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Review
. 2015 Jul 20:5:155.
doi: 10.3389/fonc.2015.00155. eCollection 2015.

Implications of the Hybrid Epithelial/Mesenchymal Phenotype in Metastasis

Mohit Kumar Jolly  1 , Marcelo Boareto  2 , Bin Huang  3 , Dongya Jia  4 , Mingyang Lu  5 , Eshel Ben-Jacob  6 , José N Onuchic  7 , Herbert Levine  8
Affiliations
Review

Implications of the Hybrid Epithelial/Mesenchymal Phenotype in Metastasis

Mohit Kumar Jolly et al. Front Oncol. .

Abstract

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.

Keywords: cancer stem cells; cancer systems biology; cell-fate decisions; intermediate EMT; partial EMT.

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Figures

Figure 1
Figure 1
EMT phenotypes and core EMT network. (A) Canonical morphological and functional characteristics of the three phenotypes – epithelial (E), hybrid epithelial/mesenchymal (E/M), and mesenchymal (M). (B) Core EMT regulatory network (shown in yellow box) consists of two interconnected mutually inhibitory feedback loops – (miR-34/SNAIL and miR-200/ZEB). Solid bars represent transcriptional inhibition, solid arrows represent transcriptional activation, and dotted lines denote miRNA-mediated regulation. Numbers mentioned alongside each regulation are the number of binding sites for that particular regulation, as experimentally determined or proposed. This core network receives inputs from a variety of signals (shown by I), modulates many cytoskeletal elements (E-cadherin, N-cadherin, Vimentin, and polarity complexes), and couples with many other cellular traits. [adapted from Refs. (22) and (41)]
Figure 2
Figure 2
Dynamic characteristics of mutually inhibitory feedback loops. (A) (left) Weak mutual inhibition between A and B allows monostability where the steady state has intermediate levels of both A and B; (middle) Strong mutual inhibition between A and B can drive one species to extremely low levels, and therefore bistability, such that the two steady states are – (high A, low B) or (1,0), and (low A, high B) or (0,1); (right) Strong mutual inhibition between A and B, coupled with strong self-activation of both A and B can enable the system to be tristable, such that the three steady states are – (high A, low B) or (1,0), and (low A, high B) or (0,1), and (medium A, medium B) or (1/2, 1/2). Red and black curves describe nullclines for A and B, and their intersections are the steady states. Green-filled circles represent stable steady states, and green hollow circles show unstable steady states. The thickness of lines representing mutual inhibition between A and B, and self-activation of A and B represent relative strength of those interactions. (B) Cartoons (corresponding to the circuit drawn in the same column) representing the potential energy of the system, where valleys represent stable steady states, and crests denote unstable steady states.
Figure 3
Figure 3
Distinct cell fates vs. quantitative trait variation of same cell fate. (A) (Left) bifurcation diagram representing variation in levels of A as an input is applied to a bistable mutually inhibitory circuit between A and B. At some threshold value of the input signal (marked by bifurcation point), the initial cell fate disappears and gives rise to two new stable steady state or cell fates. (middle) These two cell fates can be observed as different subpopulations in a FACS experiment. (right) Most cells attain one of the two cell fates, and the population distribution is bimodal with different range of values of A. (B) (left) Bifurcation diagram representing variation in levels of A as an input is applied to a monostable mutually inhibitory circuit between A and B. The circuit responds in an ultrasensitive manner but no bifurcation of cell fates observed. (middle) FACS experiments show a population with continuously varying levels of A without any sharp boundaries, hence (right) the population distribution is unimodal and broadly Gaussian.
Figure 4
Figure 4
Comparing the behavior of a phenotypic transition that has continuous state-space vs. the one with discrete state-space. (A) Behavior of a system with continuous state-space (infinite stable steady states) during a phenotypic transition as induced by an external input signal. Solid red lines show stable states, and blue dotted lines show unstable steady states. (B) Behavior of a system with discrete state-space [finite number (n = 2 here) of stable steady states (cell fates)]. Blue lines represent stable steady states, and red line denotes unstable steady states. Blue shaded region shows the range of hysteresis and bistability. Black arrows mark the levels of input where the cell switches fate (or transitions from one stable steady state to another) – X1, X2. The table presented below compares the behavior of the two scenarios depicted in (A,B). In both (A,B), green arrows denote the response of the system when the input signal is removed.
Figure 5
Figure 5
