Tahereh Kamalati

Expression of the BRK tyrosine kinase in mammary epithelial cells enhances the coupling of EGF signalling to PI 3-kinase and Akt, via erbB3 phosphorylation

A high proportion of human breast cancers, in contrast with normal mammary tissue, express the intracellular tyrosine kinase BRK. BRK expression enhances the mitogenic response of mammary epithelial cells to epidermal growth factor, and conferment of a proliferative advantage through this mechanism may account for the frequent elevation of BRK expression in tumours. Here we report that BRK expression in mammary epithelial cells, at pathologically relevant levels, results in an enhanced phosphorylation of the epidermal growth factor receptor-related receptor erbB3 in response to epidermal growth factor. As a consequence, erbB3 recruits increased levels of phosphoinositide 3-kinase, and this is associated with a potentiated activation of Akt. This effect of BRK on the regulation of phosphoinositide 3-kinase and Akt activity may account for BRKs ability to enhance mammary cell mitogenesis, and raises the possibility that breast tumours expressing BRK may acquire a resistance to pro-apoptotic signals.

ZNF366 is an estrogen receptor corepressor that acts through CtBP and histone deacetylases

The regulation of gene expression by estrogen receptor-α (ERα) requires the coordinated and temporal recruitment of diverse sets of transcriptional co-regulator complexes, which mediate nucleosome remodelling and histone modification. Using ERα as bait in a yeast two-hybrid screen, we have identified a novel ERα-interacting protein, ZNF366, which is a potent corepressor of ERα activity. The interaction between ZNF366 and ERα has been confirmed in vitro and in vivo, and is mediated by the zinc finger domains of the two proteins. Further, we show that ZNF366 acts as a corepressor by interacting with other known ERα corepressors, namely RIP140 and CtBP, to inhibit expression of estrogen-responsive genes in vivo. Together, our results indicate that ZNF366 may play an important role in regulating the expression of genes in response to estrogen.

HGF/SF in Mammary Epithelial Growth and Morphogenesis: In Vitro and In Vivo Models

HGF/SF is a multifunctional cytokine whichthrough binding to its cellular receptor, c-MET, canelicit mitogenic, morphogenic and motogenic responses intarget cells. Expression of HGF/SF and c-MET has been shown to be critical in earlyembryogenesis affecting development of many organs andtissues. The effects of HGF/SF4 on established human andmouse mammary cell lines have previously been reported. This review describes the source and targetsfor HGF/SF in both human and mouse mammary tissue anddiscusses the effects of HGF/SF on primary mouse andhuman mammary epithelial cells in vitro, detailing the individual response of the two epithelialsub-population of cells which comprise this organ.Additionally, the effects of HGF/SF overexpression onmouse mammary gland development in vivo, aresummarized.

Transient over-expression of estrogen receptor-α in breast cancer cells promotes cell survival and estrogen-independent growth

Estrogen receptor-α (ERα) positive breast cancer frequently responds to inhibitors of ERα activity, such as tamoxifen, and/or to aromatase inhibitors that block estrogen biosynthesis. However, many patients become resistant to these agents through mechanisms that remain unclear. Previous studies have shown that expression of ERα in ERα-negative breast cancer cell lines frequently inhibits their growth. In order to determine the consequence of ERα over-expression in ERα-positive breast cancer cells, we over-expressed ERα in the MCF-7 breast cancer cell line using adenovirus gene transduction. ERα over-expression led to ligand-independent expression of the estrogen-regulated genes pS2 and PR and growth in the absence of estrogen. Interestingly, prolonged culturing of these cells in estrogen-free conditions led to the outgrowth of cells capable of growth in cultures from ERα transduced, but not in control cultures. From these cultures a line, MLET5, was established which remained ERα-positive, but grew in an estrogen-independent manner. Moreover, MLET5 cells were inhibited by anti-estrogens showing that ERα remains important for their growth. Gene expression microarray analysis comparing MCF-7 cells with MLET5 highlighted apoptosis as a major functional grouping that is altered in MLET5 cells, such that cell survival would be favoured. This conclusion was further substantiated by the demonstration that MLET5 show resistance to etoposide-induced apoptosis. As the gene expression microarray analysis also shows that the apoptosis gene set differentially expressed in MLET5 is enriched for estrogen-regulated genes, our findings suggest that transient over-expression of ERα could lead to increased cell survival and the development of estrogen-independent growth, thereby contributing to resistance to endocrine therapies in breast cancer patients.

Inhibiting estrogen responses in breast cancer cells using a fusion protein encoding estrogen receptor-α and the transcriptional repressor PLZF

Estrogen receptor α (ERα) is a ligand-inducible transcription factor that acts to regulate gene expression by binding to palindromic DNA sequence, known as the estrogen response element, in promoters of estrogen-regulated genes. In breast cancer ERα plays a central role, where estrogen-regulated gene expression leads to tumor initiation, growth and survival. As an approach to silencing estrogen-regulated genes, we have studied the activities of a fusion protein between ERα and the promyelocytic leukemia zinc-finger (PLZF) protein, a transcriptional repressor that acts through chromatin remodeling. To do this, we have developed lines from the estrogen-responsive MCF-7 breast cancer cell line in which the expression of the fusion protein PLZF-ERα is conditionally regulated by tetracycline and shows that these feature long-term silencing of the expression of several well-characterized estrogen-regulated genes, namely pS2, cathepsin-D and the progesterone receptor. However, the estrogen-regulated growth of these cells is not inhibited unless PLZF-ERα expression is induced, an observation that we have confirmed both in vitro and in vivo. Taken together, these results show that PLZF-ERα is a potent repressor of estrogen-regulated gene expression and could be useful in distinguishing estrogen-regulated genes required for the growth of breast cancer cells.

