Regulators of Cancer Cell Vascular Invasion and Malignancy

Abstract

A metastasizing cancer cell will experience a series of diverse and often lethal selective pressures as it travels from a primary tumor to a distant organ site. Two crucial events in this multistep journey encompass cancer cell entry into, and later egress from, the vasculature. The first regulator detailed in this dissertation emerged from an RNAi screening procedure designed to identify genes that govern cancer cell vascular invasion. A top hit was keratin-associated protein 5-5 (Krtap5-5) and its depletion greatly reduced endothelial invasion by metastatic mammary cancer E0771 cells. Silencing Krtap5-5 destabilized keratin intermediate filaments and cells compensated by switching to vimentin intermediate filaments. Loss of key mammary keratins was accompanied by broad alterations to cytoskeletal regulators and surface markers, with a notable decrease in hemidesmosomal α6/β4-integrins. Knockdown of Krtap5-5 also negatively impacted various malignant behaviors such as cell motility, invasion into extracellular matrix, extravasation in zebrafish embryos, and lung seeding in mice. From these data we conclude that Krtap5-5 modulates cancer cell vascular invasion by controlling cytoskeletal function.

The second regulator covered in this dissertation is the mammalian Sterile20-like kinase known as MST4. It is accepted that MST4 regulates cancer cell invasion by facilitating contractility of the actomyosin cytoskeleton. Previously unknown additional functions for MST4 became evident after disrupting the gene with CRISPR/Cas9 editing in triple-negative breast cancer MDA-MB-231 cells, whereupon I discovered that MST4 is required for mitotic fidelity. Disruption of MST4 led to a subpopulation of giant aneuploid cells that resulted from successive cytokinesis failures. This aneuploid crisis was further marked by DNA mutations as well as a reduction in cell proliferation. Propagating cancer cells beyond the aneuploid crisis revealed a subpopulation that had adapted to the absence of MST4. Adapted cells retained DNA mutations amid a now normalized proliferative rate, yet migration assays suggested that the regulation of cell polarity was permanently impaired, which negatively impacted cancer cell endothelial invasion in various settings including vascular capillaries established in a microfluidic device and zebrafish embryos. We therefore conclude that genetic inhibition of MST4 has both short-term and long-term negative consequences for cancer cells.