Deaf-1 regulates epithelial cell proliferation and side-branching in the mammary gland

BACKGROUND: The transcription factor DEAF-1 has been identified as a high affinity binding partner of the LIM-only protein LMO4 that plays important roles in mammary gland development and breast cancer. Here we investigated the influence of DEAF-1 on human and mouse mammary epithelial cells both in vitro and in vivo and identified a potential target gene. RESULTS: Overexpression of DEAF-1 in human breast epithelial MCF10A cells enhanced cell proliferation in the mammary acini that develop in 3D cultures. To investigate the effects of Deaf-1 on mammary gland development and oncogenesis, we generated MMTV-Deaf-1 transgenic mice. Increased ductal side-branching was observed in young virgin mammary glands, accompanied by augmented cell proliferation. In addition, the ratio of the progesterone receptor isoforms PRA and PRB, previously implicated in regulating ductal side-branching, was altered. Affymetrix gene profiling studies revealed Rac3 as a potential target gene and quantitative RT-PCR analysis confirmed that Rac3 was upregulated by Deaf-1 in immortalized mouse mammary epithelial cells. Furthermore, MMTV-Deaf-1 transgenic mammary glands were found to have elevated levels of Rac3 mRNA, suggesting that it is a bona fide target. CONCLUSION: We have demonstrated that overexpression of Deaf-1 enhances the proliferation of human breast epithelial cells in vitro and mouse epithelial cells in vivo. Transgenic mammary glands overexpressing Deaf-1 exhibited a modest side-branching phenotype, accompanied by an increase in the number of BrdU-positive cells and a decrease in the proportion of PRA-expressing cells. Although proliferation was enhanced in Deaf-1 transgenic mice, overexpression of this gene was not sufficient to induce the formation of mammary tumors. In addition, our studies identified Rac3, encoding a small Rho-like GTPase, as a potential target of Deaf-1 in mouse mammary epithelial cells

Prostate screening uptake in Australian BRCA1 and BRCA2 carriers

Men who carry mutations in BRCA1 or BRCA2 are at increased risk for prostate cancer. However the efficacy of prostate screening in this setting is uncertain and limited data exists on the uptake of prostate screening by mutation carriers. This study prospectively evaluated uptake of prostate cancer screening in a multi-institutional cohort of mutation carriers. Subjects were unaffected male BRCA1 and BRCA2 mutation carriers, aged 40–69 years, enrolled in the Kathleen Cuningham Consortium for Research into Familial Breast Cancer (kConFab) and who had completed a mailed, self-report follow-up questionnaire 3 yearly after study entry. Of the 75 male carriers in this study, only 26 (35%) had elected to receive their mutation result. Overall, 51 (68%) did not recall having received a recommendation to have prostate screening because of their family history, but 41 (55%) had undergone a prostate specific antigen (PSA) test and 32 (43%) a digital rectal examination (DRE) in the previous 3 years. Those who were aware of their mutation result were more likely to have received a recommendation for prostate screening (43 vs. 6%, p = 0.0001), and to have had a PSA test (77 vs. 43%, p = 0.005) and a DRE (69 vs. 29%, p = 0.001) in the previous 3 years. The majority of unaffected males enrolled in kConFab with a BRCA1/2 mutation have not sought out their mutation result. However, of those aware of their positive mutation status, most have undergone at least one round of prostate screening in the previous 3 years

The future of mammary stem cell biology: the power of in vivo transplants

The recent review by Smith and Medina [1] of in vivo transplantation models and their role in investigating mammary stem cell (MaSC) biology provides comprehensive coverage of the history and complexity of the ‘gold standard ’ MaSC assay in mice. This includes a description of the pioneering studies that showed that mammary epithelial outgrowths can be generated in cleared mammary fat pads transplanted with explants or admixtures of mammary cells [2]. However, this approach clearly does not lend itself to prospective analysis of isolated subpopulations in order to identify which cells possess in vivo regenerative activity. More recently, success in obtaining complex mammary gland structures from transplanted suspensions of single cells has now made this possible [3-7]. Moreover, the regenerated structures have been shown to contain daughter cells with the same in viv