A Novel HSV-1 Virus, JS1/34.5−/47−, Purges Contaminating Breast Cancer Cells From Bone Marrow

Purpose: Oncolytic herpes simplex virus type 1 (HSV-1) vectors show considerable promise as agents for cancer therapy. We have developed a novel recombinant HSV-1 virus (JS1/34.5−/47−) for purging of occult breast cancer cells from bone marrow of patients. Here, we evaluate the therapeutic efficacy of this oncolytic virus. Experimental Design: Electron microscopy was used to determine whether human breast cancer and bone marrow cells are permissive for JS1/34.5−/47− infection. Subsequently, the biological effects of JS1/34.5−/47− infection on human breast cancer cells and bone marrow were established using cell proliferation and colony formation assays, and the efficiency of cell kill was evaluated. Finally, the efficiency of JS1/34.5−/47− purging of breast cancer cells was examined in cocultures of breast cancer cells with bone marrow as well as bone marrow samples from high-risk breast cancer patients. Results: We show effective killing of human breast cancer cell lines with the JS1/34.5−/47− virus. Furthermore, we show that treatment with JS1/34.5−/47− can significantly inhibit the growth of breast cancer cell lines without affecting cocultured mononuclear hematopoietic cells. Finally, we have found that the virus is effective in destroying disseminated tumors cells in bone marrow taken from breast cancer patients, without affecting the hematopoietic contents in these samples. Conclusion: Collectively, our data show that the JS1/34.5−/47− virus can selectively target breast cancer cells while sparing hematopoietic cells, suggesting that JS1/34.5−/47− can be used to purge contaminating breast cancer cells from human bone marrow in the setting of autologous hematopoietic cell transplantation.

Silencing of androgen-regulated genes using a fusion of AR with the PLZF transcriptional repressor

The androgen receptor (AR) is a member of the nuclear receptor superfamily of ligand-activated transcription factors and plays a key role in the development and progression of prostate cancer. Current therapies include the use of antiandrogens aimed at inhibiting the transcriptional activation of AR-regulated genes by AR. Here, we explore a strategy aimed at obtaining silencing of AR-regulated genes, based on the properties of the transcriptional repressor promyelocytic leukamia zinc-finger protein (PLZF). In order to do this, we have made a fusion protein between PLZF and AR, named PLZF-AR, and show that PLZF-AR is able to bring about silencing of genomically encoded AR-regulated genes and inhibit the androgen-regulated growth of LNCaP prostate cancer cells. Together, our results show that this strategy is able to bring about potent repression of AR-regulated responses and, therefore, could be of value in the development of new therapies for prostate cancer.

Functional consequences of cyclin D1 overexpression in human mammary luminal epithelial cells

The proliferation of eukaryotic cells is primarily regulated by a decision made during the G1 phase of the cell cycle as to remain in the cycle and divide, or to withdraw from the cycle and adopt a different cell fate. During this time, environmental signals, which regulate the synthesis of the G1 cyclins, are coupled to cell division. In this context, mammalian D-type cyclins have been shown to control progression through the G1 phase of the mammalian cell cycle. Specifically, cyclin D1 has been reported frequently to be amplified, over-tran-scribed and overexpressed in human breast carcinomas. Although the effects of cyclin D1 overexpression have been examined in human breast carcinoma cell lines, the biological consequences of cyclin D1 expression in normal human mammary epithelial cells remain to be elucidated. In this study we have stably over expressed cyclin D1 in human mammary luminal epithelial cells in order to more directly address the role of cyclin D1 in cell cycle control and tumorigenesis of the human breast. Here, we demonstrate that the effect of cyclin D1 overexpression in these cells is to reduce their growth factor dependency, as well as shorten the duration of G1 and correspondingly reduce the mean generation time. Collectively, our data indicate that deregula-tion of cyclin D1 expression in human mammary epithelial cells can provide a growth advantage and hence contribute to the oncogenic potential of these cells.

Differentiation antigens in stromal and epithelial cells of the breast.

There is a large morphological spectrum of benign and malignant diseases that are specific to the human breast. The presence of stromal tumors (phyllodes) that are not found anywhere else in the body suggests that there may be functionally distinct fibroblasts associated with the intralobular stroma. The heterogeneity of benign and malignant breast diseases also indicates that the different types of lesions may reflect origins from cells that have different degrees of commitment to the main epithelial lineages. Some benign and malignant epithelial lesions have characteristic stromal changes associated with them that point to important stromal epithelial interactions in the pathogenesis of these conditions. At the present time, however, there is very little information on the normal development of the human breast, the normal lineage patterns, and the cellular origins of benign and malignant diseases. The functional interactions between the stroma and the epithelium in the normal human breast are observational, and how perturbations of such interactions may contribute to the pathogenesis of breast diseases is even more obscure.

Erratum: Inhibiting estrogen responses in breast cancer cells using a fusion protein encoding estrogen receptor-α and the transcriptional repressor PLZF (Gene Therapy (2005) vol. 12 (452-460) 10.1038/sj.gt.3302421)

Correction to: Gene Therapy (2005) 12, 452–460. doi:10.1038/sj.gt.3302421 The above author name was published incorrectly, the correct name is given below: N Sarwar The publishers apologise for this error.