Recent preclinical and clinical data suggests enhanced metastatic fitness of hybrid epithelial/mesenchymal (E/M) phenotypes, but mechanistic details regarding their survival strategies during metastasis remain unclear. Here, we investigate immune-evasive strategies of hybrid E/M states. We construct and simulate the dynamics of a minimalistic regulatory network encompassing the known associations among regulators of EMT (epithelial-mesenchymal transition) and PD-L1, an established immune-suppressor. Our simulations for the network consisting of SLUG, ZEB1, miR-200, CDH1 and PD-L1, integrated with single-cell and bulk RNA-seq data analysis, elucidate that hybrid E/M cells can have high levels of PD-L1, similar to those seen in cells with a full EMT phenotype, thus obviating the need for cancer cells to undergo a full EMT to be immune-evasive. Specifically, in breast cancer, we show the co-existence of hybrid E/M phenotypes, enhanced resistance to anti-estrogen therapy and increased PD-L1 levels. Our results underscore how the emergent dynamics of interconnected regulatory networks can coordinate different axes of cellular fitness during metastasis.

Identifying the design principles of complex regulatory networks driving cellular decision-making remains essential to decode embryonic development as well as enhance cellular reprogramming. A well-studied network motif involved in cellular decision-making is a toggle switch—a set of two opposing transcription factors A and B, each of which is a master regulator of a specific cell fate and can inhibit the activity of the other. A toggle switch can lead to two possible states—(high A, low B) and (low A, high B)—and drives the ‘either-or' choice between these two cell fates for a common progenitor cell. However, the principles of coupled toggle switches remain unclear. Here, we investigate the dynamics of three master regulators A, B and C inhibiting each other, thus forming three-coupled toggle switches to form a toggle triad. Our simulations show that this toggle triad can lead to co-existence of cells into three differentiated ‘single positive' phenotypes—(high A, low B, low C), (low A, high B, low C) and (low A, low B, high C). Moreover, the hybrid or ‘double positive' phenotypes—(high A, high B, low C), (low A, high B, high C) and (high A, low B, high C)—can coexist together with ‘single positive' phenotypes. Including self-activation loops on A, B and C can increase the frequency of ‘double positive' states. Finally, we apply our results to understand cellular decision-making in terms of differentiation of naive CD4 + T cells into Th1, Th2 and Th17 states, where hybrid Th1/Th2 and hybrid Th1/Th17 cells have been reported in addition to the Th1, Th2 and Th17 ones. Our results offer novel insights into the design principles of a multi-stable network topology and provide a framework for synthetic biology to design tristable systems.

Translational readthrough generates proteins with extended C‐termini, which often possess distinct properties. Here, we have used various reporter assays to demonstrate translational readthrough of AGO1 mRNA. Analysis of ribosome profiling data and mass spectrometry data provided additional evidence for translational readthrough of AGO1. The endogenous readthrough product, Ago1x, could be detected by a specific antibody both in vitro and in vivo. This readthrough process is directed by a cis sequence downstream of the canonical AGO1 stop codon, which is sufficient to drive readthrough even in a heterologous context. This cis sequence has a let‐7a miRNA‐binding site, and readthrough is promoted by let‐7a miRNA. Interestingly, Ago1x can load miRNAs on target mRNAs without causing post‐transcriptional gene silencing, due to its inability to interact with GW182. Because of these properties, Ago1x can serve as a competitive inhibitor of miRNA pathway. In support of this, we observed increased global translation in cells overexpressing Ago1x. Overall, our results reveal a negative feedback loop in the miRNA pathway mediated by the translational readthrough product of AGO1.

Metastasis remains an unsolved clinical challenge. Two crucial features of metastasizing cancer cells are (a) their ability to dynamically move along the epithelial–hybrid–mesenchymal spectrum and (b) their tumor initiation potential or stemness. With increasing functional characterization of hybrid epithelial/mesenchymal (E/M) phenotypes along the spectrum, recent in vitro and in vivo studies have suggested an increasing association of hybrid E/M phenotypes with stemness. However, the mechanistic underpinnings enabling this association remain unclear. Here, we develop a mechanism-based mathematical modeling framework that interrogates the emergent nonlinear dynamics of the coupled network modules regulating E/M plasticity (miR-200/ZEB) and stemness (LIN28/let-7). Simulating the dynamics of this coupled network across a large ensemble of parameter sets, we observe that hybrid E/M phenotype(s) are more likely to acquire stemness relative to “pure” epithelial or mesenchymal states. We also integrate multiple “phenotypic stability factors” (PSFs) that have been shown to stabilize hybrid E/M phenotypes both in silico and in vitro—such as OVOL1/2, GRHL2, and NRF2—with this network, and demonstrate that the enrichment of hybrid E/M phenotype(s) with stemness is largely conserved in the presence of these PSFs. Thus, our results offer mechanistic insights into recent experimental observations of hybrid E/M phenotype(s) that are essential for tumor initiation and highlight how this feature is embedded in the underlying topology of interconnected EMT (Epithelial-Mesenchymal Transition) and stemness networks.

Epithelial-mesenchymal plasticity comprises of reversible transitions among epithelial, hybrid epithelial/mesenchymal (E/M) and mesenchymal phenotypes, and underlies various aspects of aggressive tumor progression such as metastasis, therapy resistance and immune evasion. The process of cells attaining one or more hybrid E/M phenotypes is termed as partial EMT. Cells in hybrid E/M phenotype(s) can be more aggressive than those in either fully epithelial or mesenchymal state. Thus, identifying regulators of hybrid E/M phenotypes is essential to decipher the rheostats of phenotypic plasticity and consequent accelerators of metastasis. Here, using a computational systems biology approach, we demonstrate that SLUG (SNAIL2) – an EMT-inducing transcription factor – can inhibit cells from undergoing a complete EMT and thus stabilizing them in hybrid E/M phenotype(s). It expands the parametric range enabling the existence of a hybrid E/M phenotype, thereby behaving as a phenotypic stability factor (PSF). Our simulations suggest that this specific property of SLUG emerges from the topology of the regulatory network it forms with other key regulators of epithelial-mesenchymal plasticity. Clinical data suggests that SLUG associates with worse patient prognosis across multiple carcinomas. Together, our results indicate that SLUG can stabilize hybrid E/M phenotype(s).