Victoria Hoskin

Ezrin regulates focal adhesion and invadopodia dynamics by altering calpain activity to promote breast cancer cell invasion

Ezrin regulates proper focal adhesion and invadopodia turnover by regulating calpain-1, in part by directing its proteolytic activity toward key substrates talin, FAK, and cortactin. Ezrin-deficient tumor cells show reduced lung seeding and colonization in vivo but not primary tumor growth, thus implicating ezrin as a metastasis-associated protein.

A novel role for ezrin in breast cancer angio/lymphangiogenesis

Recent evidence suggests that tumour lymphangiogenesis promotes lymph node metastasis, a major prognostic factor for survival of breast cancer patients. However, signaling mechanisms involved in tumour-induced lymphangiogenesis remain poorly understood. The expression of ezrin, a membrane cytoskeletal crosslinker and Src substrate, correlates with poor outcome in a diversity of cancers including breast. Furthermore, ezrin is essential in experimental invasion and metastasis models of breast cancer. Ezrin acts cooperatively with Src in the regulation of the Src-induced malignant phenotype and metastasis. However, it remains unclear if ezrin plays a role in Src-induced tumour angio/lymphangiogenesis. The effects of ezrin knockdown and mutation on angio/lymphangiogenic potential of human MDA-MB-231 and mouse AC2M2 mammary carcinoma cell lines were examined in the presence of constitutively active or wild-type (WT) Src. In vitro assays using primary human lymphatic endothelial cells (hLEC), an ex vivo aortic ring assay, and in vivo tumour engraftment were utilized to assess angio/lymphangiogenic activity of cancer cells. Ezrin-deficient cells expressing activated Src displayed significant reduction in endothelial cell branching in the aortic ring assay in addition to reduced hLEC migration, tube formation, and permeability compared to the controls. Intravital imaging and microvessel density (MVD) analysis of tumour xenografts revealed significant reductions in tumour-induced angio/lymphangiogenesis in ezrin-deficient cells when compared to the WT or activated Src-expressing cells. Moreover, syngeneic tumours derived from ezrin-deficient or Y477F ezrin-expressing (non-phosphorylatable by Src) AC2M2 cells further confirmed the xenograft results. Immunoblotting analysis provided a link between ezrin expression and a key angio/lymphangiogenesis signaling pathway by revealing that ezrin regulates Stat3 activation, VEGF-A/-C and IL-6 expression in breast cancer cell lines. Furthermore, high expression of ezrin in human breast tumours significantly correlated with elevated Src expression and the presence of lymphovascular invasion. The results describe a novel function for ezrin in the regulation of tumour-induced angio/lymphangiogenesis promoted by Src in breast cancer. The combination of Src/ezrin might prove to be a beneficial prognostic/predictive biomarker for early-stage metastatic breast cancer.

Combined targeting of Raf and Mek synergistically inhibits tumorigenesis in triple negative breast cancer model systems

Aberrant Ras-MAPK signaling from receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor-2 (HER2), is a hallmark of triple negative breast cancer (TNBC); thus providing rationale for targeting the Ras-MAPK pathway. Components of this EGFR/HER2-Ras-Raf-Mek-Erk pathway were co-targeted in the MDA-MB-231 and MDA-MB-468 human TNBC cell lines, and in vitro effects on signaling and cytotoxicity, as well as in vivo effects on xenograft tumor growth and metastasis were assessed. The dual EGFR/HER2 inhibitor lapatinib (LPN) displayed greater cytotoxic potency and MAPK signaling inhibition than the EGFR inhibitor erlotinib, suggesting both EGFR and HER2 contribute to MAPK signaling in this TNBC model. The Raf inhibitor sorafenib (SFN) or the Mek inhibitor U0126 suppressed MAPK signaling to a greater extent than LPN; which correlated with greater cytotoxic potency of SFN, but not U0126. However, U0126 potentiated the cytotoxic efficacy of LPN and SFN in an additive and synergistic manner, respectively. This in-series Raf-Mek co-targeting synergy was recapitulated in orthotopic mouse xenografts, where SFN and the Mek inhibitor selumitinib (AZD6244) inhibited primary tumor growth and pulmonary metastasis. Raf and Mek co-inhibition exhibits synergy in TNBC models and represent a promising combination therapy for this aggressive breast cancer type.

Regulation of HC11 mouse breast epithelial cell differentiation by the E-cadherin/Rac axis

Abstract It was previously demonstrated that differentiation of some established breast epithelial cell lines requires confluence and stimulation with hydrocortisone, insulin and prolactin inducers. We and others previously demonstrated that E‐cadherin engagement, which is favored under conditions of confluence, increases the levels and activity of the Rac small GTPase. To investigate the functional relationship between the transforming ability of Rac and its role as an integral component of the differentiative E‐cadherin signaling pathway, we introduced a mutationally activated form of Rac, RacV12, into the mouse breast epithelium‐derived cell line, HC11. Our results demonstrate that the strength of the Rac signal is key for the outcome of the differentiation process; cRac1 is critically required for differentiation, and at low levels, mutationally activated RacV12 is able to increase differentiation, presumably reinforcing the E‐cadherin/Rac differentiative signal. However, high RacV12 expression blocked differentiation concomitant with E‐cadherin downregulation, while inducing neoplastic transformation. Therefore, the intensity of the Rac signal is a central determinant in the balance between cell proliferation vs differentiation, two fundamentally opposed processes, a finding which could also have important therapeutic implications.

