BRCA1-deficient mammary tumor cells are dependent on EZH2 expression and sensitive to Polycomb Repressive Complex 2-inhibitor 3-deazaneplanocin A

10.1186/bcr2354Breast Cancer Research114R6

EZH2 and BMI1 inversely correlate with prognosis and TP53 mutation in breast cancer

Introduction PolycombGroup (PcG) proteins maintain gene repression through histone modifications and have been implicated in stem cell regulation and cancer. EZH2 is part of Polycomb Repressive Complex 2 (PRC2) and trimethylates H3K27. This histone mark recruits the BMI1-containing PRC1 that silences the genes marked by PRC2. Based on their role in stem cells, EZH2 and BMI1 have been predicted to contribute to a poor outcome for cancer patients. Methods We have analysed the expression of EZH2 and BMI1 in a well-characterised dataset of 295 human breast cancer samples. Results Interestingly, although EZH2 overexpression correlates with a poor prognosis in breast cancer, BMI1 overexpression correlates with a good outcome. Although this may reflect transformation of different cell types, we also observed a functional difference. The PcG-target genes INK4A and ARF are not expressed in tumours with high BMI1, but they are expressed in tumours with EZH2 overexpression. ARF expression results in tumour protein P53 (TP53) activation, and we found a significantly higher proportion of TP53 mutations in tumours with high EZH2. This may explain why tumours with high EZH2 respond poorly to therapy, in contrast to tumours with high BMI1. Conclusions Overall, our data highlight that whereas EZH2 and BMI1 may function in a 'linear' pathway in normal development, their overexpression has different functional consequences for breast tumourigenesi

Hormone-sensing cells require Wip1 for paracrine stimulation in normal and premalignant mammary epithelium

10.1186/bcr3381Breast Cancer Research15

Activation of the Tumor-Specific Death Effector Apoptin and Its Kinase by an N-Terminal Determinant of Simian Virus 40 Large T Antigen

Apoptin, a viral death protein derived from chicken anemia virus, displays a number of tumor-specific behaviors. In particular, apoptin is phosphorylated, translocates to the nucleus, and induces apoptosis specifically in tumor or transformed cells, whereas it is nonphosphorylated and remains primarily inactive in the cytoplasm of nontransformed normal cells. Here, we show that in normal cells apoptin can also be activated by the transient transforming signals conferred by ectopically expressed simian virus 40 (SV40) large T antigen (LT), which rapidly induces apoptin's phosphorylation, nuclear accumulation, and the ability to induce apoptosis. Further analyses with mutants of LT showed that the minimum domain capable of inducing all three of apoptin's tumor-specific properties resided in the N-terminal J domain, a sequence which is largely shared by SV40 small t antigen (st). Interestingly, the J domain in st, which lacks its own nuclear localization signal (NLS), required nuclear localization to activate apoptin. These results reveal the existence of a cellular pathway shared by conditions of transient transformation and the stable cancerous or precancerous state, and they support a model whereby a transient transforming signal confers on apoptin both the upstream activity of phosphorylation and the downstream activity of nuclear accumulation and apoptosis induction. Such a pathway may reflect a general lesion contributing to human cancers

Expression analysis of rare cellular subsets: Direct RT-PCR on limited cell numbers obtained by FACS or soft agar assays

10.2144/000114019BioTechniques544208-211BTNQ

Transcriptional Repressor Tbx3 Is Required for the Hormone-Sensing Cell Lineage in Mammary Epithelium

<div><p>The transcriptional repressor Tbx3 is involved in lineage specification in several tissues during embryonic development. Germ-line mutations in the Tbx3 gene give rise to Ulnar-Mammary Syndrome (comprising reduced breast development) and Tbx3 is required for mammary epithelial cell identity in the embryo. Notably Tbx3 has been implicated in breast cancer, which develops in adult mammary epithelium, but the role of Tbx3 in distinct cell types of the adult mammary gland has not yet been characterized. Using a fluorescent reporter knock-in mouse, we show that in adult virgin mice Tbx3 is highly expressed in luminal cells that express hormone receptors, and not in luminal cells of the alveolar lineage (cells primed for milk production). Flow cytometry identified Tbx3 expression already in progenitor cells of the hormone-sensing lineage and co-immunofluorescence confirmed a strict correlation between estrogen receptor (ER) and Tbx3 expression in situ. Using in vivo reconstitution assays we demonstrate that Tbx3 is functionally relevant for this lineage because knockdown of Tbx3 in primary mammary epithelial cells prevented the formation of ER+ cells, but not luminal ER- or basal cells. Interestingly, genes that are repressed by Tbx3 in other cell types, such as E-cadherin, are not repressed in hormone-sensing cells, highlighting that transcriptional targets of Tbx3 are cell type specific. In summary, we provide the first analysis of Tbx3 expression in the adult mammary gland at a single cell level and show that Tbx3 is important for the generation of hormone-sensing cells.</p></div

