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Myosin II isoform switching mediates invasiveness after TGF-?-induced epithelial-mesenchymal transition.

ABSTRACT: Despite functional significance of nonmuscle myosin II in cell migration and invasion, its role in epithelial-mesenchymal transition (EMT) or TGF-? signaling is unknown. Analysis of normal mammary gland expression revealed that myosin IIC is expressed in luminal cells, whereas myosin IIB expression is up-regulated in myoepithelial cells that have more mesenchymal characteristics. Furthermore, TGF-? induction of EMT in nontransformed murine mammary gland epithelial cells results in an isoform switch from myosin IIC to myosin IIB and increased phosphorylation of myosin heavy chain (MHC) IIA on target sites known to regulate filament dynamics (S1916, S1943). These expression and phosphorylation changes are downstream of heterogeneous nuclear ribonucleoprotein-E1 (E1), an effector of TGF-? signaling. E1 knockdown drives cells into a migratory, invasive mesenchymal state and concomitantly up-regulates MHC IIB expression and MHC IIA phosphorylation. Abrogation of myosin IIB expression in the E1 knockdown cells has no effect on 2D migration but significantly reduced transmigration and macrophage-stimulated collagen invasion. These studies indicate that transition between myosin IIC/myosin IIB expression is a critical feature of EMT that contributes to increases in invasive behavior.

SUBMITTER: Beach JR

PROVIDER: S-EPMC3207690 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Myosin II isoform switching mediates invasiveness after TGF-β-induced epithelial-mesenchymal transition.

Beach Jordan R JR Hussey George S GS Miller Tyler E TE Chaudhury Arindam A Chaudhury Arindam A Patel Purvi P Monslow James J Zheng Qiao Q Keri Ruth A RA Reizes Ofer O Bresnick Anne R AR Howe Philip H PH Egelhoff Thomas T TT

Proceedings of the National Academy of Sciences of the United States of America 20111024 44

Despite functional significance of nonmuscle myosin II in cell migration and invasion, its role in epithelial-mesenchymal transition (EMT) or TGF-β signaling is unknown. Analysis of normal mammary gland expression revealed that myosin IIC is expressed in luminal cells, whereas myosin IIB expression is up-regulated in myoepithelial cells that have more mesenchymal characteristics. Furthermore, TGF-β induction of EMT in nontransformed murine mammary gland epithelial cells results in an isoform swi ...[more]

PMID: 22025714

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Project description:A growing body of evidence supports that the epithelial-to-mesenchymal transition (EMT), which occurs during cancer development and progression, has a crucial role in metastasis by enhancing the motility of tumor cells. Transforming growth factor-β (TGF-β) is known to induce EMT in a number of cancer cell types; however, the mechanism underlying this transition process is not fully understood. In this study we have demonstrated that TGF-β upregulates the expression of tumor suppressor protein Par-4 (prostate apoptosis response-4) concomitant with the induction of EMT. Mechanistic investigations revealed that exogenous treatment with each TGF-β isoform upregulates Par-4 mRNA and protein levels in parallel levels of phosphorylated Smad2 and IκB-α increase. Disruption of TGF-β signaling by using ALK5 inhibitor, neutralizing TGF-β antibody or phosphoinositide 3-kinase inhibitor reduces endogenous Par-4 levels, suggesting that both Smad and NF-κB pathways are involved in TGF-β-mediated Par-4 upregulation. NF-κB-binding sites in Par-4 promoter have previously been reported; however, using chromatin immunoprecipitation assay we showed that Par-4 promoter region also contains Smad4-binding site. Furthermore, TGF-β promotes nuclear localization of Par-4. Prolonged TGF-β3 treatment disrupts epithelial cell morphology, promotes cell motility and induces upregulation of Snail, vimentin, zinc-finger E-box binding homeobox 1 and N-Cadherin and downregulation of Claudin-1 and E-Cadherin. Forced expression of Par-4, results in the upregulation of vimentin and Snail expression together with increase in cell migration. In contrast, small interfering RNA-mediated silencing of Par-4 expression results in decrease of vimentin and Snail expression and prevents TGF-β-induced EMT. We have also uncovered a role of X-linked inhibitor of apoptosis protein in the regulation of endogenous Par-4 levels through inhibition of caspase-mediated cleavage. In conclusion, our findings suggest that Par-4 is a novel and essential downstream target of TGF-β signaling and acts as an important factor during TGF-β-induced EMT.

Project description:Aberrant T-cell factor (TCF) transcription is implicated in the majority of colorectal cancers (CRCs). TCF transcription induces epithelial-mesenchymal transition (EMT), promoting a tumor-initiating cell (TIC) phenotype characterized by increased proliferation, multidrug resistance (MDR), invasion and metastasis. The data presented herein characterize topoisomerase II? (TopoII?) as a required component of TCF transcription promoting EMT. Using chromatin immunoprecipitation (ChIP) and protein co-immunoprecipitation (co-IP) studies, we show that TopoII? forms protein-protein interactions with ?-catentin and TCF4 and interacts with Wnt response elements (WREs) and promoters of direct target genes of TCF transcription, including: MYC, vimentin, AXIN2 and LEF1. Moreover, both TopoII? and TCF4 ChIP with the N-cadherin promoter, which is a new discovery indicating that TCF transcription may directly regulate N-cadherin expression. TopoII? N-terminal ATP-competitive inhibitors, exemplified by the marine alkaloid neoamphimedine (neo), block TCF activity in vitro and in vivo. Neo effectively inhibits TopoII? and TCF4 from binding WREs/promoter sites, whereas protein-protein interactions remain intact. Neo inhibition of TopoII?-dependent TCF transcription also correlates with significant antitumor effects in vitro and in vivo, including the reversion of EMT, the loss of TIC-mediated clonogenic colony formation, and the loss of cell motility and invasion. Interestingly, non-ATP-competitive inhibitors of TopoII?, etoposide and merbarone, were ineffective at preventing TopoII?-dependent TCF transcription. Thus, we propose that TopoII? participation in TCF transcription may convey a mechanism of MDR to conventional TopoII? inhibitors. However, our results indicate that TopoII? N-terminal ATP-binding sites remain conserved and available for drug targeting. This article defines a new strategy for targeted inhibition of TCF transcription that may lead to effective therapies for the treatment of CRC and potentially other Wnt-dependent cancers.

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Myosin II isoform switching mediates invasiveness after TGF-?-induced epithelial-mesenchymal transition.

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Myosin II isoform switching mediates invasiveness after TGF-β-induced epithelial-mesenchymal transition.

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