Dushanthi Pinnaduwage

Met induces mammary tumors with diverse histologies and is associated with poor outcome and human basal breast cancer

Elevated MET receptor tyrosine kinase correlates with poor outcome in breast cancer, yet the reasons for this are poorly understood. We thus generated a transgenic mouse model targeting expression of an oncogenic Met receptor (Metmt) to the mammary epithelium. We show that Metmt induces mammary tumors with multiple phenotypes. These reflect tumor subtypes with gene expression and immunostaining profiles sharing similarities to human basal and luminal breast cancers. Within the basal subtype, Metmt induces tumors with signatures of WNT and epithelial to mesenchymal transition (EMT). Among human breast cancers, MET is primarily elevated in basal and ERBB2-positive subtypes with poor prognosis, and we show that MET, together with EMT marker, SNAIL, are highly predictive of poor prognosis in lymph node-negative patients. By generating a unique mouse model in which the Met receptor tyrosine kinase is expressed in the mammary epithelium, along with the examination of MET expression in human breast cancer, we have established a specific link between MET and basal breast cancer. This work identifies basal breast cancers and, additionally, poor-outcome breast cancers, as those that may benefit from anti-MET receptor therapies.

A role for the TGFβ-Par6 polarity pathway in breast cancer progression

The role of polarity signaling in cancer metastasis is ill defined. Using two three-dimensional culture models of mammary epithelial cells and an orthotopic mouse model of breast cancer, we reveal that Par6 signaling, which is regulated directly by TGFβ, plays a role in breast cancer metastasis. Interference with Par6 signaling blocked TGFβ-dependent loss of polarity in acini-like structures formed by non-transformed mammary cells grown in three-dimensional structures and suppressed the protrusive morphology of mesenchymal-like invasive mammary tumor cells without rescuing E-cadherin expression. Moreover, blockade of Par6 signaling in an in vivo orthotopic model of metastatic breast cancer induced the formation of ZO-1-positive epithelium-like structures in the primary tumor and suppressed metastasis to the lungs. Analysis of the pathway in tissue microarrays of human breast tumors further revealed that Par6 activation correlated with markers of the basal carcinoma subtype in BRCA1-associated tumors. These studies thus reveal a key role for polarity signaling and the control of morphologic transformation in breast cancer metastasis.

The combination of p53 mutation and neu/erbB-2 amplification is associated with poor survival in node-negative breast cancer

PURPOSE Increases in neu/erbB-2 have been implicated in breast cancer prognosis, but do not predict all recurrences. On the basis of evidence that p53 mutation is involved in the development of human neoplasia, we examined the prognostic value of p53 alterations in combination with neu/erbB-2 amplification. PATIENTS AND METHODS A consecutive series of women were observed for recurrence and death (median follow-up of 85 months) and tumors from 543 individuals were analyzed for p53 mutation status and neu/erbB-2 amplification. Exons 4 through 10 of the p53 gene were analyzed by single-stranded conformational polymorphism and mutations were confirmed by DNA sequencing. The association of p53 mutation status and neu/erbB-2 amplification with risk of recurrence and death was examined in survival analyses with traditional and histologic markers as prognostic factors. RESULTS p53 mutations occurred in 24.5% of the axillary node-negative breast carcinomas. Mutations were more frequent in carcinomas with neu/erbB-2 amplification: 38.9% compared with only 20.9% in those without neu/erbB-2 amplification. We found elevated risks of disease recurrence and overall mortality in patients with both p53 mutation and neu/erbB-2 amplification in their tumor compared with patients with neither or only one of the alterations. This increase persisted with adjustment for other prognostic factors (relative risk, 2.32; P =.002 for recurrence; relative risk, 2.22; P =.004 for death). CONCLUSION Evaluation of tumors for p53 mutations may be beneficial to identify women at higher risk of disease recurrence and death when the tumor has neu/erbB-2 amplification, but in the absence of neu/erbB-2 amplification, the presence of p53 mutation may not provide additional independent prognostic information.

