Abstract 3940: Ezrin is a key regulator in Src-induced angiogenesis and lymphangiogenesis in breast cancer.
Authors: Ghaffari Abdi , Hoskin Victoria , Szeto Alvin , Hum Maaike , Liaghati Navid , Nakatsu Kanji , Elliott Bruce E. 
Published: August 2014

Abstract
Src kinase plays a critical intrinsic role in metastasis through disruption of cell-cell junctions and focal adhesions, and extrinsically by upregulation of paracrine pro-angiogenic factors such as VEGF. Ezrin, a Src substrate and membrane cytoskeletal crosslinker, acts cooperatively with Src in the regulation of malignant phenotypes in carcinoma cells. Ezrin expression has been associated with increased metastatic potential in various human cancers and it has also been identified as a potential prognostic/predictive biomarker for an invasive subtype of breast cancer. To examine the extrinsic role of ezrin in Src-induced tumour vascularization we performed Matrigel plug assays by injecting human mammary carcinoma cells subcutaneously into the flanks of immunodeficient Rag2-/-γc-/- and nude mice. On day 12 post-injection, intravital imaging revealed a potent angiogenic response in MDA-MB-231 cells expressing activated Src (MDASrc). However, ezrin knockdown (KD) in MDASrc cells was sufficient to reverse Src-induced tumour vascularization. Microvascular density, vascular cross-sectional area, and blood (CD31) and lymphatic (Lyve-1 & podoplanin) vessel immunostaining of harvested Matrigel plugs (day 12) further confirmed the intravital results. In addition, ectopic expression of a mutant ezrin (Y477F), which is non-phosphorylatable by Src, also caused significant reduction in tumour neovascularization. We further assessed the angiogenic activity of conditioned medium collected from various cell lines by performing ex vivo aortic ring assays. Again, Src-induced endothelial cells branching in MDASrc group was significantly reduced following ezrin KD. To assess lymphangiogenesis, we co-cultured primary lymphatic endothelial cells (hLEC) with MDASrc in presence and absence of ezrin. Ezrin KD significantly reduced Src-induced hLEC tube formation, migration, and permeability in vitro. Results from conditioned media and co-culture assays also suggested the involvement of paracrine angio- and lymphangio-genic factors regulated by Src/ezrin signaling axis in cancer cells. Western blot analysis of cell lysates revealed that ezrin KD was sufficient to block Src-induced Stat3 activation and VEGF-A and -C expression in MDASrc cells. In conclusion, our study provides a novel mechanism for the role of ezrin in breast cancer metastasis as a key regulator in Src-induced angio- and lymphangio-genesis, and could lead to improved drug targeting of metastatic disease.
Citation Format: Abdi Ghaffari, Victoria Hoskin, Alvin Szeto, Maaike Hum, Navid Liaghati, Kanji Nakatsu, Bruce E. Elliott. Ezrin is a key regulator in Src-induced angiogenesis and lymphangiogenesis in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3940. doi:10.1158/1538-7445.AM2013-3940