Mutation analysis of FANCD2, BRIP1/BACH1, LMO4 and SFN in familial breast cancer

INTRODUCTION: Mutations in known predisposition genes account for only about a third of all multiple-case breast cancer families. We hypothesized that germline mutations in FANCD2, BRIP1/BACH1, LMO4 and SFN may account for some of the unexplained multiple-case breast cancer families. METHODS: The families used in this study were ascertained through the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab). Denaturing high performance liquid chromatography (DHPLC) analysis of the coding regions of these four genes was conducted in the youngest affected cases of 30 to 267 non-BRCA1/2 breast cancer families. In addition, a further 399 index cases were also screened for mutations in two functionally significant regions of the FANCD2 gene and 253 index cases were screened for two previously reported mutations in BACH1 (p. P47A and p. M299I). RESULTS: DHPLC analysis of FANCD2 identified six silent exonic variants, and a large number of intronic variants, which tagged two common haplotypes. One protein truncating variant was found in BRIP1/BACH1, as well as four missense variants, a silent change and a variant in the 3' untranslated region. No missense or splice site mutations were found in LMO4 or SFN. Analysis of the missense, silent and frameshift variants of FANCD2 and BACH1 in relatives of the index cases, and in a panel of controls, found no evidence suggestive of pathogenicity. CONCLUSION: There is no evidence that highly penetrant exonic or splice site mutations in FANCD2, BRIP1/BACH1, LMO4 or SFN contribute to familial breast cancer. Large scale association studies will be necessary to determine whether any of the polymorphisms or haplotypes identified in these genes contributes to breast cancer risk

Cell fate takes a slug in BRCA1-associated breast cancer

Understanding why BRCA1 mutation carriers have a predilection for developing clinically aggressive basal-like breast tumors could inform the development of targeted treatment or prevention strategies. Analysis of both mouse and human mammary epithelial cells has identified a role for BRCA1 in orchestrating differentiation. The ability to isolate discrete epithelial subpopulations from mammary tissue has recently directed attention to luminal progenitor cells - the descendants of mammary stem cells - as the likely 'cells-of-origin' in BRCA1-associated breast cancer. A new publication has confirmed the importance of aberrant luminal cells as key culprits and provided insights on how BRCA1 haploinsufficiency biases luminal cells toward a basal-like fate through aberrant expression of the transcription factor SLUG

Resident macrophages influence stem cell activity in the mammary gland

Introduction Macrophages in the mammary gland are essential for morphogenesis of the ductal epithelial tree and have been implicated in promoting breast tumor metastasis. Although it is well established that macrophages influence normal mammopoiesis, the mammary cell types that these accessory cells influence have not been determined. Here we have explored a role for macrophages in regulating mammary stem cell (MaSC) activity, by assessing the ability of MaSCs to reconstitute a mammary gland in a macrophage-depleted fat pad. Methods Two different in vivo models were used to deplete macrophages from the mouse mammary fat pad, allowing us to examine the effect of macrophage deficiency on the mammary repopulating activity of MaSCs. Both the Csf1(op/op) mice and clodronate liposome-mediated ablation models entailed transplantation studies using the MaSC-enriched population. Results We show that mammary repopulating ability is severely compromised when the wild-type MaSC-enriched subpopulation is transplanted into Csf1(op/op) fat pads. In reciprocal experiments, the MaSC-enriched subpopulation from Csf1(op/op) glands had reduced regenerative capacity in a wildtype environment. Utilizing an alternative strategy for selective depletion of macrophages from the mammary gland, we demonstrate that co-implantation of the MaSC-enriched subpopulation with clodronate-liposomes leads to a marked decrease in repopulating frequency and outgrowth potential. Conclusions Our data reveal a key role for mammary gland macrophages in supporting stem/progenitor cell function and suggest that MaSCs require macrophage-derived factors to be fully functional. Macrophages may therefore constitute part of the mammary stem cell nich

Role of Engrailed-2 (EN2) as a prostate cancer detection biomarker in genetically high risk men

Controversy surrounds the use of PSA as a biomarker for prostate cancer detection, leaving an unmet need for a novel biomarker in this setting; urinary EN2 may identify individuals with clinically relevant prostate cancer. Male BRCA1 and BRCA2 mutation carriers are at increased risk of clinically significant prostate cancer and may benefit from screening. Urine samples from 413 BRCA1 and BRCA2 mutation carriers and controls were evaluated. Subjects underwent annual PSA screening with diagnostic biopsy triggered by PSA > 3.0 ng/ml; 21 men were diagnosed with prostate cancer. Urinary EN2 levels were measured by ELISA and had a sensitivity of 66.7% and specificity of 89.3% for cancer detection. There was no statistically significant difference in EN2 levels according to genetic status or Gleason score. Urinary EN2 may be useful as a non-invasive early biomarker for prostate cancer detection in genetically high-risk individuals

Nipple aspiration and ductal lavage in women with a germline BRCA1 or BRCA2 mutation

