Breast Cancer Research - Latest articles

ErbB3 is required for ductal morphogenesis in the mouse mammary gland

2008-11-18

IntroductionThe receptor ErbB3/HER3 is often overexpressed in human breast cancers, frequently in conjunction with overexpression of the proto-oncogene ERBB2/HER2/NEU. Although the prognostic/predictive value of ErbB3 expression in breast cancer is unclear, ErbB3 is known to contribute to therapeutic resistance. Understanding ErbB3 functions in the normal mammary gland will help explain its role in cancer etiology and as a modulator of signalling responses to the mammary oncogene ERBB2. Methods: To investigate the roles of ErbB3 in mouse mammary gland development, we transplanted mammary buds from ErbB3-/- embryos into the cleared mammary fat pads of wild-type immunocompromised mice. Effects on ductal outgrowth were analyzed at 4 weeks, 7 weeks and 20 weeks post-transplantation for total ductal outgrowth, branch density and number and area of terminal end buds. Sections of glands containing terminal end buds were analyzed for number and epithelial area of terminal end buds. Terminal end buds were also analyzed for presence of mitotic figures, apoptotic figures, BrdU incorporation, and expression of E-cadherin, P-cadherin, alpha-smooth muscle actin, and cleaved caspase-3. Results: The mammary ductal trees developed from ErbB3-/- buds only partly filled the mammary fat pad. In contrast to similar experiments with ErbB2-/- mammary buds, this phenotype was maintained through adulthood, pregnancy, and parturition. In addition, in contrast to similar work with ErbB4-/- mammary buds, lobuloalveolar development of ErbB3-/- transplanted glands was normal. The ErbB3-/- mammary outgrowth defect was associated with a decrease in the size of the terminal end buds, and increases in branch density, the number of terminal end buds, and in the number of luminal spaces. Proliferation rates were not affected by the lack of ErbB3, but there was an increase in apoptosis in ErbB3-/- terminal end buds. Conclusions: Endogenous ErbB3 regulates morphogenesis of mammary epithelium.

Interleukin-17 expression by breast cancer associated macrophages: IL-17 promotes invasiveness of breast cancer cell lines

2008-11-17

IntroductionInterleukin (IL)-17 plays an important role in autoimmunity, promoting auto-immunity, inflammation and invasion in multiple sclerosis, rheumatoid arthritis and type-I diabetes. However, its role in cancer is unclear as there are few studies examining IL-17 protein expression in cancer. Therefore, we examined IL-17 protein expression in human breast cancer and modelled its potential biological significance in vitro. Methods: Immunohistochemistry was used to determine IL-17 expression in breast cancers. Matrigel invasion assays were employed to examine the effect of IL-17 on cancer cell invasion by a panel of breast cancer cell lines. The role of matrix metalloproteinases (MMPs) was investigated with selective antagonists and immunoassays for MMPs-2, 3, 9 and tissue inhibitor of MMP (TIMP1). Results: IL-17 expressing cells with macrophage morphology were identified in the peri-tumoural area of a proportion of patients (8/19). Macrophages were confirmed by CD68 staining on serial sections. With the exception of occasional lymphocytes, one patient with rare multinucleate giant cells and one patient with occasional expression of IL-17 in tumour cells, no other IL-17+ cells were detected. Addition of IL-17 to cell lines in vitro stimulated marked invasion of Matrigel. In contrast, IL-17 did not promote the invasion of MCF7 or T47D cell lines. Invasion was initially thought to be dependent on MMPs as evidenced by the broad-spectrum MMPs inhibitor, GM6001, and selective antagonists of MMP2/9 and MMP3. However measurement of MMPs-2, 3 and 9 and TIMP1 secretion failed to reveal any changes in expression following IL-17 exposure. In contrast, tumour necrosis factor (TNF) promoted secretion of MMPs but IL-17 did not augment TNF, indicating that IL-17 acts via an independent mechanism. Conclusions: This is the first study to describe in situ expression of IL-17 protein in human breast tumours and propose a direct association between IL-17 and breast cancer invasion. The precise effectors of IL-17 dependent invasion remain to be characterised but could include a range of proteases such as a disintegrin and metalloproteinase (ADAM) or astacins. Nevertheless, this work identifies a novel potential mechanism for breast cancer invasion and tumour progression, the prognostic implication of which is currently under investigation.

