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  • Resistance to endocrine therapy: are breast cancer stem cells the culprits?

    O'Brien, Ciara S; Howell, Sacha J; Farnie, Gillian; Clarke, Robert B; Breast Biology Group, School of Cancer and Imaging Sciences, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK. (2009-03)
    From a developmental point of view, tumors can be seen as aberrant versions of their tissue of origin. For example, tumors often partially retain differentiation markers of their tissue of origin and there is evidence that they contain cancer stem cells (CSCs) that drive tumorigenesis. In this review, we summarise current evidence that breast CSCs may partly explain endocrine resistance in breast cancer. In normal breast, the stem cells are known to possess a basal phenotype and to be mainly ERalpha-. If the hierarchy in breast cancer reflects this, the breast CSC may be endocrine resistant because it expresses very little ERalpha and can only respond to treatment by virtue of paracrine influences of neighboring, differentiated ERalpha+ tumor cells. Normal breast epithelial stem cells are highly dependent on the EGFR and other growth factor receptors and it may be that the observed increased growth factor receptor expression in endocrine-resistant breast cancers reflects an increased proportion of CSCs selected by endocrine therapies. There is evidence from a number of studies that breast CSCs are ERalpha- and EGFR+/HER2+, which would support this view. CSCs also express mesenchymal genes which are suppressed by ERalpha expression, further indicating the mutual exclusion between ERalpha+ cells and the CSCs. As we learn more about CSCs, differentiation and the expression and functional activity of the ERalpha in these cells in diverse breast tumor sub-types, it is hoped that our understanding will lead to new modalities to overcome the problem of endocrine resistance in the clinic.
  • Normal breast tissue implanted into athymic nude mice identifies biomarkers of the effects of human pregnancy levels of estrogen.

    Blance, Rognvald N; Sims, Andrew H; Anderson, Elizabeth; Howell, Anthony; Clarke, Robert B; Breast Biology Group, School of Cancer and Imaging Sciences, Paterson Institute for Cancer Research, University of Manchester, Manchester, United Kingdom. (2009-03)
    We have generated a novel model system for the study of estrogen intervention in normal breast tissue. Nulliparous human breast tissue was implanted into immunocompromised nude mice and treated with high-dose estrogen to simulate the effects of pregnancy. Treatment of mice with human mid-pregnancy levels of 17beta-estradiol for a period of 4 weeks was followed by 4 weeks of withdrawal to mimic involution. Gene expression in the xenograft tissue was then analyzed by real-time reverse transcription-PCR to identify differences between treated and control tissues. Ten genes previously identified as altered by pregnancy in rodent models were found to be differentially expressed in human breast tissue with a > or =1.8-fold up-regulation of CDC42, TGFbeta3, DCN, KRT14, LTF, and AREG and a > or =0.7-fold down-regulation of STAT1, CTGF, IGF1, and VAMP1. Immunohistochemical analysis of archival paraffin-embedded adult premenopausal human breast tissue specimens identified a significantly lower level of expression of STAT1 (P < 0.05, Mann-Whitney U test) in parous compared with age-matched nulliparous tissue (median of 24% compared with 42% epithelial cells positive). We conclude that many of the pregnancy-induced breast cancer-protective changes observed in rodent models also occur in human breast tissue following intervention using human pregnancy levels of estrogen and that STAT1 expression is a potential biomarker of parity-induced breast cancer protection in the human breast.
  • The origin of estrogen receptor alpha-positive and alpha-negative breast cancer.

    Clarke, Robert B; Sims, Andrew H; Howell, Anthony; Breast Biology Group, Division of Cancer Studies, University of Manchester Christie Hospital (NHS) Trust, Manchester, UK. (2008)
  • Prolactin receptor antagonism reduces the clonogenic capacity of breast cancer cells and potentiates doxorubicin and paclitaxel cytotoxicity.

    Howell, Sacha J; Anderson, Elizabeth; Hunter, Tom; Farnie, Gillian; Clarke, Robert B; Breast Biology Group, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. (2008)
    INTRODUCTION: Exogenous prolactin is mitogenic and antiapoptotic in breast cancer cells, and overexpression of autocrine prolactin cDNA in breast cancer cell lines has been shown to stimulate their growth and to protect against chemotherapy-induced apoptosis. We examined the effects of the 'pure' prolactin receptor antagonist Delta1-9-G129R-hPrl (Delta1-9) on the breast cancer cell number and clonogenicity, alone and in combination with chemotherapy. METHODS: The effects of doxorubicin, paclitaxel and Delta1-9 on the growth of breast cancer cell lines (MCF-7, T47D, MDA-MB-453, MDA-MB-468 and SK-BR-3) in monolayer culture were assessed by the sulphorhodamine B assay. Effects on clonogenicity were assessed by soft agar assay for the cell lines and by the mammosphere assay for disaggregated primary ductal carcinoma in situ samples. Dual-fluorescence immunocytochemistry was used to identify subpopulations of cells expressing the prolactin receptor and autocrine prolactin. RESULTS: Delta1-9 as a single agent had no effect on the cell number in monolayer culture, but potentiated the cytotoxic effects of doxorubicin and paclitaxel. Doxorubicin accordingly induced expression of prolactin mRNA and protein in all five breast cancer cell lines tested. Delta1-9 alone inhibited the clonogenicity in soft agar of cell lines by ~90% and the mammosphere forming efficiency of six disaggregated primary ductal carcinoma in situ samples by a median of 56% (range 32% to 88%). Subpopulations of cells could be identified in the cell lines based on the prolactin receptor and prolactin expression. CONCLUSION: Autocrine prolactin appears to act as an inducible survival factor in a clonogenic subpopulation of breast cancer cells. The rational combination of cytotoxics and Delta1-9 may therefore improve outcomes in breast cancer therapy by targeting this cell population.
  • Tumour-promoting activity of altered WWP1 expression in breast cancer and its utility as a prognostic indicator.

    Nguyen Huu, Ngoc Sa; Ryder, W David J; Zeps, N; Flasza, Marzena; Chiu, M; Hanby, A M; Poulsom, R; Clarke, Robert B; Baron, M; University of Manchester, Faculty of Life Sciences, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK. (2008-09)
    WWP1 is a ubiquitin ligase, associated with the post-translational regulation of several tumour-promoting and tumour suppressor proteins. Here we show that WWP1 expression is up-regulated in a subset of breast tumour cell lines and primary breast tumours. We overexpressed WWP1 in MCF10A breast epithelial cells and demonstrated increased cell growth and anchorage-independent colony formation. RNAi knockdown of WWP1 expression in T47D and MCF7 breast tumour cell lines reduced anchorage-independent colony formation. We used WWP1 protein expression levels, in combination with its sub-cellular localization, to classify breast tumours into four categories. Surprisingly, a category with low/absent WWP1 expression displayed a consistently worse prognosis compared with WWP1-expressing tumours. Importantly, the association with disease-free survival was independent of the status of other commonly used prognostic indicators. Thus, WWP1 is a prognostic marker and may be a potential therapeutic target for a subset of breast tumours.