INTRODUCTION: The aim of this study was to collect serial samples of nipple aspirate (NA) and ductal lavage (DL) fluid from women with germline BRCA1/2 mutations in order to create a biorepository for use in identifying biomarkers of breast cancer risk. METHODS: Between March 2003 and February 2005, 52 women with germline BRCA1 or BRCA2 mutations (median age 43 years, range 27 to 65 years) were scheduled for six-monthly NA, DL and venesection. DL was attempted for all NA fluid-yielding (FY) and any non-FY ducts that could be located at each visit. RESULTS: Twenty-seven (52%) women were postmenopausal, predominantly (19/27) from risk reducing bilateral salpingo-oophorectomy (BSO). FY ducts were identified in 60% of all women, 76% of premenopausal women versus 44% of postmenopausal (P = 0.026). Eighty-five percent of women had successful DL. Success was most likely in women with FY ducts (FY 94% versus non-FY 71% (P = 0.049). DL samples were more likely to be cellular if collected from FY ducts (FY 68% versus non-FY 43%; P = 0.037). Total cell counts were associated with FY status (FY median cell count 30,996, range 0 to >1,000,000 versus non-FY median cell count 0, range 0 to 173,577; P = 0.002). Four women (8%) had ducts with severe atypia with or without additional ducts with mild epithelial atypia; seven others had ducts with mild atypia alone (11/52 (21%) in total). Median total cell count was greater from ducts with atypia (105,870, range 1920 to >1,000,000) than those with no atypia (174, 0 to >1,000,000; P ≤ 0.001). CONCLUSION: It is feasible to collect serial NA and DL samples from women at high genetic risk of breast cancer, and we are creating a unique, prospective collection of ductal samples that have the potential to be used for discovery of biomarkers of breast cancer risk and evaluate the ongoing effects of risk reducing BSO. DL cellular atypia was not predictive of a current breast cancer and longer follow up is needed to determine whether atypia is an additional marker of future breast cancer risk in this population already at high genetic risk of breast cancer

Transcriptome analyses of mouse and human mammary cell subpopulations reveal multiple conserved genes and pathways

INTRODUCTION: Molecular characterization of the normal epithelial cell types that reside in the mammary gland is an important step toward understanding pathways that regulate self-renewal, lineage commitment, and differentiation along the hierarchy. Here we determined the gene expression signatures of four distinct subpopulations isolated from the mouse mammary gland. The epithelial cell signatures were used to interrogate mouse models of mammary tumorigenesis and to compare with their normal human counterpart subsets to identify conserved genes and networks. METHODS: RNA was prepared from freshly sorted mouse mammary cell subpopulations (mammary stem cell (MaSC)-enriched, committed luminal progenitor, mature luminal and stromal cell) and used for gene expression profiling analysis on the Illumina platform. Gene signatures were derived and compared with those previously reported for the analogous normal human mammary cell subpopulations. The mouse and human epithelial subset signatures were then subjected to Ingenuity Pathway Analysis (IPA) to identify conserved pathways. RESULTS: The four mouse mammary cell subpopulations exhibited distinct gene signatures. Comparison of these signatures with the molecular profiles of different mouse models of mammary tumorigenesis revealed that tumors arising in MMTV-Wnt-1 and p53-/- mice were enriched for MaSC-subset genes, whereas the gene profiles of MMTV-Neu and MMTV-PyMT tumors were most concordant with the luminal progenitor cell signature. Comparison of the mouse mammary epithelial cell signatures with their human counterparts revealed substantial conservation of genes, whereas IPA highlighted a number of conserved pathways in the three epithelial subsets. CONCLUSIONS: The conservation of genes and pathways across species further validates the use of the mouse as a model to study mammary gland development and highlights pathways that are likely to govern cell-fate decisions and differentiation. It is noteworthy that many of the conserved genes in the MaSC population have been considered as epithelial-mesenchymal transition (EMT) signature genes. Therefore, the expression of these genes in tumor cells may reflect basal epithelial cell characteristics and not necessarily cells that have undergone an EMT. Comparative analyses of normal mouse epithelial subsets with murine tumor models have implicated distinct cell types in contributing to tumorigenesis in the different models

Frequency of the ATM IVS10-6T→G variant in Australian multiple-case breast cancer families

BACKGROUND: Germline mutations in the genes BRCA1 and BRCA2 account for only a proportion of hereditary breast cancer, suggesting that additional genes contribute to hereditary breast cancer. Recently a heterozygous variant in the ataxia–telangiectasia mutated (ATM) gene, IVS10-6T→G, was reported by an Australian multiple-case breast cancer family cohort study (the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer) to confer a substantial breast cancer risk. Although this variant can result in a truncated ATM product, its clinical significance as a high-penetrance breast cancer allele or its role as a low-penetrance risk-modifier is controversial. METHODS: We determined the frequency of ATM IVS10-6T→G variants in a cohort of individuals affected by breast and/or ovarian cancer who underwent BRCA1 and BRCA2 genetic testing at four major Australian familial cancer clinics. RESULTS: Seven of 495 patients (1.4%) were heterozygous for the IVS10-6T→G variant; the carrier rate in unselected Australian women with no family history of breast cancer is reported to be 6 of 725 (0.83%) (P = 0.4). Two of the seven probands also harboured a pathogenic BRCA1 mutation and one patient had a BRCA1 unclassified variant of uncertain significance. CONCLUSION: These findings indicate that the ATM IVS10-6T→G variant does not seem to occur at a significantly higher frequency in affected individuals from high-risk families than in the general population. A role for this variant as a low-penetrance allele or as a modifying gene in association with other genes (such as BRCA1) remains possible. Routine testing for ATM IVS10-6T→G is not warranted in mutation screening of affected individuals from high-risk families