Heat shock protein-90-alpha, a prolactin-STAT5 target gene identified in breast cancer cells, is involved in apoptosis regulation

2008-11-13

IntroductionThe prolactin-Janus-kinase-2-signal transducer and activator of transcription-5 (JAK2-STAT5) pathway is essential for the development and the functional differentiation of the mammary gland. The pathway also has important roles in mammary tumourigenesis. Prolactin regulated target genes are not yet well defined in tumour cells, and we undertook the first large genetic screen of breast cancer cells treated with or without exogenous prolactin. We hypothesize that the identification of these genes should yield insights into the mechanisms by which prolactin participates in cancer formation or progression and possibly how it also regulates normal mammary gland development. Methods: We used subtractive hybridization to identify a number of prolactin-regulated genes in the human mammary carcinoma cell line SKBR3. Northern analysis and luciferase assays identified the gene encoding heat shock protein 90-alpha (HSP90A) as a prolactin-JAK2-STAT5 target gene, whose function was characterized using apoptosis assays. Results: We identified a number of new prolactin-regulated genes in breast cancer cells. Focusing on HSP90A, we determined that prolactin increased HSP90A mRNA in cancerous human breast SKBR3 cells and that STAT5B preferentially activated the HSP90A promoter in reporter gene assays. Both prolactin and its downstream protein effector, HSP90-alpha, promote survival, as shown by apoptosis assays and by the addition of the heat shock protein 90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), in both untransformed HC11 mammary epithelial cells and SKBR3 breast cancer cells. The constitutive expression of HSP90A, however, sensitized differentiated HC11 cells to starvation-induced wild-type p53-independent apoptosis. Interestingly, in SKBR3 breast cancer cells, HSP90-alpha promoted survival in the presence of serum but appeared to have little effect during starvation. Conclusions: In addition to identifying new prolactin regulated genes in breast cancer cells, we found that prolactin-JAK2-STAT5 induces expression of the HSP90A gene, which encodes the master chaperone of cancer. This identifies one mechanism by which prolactin contributes to breast cancer. Increased HSP90-alpha expression in breast cancer is correlated with increased cell survival and poor prognosis and HSP90-alpha inhibitors are being tested in clinical trials as a breast cancer treatment. Our results also indicate that HSP90-alpha promotes survival depending upon the cellular conditions and state of cellular transformation.

A comprehensive analysis of prognostic signatures reveals the high predictive capacity of Proliferation, Immune response and RNA splicing modules in breast cancer.

2008-11-13

IntroductionSeveral gene expression signatures have been proposed which have been demonstrated to be predictive of outcome in breast cancer. Here we address the following issues: 1) Do these signatures perform similarly? 2) Are there (common) molecular processes reported by these signatures? 3) Can better prognostic predictors be constructed based on these identified molecular processes? Methods: We performed a comprehensive analysis of the performance of nine gene expression signatures on seven different breast cancer datasets. To better characterize the functional processes associated with these signatures, we enlarged each signature by including all probes with a significant correlation to at least one of the genes in the original signature. The enrichment of functional groups was assessed using four ontology databases. Results: The classification performance of the nine gene expression signatures is very similar in terms of assigning a sample to either a 'poor' or 'good' outcome group. Nevertheless the concordance in classification at the sample level is low, with only 50% of the breast cancer samples classified in the same outcome group by all classifiers. The predictive accuracy decreases with the number of 'poor' outcome assignments given to a sample. The best classification performance was obtained for the group of patients with only 'good' outcome assignments. Enrichment analysis of the enlarged signatures revealed 11 functional modules with prognostic ability. The combination of the RNA-splicing and Immune modules resulted in a classifier with high prognostic performance on an independent validation set. Conclusions: This study revealed that the nine signatures perform similarly but exhibit a large degree of discordance in prognostic group assignment. Functional analyses indicate that proliferation is a common cellular process, but that other functional categories are also enriched and show independent prognostic ability. We provide new evidence of the potentially promising prognostic impact of Immunity and RNA-splicing processes in breast cancer.

Local control by radiotherapy: is that all there is?

Silvia C Formenti and Sandra Demaria — 2008-11-05

Radiotherapy is a local treatment modality employed in breast cancer to reduce local recurrence following surgery. The observed association of optimal local control with improved survival was not expected in a disease characterized by early systemic spread. The underlying mechanisms whereby the application of ionizing radiation to the primary tumor site can have systemic effects remain unclear and are the subject of much debate. In the present article we discuss the hypothesis that radiotherapy has unique biological effects and that, in addition to killing residual neoplastic cells after surgery is performed, it might favorably alter the microenvironment at the primary tumor site during the process of wound healing and the development of antitumor immune responses.

