Abstract
Abstract Background: The capacity of T-cell lymphocytes to migrate and localize in tissues is important in their protective function against infectious agents, however, the ability of these cells to migrate and infiltrate the tumor microenvironment is also a major contributing factor in the development of cancer and cancer metastasis. T-cell migration requires ligand (ICAM-1)/integrin(LFA-1) interaction, activating intracellular signaling pathways which result in a distinct polarized morphology, with an actin-rich lamellipodium and microtubule-rich uropod. Microtubule-targeting agents (MTAs) are amongst some of the most widely used anti-cancer drugs and several have been shown to exhibit anti-inflammatory activity. Combretastatin (CA) A-4 is a MT-destabilizing agent that possesses potent anti-tumor and anti-vascular properties both in vitro and in vivo. CA-432, a novel cis-restriciting analogue containing a β-lactam ring, has recently been synthesized to circumvent the problem of CA-4 isomerisation from a biologically active cis-conformation to a more thermodynamically stable but inactive trans-isomer. Methods: HuT-78 cell line and peripheral blood lymphocyte (PBL) T-cells were cultured. In this study, the effect of CA-4 and CA-432 on the migratory polarity of T-cells was explored using high content analysis (HCA). The active migration of PBL T-cells was studied using a transwell assay. The mechanistic and signaling pathways involved were examined using western blotting and confocal microscopy. Cytoxicity of both compounds was tested using a viability assay and flow cytometry. Results: CA-4 and CA-432 treated cells displayed altered T-cell migratory polarity in HuT-78 and peripheral blood T-lymphocyte (PBTL) cells and inhibited active migration of PBTLs. Both compounds induced activation of the RhoA / RhoA associated kinase (ROCK) signaling pathway, leading to the phosphorylation of myosin light chain (MLC), acto-myosin contractility and impaired migration. Disruption of the MT network of T-cells through CA-induced MT depolymerisation was associated with reduced acetylated tubulin expression and decreased MT stability. GEF-H1 is a MT-associated nucleotide exchange factor which activates RhoA upon release from MTs, The siRNA-mediated depletion of GEF-H1 in Hut-78 T cells prevented CA-induced phosphorylation of MLC and attenuated the formation of actin-rich membrane protrusions and cell contractility. Conclusions: These results suggest an important role for a GEF-H1/RhoA/ROCK/MLC signaling pathway in mediating CA-induced contractility of T cells. Therapeutic agents inhibiting cell migration may open new avenues in the treatment of cancer and metastasis. Citation Format: Jade Kirstin Pollock, Navin K. Verma, Mary J. Meegan, Daniela Zisterer. Combretastatin (CA)-4 and its novel analogue CA-432 impair T-cell migration through the Rho/ROCK signalling pathway. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B81.
Published Version
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