Tbx3 is required for the generation of hormone-sensing cells.

<p>(A) Primary MECs from wildtype or KI mice were transduced with a non-silencing lentiviral vector (control) or with two independent short hairpins against Tbx3 (sh-1 and sh-2). Cells were injected into mammary fat pads devoid of endogenous epithelium and outgrowths were analyzed 8–10 weeks later for the identity of lentivirally transduced cells (recognized by tGFP expression). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110191#pone.0110191.s004" target="_blank">File S4B</a> for a schematic experimental design. Each bar represents one fat pad and 46 to 569 tGFP+ luminal cells were counted per fat pad. There is a significant bias against the formation of HS cells by cells with Tbx3 knockdown (Chi square of shRNA versus control transplant <0.01). (B) Paraffin section of a cleared mammary fat pad transplanted with MECs that were exposed to the non-silencing control vector. Transduced cells are identified with an antibody staining against tGFP (green), luminal cells are identified by cytokeratin 8 (blue) and HS cells are identified by the estrogen receptor (ER, red). White arrow indicates transduced cells contributing to the hormone-sensing lineage. (C) Paraffin section of a mammary fat pad transplanted with MECs exposed to the first short hairpin against Tbx3. White arrow head indicated transduced cells in the luminal alveolar (ER-negative) lineage. The background of immunohistochemistry is higher in transplanted samples (arguably due to fibrosis). Where ER staining was ambiguous due to high background, we used progesterone receptor (PR, red) staining as an alternative marker for HS cells. (D) Paraffin section of a mammary fat pad transplanted with MECs exposed to the second short hairpin against Tbx3. White arrow head indicates transduced cells in the luminal alveolar (ER-negative) lineage. White scale bar is 20 µm and yellow scale bar is 10 µm.</p

Tbx3 marks the hormone sensing lineage, including ER+ progenitor cells.

<p>(A) Combined density/contour plot of mammary epithelial cells (from a pool of 5 Tbx3^+/Venus mice) separated into basal (red) and luminal (blue) cells based on CD24 and alpha6-integrin (CD49f) expression. (B) Colony forming potential of 1000 sorted cells from each population, representative of two independent experiments. (C) Histogram of luminal mammary epithelial cells Venus^High (luminal) and Venus^Low (luminal) cells sorted for colony forming assay (from a pool of 5 Tbx3^+/Venus mice per experiment). (D) Colony forming potential of 1000 sorted cells from each population, representative of two independent experiments. (E) FACS profile of hormone-sensing (CD49b^high and CD49b^low) and alveolar progenitor cells that were used for colony assays. Cells are color-coded based on Venus expression (green = Venus^High, grey = Venus^Low). (F) Fold change in Tbx3, progesterone receptor (PR) and estrogen receptor (ER) mRNA expression of sorted CD49b^high and CD49b^low hormone-sensing cells and alveolar progenitor cells, relative to CD49b^low hormone-sensing cells (dark purple bar). Fold change in Elf5 and cKit mRNA expression is shown relative to luminal alveolar cells (orange bar). (G) Colony forming potential of 1000 sorted luminal cells: CD49b^low and CD49b^high hormone-sensing cells and alveolar progenitor cells. (H) Quantification of colony forming assays with HS cells (Sca1^highCD49b^low), HS progenitor cells (Sca1^highCd49b^high) and alveolar progenitor cells (Sca1^lowCD49b^high). Bars represent the mean of three independent pools of 5–6 adult virgin Tbx3^+/Venus animals ± SD. HS progenitor cells form more colonies than HS cells (p = 0.02, paired t-test) and there is no significant difference in colony forming potential between HS progenitor and alveolar progenitor cells (p = 0.21, paired t-test).</p