Sandip K. SenGupta

The membrane cytoskeletal crosslinker ezrin is required for metastasis of breast carcinoma cells

IntroductionThe membrane cytoskeletal crosslinker ezrin participates in several functions including cell adhesion, motility and cell survival, and there is increasing evidence that it regulates tumour progression. However, the role played by ezrin in breast cancer metastasis has not been clearly delineated.MethodsWe examined the role of ezrin in metastasis using a highly metastatic murine mammary carcinoma cell line, namely AC2M2. Stable cell clones that overexpress wild-type ezrin or a dominant-negative amino-terminal domain of ezrin were selected. They were then tested for cell motility and invasion in vitro, and metastasis in a mouse in vivo tumour transplantation model.ResultsParental AC2M2 cells and cells overexpressing wild-type ezrin were transplanted into the mammary fat pad of syngeneic recipient mice; these animals subsequently developed lung metastases. In contrast, expression of the dominant-negative amino-terminal ezrin domain markedly inhibited lung metastasis. Consistent with this effect, we observed that the expression of amino-terminal ezrin caused strong membrane localization of cadherin, with increased cell–cell contact and a decrease in cell motility and invasion, whereas cells expressing wild-type ezrin exhibited strong cytoplasmic expression of cadherins and pseudopodia extensions. In addition, inhibitors of phosphatidylinositol 3-kinase and c-Src significantly blocked cell motility and invasion of AC2M2 cells expressing wild-type ezrin. We further found that overexpression of amino-terminal ezrin reduced levels of Akt pS473 and cytoskeletal-associated c-Src pY418 in AC2M2 cells, which contrasts with the high levels of phosphorylation of these proteins in cells expressing wild-type ezrin. Phosphorylated Erk1/2 was also reduced in amino-terminal ezrin expressing cells, although a mitogen-activated protein kinase kinase (MEK) inhibitor had no detectable effect on cell motility or invasion in this system.ConclusionOur findings indicate that ezrin is required for breast cancer metastasis, and that c-Src and phosphatidylinositol 3-kinase/Akt are effectors of ezrin in the cell motility and invasion stages of the metastatic process. Together, these results suggest that blocking ezrin function may represent a novel and effective strategy for preventing breast cancer metastasis.

Increased risk of breast cancer associated with long-term shift work in Canada

Objectives Long-term night work has been suggested as a risk factor for breast cancer; however, additional studies with more comprehensive methods of exposure assessment to capture the diversity of shift patterns are needed. As well, few previous studies have considered the role of hormone receptor subtype. Methods Relationships between night shift work and breast cancer were examined among 1134 breast cancer cases and 1179 controls, frequency-matched by age in Vancouver, British Columbia, and Kingston, Ontario. Self-reported lifetime occupational histories were assessed for night shift work, and hormone receptor status obtained from tumour pathology records. Results With approximately one-third of cases and controls ever employed in night shift work, associations with duration demonstrated no relationship between either 0–14 or 15–29 years, while an association was apparent for ≥30 years (OR=2.21, 95% CI 1.14 to 4.31). This association with long-term night shift work is robust to alternative definitions of prolonged shift work, with similar results for both health and non-health care workers. Conclusions Long-term night shift work in a diverse mix of occupations is associated with increased breast cancer risk and not limited to nurses, as in most previous studies.

Long-term outcomes of hypofractionation versus conventional radiation therapy after breast-conserving surgery for ductal carcinoma in situ of the breast.

PURPOSE Whole-breast radiation therapy (XRT) after breast-conserving surgery (BCS) for ductal carcinoma in situ (DCIS) may decrease the risk of local recurrence, but the optimal dose regimen remains unclear. Past studies administered 50 Gy in 25 fractions (conventional); however, treatment pattern studies report that hypofractionated (HF) regimens (42.4 Gy in 16 fractions) are frequently used. We report the impact of HF (vs conventional) on the risk of local recurrence after BCS for DCIS. METHODS AND MATERIALS All women with DCIS treated with BCS and XRT in Ontario, Canada from 1994 to 2003 were identified. Treatment and outcomes were assessed through administrative databases and validated by chart review. Survival analyses were performed. To account for systematic differences between women treated with alternate regimens, we used a propensity score adjustment approach. RESULTS We identified 1609 women, of whom 971 (60%) received conventional regimens and 638 (40%) received HF. A total of 489 patients (30%) received a boost dose, of whom 143 (15%) received conventional radiation therapy and 346 (54%) received HF. The median follow-up time was 9.2 years. The median age at diagnosis was 56 years (interquartile range [IQR], 49-65 years). On univariate analyses, the 10-year actuarial local recurrence-free survival was 86% for conventional radiation therapy and 89% for HF (P=.03). On multivariable analyses, age <45 years (hazard ratio [HR] = 2.4; 95% CI: 1.6-3.4; P<.0001), high (HR=2.9; 95% CI: 1.2-7.3; P=.02) or intermediate nuclear grade (HR=2.7; 95% CI: 1.1-6.6; P=.04), and positive resection margins (HR=1.4; 95% CI: 1.0-2.1; P=.05) were associated with an increased risk of local recurrence. HF was not significantly associated with an increased risk of local recurrence compared with conventional radiation therapy on multivariate analysis (HR=0.8; 95% CI: 0.5-1.2; P=.34). CONCLUSIONS The risk of local recurrence among individuals treated with HF regimens after BCS for DCIS was similar to that among individuals treated with conventional radiation therapy.

