Abstract A47: Reducing actin contraction in E-cadherin inactivated mammary epithelial cells leads to the development of invasive lobular carcinoma
Authors: Schipper Koen , Nethe Micha , Kas Sjors M. , Ruiter Julian R. de , Drenth Anne Paulien , Burg Eline van der , Boon Ute , Schut Eva , Jonkers Jos 
Published: May 2018

Invasive lobular carcinoma (ILC) accounts for 10-15% of breast cancer cases in women. One of the characteristics of ILC is the loss of intercellular adhesion, which occurs as a consequence of inactivation of E-cadherin (CDH1). Inactivation of E-cadherin often occurs through truncating mutations followed by loss of heterozygosity. E-cadherin is a master regulator of cell-cell interactions and thereby functions as an important tumor suppressor. Nevertheless, loss of E-cadherin by itself does not induce mammary tumor formation in mice. Moreover, somatic inactivation of E-cadherin in mouse mammary epithelium resulted in outgrowth mammary ducts with only E-cadherin proficient mammary epithelial cells, suggesting that loss of E-cadherin by itself is not tolerated. Why the inactivation of E-cadherin is not tolerated in mammary epithelial cells remains unclear.

To uncover the fate of mammary epithelial cells upon inactivation of E-cadherin, a WapCre;Cdh1F/F;mTmg mouse model was generated. The introduction of the mTmG reporter allows for the identification and tracking of Cre-driven E-cadherin inactivated mammary epithelial cells. Analysis of the mammary ducts of these mice revealed that at least a portion of E-cadherin inactivated mammary epithelial cells survive. At 12 weeks of age clusters of E-cadherin inactivated mammary epithelial cells were found nested between the myoepithelial cells and the basal membrane. Despite the fact that they survive, these cells appear dormant since these clusters do not increase in size over time. Using in vivo imaging of WapCre;Cdh1F/F;mTmg mice, it becomes clear that the E-cadherin inactivated cells are motile and show a high degree of membrane blebbing. The E-cadherin inactivated cells have increased cortical accumulation of MYH9 and myosin light chain phosphorylation, which indicate increased actin contraction.

Recently we identified PPP1R12A/B, TP53BP2, and MYH9 as drivers of ILC in an in vivo insertional mutagenesis screen. The identified drivers did not have any effect on the PI3 kinase and MAP kinase pathways nor did they alter P53 activation. They did, however, allow survival of E-cadherin inactivated mammary cells in vitro and in vivo. PPP1R12A/B and MYH9 are primarily known for their critical role in actin contractility. Interestingly, overexpression of the TRP53BP2 truncation variant identified in the screen reduced myosin light chain phosphorylation in a similar manner as the PPP1R12A truncation variant. To provide more evidence that the reduction of actin contraction allows rescue of E-cadherin inactivated cells, we inhibited actin contraction pharmacologically. Both exposure to a Rho kinase inhibitor as well as blebbistatin (inhibits non-muscle myosin ATPase activity) allowed for the growth of E-cadherin inactivated primary epithelial cells.

Overall our data suggest that the inactivation of E-cadherin in mammary epithelial cells leads to aberrant actin contraction preventing oncogenic transformation. By reducing actin contraction, it allows for the many benefits of losing E-cadherin to drive the development of ILC. We are currently investigating through which signaling routes the increase in actin contraction prevents growth.

Citation Format: Koen Schipper, Micha Nethe, Sjors M. Kas, Julian R. de Ruiter, Anne Paulien Drenth, Eline van der Burg, Ute Boon, Eva Schut, Jos Jonkers. Reducing actin contraction in E-cadherin inactivated mammary epithelial cells leads to the development of invasive lobular carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A47.