Population distribution or multimodality in EMT response. (A) (Middle) bifurcation of ZEB mRNA levels in response to protein SNAIL (EMT-inducing signal) for the miR-200/ZEB/SNAIL circuit (shown at extreme left). For a certain range of SNAIL values (marked by green rectangle), cells can attain any of the three phenotypes – E, M, and E/M, giving rise to a trimodal population distribution as shown in FACS figure (left). For a different range of SNAIL values (marked by orange rectangle), cells can adopt either E/M or M phenotype, and be distributed in a bimodal manner in FACS figure (right). (B) Bifurcation of ZEB mRNA levels in response to protein SNAIL (EMT-inducing signal) for the miR-200/ZEB/SNAIL/OVOL circuit (shown at extreme left). For a certain range of SNAIL values (marked by yellow rectangle), cells can adopt either E/M or E phenotype, and be distributed in a bimodal manner (FACS figure, left); and for a different range (marked by brown rectangle) all cells are likely to be in E/M phenotype as shown in FACS figure (right). Importantly, as compared to the behavior of miR-200/ZEB/SNAIL circuit, miR-200/ZEB/SNAIL/OVOL circuit allows the existence of new phases (combinations of phenotypes) such as {E/M} and {E, E/M}, and precludes the existence of phases {E, E/M, M}.
Figure 6
Figure 6
Landscape of cellular shape plasticity during carcinoma metastasis. (A) Cartoon representation of different cell shapes/phenotypes with their respective places on the two-dimensional space of levels of active RhoA (RhoA-GTP) and active Rac1 (Rac1-GTP). As miR-34 and miR-200 inhibit both RhoA and Rac1, both epithelial and hybrid E/M phenotypes have low levels of active forms of RhoA and Rac1. The (high RhoA-GTP, low Rac1-GTP) profile associates with amoeboid (A) morphology with blebs [blebby amoeboid (BA)], whereas (low RhoA-GTP, high Rac1-GTP) associates with mesenchymal (M) shape – cells with lamellopodia or filopodia (LAM or FIL). Cells with (high RhoA-GTP, high Rac1-GTP) adopt a hybrid A/M morphology that can be manifested in multiple ways – lamellipoida with blebs (LB), lobopodia (LP), and pseudopodal amoeboid (PA). Transitions among E, E/M, and M phenotypes (EMT/MET) are represented by orange arrows, those between amoeboid and mesenchymal morphologies – A, A/M, and M – are denoted by blue arrows, and transitions between E/M and A phenotypes – CAT/ACT – are denoted by black arrows. (B) Circuits showing the coupling of core EMT circuit with RhoA and Rac1 – the two GTPases that are critical in regulating cell shape. They inhibit the GTP loading (switching from inactive GDP-bound state to active GTP-bound state) of each other and promote that of themselves (shown by dotted lines). Also, RhoA can activate itself indirectly on a transcription level (solid black lines) (see Ref. (74) and references therein). The microRNAs miR-34 and miR-200 inhibit the translation of RhoA and Rac1. Figure adapted from Ref. (74).
Figure 7
Figure 7
Association of partial EMT with stemness. (A) “EMT gradient” model proposed by Ombrato and Malanchi (134) where stemness is maintained within a window between a fully differentiated epithelial cell and a fully de-differentiated mesenchymal cell [Figure adapted from Ref. (134), cartoons included]. (B) Stemness or tumor-initiating potential of E, E/M, and M phenotype, or variation of stemness during EMT progression (brown line).
Figure 8
Figure 8
Cell–cell communication and partial EMT. (A) Coupling of EMT circuit with Notch circuit. Notch pathway, when activated by Jagged or Delta, belonging to neighboring cell, can activate Jagged and Notch, but inhibit Delta. EMT circuit couples with Notch circuit in many ways – miR-200 inhibits Jagged1, miR-34 inhibits both Notch and Delta, and NICD can activate SNAIL to drive EMT. (B) Notch-Delta signaling between two cells induces opposite fates in them – one cell behaves as a Sender (high Delta, low Notch) and the other a Receiver (high Notch, low Delta). Due to this lateral inhibition, it can promote “salt-and-pepper” based patterns. (C) Notch-Jagged signaling between two cells induces similar fates in them – lateral induction – and thus leads to patterns with all cells with the same fate. (D) (Left) cells in a partial EMT and interacting via N-D signaling might not be spatially close to each other, because N-D signaling inhibits two neighbors to adopt the same fate. (right) Cells in a partial EMT and interacting via N-J signaling can mutually stabilize the E/M phenotype and stay together as a cluster. Figure adapted from Ref. (173).
Figure 9
Figure 9
Interplay between Notch-Jagged signaling, Partial EMT or CTC clusters, stemness, and therapy/drug resistance. Cells in a hybrid E/M or partial EMT phenotype (as present in CTC clusters) can possess a much higher tumor-initiation potential (“stemness”) and drug resistance as compared to a completely mesenchymal phenotype. These cells can maintain their “metastable” hybrid E/M phenotype via Notch-Jagged signaling that promotes lateral stabilization (maintenance of same cell fate in neighboring cells) and/or lateral induction (propagation of the same fate as of its own to the neighbor) among a population of cells. This lateral induction can also be utilized to propagate drug resistance among a small subpopulation of cells known as “Cancer Stem Cells” (CSCs).
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