Impact of Boost Radiation in the Treatment of Ductal Carcinoma In Situ: A Population-Based Analysis

PURPOSE To report the outcomes of a population of women with ductal carcinoma in situ (DCIS) treated with breast-conserving surgery and radiation and to evaluate the independent effect of boost radiation on the development of local recurrence. METHODS AND MATERIALS All women diagnosed with DCIS and treated with breast-conserving surgery and radiation therapy in Ontario from 1994 to 2003 were identified. Treatments and outcomes were identified through administrative databases and validated by chart review. The impact of boost radiation on the development of local recurrence was determined using survival analyses. RESULTS We identified 1895 cases of DCIS that were treated by breast-conserving surgery and radiation therapy; 561 patients received boost radiation. The cumulative 10-year rate of local recurrence was 13% for women who received boost radiation and 12% for those who did not (P=.3). The 10-year local recurrence-free survival (LRFS) rate among women who did and who did not receive boost radiation was 88% and 87%, respectively (P=.27), 94% and 93% for invasive LRFS (P=.58), and was 95% and 93% for DCIS LRFS (P=.31). On multivariable analyses, boost radiation was not associated with a lower risk of local recurrence (hazard ratio = 0.82, 95% confidence interval 0.59-1.15) (P=.25). CONCLUSIONS Among a population of women treated with breast-conserving surgery and radiation for DCIS, additional (boost) radiation was not associated with a lower risk of local or invasive recurrence.

Age at diagnosis predicts local recurrence in women treated with breast-conserving surgery and postoperative radiation therapy for ductal carcinoma in situ: a population-based outcomes analysis

PURPOSE The main goal of treating ductal carcinoma in situ (dcis) is to prevent the development of invasive breast cancer. Most women are treated with breast-conserving surgery (bcs) and radiotherapy. Age at diagnosis may be a risk factor for recurrence, leading to concerns that additional treatment may be necessary for younger women. We report a population-based study of women with dcis treated with bcs and radiotherapy and an evaluation of the effect of age on local recurrence (lr). METHODS All women diagnosed with dcis in Ontario from 1994 to 2003 were identified. Treatments and outcomes were collected through administrative databases and validated by chart review. Women treated with bcs and radiotherapy were included. Survival analyses were performed to evaluate the effect of age on outcomes. RESULTS We identified 5752 cases of dcis; 1607 women received bcs and radiotherapy. The median follow-up was 10.0 years. The 10-year cumulative lr rate was 27% for women younger than 45 years, 14% for women 45-50 years, and 11% for women more than 50 years of age (p < 0.0001). The 10-year cumulative invasive lr rate was 22% for women younger than 45 years, 10% for women 45-50 years, and 7% for women more than 50 years of age (p < 0.0001). On multivariate analyses, young age (<45 years) was significantly associated with lr and invasive lr [hazard ratio (hr) for lr: 2.6; 95% confidence interval (ci): 1.9 to 3.7; p < 0.0001; hr for invasive lr: 3.0; 95% ci: 2.0 to 4.4; p < 0.0001]. An age of 45-50 years was also significantly associated with invasive lr (hr: 1.6; 95% ci: 1.0 to 2.4; p = 0.04). CONCLUSIONS Age at diagnosis is a strong predictor of lr in women with dcis after treatment with bcs and radiotherapy.

Implementation of a Canadian external quality assurance program for breast cancer biomarkers: an initiative of Canadian Quality Control in immunohistochemistry (cIQc) and Canadian Association of Pathologists (CAP) National Standards Committee/Immunohistochemistry.

Immunohistochemistry results for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 are used to guide breast carcinoma patient management and it is essential to monitor these tests in external quality assurance (EQA) programs. Canadian Immunohistochemistry Quality Control is a web-based program with novel approach to EQA. Canadian Immunohistochemistry Quality Control RUN2 included tissue microarray slides with 38 samples tested by 18 immunohistochemical laboratories. Deidentified results were posted for viewing at www.ciqc.ca including all used protocols matched with scanned slides for virtual microscopy and garrattograms. Sensitivity, specificity, Kendall W test (concordance between laboratories), and κ statistics (agreement with designated reference values) were calculated. Kappa values were within the target range (>0.8, or “near perfect” agreement) for 85% results. Kendall coefficient was 0.942 for estrogen receptor, 0.930 for progesterone receptor, and 0.958 for human epidermal growth factor receptor 2. The anonymous participation, quick feedback, and unrestricted full access in EQA results provides rapid insight into technical or interpretive deficiencies, allowing appropriate corrective action to be taken whereas the use of tissue microarrays enables meaningful statistical analysis.

