Abnormal cell division leading to the gain or loss of entire chromosomes and consequent genetic instability is a hallmark of cancer. Centromere protein –A (CENPA) is a centromere-specific histone-H3-like variant gene involved in regulating chromosome segregation during cell division. CENPA is one of the genes included in some of the commercially available RNA based prognostic assays for breast cancer (BCa)—the 70 gene signature MammaPrint^® and the five gene Molecular Grade Index (MGI^SM). Our aim was to assess the immunohistochemical (IHC) expression of CENPA in normal and malignant breast tissue. Clinically annotated triplicate core tissue microarrays of 63 invasive BCa and 20 normal breast samples were stained with a monoclonal antibody against CENPA and scored for percentage of visibly stained nuclei. Survival analyses with Kaplan–Meier (KM) estimate and Cox proportional hazards regression models were applied to assess the associations between CENPA expression and disease free survival (DFS). Average percentage of nuclei visibly stained with CENPA antibody was significantly higher (p = 0.02) in BCa than normal tissue. The 3-year DFS in tumors over-expressing CENPA (>50% stained nuclei) was 79% compared to 85% in low expression tumors ( 60.07; p = 0.06) within our small cohort. To the best of our knowledge, this is the first published report evaluating the implications of increased IHC expression of CENPA in paraffin embedded breast tissue samples. Our finding that increased CENPA expression may be associated with shorter DFS in BCa supports its exploration as a potential prognostic biomarker.

Limited understanding of the cancer biology of metastatic sites is a major factor contributing to poor outcomes in cancer patients. The regional lymph nodes are the most common site of metastasis in most solid cancers and their involvement is a strong predictor of relapse in breast cancer (BC). We have previously shown that ezrin, a cytoskeletal–membrane linker protein, is associated with lymphovascular invasion and promotes metastatic progression in BC. However, the efficacy of pharmacological inhibition of ezrin in blocking cancer cell migration and metastasis remains unexplored in BC. We quantified ezrin expression in a BC tissue microarray (n = 347) to assess its correlation with risk of relapse. Next, we developed a quantitative intravital microscopy (qIVM) approach, using a syngeneic lymphatic reporter mouse tumor model, to investigate the effect of systemic ezrin inhibition on cancer cell migration and metastasis. We show that ezrin is expressed at significantly higher levels in lymph node metastases compared to matched primary tumors, and that a high tumor ezrin level is associated with increased risk of relapse in BC patients with regional disease. Using qIVM, we observe a subset of cancer cells that retain their invasive and migratory phenotype at the tumor-draining lymph node. We further show that systemic inhibition of ezrin, using a small molecule compound (NSC668394), impedes the migration of cancer cells in vivo. Furthermore, systemic ezrin inhibition leads to reductions in metastatic burden at the distal axillary lymph node and lungs. Our findings demonstrate that the tumor ezrin level act as an independent biomarker in predicting relapse and provide a rationale for therapeutic targeting of ezrin to reduce the metastatic capacity of cancer cells in high-risk BC patients with elevated ezrin expression.

Background: The membrane cytoskeletal crosslinker, ezrin, a member of the ERM family of proteins, is frequently over-expressed in human breast cancers, and is required for motility and invasion of epithelial cells. Our group previously showed that ezrin acts co-operatively with the non-receptor tyrosine kinase, Src, in deregulation of cell-cell contacts and scattering of epithelial cells. In particular, ezrin phosphorylation on Y477 by Src is specific to ezrin within the ERM family, and is required for HGF-induced scattering of epithelial cells. We therefore sought to examine the role of Y477 phosphorylation in ezrin on tumor progression. Methods: Using a highly metastatic mouse mammary carcinoma cell line (AC2M2), we tested the effect of over-expressing a non-phosphorylatable form of ezrin (Y477F) on invasive colony growth in 3-dimensional Matrigel cultures, and on local invasion and metastasis in an orthotopic engraftment model. Results: AC2M2 cells over-expressing Y477F ezrin exhibited delayed migration in vitro, and cohesive round colonies in 3-dimensional Matrigel cultures, compared to control cells that formed invasive colonies with branching chains of cells and numerous actin-rich protrusions. Moreover, over-expression of Y477F ezrin inhibits local tumor invasion in vivo. Whereas orthotopically injected wild type AC2M2 tumor cells were found to infiltrate into the abdominal wall and visceral organs within three weeks, tumors expressing Y477F ezrin remained circumscribed, with little invasion into the surrounding stroma and abdominal wall. Additionally, Y477F ezrin reduces the number of lung metastatic lesions. Conclusions: Our study implicates a role of Y477 ezrin, which is phosphorylated by Src, in regulating local invasion and metastasis of breast carcinoma cells, and provides a clinically relevant model for assessing the Src/ezrin pathway as a potential prognostic/predictive marker or treatment target for invasive human breast cancer.

