(662b) Probing the Role of Cancer Lipid Microenvironment in the Regulation of Notch Cleavage By Gamma-Secretase

This work is directed at the development of a new molecular analysis platform to investigate the role of lipid microenvironment in the regulation of Notch/γ-secretase signaling in prostate cancer (PCa). In prostate cancer (PCa), notch activation is controlled in part by γ-secretase cleavage and modulation of this enzyme has been shown to have chemotherapeutic potential.

γ-secretase is a core intramembrane protease in the Notch signaling pathway, which plays a significant role in cell fate determination by controlling cell differentiation, proliferation, and apoptosis. This pathway can be modulated by means of pharmacological and biologic agents targeted at γ-secretase. In prostate cancer, enhanced Notch activation has been associated with initiation of tumors, as well as their progression and treatment resistance. Further complicating Notch signaling in PCa, the connection between lipid dysregulation and prostate cancer is well documented and aberrant lipid distributions are found in the expression of the malignant phenotype. There is dysregulation of cholesterol metabolism at the cellular level, leading to changes in membrane organization. At present, it is unclear how Notch signaling is regulated in PCa and the lipid microenvironment of γ-secretase could play a key role.

We hypothesize that cancer-specific lipid compositions give rise to changes in cholesterol-rich domain structures that alter the mobility, local concentrations, and microenvironment of the enzyme and its Notch substrates, affecting cleavage activity and and inhibition. Control of lipid phase separation and composition in our proposed supported biomembrane systems forms the basis of a new in vitro methodology to investigate the influence of PCa lipid microenvironment onγ-secretase cleavage of Notch substrates.

We will outline our studies on probing the effect of lipid composition on phase partitioning and lateral mobility of γ-secretase and Notch1 substrate in supported biomembranes. We have characterized the structures, phase colocalization, and membrane protein (MP) orientations by employing correlative optical and atomic-force microscopy from the micro- to the nanoscale in spherical Proteolipobead (PLB) and planar formats. We have probed the phase partitioning of enzyme and substrates and have used a combination of biomembrane mobility methods to examine the diffusivities of Notch substrates and γ-secretase. We will present our studies investigating the influence of cancer-relevant lipid composition and phase separation on γ-secretase cleavage activity. We will present data from lipid formulations defined by PCa lipidomics and published studies of prostate cancer cell lines.