(648f) Probing the Quantitative Biology of ?-Secretase/Notch Signaling in Breast Cancer: Coupling Biophysical Findings with Spatial-Reaction Diffusion Modeling

The overall objective of this research is to investigate the spatiotemporal attributes of the γ-secretase/Notch pathway and inhibition in triple negative breast cancer (TNBC) and provide a molecular basis for therapeutic development. The mortality from TNBC in African American (AA) women is higher than in White American (WA) or European-American (EA) women. Recent studies have indicated that Notch signaling plays an important role in this racial disparity. Notch signaling has been associated with tumor transformation, proliferation, survival, angiogenesis and metastasis. We have shown that γ-secretase cleavage of Notch is highly regulated in breast cancer cells (Villa et al (2014) 8, 1077-1092). Our other recent studies have shown that subtle changes in membrane lipid composition affect the distribution of Notch substrates and γ-secretase and thus enzymatic activity(Barros et al. (2020) Langmuir 36, 23, 6569–6579). At this point, ethnic differences in γ-secretase/Notch signaling and its role in the higher incidence of and poorer outcomes of AA from TNBC are poorly understood.

We hypothesize that γ-secretase plays a major role in the control of Notch signaling in TNBC and contributes to this racial disparity. The emergence of Notch signaling as a therapeutic target of interest in TNBC has spurred interest in this pathway as a potential suspect in the racially disparate burden in TNBC. At this point, ethnic differences in Notch signaling and its role in the higher incidence of and poorer outcomes of African-americans from TNBC remain understudied. In this work we have characterized the Notch and γ-secretase pathways in triple negative breast cancer (TNBC) lipid microenvironments using TNBC-giant plasma membrane vesicles (GPMVs) and TNBC specific lipid formulations in order to examine potential racial disparities at the molecular level in supported bilayer systems. GPMVs were formed from panels of AA and EA TNBC cell lines and enzyme kinetics studies and relevant membrane protein diffusivity measurements were undertaken.

We are comparing these results to our earlier studies of breast cancer cell lines using the same chemical biologic methods and gamma-secretase inhibitors (GSIs) (Villa et al (2014) 8, 1077-1092) To further interpret and understand the spatiotemporal distribution and reporter staining patterns of γ-secretase and the Notch substrates and fragments we have implemented spatial reaction-diffusion computational models. The core element of this modeling is the integration of biophysical measurements with the spatial proteomics of the panels of AA and EA TNBC cell lines accomplished via correlative airyscan confocal and electron microscopy. This method is being used to interpret the spatial buildup/loading of the relevant signaling-involved molecules under enzyme inhibition. This work will facilitate the examination of racial disparities in Notch and γ-secretase pathways at the molecular level in TNBCs and will allow for the examination of how γ-secretase lipid microenvironment is coupled to the mechanism in the context of Notch signaling in TNBC. We believe that this work could lead to the basis for new racially-distinct therapeutic strategies based on γ-secretase inhibition characteristics.