Abstract 5914: A soft microenvironment protects from failure of midbody abscission and multinucleation downstream of EMT initiators

Abstract

This study investigates how increased stiffness of the tumor microenvironment can induce cellular multinucleation, an easily observable marker of polyploidy. Up to 37% percent of tumors exhibit whole-genome doubling, which typically precedes other somatic copy number alterations. Additionally, induction of tetraploidy in human cells promotes increased tolerance for mutation, resistance to chemotherapeutic drugs, and transformation in culture. Tumors are inherently stiffer than normal tissue, and this property has been shown to affect cell growth and proliferation. Similarly, cell cycle errors have long been linked to chromosomal abnormalities. Here, we used engineered two-dimensional substrata that mimic the stiffness of tumor and normal microenvironments to investigate how matrix stiffness regulates multinucleation in mammary epithelial cells. Multinucleation was quantified by staining with Hoescht to visualize the nuclei. Timelapse microscopy enabled visualization of the process by which cells become multinucleated. Changes in gene expression were determined by quantitative RT-PCR. Cells cultured on “stiff” substrata, representing tumor tissue, showed a nearly 14-fold increase in multinucleation compared to cells cultured on “soft” substrata, representing normal tissue. We found that multinucleation was regulated in part by signaling downstream of matrix metalloproteinase-3 (MMP3), which is commonly upregulated in cancer and known to induce epithelial-mesenchymal transition (EMT). This signaling depended on expression of the Rac1 splice variant, Rac1b, production of ROS, and expression of Snail. Under all conditions, cells cultured on soft substrata maintained a low frequency of multinucleation. Multinucleation on stiff substrata primarily resulted from midbody abscission failure. A soft microenvironment protected the stability of the genome in epithelial cells by preventing midbody stability, which depended on septin 4, a novel target of Snail. Importantly, we found that transforming growth factor-β (TGFβ), another EMT-inducer, also caused multinucleation downstream of Snail, which was prevented by culture on soft substrata. Our data thus suggest that tissue stiffening during tumorigenesis synergizes with oncogenic signaling to promote genomic abnormalities that drive cancer progression. Further, our results suggest that EMT-related signaling pathways are associated with disease progression not necessarily because they induce metastasis, but because they induce genomic instability. Note: This abstract was not presented at the meeting. Citation Format: Allison K. Simi, Alisya A. Anlas, Sherry X. Zhang, Tiffaney Hsia, Derek C. Radisky, Celeste M. Nelson. A soft microenvironment protects from failure of midbody abscission and multinucleation downstream of EMT initiators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5914. doi:10.1158/1538-7445.AM2017-5914