h Phenotypic plasticity involves a process in which cells transiently acquire phenotypic traits of another lineage. Two commonly studied types of phenotypic plasticity are epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET). In carcinomas, EMT drives invasion and metastatic dissemination, while MET is proposed to play a role in metastatic colonization. Phenotypic plasticity in sarcomas is not well studied; however, there is evidence that a subset of sarcomas undergo an MET-like phenomenon. While the exact mechanisms by which these transitions occur remain largely unknown, it is likely that some of the same master regulators that drive EMT and MET in carcinomas also act in sarcomas. In this study, we combined mathematical models with bench experiments to identify a core regulatory circuit that controls MET in sarcomas. This circuit comprises the microRNA 200 (miR-200) family, ZEB1, and GRHL2. Interestingly, combined expression of miR-200s and GRHL2 further upregulates epithelial genes to induce MET. This effect is phenocopied by downregulation of either ZEB1 or the ZEB1 cofactor, BRG1. In addition, an MET gene expression signature is prognostic for improved overall survival in sarcoma patients. Together, our results suggest that a miR-200, ZEB1, GRHL2 gene regulatory network may drive sarcoma cells to a more epithelial-like state and that this likely has prognostic relevance. P henotypic plasticity is defined as the reversible conversion of cellular phenotypes from one state to another. The two most commonly studied types of plasticity are epithelial-mesenchymal transition (EMT) and the reverse, mesenchymal-epithelial transition (MET). These phenotypic transitions play important roles in normal development (reviewed in references 1 to 4) and wound healing (reviewed in reference 5); however, similar pathways and gene expression programs can also be coopted by the cell during fibrosis (reviewed in references 6 to 8) and carcinoma progression (reviewed in references 9 to 12). In the context of carcinoma progression, a subset of cells within the tumor are thought to undergo an EMT, which enables those cells to break free from the tumor mass via loss of cell-cell adhesions (13,14) and upregulate invasive programs that facilitate dissemination (13). In addition to these phenotypic changes, EMT also contributes to alterations in cancer cell metabolism (10), drug resistance (15, 16), tumor initiation ability (17, 18), and perhaps even host immune evasion (19). EMT is often accompanied by downregulation of proliferation (20, 21), and, in some cases, MET is important for reinitiation of proliferation during metastatic colonization (22). It is important to note that as the field of phenotypic plasticity has matured, particularly in the context of carcinoma progression, EMT and MET have become recognized as more of a spectrum of phenotypes, rather than discrete states of fully differentiated epithelial and mesenchymal phenotypes. These metastable, or hybrid, E/M transition states have been obs...

E-cadherin, an epithelial-specific cell-cell adhesion molecule, plays multiple roles in maintaining adherens junctions, regulating migration and invasion, and mediating intracellular signaling. Downregulation of E-cadherin is a hallmark of epithelial-mesenchymal transition (EMT) and correlates with poor prognosis in multiple carcinomas. Conversely, upregulation of E-cadherin is prognostic for improved survival in sarcomas. Yet, despite the prognostic benefit of E-cadherin expression in sarcoma, the mechanistic significance of E-cadherin in sarcomas remains poorly understood. Here, by combining mathematical models with wet-bench experiments, we identify the core regulatory networks mediated by E-cadherin in sarcomas, and decipher their functional consequences. Unlike in carcinomas, E-cadherin overexpression in sarcomas does not induce a mesenchymal-epithelial transition (MET). However, E-cadherin acts to reduce both anchorage-independent growth and spheroid formation of sarcoma cells. Ectopic E-cadherin expression acts to downregulate phosphorylated CREB (p-CREB) and the transcription factor, TBX2, to inhibit anchorage-independent growth. RNAi-mediated knockdown of TBX2 phenocopies the effect of E-cadherin on p-CREB levels and restores sensitivity to anchorage-independent growth in sarcoma cells. Beyond its signaling role, E-cadherin expression in sarcoma cells can also strengthen cell-cell adhesion and restricts spheroid growth through mechanical action. Together, our results demonstrate that E-cadherin inhibits sarcoma aggressiveness by preventing anchorage-independent growth.