Functional genomic analysis of drug sensitivity pathways to guide adjuvant strategies in breast cancer

Charles Swanton, Zoltan Szallasi, James D Brenton and Julian Downward — 2008-10-31

The widespread introduction of high throughput RNA interference screening technology has revealed tumour drug sensitivity pathways to common cytotoxics such as paclitaxel, doxorubicin and 5-fluorouracil, targeted agents such as trastuzumab and inhibitors of AKT and Poly(ADP-ribose) polymerase (PARP) as well as endocrine therapies such as tamoxifen. Given the limited power of microarray signatures to predict therapeutic response in associative studies of small clinical trial cohorts, the use of functional genomic data combined with expression or sequence analysis of genes and microRNAs implicated in drug response in human tumours may provide a more robust method to guide adjuvant treatment strategies in breast cancer that are transferable across different expression platforms and patient cohorts.

Prospective study of physical activity and risk of postmenopausal breast cancer

2008-10-31

IntroductionTo prospectively examine the relation of total, vigorous and non-vigorous physical activity to postmenopausal breast cancer risk. Methods: We studied 32,269 women enrolled in the Breast Cancer Detection Demonstration Project Follow-up Study. Usual physical activity (including household, occupational and leisure activities) throughout the previous year was assessed at baseline using a self-administered questionnaire. Postmenopausal breast cancer cases were identified through self-reports, death certificates and linkage to state cancer registries. A Cox proportional hazards regression was used to estimate the relative risk and 95% confidence intervals of postmenopausal breast cancer associated with physical activity. Results: During 269,792 person-years of follow-up from 1987 to 1998, 1506 new incident cases of postmenopausal breast cancer were ascertained. After adjusting for potential risk factors of breast cancer, a weak inverse association between total physical activity and postmenopausal breast cancer was suggested (relative risk comparing extreme quintiles = 0.87; 95% confidence interval = 0.74 to 1.02; p for trend = 0.21). That relation was almost entirely contributed by vigorous activity (relative risk comparing extreme categories = 0.87; 95% confidence interval = 0.74 to 1.02; p for trend = 0.08). The inverse association with vigorous activity was limited to women who were lean (ie, body mass index <25.0 kg/m2: relative risk = 0.68; 95% confidence interval = 0.54 to 0.85). In contrast, no association with vigorous activity was noted among women who were overweight or obese (ie, body mass index ≥ 25.0 kg/m2: relative risk = 1.18; 95% confidence interval = 0.93 to 1.49; p for interaction = 0.008). Non-vigorous activity showed no relation to breast cancer (relative risk comparing extreme quintiles = 1.02; 95% confidence interval = 0.87 to 1.19; p for trend = 0.86). The physical activity and breast cancer relation was not specific to a certain hormone receptor subtype. Conclusions: In this cohort of postmenopausal women, breast cancer risk reduction appeared to be limited to vigorous forms of activity; it was apparent among normal weight women but not overweight women, and the relation did not vary by hormone receptor status. Our findings suggest that physical activity acts through underlying biological mechanisms that are independent of body weight control.

Serial analysis of gene expression of lobular carcinoma in situ identifies down regulation of claudin 4 and overexpression of matrix metalloproteinase 9

2008-10-27

IntroductionAlthough lobular carcinoma in situ (LCIS) has traditionally been viewed as a marker of breast cancer risk, recent clinical, pathological and genetic analyses have supported the concept that LCIS is a low risk, direct precursor of invasive lobular carcinoma. Global gene expression profiling of LCIS has not been performed. Methods: We analysed the comprehensive gene expression profile of a unique case of mass-forming LCIS using serial analysis of gene expression (SAGE). This SAGE library is publicly available online. By comparing the gene expression profile of LCIS to that of benign breast epithelium and stroma, we identified several genes up and down regulated in LCIS. Differential expression of selected genes not previously studied in LCIS was validated at the protein level by immunohistochemistry and at the RNA level by quantitative reverse transcriptase PCR (RT-PCR). Results: We identified down regulation of claudin 4 and overexpression of matrix metalloproteinase 9 in LCIS relative to normal breast epithelium and stroma. We validated these findings by immunohistochemistry in a separate series of 11 and 19 LCIS cases, respectively. Overexpression of matrix metalloproteinase 9 was further confirmed by quantitative RT-PCR analysis of the index case. Conclusions: We have created the first global gene expression profile of LCIS, and demonstrated down regulation of cell junction proteins (an expected result) and overexpression of matrix metalloproteinase 9 (an unexpected result). Additional analysis of this data made available as an online resource should facilitate further molecular characterisation of LCIS.

