Abstract C39: Building a novel molecular model for Twist1-induced epithelial dissemination
Authors: Shamir Eliah R. , Tran Phuoc T. , Ewald Andrew J. 
Published: August 2014

Most solid tumors originate from epithelial cells, and their metastasis represents the major cause of death among cancer patients. Because each tumor contains many mutations, it has been difficult to isolate how individual genetic events contribute to a tumor cell's ability to disseminate. Two posited mechanisms for metastasis are loss of cell-cell adhesion and activation of an epithelial-mesenchymal transition (EMT). These molecular models converge on the cell adhesion gene E-cadherin (Cdh1). E-cadherin is frequently mutated in human cancers, and it can also be transcriptionally repressed during EMT. In turn, expression of EMT transcription factors in multiple epithelial cancers has been correlated with increased invasion and metastasis. However, the acute consequences of E-cadherin loss or EMT activator expression in otherwise normal epithelial tissues remain incompletely understood.

We therefore utilized a combination of organotypic culture, Cre-lox based gene deletion, inducible expression systems, and time-lapse imaging to test the sufficiency of single genetic perturbations to induce dissemination in primary murine mammary epithelium. E-cadherin deletion induced loss of simple epithelial architecture and inhibited branching morphogenesis in 3D culture and in vivo. Furthermore, E-cadherin loss acutely induced collective invasion past basement membrane. However, E-cadherin- cells remained non-protrusive, adhered to one another, and rarely disseminated. Conversely, expression of the EMT transcription factor Twist1 induced rapid dissemination of individual epithelial cells into the extracellular matrix (ECM), and disseminated cells proliferated to form secondary sites. Surprisingly, Twist1+ cells were observed to have membrane-localized E-cadherin during dissemination and during migration in the ECM. Our data thus provide distinct roles for E-cadherin and Twist1 in the early cellular processes of metastasis. We are currently testing whether shRNA knockdown of E-cadherin synergizes with Twist1 to further enhance dissemination.

To date, we have demonstrated the sufficiency of constitutive Twist1 expression to induce dissemination of both major mammary cell types: inner luminal epithelial cells and outer myoepithelial cells. Moreover, we observe abnormal localization of myoepithelial cells and basement membrane to the inside of the tissue. However, it remains unclear why some Twist1-expressing cells disseminate while many others remain in the epithelium. We hypothesize that intrinsic, cell type-specific factors influence disseminative capacity. We next seek to test the sufficiency of Twist1 to induce cell dissemination and secondary site formation in distinct mammary epithelial subpopulations. We have developed mouse models that use cell-type specific promoters to drive Twist1 expression in myoepithelial, luminal progenitor, and differentiated luminal lineages. We anticipate that differences across cell types may help identify the subpopulations within a heterogeneous tumor with the highest metastatic potential. Furthermore, we expect that extrinsic factors, such as normal myoepithelial cells, may restrict dissemination of Twist1-expressing luminal cells. Understanding such modulation of Twist1's phenotypic outcomes could suggest new approaches to suppressing metastasis without necessitating direct inhibition of Twist1, itself an “undruggable” target.

Citation Format: Eliah R. Shamir, Phuoc T. Tran, Andrew J. Ewald. Building a novel molecular model for Twist1-induced epithelial dissemination. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C39.