Mohit Kumar Jolly

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) – 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.

Tumor Budding: The Name is EMT. Partial EMT.

Tumor budding is a histological phenomenon encountered in various cancers, whereby individual malignant cells and/or small clusters of malignant cells are seen in the tumor stroma. Postulated to be mirror epithelial-mesenchymal transition, tumor budding has been associated with poor cancer outcomes. However, the vast heterogeneity in its exact definition, methodology of assessment, and patient stratification need to be resolved before it can be routinely used as a standardized prognostic feature. Here, we discuss the heterogeneity in defining and assessing tumor budding, its clinical significance across multiple cancer types, and its prospective implementation in clinical practice. Next, we review the emerging evidence about partial, rather than complete, epithelial-mesenchymal phenotype at the tumor bud level, and its connection with tumor proliferation, quiescence, and stemness. Finally, based on recent literature, indicating a co-expression of epithelial and mesenchymal markers in many tumor buds, we posit tumor budding to be a manifestation of this hybrid epithelial/mesenchymal phenotype displaying collective cell migration.

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.

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.

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.

The three-way switch operation of Rac1/RhoA GTPase-based circuit controlling amoeboid-hybrid-mesenchymal transition.

Metastatic carcinoma cells exhibit at least two different phenotypes of motility and invasion - amoeboid and mesenchymal. This plasticity poses a major clinical challenge for treating metastasis, while its underlying mechanisms remain enigmatic. Transitions between these phenotypes are mediated by the Rac1/RhoA circuit that responds to external signals such as HGF/SF via c-MET pathway. Using detailed modeling of GTPase-based regulation to study the Rac1/RhoA circuits dynamics, we found that it can operate as a three-way switch. We propose to associate the circuits three possible states to the amoeboid, mesenchymal and amoeboid/mesenchymal hybrid phenotype. In particular, we investigated the range of existence of, and the transition between, the three states (phenotypes) in response to Grb2 and Gab1 - two downstream adaptors of c-MET. The results help to explain the regulation of metastatic cells by c-MET pathway and hence can contribute to the assessment of possible clinical interventions.

Toward Decoding the Principles of Cancer Metastasis Circuits

Understanding epithelial-mesenchymal transitions (EMT) during cancer metastasis remains a major challenge in modern biology. Recent observations of cell behavior together with progress in mapping the underlying regulatory genetic networks led to new understandings of carcinoma metastasis. It is now established that the genetic network that regulates the EMT also enables an epithelial-mesenchymal hybrid phenotype. These hybrid cells possess mixed carcinoma epithelial and mesenchymal characteristics that enable specialized capabilities such as collective cell migration. On the gene network perspective, a four-component decision unit composed of two highly interconnected chimeric modules--the miR34/SNAIL and the miR200/ZEB mutual-inhibition feedback circuits--regulates the coexistence of and transitions between the different phenotypes. Here, we present a new tractable theoretical framework to model and decode the underlying principles governing the operation of the regulatory unit. Our approach connects the knowledge about intracellular pathways with observations of cellular behavior and advances toward understanding the logic of cancer decision-making. We found that the miR34/SNAIL module acts as an integrator while the miR200/ZEB module acts as a three-way switch. Consequently, the combined unit can give rise to three phenotypes (stable states): (i) a high miR200 and low ZEB, or (1, 0) state; (ii) a low miR200 and high ZEB, or (0, 1) state; and (iii) a medium miR200 and medium ZEB, or (½, ½) state. We associate these states with the epithelial, mesenchymal, and hybrid phenotypes, respectively. We reflect on the consistency between our theoretical predictions and recent observations in several types of carcinomas and suggest new testable predictions. See all articles in this Cancer Research section,

EMT and MET: necessary or permissive for metastasis?

Epithelial‐to‐mesenchymal transition (EMT) and its reverse mesenchymal‐to‐epithelial transition (MET) have been suggested to play crucial roles in metastatic dissemination of carcinomas. These phenotypic transitions between states are not binary. Instead, carcinoma cells often exhibit a spectrum of epithelial/mesenchymal phenotype(s). While epithelial/mesenchymal plasticity has been observed preclinically and clinically, whether any of these phenotypic transitions are indispensable for metastatic outgrowth remains an unanswered question. Here, we focus on epithelial/mesenchymal plasticity in metastatic dissemination and propose alternative mechanisms for successful dissemination and metastases beyond the traditional EMT/MET view. We highlight multiple hypotheses that can help reconcile conflicting observations, and outline the next set of key questions that can offer valuable insights into mechanisms of metastasis in multiple tumor models.