MECHANICAL REGULATION OF GENOMIC INSTABILITY IN CANCER

Abstract

It is expected that 1 in 3 people in the United States will develop cancer in their lifetime, and it remains the second leading cause of death worldwide. The research that drove most current cancer therapies focused primarily on the cancer cells themselves, even though tumor growth and progression are critically influenced by signals from the local microenvironment. For example, tissue stiffening is an inherent feature of tumorigenesis, and induces changes in the organization and proliferation of surrounding cells. Its ubiquity aids diagnosis; stiffness correlates with density, which is detected by x-rays in mammograms. This dissertation explores how tissue stiffness regulates multinucleation, a phenotype that is found in more than one third of tumors and is associated with invasion, chemotherapeutic resistance, and increased tolerance for mutation. Importantly, multinucleated cells are an intermediate to aneuploidy. Defined as an abnormal number of chromosomes, aneuploidy is found in over 85% of tumors, and has been shown to induce tumorigenesis in vivo. We used two-dimensional engineered substrata to investigate the effects of substratum stiffness on multinucleation in mammary epithelial cells.

We found that increased stiffness induces multinucleation, regulated in part by signaling downstream of matrix metalloproteinase-3 (MMP3). MMP3 is commonly upregulated in cancer and known to induce epithelial-mesenchymal transition (EMT), a process believed to initiate metastasis. Signaling includes expression of the key EMT effector and transcription factor, Snail. Similarly, we found that transforming growth factor-β (TGFβ), another EMT-inducer, also causes multinucleation downstream of Snail. Under all conditions, cells cultured on soft substrata maintain a low frequency of multinucleation.

Following these findings, we investigated the specific mechanisms by which stiffness and EMT signaling induce multinucleation. We found that elevated levels of septin-6, a novel target of Snail, results in midbody persistence, abscission failure, and multinucleation. A soft microenvironment protects cells from becoming multinucleated by preventing Snail-induced upregulation of septin-6. Consistently, we observed elevated expression of Snail and septin-6, as well as multinucleation, in high density regions of a human patient sample of metaplastic carcinoma of the breast, a rare classification characterized by active EMT.

Taken together, these data suggest that tissue stiffening during tumorigenesis synergizes with oncogenic signaling to promote genomic abnormalities that drive cancer progression, and provide the first evidence that mechanical stiffness of the microenvironment can play a direct causal role in altering the genome. Further, our results suggest that EMT-related signaling pathways are associated with disease progression not necessarily because they induce metastasis, but because they induce multinucleation. Thus we speculate that stiffness and EMT signaling are relevant in early stages of breast cancer, and that within these data are unused biomarkers and targets for new therapeutics.