Among women worldwide, breast cancer is the most commonly diagnosed cancer, and the second leading cause of cancer-related deaths. Improved understanding of breast tumourigenesis may facilitate the development of more effective therapies. Peroxisome proliferator-activated receptor (PPAR)γ is a transcription factor that regulates genes involved in insulin sensitivity and adipogenesis. Previously, we showed, using 7,12-dimethylbenz [a] anthracene (DMBA)-treated haploinsufficient PPARγ mice, that PPARγ suppresses breast tumour progression; however, the PPARγ expressing cell types and mechanisms involved remain to be clarified. Here, the role of PPARγ expression and activation in mammary epithelial cells (MG) with respect to DMBA-mediated breast tumourigenesis was investigated. PPARγ MG knockout (PPARγ-MG KO) mice and their congenic, wild-type controls (PPARγ-WT) were treated once a week for six weeks by oral gavage with 1 mg DMBA dissolved in corn oil and maintained on a normal chow diet. At week 7, mice were randomly divided into those maintained on a normal chow diet (DMBA Only; PPARγ-WT: n = 25 and PPARγ-MG KO: n = 39) or those receiving a diet supplemented with the PPARγ ligand, rosiglitazone (ROSI, 4 mg/kg/day) (DMBA + ROSI; PPARγ-WT: n = 34 and PPARγ-MG KO: n = 17) for the duration of the 25-week study. Compared to DMBA Only-treated PPARγ-WTs, both breast tumour susceptibility and serum levels of proinflammatory and chemotactic cytokines, namely IL-4, eotaxin, GM-CSF, IFN-γ, and MIP-1α, were decreased among PPARγ-MG KOs. Cotreatment with ROSI significantly reduced breast tumour progression among PPARγ-WTs, correlating with increased BRCA1 and decreased VEGF and COX-2 protein expression levels in breast tumours; whereas, surprisingly DMBA + ROSI-treated PPARγ-MG KOs showed increased breast tumourigenesis, correlating with activation of COX-2. These novel data suggest MG-specific PPARγ expression and signaling is critical during breast tumourigenesis, and may serve as a strong candidate predictive biomarker for response of breast cancer patients to the use of therapeutic strategies that include PPARγ ligands.

Introduction: 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. Methods: 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. Results: 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. Conclusions: 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.

Mastectomy is effective treatment for ductal carcinoma in situ (DCIS) but some women will develop chest wall recurrence. Most chest wall recurrences that develop after mastectomy are invasive cancer and are associated with poorer prognosis. Past studies have been unable to identify factors predictive of chest wall recurrence. Therefore, it remains unclear if a subset exists of women with DCIS treated by mastectomy experience a high rate of recurrence in whom more aggressive treatment may be of benefit. We report outcomes of all women in Ontario (N = 1,546) diagnosed with pure DCIS from 1994 to 2003 treated with mastectomy without radiotherapy and evaluate factors associated with the development of chest wall recurrence. Treatments and outcomes were validated by chart review. Proportional differences were compared using Chi square analyses. Survival analyses were used to study the development of chest wall recurrence in relation to patient and tumor characteristics. Median follow-up was 10.1 years. Median age was 57.1 years. 36 patients (2.3%) developed chest wall recurrence. The 10-year actuarial chest wall recurrence-free survival rates and invasive chest wall recurrence-free survival rates were 97.6 and 98.6%, respectively. There was no difference in cumulative 10 year rates of chest wall recurrence by age at diagnosis (50 years = 2.1%; p = 0.19), nuclear grade (high = 3.0%, intermediate = 1.4%, low = 1.0%, unreported = 2.5%; p = 0.41), or among women with close or positive resection margins (positive = 3.0%, 2 mm or less = 1.4%, >2 mm = 1.5%, unreported = 2.8%; p = 0.51). On univariate and multivariable analysis, none of the factors were significantly associated with the development of chest wall recurrence. In this population cohort, individuals treated by mastectomy experienced low rates of chest wall recurrence. We did not identify a subset of patients with a high rate of chest wall recurrence, including those with positive margins.