Abstract P5-01-01: Real-time imaging of lymph node metastasis in response to systemic ezrin inhibitor treatment in breast cancer

Lymph node (LN) metastasis is a key driver of recurrence and survival in breast cancer (BC) patients. However, the mechanisms of metastatic dissemination of tumour cells from LNs to distant sites and their predictors of response to systemic therapy remain poorly understood, mainly due to a lack of non-invasive in vivo imaging models. We have recently described ezrin, a pro-metastatic crosslinker protein, as a regulator of tumour lymphangiogenesis and metastasis in BC (Breast Cancer Res. 2014; 16(5): 438). Furthermore, we demonstrated significant association of high ezrin expression with lymphovascular invasion in a cohort (n=63) of premenopausal patients with invasive BC (p =0.024). These findings prompted us to examine the role of ezrin in migration and invasion of metastatic tumour cells in LNs and their response to ezrin-targeted therapy. Using a locally accrued LN positive patient cohort (n=94), we demonstrated a significant association between high ezrin levels and reduced recurrence-free survival (univariate Log-rank test, p=0.033), suggesting that ezrin is a potential predictor of relapse in LN positive BC. To address the mechanistic role of ezrin in LN metastasis, we developed a novel intravital imaging model using a lymphatic reporter transgenic mouse (B6-prox1-mOrange2-pA-BAC) to examine the response of tumour-draining LN to anti-ezrin systemic therapy in real time. Next, we tested the effects of a small molecule ezrin inhibitor (NSC668394) in vitro and observed significant suppression of ezrin activation (p-T567) and cancer cell invasive phenotype. Intravital imaging of inguinal LN metastases, derived from subcutaneously implanted breast adenocarcinoma E0771-LMV (lung metastatic variant) cells, demonstrated significant reduction in mobility and invasiveness (Mann Whitney, p (Supported by CRS, CIHR, CBCF, BCAK, Queen9s SRC). Citation Format: Ghaffari A, Hoskin V, Mullins G, Greer P, Kiefer F, Madarnas Y, SenGupta S, Elliott B. Real-time imaging of lymph node metastasis in response to systemic ezrin inhibitor treatment in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-01-01.

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

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.

Abstract 3940: Ezrin is a key regulator in Src-induced angiogenesis and lymphangiogenesis in breast cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC 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

Ezrin is required for adhesion and migration in invasive breast cancer

Background Deregulation of focal adhesion (FA) dynamics has been found to contribute to tumour progression by promoting cancer cell migration and invasion. Both focal adhesion kinase (FAK) and Src are involved in FA formation, and both interact with the scaffolding protein ezrin, which our group has shown to be required for breast cancer metastasis. Our aim was to assess the role of the membrane-cytoskeletal linker protein ezrin in Src-induced adhesion and migration in the human invasive breast cancer cell line MDA-MB-231.

Abstract 535A: The tyrosine kinase Src co-operates with the ERM family member ezrin to promote Met activation and ECM degradation in a breast epithelial cell model

Over-expression and aberrant activation of the Met proto-oncogene frequently occurs in invasive breast cancer, and is linked with poor patient prognosis. We and others have shown that the tyrosine kinase Src mediates cell-adhesion dependent activation of Met in the absence of ligand (JCB 107:1168-81, 2009). We have also shown that Src acts co-operatively with the membrane cytoskeletal crosslinker ezrin to disrupt cell-cell contacts, and to increase cell spreading and scattering, all characteristics of epithelial-to-mesenchymal transition (EMT) (JCB 92:16-28, 2004). We therefore examined the role of Src and ezrin in Met activation and invasion of breast epithelial cells. As a model, we used the HC11 breast epithelial cell line expressing various combinations of activated Src and ezrin mutants, as well as a dominant negative ezrin mutant. In contrast to activated Src, which promotes the phosphorylation and hence activation of Met and cell spreading, activated ezrin alone induces the formation of numerous filopodia-like structures, but not Met phosphorylation or cell spreading. Co-activation of Src and ezrin produces a combination of both cell spreading and filopodia-like structures, and strongly increases Met phosphorylation over levels resulting from activated Src alone. Interestingly, Src/ezrin co-operativity dramatically increases tyrosine-phosphorylation of cortactin and MMP2 expression, resulting in the formation of invadopodia and extracellular matrix degradation, a hallmark feature of invading cancer cells. However expression of a dominant negative ezrin domain (aa1-309) abolishes Src and ezrin-induced morphological phenotypes, as well as Src-dependent Met phosphorylation and invadopodia formation, suggesting an important requirement for ezrin in these processes. These findings implicate the Src/ezrin axis in regulating adhesion-dependent Met activation and invadopodia formation, and may provide important clues toward treatment strategies for invasive breast tumors which exploit this signaling pathway (Supported by CBCRA and CIHR). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 535A.