Mechanism of action of the vascular disrupting agent OXi8006 on activated endothelial cell signaling

Abstract

Tumor cell survival, growth, and metastasis require a functioning vascular network to provide oxygen and nutrient supply. While the endothelium of normal, remodeled blood vessels is largely quiescent, the neovasculature of tumors is immature, less differentiated, distinct in morphology, lacking in pericyte support, more responsive to angiogenic cell signaling, and exhibits increased permeability. Consequently, tumor vasculature offers a unique, potentially selective target for anticancer therapy. The goal of treatment with vascular disrupting agents (VDAs) is to instigate rapid, widespread disruption of existing tumor vasculature, leading to blood flow shutdown, followed by induction of microenvironmental hypoxia and profound tumor necrosis. This mechanism is dissimilar from that of angiogenesis inhibiting agents (AIAs), which prevent the growth of new blood vessels. Not all VDAs are inhibitors of tubulin polymerization, but most promising pre‐clinical VDA candidates function by interfering with microtubule dynamics through binding to the colchicine site on the tubulin heterodimer in endothelial cells lining tumor‐associated vasculature. This interaction causes rapid morphological changes, which results in increased vessel permeability, cell detachment, irreversible vessel occlusion and wall damage. Understanding the signaling mechanism of drug action at the molecular level is essential for predicting potential off target effects, and providing information to circumvent drug resistance. Previously, we have demonstrated that treatment with the VDA OXi8006 in activated human umbilical vein endothelial cells (HUVECs), a model for tumor vessel endothelium, resulted in profound restructuring of cytoskeletal networks, indicated by microtubule collapse followed closely by focal adhesion‐anchored actin stress fiber formation and correlated to an increase in phosphorylation of myosin light chain, focal adhesion kinase, and the filamentous actin severing protein, cofilin. Cytoskeletal crosstalk subsequent to vascular disruption, was modulated by the intramolecular switch, RhoA, and its downstream effector RhoA kinase (ROCK). In the present study, we have investigated the connection between microtubule catastrophe and activation of the GTP‐ase RhoA. Two guanine nucleotide exchange factors (GEFs) that associate with microtubules, GEF‐H1 and p190RhoGEF (RGNEF) were independently knocked down (KD) by siRNA in HUVECs, confirmed by western blotting and confocal microscopy. OXi8006 treatment of GEF‐H1 KD HUVECs resulted in microtubule collapse, yet downstream events associated with VDA treatment were abrogated or greatly diminished. Contrastingly, OXi8006 mediated collapse of microtubules in p190RhoGEF KD HUVECs was followed by actin stress fiber formation and an increase in phosphorylation of myosin light chain, focal adhesion kinase, and cofilin, similar to responses observed in control HUVECs. Our data confirm the role of GEF‐H1, but not p190RhoGEF in mediating activation of RhoA in OXi8006 treated endothelial cells.Support or Funding InformationCPRIT RP140399, National Institutes of Health National Cancer Institute 5R01CA140674This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.