Tumoral Lymphocytic Infiltration and Expression of the Chemokine CXCL10 in Breast Cancers from the Ontario Familial Breast Cancer Registry

Purpose: Breast carcinomas, including basal and hereditary cases, often present with a prominent tumoral lymphocytic infiltrate. Chemokines could play a role in attracting these cells and contribute to tumor progression. We explored tumoral expression of CXCL10 and determined the relationship between CXCL10 and lymphocytic infiltrate in a cohort of breast cancers. Experimental Design: Using tissue microarrays of 364 breast tumors, we evaluated expression of CXCL10 and its receptor, CXCR3, in relation to histopathologic features, biomarkers, and lymphocyte markers. In addition, we overexpressed CXCL10 and CXCR3 in MCF7 breast cancer cells and monitored T-lymphocyte migration and invasion. Results: Forty-five percent of tumors expressed CXCL10, and a significant association was found with CXCR3 and lymphocytic infiltrate. Further characterization of the lymphocytic infiltrate revealed an association with CXCL10 expression for peritumoral CD4+ and CD8+ lymphocytes. CD8+ intratumoral lymphocytes, FOXP3+ regulatory T cells (Tregs), and T-BET+ TH1 cells were associated with BRCA1 and basal tumors. Conditioned media from MCF7 cells overexpressing both CXCL10 and CXCR3 increased T-lymphocyte migration and invasion. Conclusions: Our findings suggest that CXCL10 may act in a paracrine manner, affecting the tumor microenvironment, and in an autocrine manner, acting on the tumor cells themselves and may play a role in tumor invasiveness and progression. The CXCL10-CXCR3 axis can serve as a potential target in BRCA1 and basal breast cancers, which present with a prominent lymphocytic infiltrate and a poor prognosis. Clin Cancer Res; 19(2); 336–46. ©2012 AACR.

Met synergizes with p53 loss to induce mammary tumors that possess features of claudin-low breast cancer

Significance Triple-negative breast cancers lack targeted therapies and are subdivided into molecular subtypes, including basal and claudin-low. Preclinical models representing these subtypes are limited. We have developed a murine model in which mammary gland expression of a receptor tyrosine kinase (MET) and loss of tumor suppressor gene p53 (Trp53), synergize to promote tumors with pathological and molecular features of claudin-low breast cancer. These tumors require MET signaling for proliferation, as well as mesenchymal characteristics, which are key features of claudin-low biology. This work associates MET expression and p53 loss with claudin-low breast cancers and highly proliferative breast cancers of poor outcome. Triple-negative breast cancer (TNBC) accounts for ∼20% of cases and contributes to basal and claudin-low molecular subclasses of the disease. TNBCs have poor prognosis, display frequent mutations in tumor suppressor gene p53 (TP53), and lack targeted therapies. The MET receptor tyrosine kinase is elevated in TNBC and transgenic Met models (Metmt) develop basal-like tumors. To investigate collaborating events in the genesis of TNBC, we generated Metmt mice with conditional loss of murine p53 (Trp53) in mammary epithelia. Somatic Trp53 loss, in combination with Metmt, significantly increased tumor penetrance over Metmt or Trp53 loss alone. Unlike Metmt tumors, which are histologically diverse and enriched in a basal-like molecular signature, the majority of Metmt tumors with Trp53 loss displayed a spindloid pathology with a distinct molecular signature that resembles the human claudin-low subtype of TNBC, including diminished claudins, an epithelial-to-mesenchymal transition signature, and decreased expression of the microRNA-200 family. Moreover, although mammary specific loss of Trp53 promotes tumors with diverse pathologies, those with spindloid pathology and claudin-low signature display genomic Met amplification. In both models, MET activity is required for maintenance of the claudin-low morphological phenotype, in which MET inhibitors restore cell-cell junctions, rescue claudin 1 expression, and abrogate growth and dissemination of cells in vivo. Among human breast cancers, elevated levels of MET and stabilized TP53, indicative of mutation, correlate with highly proliferative TNBCs of poor outcome. This work shows synergy between MET and TP53 loss for claudin-low breast cancer, identifies a restricted claudin-low gene signature, and provides a rationale for anti-MET therapies in TNBC.