42E-cadherin, an epithelial-specific cell-cell adhesion molecule, plays multiple roles in maintaining 43 adherens junctions, regulating migration and invasion, and mediating intracellular signaling. 44 Downregulation of E-cadherin is a hallmark of epithelial-mesenchymal transition (EMT) and 45 correlates with poor prognosis in multiple carcinomas. Conversely, upregulation of E-cadherin is 46 prognostic for improved survival in sarcomas. Yet, despite the prognostic benefit of E-cadherin 47 expression in sarcoma, the mechanistic significance of E-cadherin in sarcomas remains poorly 48 understood. Here, by combining mathematical models with wet-bench experiments, we identify 49 the core regulatory networks mediated by E-cadherin in sarcomas, and decipher their functional 50 consequences. Unlike in carcinomas, E-cadherin overexpression in sarcomas does not induce a 51 mesenchymal-epithelial transition (MET). However, E-cadherin acts to reduce both anchorage-52 independent growth and spheroid formation of sarcoma cells. Ectopic E-cadherin expression acts 53 to downregulate phosphorylated CREB (p-CREB) and the transcription factor, TBX2, to inhibit 54 anchorage-independent growth. RNAi-mediated knockdown of TBX2 phenocopies the effect of 55 E-cadherin on p-CREB levels and restores sensitivity to anchorage-independent growth in 56 sarcoma cells. Beyond its signaling role, E-cadherin expression in sarcoma cells can also 57 strengthen cell-cell adhesion and restricts spheroid growth through mechanical action. Together, 58 our results demonstrate that E-cadherin inhibits sarcoma aggressiveness by preventing 59 anchorage-independent growth. 60 61 62 63 64 65 66 67 68 69 70 71Sarcomas -deadly cancers that arise from tissues of a mesenchymal lineage -are highly 72 aggressive, with five year survival rates of just 66% (1). Despite their mesenchymal origin, some 73 sarcomas undergo phenotypic plasticity in which they gain "epithelial-like" traits (2)(3)(4). While 74 this transition to a more epithelial-like state is now being recognized as a feature of multiple 75 subtypes of soft tissue sarcoma and osteosarcoma (2)(3)(4), there are also a number of sarcoma 76 subtypes that are classically known to exhibit epithelioid features pathologically, including 77 synovial sarcoma (5), epithelioid sarcoma (6), and adamantinoma (7). One might expect the 78 acquisition of epithelial-like traits to be of little relevance in mesenchymal tumors, yet that is not 79 the case. Phenotypic plasticity is clinically important in sarcoma patients: Sarcoma patients 80 whose tumors express epithelial-like biomarkers have improved outcomes relative to patients 81 with more "mesenchymal-like" tumors (2)(3)(4)8). 82 Phenotypic plasticity observed in sarcomas is reminiscent of the phenomenon of 83 epithelial plasticity in carcinomas. Epithelial plasticity refers to reversible transitions between 84 epithelial and mesenchymal phenotypes. In carcinomas, the phenotypic transition to a more 85 mesenchymal-like state via an epithelial-mesenchyma...

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic plasticity drives invasion, dissemination and metastasis. Indeed, while most of the studies of phenotypic plasticity have been in the context of epithelial-derived carcinomas, it turns out sarcomas, which are mesenchymal in origin, also exhibit phenotypic plasticity, with a subset of sarcomas undergoing a phenomenon that resembles a mesenchymal-epithelial transition (MET). Here, we developed a method comprising the miR-200 family and grainyhead-like 2 (GRHL2) to mimic this MET-like phenomenon observed in sarcoma patient samples.We sequentially express GRHL2 and the miR-200 family using cell transduction and transfection, respectively, to better understand the molecular underpinnings of these phenotypic transitions in sarcoma cells. Sarcoma cells expressing miR-200s and GRHL2 demonstrated enhanced epithelial characteristics in cell morphology and alteration of epithelial and mesenchymal biomarkers. Future studies using these methods can be used to better understand the phenotypic consequences of MET-like processes on sarcoma cells, such as migration, invasion, metastatic propensity, and therapy resistance.

<p>S2. Ectopic E-cadherin expression in sarcoma cells does not alter EMT.</p>

<p>Supplementary Data</p>

<p>S4. E-cadherin induces E- to N-cadherin switching in sarcoma cells.</p>

<p>S3. CREB knockdown indicates it is downstream of E-cadherin and TBX2.</p>