Selective segregation of DNA strands persists in long label retaining mammary cells during pregnancy

Brian W Booth, Corinne A Boulanger and Gilbert H Smith — 2008-10-24

IntroductionDuring pregnancy the mammary epithelial compartment undergoes extreme proliferation and differentiation facilitated by stem/progenitor cells. Mouse mammary epithelium in non-pregnant mice contain long label-retaining epithelial cells (LREC) that divide asymmetrically and retain their template DNA strands. The role of LREC during alveogenesis has not been determined. Methods: Immunohistochemistry and autoradiography was performed on murine mammary glands that had been labeled with 5-bromodeoxyuridine (5BrdU) during allometric ductal growth to investigate the co-expression of DNA label-retention and estrogen receptor-alpha (ERalpha) or progesterone receptor (PR) during pregnancy. A second DNA label ([3H]-thymidine) was administered during pregnancy to identify label-retaining cells (LRC), which subsequently enter the cell cycle. Using this methodology allowed us to investigate the co-localization of 5BrdU with smooth muscle actin, CD31, cytokeratin and desmin in periductal or peri-acinar LRC in mammary tissue from pregnant mice subsequent to a long chase period to identify label-retaining cells. Results: ERalpha-positive (ERalpha+) and PR-positive (PR+) cells represented roughly 30-40% of the LREC which is <1.0% of the epithelial subpopulation. Pregnancy altered the percent of LREC expressing ERalpha. LRC situated in periductal or peri-acinar positions throughout the gland do not express epithelial, endothelial or myoepithelial markers and these undefined LRC persist throughout pregnancy. Additionally, new cycling LREC ([3H]-thymidine-retaining) appear during alveologenesis and LRC found in other tissue types (e.g. endothelium and nerve) within the mammary fat pad become double-labeled during pregnancy, an indication that they may also divide asymmetrically. Conclusions: Our results support the premise that there is a subpopulation of LREC in the mouse mammary gland that persists during alveologenesis. These cells react to the hormonal cues during pregnancy and enter the cell cycle while continuing to retain, selectively, their original template DNA. In addition, non-epithelial LRC are found in periductal or peri-acinar positions. These LRC also enter the cell cycle during pregnancy. During alveologenesis, newly created label-retaining (3H-thymidine) epithelial cells appear within the expanding alveoli and continue to cycle and retain their original template DNA (3H-thymidine) strands as determined by a second pulse of 5BrdU.

Novel image analysis approach for quantifying expression of nuclear proteins assessed by immunohistochemistry: application to measurement of oestrogen and progesterone receptor levels in breast cancer

2008-10-23

IntroductionManual interpretation of immunohistochemistry (IHC) is a subjective, time-consuming and variable process, with an inherent intra-observer and inter-observer variability. Automated image analysis approaches offer the possibility of developing rapid, uniform indicators of IHC staining. In the present article we describe the development of a novel approach for automatically quantifying oestrogen receptor (ER) and progesterone receptor (PR) protein expression assessed by IHC in primary breast cancer. Methods: Two cohorts of breast cancer patients (n = 743) were used in the study. Digital images of breast cancer tissue microarrays were captured using the Aperio ScanScope XT slide scanner (Aperio Technologies, Vista, CA, USA). Image analysis algorithms were developed using MatLab 7 (MathWorks, Apple Hill Drive, MA, USA). A fully automated nuclear algorithm was developed to discriminate tumour from normal tissue and to quantify ER and PR expression in both cohorts. Random forest clustering was employed to identify optimum thresholds for survival analysis. Results: The accuracy of the nuclear algorithm was initially confirmed by a histopathologist, who validated the output in 18 representative images. In these 18 samples, an excellent correlation was evident between the results obtained by manual and automated analysis (Spearman's ρ = 0.9, P < 0.001). Optimum thresholds for survival analysis were identified using random forest clustering. This revealed 7% positive tumour cells as the optimum threshold for the ER and 5% positive tumour cells for the PR. Moreover, a 7% cutoff level for the ER predicted a better response to tamoxifen than the currently used 10% threshold. Finally, linear regression was employed to demonstrate a more homogeneous pattern of expression for the ER (R = 0.860) than for the PR (R = 0.681). Conclusions: In summary, we present data on the automated quantification of the ER and the PR in 743 primary breast tumours using a novel unsupervised image analysis algorithm. This novel approach provides a useful tool for the quantification of biomarkers on tissue specimens, as well as for objective identification of appropriate cutoff thresholds for biomarker positivity. It also offers the potential to identify proteins with a homogeneous pattern of expression.