy the 'metastatic sweet spot'.

Quantifying Cancer Epithelial-Mesenchymal Plasticity and its Association with Stemness and Immune Response.

Cancer cells can acquire a spectrum of stable hybrid epithelial/mesenchymal (E/M) states during epithelial-mesenchymal transition (EMT). Cells in these hybrid E/M phenotypes often combine epithelial and mesenchymal features and tend to migrate collectively commonly as small clusters. Such collectively migrating cancer cells play a pivotal role in seeding metastases and their presence in cancer patients indicates an adverse prognostic factor. Moreover, cancer cells in hybrid E/M phenotypes tend to be more associated with stemness which endows them with tumor-initiation ability and therapy resistance. Most recently, cells undergoing EMT have been shown to promote immune suppression for better survival. A systematic understanding of the emergence of hybrid E/M phenotypes and the connection of EMT with stemness and immune suppression would contribute to more effective therapeutic strategies. In this review, we first discuss recent efforts combining theoretical and experimental approaches to elucidate mechanisms underlying EMT multi-stability (i.e., the existence of multiple stable phenotypes during EMT) and the properties of hybrid E/M phenotypes. Following we discuss non-cell-autonomous regulation of EMT by cell cooperation and extracellular matrix. Afterwards, we discuss various metrics that can be used to quantify EMT spectrum. We further describe possible mechanisms underlying the formation of clusters of circulating tumor cells. Last but not least, we summarize recent systems biology analysis of the role of EMT in the acquisition of stemness and immune suppression.