Simi, Allison K.

Abstract Metastasis, the leading cause of mortality in cancer patients, depends upon the ability of cancer cells to invade into the extracellular matrix that surrounds the primary tumor and to escape into the vasculature. To investigate the features of the microenvironment that regulate invasion and escape, we generated solid microtumors of MDA-MB-231 human breast carcinoma cells within gels of type I collagen. The microtumors were formed at defined distances adjacent to an empty cavity, which served as an artificial vessel into which the constituent tumor cells could escape. To define the relative contributions of matrix degradation and cell proliferation on invasion and escape, we used pharmacological approaches to block the activity of matrix metalloproteinases (MMPs) or to arrest the cell cycle. We found that blocking MMP activity prevents both invasion and escape of the breast cancer cells. Surprisingly, blocking proliferation increases the rate of invasion but has no effect on that of escape. We found that arresting the cell cycle increases the expression of MMPs, consistent with the increased rate of invasion. To gain additional insight into the role of cell proliferation in the invasion process, we generated microtumors from cells that express the fluorescent ubiquitination-based cell cycle indicator. We found that the cells that initiate invasions are preferentially quiescent, whereas cell proliferation is associated with the extension of invasions. These data suggest that matrix degradation and cell proliferation are coupled during the invasion and escape of human breast cancer cells and highlight the critical role of matrix proteolysis in governing tumor phenotype.

Abstract Multinucleation is found in more than one third of tumors and is linked to increased tolerance for mutation, resistance to chemotherapy, and invasive potential. The integrity of the genome depends on proper execution of the cell cycle, which can be altered through mechanotransduction pathways as the tumor microenvironment stiffens during tumorigenesis. Here, we show that signaling downstream of matrix metalloproteinase-3 (MMP3) or TGFβ, known inducers of epithelial–mesenchymal transition (EMT), also promotes multinucleation in stiff microenvironments through Snail-dependent expression of the filament-forming protein septin-6, resulting in midbody persistence, abscission failure, and multinucleation. Consistently, we observed elevated expression of Snail and septin-6 as well as multinucleation in a human patient sample of metaplastic carcinoma of the breast, a rare classification characterized by deposition of collagen fibers and active EMT. In contrast, a soft microenvironment protected mammary epithelial cells from becoming multinucleated by preventing Snail-induced upregulation of septin-6. Our data suggest that tissue stiffening during tumorigenesis synergizes with oncogenic signaling to promote genomic abnormalities that drive cancer progression. Significance: These findings reveal tissue stiffening during tumorigenesis synergizes with oncogenic signaling to promote genomic abnormalities that drive cancer progression. Cancer Res; 78(9); 2277–89. ©2018 AACR.

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

Abstract This study investigates how increased stiffness of the tumor microenvironment (TME) can facilitate cellular multinucleation. Multinucleation is an easily observable marker for polyploidy, facilitating the quantification of this phenotype. Polyploidy is commonly an intermediate to aneuploidy, defined as an abnormal number of chromosomes, a phenotype found in 85% of solid cancers that can drive tumorigenesis. 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 promoted 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 tumor and normal microenvironments to investigate how matrix stiffness regulates multinucleation in mammary epithelial cells. 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 MMP3, a protease commonly upregulated in cancer. This signaling depended on expression of the Rac1 splice variant, Rac1b, production of reactive oxygen species (ROS), and expression of Snail. Under all conditions, cells cultured on soft substrata maintained a low frequency of multinucleation. We also investigated biochemical effectors downstream of Snail. Aurora A kinase (Aurora A) is a mitotic regulator commonly elevated in cancer that has been shown to induce multinucleation in mammary epithelial cells and increase tumor incidence in mice. We found using microarray analysis that MMP3 increases the levels of Aurora A in mammary epithelial cells. We further discovered that upregulation of Aurora A in response to MMP3 signaling was limited to one transcript variant. These data suggest that an increase in multinucleation is driven by the stiffening of the tissue during tumorigenesis, in part by MMP3-induced signaling and regulation of Aurora A splicing. More broadly, these data suggest a key role for the mechanical properties of the tumor microenvironment in cancer progression. Citation Format: Allison K. Simi, Derek C. Radisky, Celeste M. Nelson. Tissue stiffness regulates multinucleation in mammary epithelial cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5097.