Mechanisms Utilized During the Survival of Extracellular Matrix Detached Mammary Epithelial Cells

Normal epithelial cells require attachment to the extracellular matrix (ECM) in order to proliferate and survive. Binding to the ECM facilitates the activation of pathways responsible for ATP production and cell survival. When detached from the ECM, normal cells will upregulate apoptotic pathways and will be systematically removed. This detachment-induced apoptosis is known as anoikis. ECM detachment in normal epithelial cells disrupts cellular signaling derived from integrin-mediated basal attachment as well as cadherin assisted signaling from lateral cell contacts. It has been well established by countless studies that the overexpression of oncogenes can facilitate the ability of ECM-detached cells to avoid anoikis. ErbB2 and Ras are two oncogenes that have been implicated in the ability of cancer cells to avoid anoikis. ErbB2 is an orphan member of the EGFR family of tyrosine kinases and is a potent oncogene found in 30% of all breast cancer tissue. Previous studies in our laboratory have found a direct correlation between upregulation of survival pathways in ECM detached cells and expression of ErbB2. Mutated Ras is found in up to 30% of all human cancers and is commonly found overexpressed in a multitude of cancers such as pancreatic and colon cancers. Although it is rare in breast cancers and found mutated in only 5% of breast cancer diagnoses, Ras signaling can be driven considerably by multiple growth factors, such as ErbB2. This makes Ras-driven signaling an important factor in disease progression. Previous literature has shown that ErbB2 and Ras regulate cytochrome c release from the mitochondria by regulating the action of Bcl-2 proteins via the stimulation of multiple survival kinase pathways. My studies have found that the overexpression of ErbB2 in non-malignant mammary epithelial cells (MCF-10A) results in substantial aggregation of detached cells and the inhibition of caspase activation. These data suggest that ErbB2 plays a role in protecting detached cells from anoikis and this activity may be linked to cell-cell adhesion in suspension. We found that the disruption of cell-cell contacts in ErbB2-overexpressing MCF-10A cells (using methylcellulose or by antagonizing the formation of adherens junctions) induces caspase activation.Furthermore, in cells that form large aggregates, ErbB2 can interact with E-cadherin and EGFR in a fashion that prevents the internalization of EGFR and ultimately its lysosome mediated degradation. The disruption of aggregation causes the ErbB2/EGFR/E-cadherin complex to fail and subsequently results in EGFR degradation in the lysosome. This degradation of EGFR diminishes signaling through the MAPK pathway, preventing the induction of anoikis.In summary, these data suggest that oncogenic signaling through ErbB2 promotes the formation of cellular aggregates that function to prevent EGFR from degradation and subsequently to block the induction of anoikis.

My studies have also uncovered that the overexpression of oncogenic Ras results in decreased sensitivity to exogenous cytochrome c in ECM-detachment compared to controls. This decreased sensitivity was observed despite the fact that there is an upregulation of apoptosomal proteins in Ras cells in a MAPK dependent manner. This suggests that Ras-hyperactivated cells are uniquely resistant to cytochrome c mediated cell death in ECM-detachment compared to controls. Additionally, my studies have observed that the resistance to cytochrome c in ECM-detached Ras cells is not conferred to ECM-attached cells. Upon investigation of apoptosomal proteins, it was found that Ras-hyperactivated cells in ECM-attachment exhibit an induction in apoptosomal proteins compared to control cells. These data suggest that Ras-hyperactivated cells may be uniquely susceptible to cytochrome c mimetic therapies in ECM-attachment.