Distinguishing luminal breast cancer subtypes by Ki67, progesterone receptor or TP53 status provides prognostic information

The objectives of this study were to determine the prognostic significance of subgrouping estrogen receptor (ER)-positive breast tumors into low- and high-risk luminal categories using Ki67 index, TP53, or progesterone receptor (PR) status. The study group comprised 540 patients with lymph node negative, invasive breast carcinoma. Luminal A subtype was defined as being ER positive, HER2 negative, and Ki67 low (<14% cells positive) and luminal B subtype as being ER positive, HER2 negative, and Ki67 high (≥14% cells positive). Luminal tumors were also subgrouped into risk categories based on the PR and TP53 status. Survival analysis was performed. Patients with luminal B tumors (n=173) had significantly worse disease-free survival compared to those with luminal A tumors (n=186) (log rank P-value=0.0164; univariate Cox regression relative risk 2.00; 95% CI, 1.12–3.58; P=0.0187). Luminal subtype remained an independent prognostic indicator on multivariate analysis including traditional prognostic factors (relative risk 2.12; 95% CI, 1.16–3.88; P=0.0151). Using TP53 status or PR negativity rather than Ki67 to classify ER-positive luminal tumors gave similar outcome results to those obtained using the proliferation index. However, it was a combination of the three markers, which proved the most powerful prognostically. Ki67 index, TP53 status, or PR negativity can be used to segregate ER-positive, HER2-negative tumors into prognostically meaningful subgroups with significantly different clinical outcomes. These biomarkers particularly in combination may potentially be used clinically to guide patient management.

Prognostic Effect of Basal-Like Breast Cancers Is Time Dependent: Evidence from Tissue Microarray Studies on a Lymph Node–Negative Cohort

Purpose: To determine whether data obtained from tissue microarrays (TMA) of a prospectively accrued node-negative breast cancer cohort are prognostically informative, we compared data derived from TMA with previously determined molecular markers. Subsequent to this validation, we examined outcome in specific subgroups defined using TMA data. Experimental Design: A consecutive series of 1,561 patients were followed for recurrence (median follow-up of 107 months). Estrogen receptor, progesterone receptor, p53, and HER2 expression, examined using TMA constructed from 887 tumors, was compared with status evaluated previously by biochemical and molecular methods. The associations with risk of recurrence were examined for biomarkers as well as for HER2, luminal, and basal subgroups defined by immunohistochemical expression. Results: In line with earlier molecular studies, a significant risk of recurrence was found in patients with HER2 overexpression (relative risk = 2.30; P = 0.002) and p53-positive tumors (relative risk = 1.81; P = 0.005) in univariate Cox model analysis. Although complete concordance between methodologies was not observed for estrogen receptor and progesterone receptor, their associations with disease-free survival were consistent with established prognostic findings. Patients with basal-type tumors fared worse within 36 months of diagnosis but not thereafter. Conclusions: This study shows the clinical validity of TMA in evaluating the importance of prognostic markers in this cohort. Furthermore, it shows a marked time-dependent effect in tumor subgroups, most notable within the basal subgroup. Our data suggest that patients with basal-like tumors may be broadly separable into two clinically distinctive groups: those likely to experience disease recurrence in the short term and those that will experience long-term survival.

Clinical–pathologic significance of cancer stem cell marker expression in familial breast cancers

Human breast cancer cells with a CD44+/CD24−/low or ALDH1+ phenotype have been demonstrated to be enriched for cancer stem cells (CSCs) using in vitro and in vivo techniques. The aim of this study was to determine the association between CD44+/CD24−/low and ALDH1 expression with clinical–pathologic tumor characteristics, tumor molecular subtype, and survival in a well characterized collection of familial breast cancer cases. 364 familial breast cancers from the Ontario Familial Breast Cancer Registry (58 BRCA1-associated, 64 BRCA2-associated, and 242 familial non-BRCA1/2 cancers) were studied. Each tumor had a centralized pathology review performed. TMA sections of all tumors were analyzed for the expression of ER, PR, HER2, CK5, CK14, EGFR, CD44, CD24, and ALDH1. The Chi square test or Fisher’s exact test was used to analyze the marker associations with clinical–pathologic tumor variables, molecular subtype and genetic subtype. Analyses of the association of overall survival (OS) with marker status were conducted using Kaplan–Meier plots and log-rank tests. The CD44+/CD24−/low and ALDH1+ phenotypes were identified in 16% and 15% of the familial breast cancer cases, respectively, and associated with high-tumor grade, a high-mitotic count, and component features of the medullary type of breast cancer. CD44+/CD24−/low and ALDH1 expression in this series were further associated with the basal-like molecular subtype and the CD44+/CD24−/low phenotype was independently associated with BRCA1 mutational status. The currently accepted breast CSCs markers are present in a minority of familial breast cancers. Whereas the presence of these markers is correlated with several poor prognostic features and the basal-like subtype of breast cancer, they do not predict OS.

