Mapping endothelial functional phenotype in cancer by unveiling the kinase and phosphatase pathways

Abstract

Endothelial cells (EC) are critical to the tumor ecosystem, lining the blood vessels that control nutrient transport while also regulating homeostasis. Normalization of vessel structure has shown clinical promise, but EC regulation is known to be state-dependent: while proliferative ECs stimulate growth, quiescent ECs can inhibit it. We studied the functional and phosphorylative transformations of EC state in cancer to elucidate further targets for EC normalization. Confluent ECs cultured in breast cancer cell conditioned media displayed marked elongation and impaired wound healing. Given the well-established relationship between cytoskeletal reorganization and phosphorylative regulation, we estimated kinase and phosphatase activity by quantifying phosphorylation of downstream targets using mass spectrometry.

Of the 152 kinases and phosphatases analyzed across 62 phosphoenzyme families, 9 families were categorized as significant drivers of dysfunction, and potential targets for normalization. Using inhibitors, we functionally validated six of the most significant in morphology and wound healing. The most promising candidate target for normalization was Akt, whose inhibition restored the control phenotype in both assays. Counter to much of the literature, Src activity was decreased in cancer-conditioned transformation and Src inhibition in control cells induced a dysfunctional phenotype. These data prompt further investigation of Akt and caution with regard to Src as targets for inducing cancer homeostasis. Further, the inhibitors overall charted a continuum of EC phenotypes, highlighting the need for further exploration of the complex relationships between EC phenotype, transformation, and regulation.

The framework presented in this thesis that maps functional changes to phosphoenzyme drivers can be readily applied to other models, and the comprehensive library of phosphoenzyme activity developed will shed light on how existing cancer-targeting inhibitors affect tumor endothelium.