Abstract A091: Ezrin functions as a metastasis-associated protein by regulating multiple steps involved in breast cancer cell dissemination

Abstract

Abstract The membrane cytoskeleton cross-linker ezrin, is frequently up-regulated in many aggressive cancer types including breast, and is linked to metastatic progression. However, the underlying molecular mechanisms that delineate how ezrin may be involved in the cancer cell dissemination process remain unclear. In this study, we sought to determine the precise role of ezrin in several key components of the metastatic cascade, namely angiogenesis, cell migration, invasion, and lung seeding, in order to gain a comprehensive understanding of the function of ezrin as a metastasis-associated protein. By depleting ezrin expression in MDA-MB-231 invasive breast carcinoma cells, we demonstrate using ex vivo aortic ring and in vivo Matrigel plug assays that ezrin is required for promoting angiogenesis, thereby providing a critical escape route for tumor cells. We further show that the endogenous levels of vascular endothelial growth factor-A (VEGF-A), a potent angiogenic regulator, are significantly reduced in ezrin-depleted cells. Interestingly, secretion of interleukin-6 (IL-6), a known regulator of VEGF-A expression and myeloid cell recruitment, and activation of its downstream effector signal transducer and activator of transcription 3 (Stat3) were also markedly inhibited in these cells, thus suggesting a critical role for ezrin in mediating angiogenesis and potentially pre-metastatic niche priming. Using real-time microscopy, we found that ezrin-deficient cells displayed impaired focal adhesion and invadopodia dynamics, resulting in increased cell-ECM attachment, reduced migration and invasion, though no change in proteolysis was observed. Furthermore, ezrin-depleted cells exhibited significantly less directionality in their movement and were defective in their ability to migrate through an endothelial cell barrier by affecting tight junction permeability. These findings suggest that ezrin may be involved in facilitating intra/extravasation. One of the final stages of cancer cell dissemination is colonization at a distant organ site. Indeed, in vivo lung seeding experiments revealed that fewer ezrin-depleted cells remained in the lung 24 h post-injection, and ultimately led to a reduction in the number of tumor nodules. Collectively, our results unveil a novel coordinate role for ezrin in regulating metastatic progression, and provide important insight in evaluating ezrin as a potential prognostic/predictive marker for metastatic relapse in human breast cancers. (Supported by Canadian Institutes of Health Research, CIHR; Canadian Breast Cancer Foundation Doctoral Fellowship Program; and the Terry Fox Training Program in Transdisciplinary Cancer Research). Citation Format: Victoria Hoskin, Abdi Ghaffari, Alvin Szeto, Bruce E. Elliott. Ezrin functions as a metastasis-associated protein by regulating multiple steps involved in breast cancer cell dissemination. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A091.