The interaction of PP1 with BRCA1 and analysis of their expression in breast tumors

BackgroundThe breast cancer susceptibility gene, BRCA1, is implicated in multiple cellular processes including DNA repair, the transactivation of genes, and the ubiquitination of proteins; however its precise functions remain to be fully understood. Identification and characterization of BRCA1 protein interactions may help to further elucidate the function and regulation of BRCA1. Additionally, detection of changes in the expression levels of BRCA1 and its interacting proteins in primary human breast tumors may further illuminate their role in the development of breast cancer.MethodsWe performed a yeast two-hybrid study to identify proteins that interact with exon11 of BRCA1 and identified Protein Phosphatase 1β (PP1β), an isoform of the serine threonine phosphatase, PP1. GST-pull down and co-immunoprecipitation assays were performed to further characterize this interaction. Additionally, Real-Time PCR was utilized to determine the expression of BRCA1, PP1α, β and γ in primary human breast tumors and normal breast tissue to identify alterations in the expression of these genes in breast cancer.ResultsPP1 and BRCA1 co-immunoprecipitate and the region within BRCA1 as well as the specific PP1 interacting domain mediating this interaction were identified. Following mRNA expression analysis, we identified low levels of BRCA1 and variable levels of PP1α and β in primary sporadic human breast tumors. Furthermore, BRCA1, PP1β and PP1γ were significantly higher in normal tissue specimens (BRCA1 p = 0.01, PP1β: p = 0.03, PP1γ, p = 1.9 × 10-6) compared to sporadic breast tumor samples. Interestingly, we also identified that ER negative tumors are associated with low levels of PP1α expression.ConclusionThe identification and characterization of the interaction of BRCA1 with PP1 and detection of changes in the expression of PP1 and genes encoding other BRCA1 associated proteins identifies important genetic pathways that may be significant to breast tumorigenesis. Alterations in the expression of genes, particularly phosphatases that operate in association with BRCA1, could negatively affect the function of BRCA1 or BRCA1 associated proteins, contributing to the development of breast cancer.

Mena calc , a quantitative method of metastasis assessment, as a prognostic marker for axillary node-negative breast cancer

BackgroundMenacalc is an immunofluorescence-based, quantitative method in which expression of the non-invasive Mena protein isoform (Mena11a) is subtracted from total Mena protein expression. Previous work has found a significant positive association between Menacalc and risk of death from breast cancer. Our goal was to determine if Menacalc could be used as an independent prognostic marker for axillary node-negative (ANN) breast cancer.MethodsAnalysis of the association of Menacalc with overall survival (death from any cause) was performed for 403 ANN tumors using Kaplan Meier survival curves and the univariate Cox proportional hazards (PH) model with the log-rank or the likelihood ratio test. Cox PH models were used to estimate hazard ratios (HRs) for the association of Menacalc with risk of death after adjustment for HER2 status and clinicopathological tumor features.ResultsHigh Menacalc was associated with increased risk of death from any cause (P = 0.0199, HR (CI) = 2.18 (1.19, 4.00)). A similarly elevated risk of death was found in the subset of the Menacalc cohort which did not receive hormone or chemotherapy (n = 142) (P = 0.0052, HR (CI) = 3.80 (1.58, 9.97)). There was a trend toward increased risk of death with relatively high Menacalc in the HER2, basal and luminal molecular subtypes.ConclusionsMenacalc may serve as an independent prognostic biomarker for the ANN breast cancer patient population.