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’.

Epithelial–mesenchymal transition (EMT) plays key roles during embryonic development, wound healing and cancer metastasis. Cells in a partial EMT or hybrid epithelial/mesenchymal (E/M) phenotype exhibit collective cell migration, forming clusters of circulating tumour cells—the primary drivers of metastasis. Activation of cell–cell signalling pathways such as Notch fosters a partial or complete EMT, yet the mechanisms enabling cluster formation remain poorly understood. Using an integrated computational–experimental approach, we examine the role of Numb—an inhibitor of Notch intercellular signalling—in mediating EMT and clusters formation. We show via an mathematical model that Numb inhibits a full EMT by stabilizing a hybrid E/M phenotype. Consistent with this observation, knockdown of Numb in stable hybrid E/M cells H1975 results in a full EMT, thereby showing that Numb acts as a brake for a full EMT and thus behaves as a ‘phenotypic stability factor' by modulating Notch-driven EMT. By generalizing the mathematical model to a multi-cell level, Numb is predicted to alter the balance of hybrid E/M versus mesenchymal cells in clusters, potentially resulting in a higher tumour-initiation ability. Finally, Numb correlates with a worse survival in multiple independent lung and ovarian cancer datasets, hence confirming its relationship with increased cancer aggressiveness.

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.

Extracting dynamical information from single‐cell transcriptomics is a novel task with the promise to advance our understanding of cell state transition and interactions between genes. Yet, theory‐oriented, bottom‐up approaches that consider differences among cell states are largely lacking. Here, we present spliceJAC, a method to quantify the multivariate mRNA splicing from single‐cell RNA sequencing (scRNA‐seq). spliceJAC utilizes the unspliced and spliced mRNA count matrices to constructs cell state‐specific gene–gene regulatory interactions and applies stability analysis to predict putative driver genes critical to the transitions between cell states. By applying spliceJAC to biological systems including pancreas endothelium development and epithelial–mesenchymal transition (EMT) in A549 lung cancer cells, we predict genes that serve specific signaling roles in different cell states, recover important differentially expressed genes in agreement with pre‐existing analysis, and predict new transition genes that are either exclusive or shared between different cell state transitions.

Migration from the primary tumor is a crucial step in the metastatic cascade. Cells with various degrees of adhesion and motility migrate and are launched into the bloodstream as single circulating tumor cells (CTC) or multicellular CTC clusters. The frequency and size distributions of these clusters have been recently measured, but the underlying mechanisms enabling these different modes of migration remain poorly understood. We present a biophysical model that couples the phenotypic plasticity enabled by the epithelial–mesenchymal transition (EMT) and cell migration to explain the modes of individual and collective cancer cell migration. This reduced physical model captures how cells undergo a transition from individual migration to collective cell migration and robustly recapitulates CTC cluster fractions and size distributions observed experimentally across several cancer types, thus suggesting the existence of common features in the mechanisms underlying cancer cell migration. Furthermore, we identify mechanisms that can maximize the fraction of CTC clusters in circulation. First, mechanisms that prevent a complete EMT and instead increase the population of hybrid epithelial/mesenchymal (E/M) cells are required to recapitulate CTC size distributions with large clusters of 5 to 10 cells. Second, multiple intermediate E/M states give rise to larger and heterogeneous clusters formed by cells with different epithelial–mesenchymal traits. Overall, this biophysical model provides a platform to continue to bridge the gap between the molecular and biophysical regulation of cancer cell migration and highlights that a complete EMT might not be required for metastasis. Significance: A biophysical model of cancer cell invasion integrates phenotypic heterogeneity and cell migration to interpret experimental observations of circulating tumor cell clusters and provides new predictions.

Folded proteins show a high degree of structural order and undergo (fairly constrained) collective motions related to their functions. On the other hand, intrinsically disordered proteins (IDPs), while lacking a well-defined three-dimensional structure, do exhibit some structural and dynamical ordering, but are less constrained in their motions than folded proteins. The larger structural plasticity of IDPs emphasizes the importance of entropically driven motions. Many IDPs undergo function-related disorder-to-order transitions driven by their interaction with specific binding partners. As experimental techniques become more sensitive and become better integrated with computational simulations, we are beginning to see how the modest structural ordering and large amplitude collective motions of IDPs endow them with an ability to mediate multiple interactions with different partners in the cell. To illustrate these points, here, we use Prostate-associated gene 4 (PAGE4), an IDP implicated in prostate cancer (PCa) as an example. We first review our previous efforts using molecular dynamics simulations based on atomistic AWSEM to study the conformational dynamics of PAGE4 and how its motions change in its different physiologically relevant phosphorylated forms. Our simulations quantitatively reproduced experimental observations and revealed how structural and dynamical ordering are encoded in the sequence of PAGE4 and can be modulated by different extents of phosphorylation by the kinases HIPK1 and CLK2. This ordering is reflected in changing populations of certain secondary structural elements as well as in the regularity of its collective motions. These ordered features are directly correlated with the functional interactions of WT-PAGE4, HIPK1-PAGE4 and CLK2-PAGE4 with the AP-1 signaling axis. These interactions give rise to repeated transitions between (high HIPK1-PAGE4, low CLK2-PAGE4) and (low HIPK1-PAGE4, high CLK2-PAGE4) cell phenotypes, which possess differing sensitivities to the standard PCa therapies, such as androgen deprivation therapy (ADT). We argue that, although the structural plasticity of an IDP is important in promoting promiscuous interactions, the modulation of the structural ordering is important for sculpting its interactions so as to rewire with agility biomolecular interaction networks with significant functional consequences.