Vascularity demonstrated by doppler ultrasound and immunohistochemistry in invasive ductal carcinoma of the breast

BackgroundVascularity is an important determinant of a tumours ability to grow and disseminate. Breast tumour vascularity can be determined with doppler ultrasound (US) and by counting the vessels microscopically (microvascular density — MVD). The biologic characteristics of tumours based on their vascularity have not been extensively studied.MethodPreoperative US was performed on 207 patients with invasive ductal breast carcinomas (IDC). MVD was assessed immunohistochemically using polyclonal antisera against factor VIII and the proliferation rate was measured with Ki-67 polyclonal antisera. Histologic tumour characteristics and oestrogen receptor (ER) status were determined. Thermography was performed on 174 of the patients.ResultsTwenty-five percent of IDC demonstrated US-vascularity. US-vascular tumours were more likely to be node positive, and had a higher mitotic rate than avascular cancers. US-vascularity was more common in tumours with MVD greater than 80 vessels/250x field than those with fewer vessels. The proliferation rate, histologic grade III, and nuclear grade III were higher and ER positivity lower, but the differences were not statistically significant. US-vascular cancers were associated with significantly more thermographic abnormalities. The cancer recurrence rate at three years was higher in patients with vascular cancers although the difference was not statistically significant.ConclusionUS appears to be a simple, non-invasive method of identifying vascular cancers associated with factors indicating a poor prognosis.

Loss of PPARγ expression in mammary secretory epithelial cells creates a pro-breast tumorigenic environment

Breast cancer is the leading cause of new cancer diagnoses among women. Using peroxisome proliferator‐activated receptor (PPAR)γ(+/−) mice, we showed normal expression of PPARγ was critical to stop 7,12‐dimethylbenz[a]anthracene (DMBA)‐induced breast tumorigenesis. PPARγ is expressed in many breast cell types including mammary secretory epithelial (MSE) cells. MSEs proliferate as required during pregnancy, and undergo apoptosis or reversible transdifferentiation during involution once lactation is complete. Thus, MSE‐specific loss of PPARγ was hypothesized to enhance DMBA‐mediated breast tumorigenesis. To test this, MSE cell‐specific PPARγ knockout (PPARγ‐MSE KO) and control (PPARγ‐WT) mice were generated, mated and allowed to nurse for three days. One week after involution, dams were treated with DMBA to initiate breast tumors, and randomized on week 7 to continue receiving a normal chow diet (DMBA Only: PPARγ‐WT, n = 15; PPARγ‐MSE KO, n = 25) or one supplemented with a PPARγ activating drug (DMBA + ROSI: PPARγ‐WT, n = 17; PPARγ‐MSE KO, n = 24), and monitored for changes in breast tumor outcomes. PPARγ‐MSE KOs had significantly lower overall survival and decreased mammary tumor latency as compared to PPARγ‐WT controls. PPARγ activation significantly reduced DMBA‐mediated malignant mammary tumor volumes irrespective of genotype. MSE‐specific PPARγ loss resulted in decreased mammary gland expression of PTEN and Bax, increased superoxide anion production, and elevated serum eotaxin and RANTES, creating a protumorigenic environment. Moreover, PPARγ activation in MSEs delayed mammary tumor growth in part by down‐regulating Cox‐1, Cox‐2 and cyclin D1. Collectively, these studies highlight a protective role of MSE‐specific PPARγ during breast tumorigenesis, and support a novel chemotherapeutic role of PPARγ activation in breast cancer.

Stromal Adipocyte PPARγ Protects Against Breast Tumorigenesis

Peroxisome proliferator-activated receptor (PPAR)γ regulates the expression of genes essential for fat storage, primarily through its activity in adipocytes. It also has a role in carcinogenesis. PPARγ normally stops the in vivo progression of 7,12-dimethylbenz[a]anthracene (DMBA)-mediated breast tumours as revealed with PPARγ haploinsufficient mice. Since many cell types associated with the mammary gland express PPARγ, each with unique signal patterns, this study aimed to define which tissues are required for PPARγ-dependent antitumour effects. Accordingly, adipocyte-specific PPARγ knockout (PPARγ-A KO) mice and their wild-type (PPARγ-WT) controls were generated, and treated with DMBA for 6 weeks to initiate breast tumorigenesis. On week 7, mice were randomized to continue on normal chow diet or one supplemented with rosiglitazone (ROSI), and followed for 25 weeks for tumour outcomes. In PPARγ-A KO versus PPARγ-WT mice, malignant mammary tumour incidence was significantly higher and mammary tumour latency was decreased. DMBA + ROSI treatment reduced average mammary tumour volumes by 50%. Gene expression analyses of mammary glands by quantitative real-time polymerase chain reaction and immunofluorescence indicated that untreated PPARγ-A KOs had significantly decreased BRCA1 expression in mammary stromal adipocytes. Compared with PPARγ-WT mice, serum leptin levels in PPARγ-A KOs were also significantly higher throughout the study. Together, these data are the first to suggest that in vivo PPARγ expression in mammary stromal adipocytes attenuates breast tumorigenesis through BRCA1 upregulation and decreased leptin secretion. This study supports a protective effect of activating PPARγ as a novel chemopreventive therapy for breast cancer.

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.