Prospective isolation and characterization of committed and multipotent progenitors from immortalized mouse mammary epithelial cells with morphogenic potential.

INTRODUCTION: Utilizing single-cell cloning of the COMMA-D cell line engineered to express ß-galactosidase (CDß) cell line, which exhibits normal in vivo morphogenesis, distinct multipotent, ductal-limited, alveolar-limited and luminal-restricted progenitors, have been isolated and characterized. METHODS: A single-cell suspension of CDß cells was stained using Hoechst dye 33342, followed by analysis and sorting. Cells that effluxed the dye appeared on the left side of a FACS analysis panel and were referred to as side population (SP) cells. Cells that retained the dye appeared on the right side and were referred to as non-SP (NSP) cells. Cells from both SP and NSP regions were sorted and analyzed for outgrowth potential. Additionally, individual clones were derived from single cells sorted from each region. RESULTS: There was no difference in the outgrowth potential of the SP vs. NSP cells when 5,000 cells per fat pad were transplanted. However, individual clones derived from single cells sorted from either SP or NSP regions had varying growth potential. A total of nine clones were identified, four of which possessed in vivo mammary outgrowth potential and five of which lacked in vivo outgrowth potential. Two of the clones formed mammary lobuloalveolar structures that contained both ducts and alveoli and were termed multipotent. Two of the clones generated either ductal-only or alveolar-only structures and were referred to as ductal-limited or alveolar-limited progenitor clones, respectively. The ability to expand the clones in vitro allowed for the characterization of their unique molecular phenotypes. Among the mammary-specific markers tested, high cytokeratin 5 (CK5) expression was the only marker that correlated with the clones' outgrowth potential. Among the clones that did not show any in vivo outgrowth potential when transplanted alone, one clone showed in vivo growth and incorporated into the mammary lumen when mixed with normal mammary epithelial cells. This clone also showed the highest in vitro expression of CK8 and Elf5and may represent a luminal-restricted progenitor clone. In addition, six "biclones," each made from an SP cell plus an NSP cell, were analyzed. Of these six, three exhibited lobuloalveolar growth. CONCLUSIONS: Distinct immortalized mammary progenitors have been isolated and characterized. Importantly, the results of this study provide further evidence for the existence of distinct ductal and alveolar mammary progenitors.

Pathways contributing to development of spontaneous mammary tumors in BALB/c-Trp53+/- mice.

Mutation and loss of function in p53 are common features among human breast cancers. Here we use BALB/c-Trp53+/- mice as a model to examine the sequence of events leading to mammary tumors. Mammary gland proliferation rates were similar in both BALB/c-Trp53+/- mice and wild-type controls. In addition, sporadic mammary hyperplasias were rare in BALB/c-Trp53+/- mice and not detectably different from those of wild-type controls. Among the 28 mammary tumors collected from BALB/c-Trp53+/- mice, loss of heterozygosity for Trp53 was detected in more than 90% of invasive mammary tumors. Transplantation of Trp53+/- ductal hyperplasias also indicated an association between loss of the wild-type allele of Trp53 and progression to invasive carcinomas. Therefore, loss of p53 function seems to be a rate-limiting step in progression. Moreover, expression of biomarkers such as estrogen receptor alpha, progesterone receptor, Her2/Neu, and activated Notch1 varied among mammary tumors, suggesting that multiple oncogenic lesions collaborate with loss of p53 function. Expression of biomarkers was retained when tumor fragments were transplanted to syngeneic hosts. Tumors expressing solely luminal or basal keratins were also observed (27 and 11%, respectively), but the largest class of tumors expressed both luminal and basal keratins (62%). Overall, this panel of transplantable tumors provides a resource for detailed evaluation of the cell lineages undergoing transformation and preclinical testing of therapeutic agents targeting a variety of oncogenic pathways including cancer stem cells.

Overexpression of Separase induces aneuploidy and mammary tumorigenesis.

Separase is an endopeptidase that separates sister chromatids by cleaving cohesin Rad21 during the metaphase-to-anaphase transition. Conditional expression of Separase in tetracycline-inducible diploid FSK3 mouse mammary epithelial cells with both p53 WT and mutant (Ser-233-234) alleles of unknown physiological significance develops aneuploidy within 5 days of Separase induction in vitro. Overexpression of Separase induces premature separation of chromatids, lagging chromosomes, and anaphase bridges. In an in vivo mouse mammary transplant model, induction of Separase expression in the transplanted FSK3 cells for 3-4 weeks results in the formation of aneuploid tumors in the mammary gland. Xenograft studies combined with histological and cytogenetic analysis reveal that Separase-induced tumors are clonal in their genomic complements and have a mesenchymal phenotype suggestive of an epithelial-mesenchymal transition. Induction of Separase resulted in trisomies for chromosomes 8, 15, and 17; monosomy for chromosome 10; and amplification of the distal region of chromosomes 8 and 11. Separase protein is found to be significantly overexpressed in human breast tumors compared with matched normal tissue. These results collectively suggest that Separase is an oncogene, whose overexpression alone in mammary epithelial cells is sufficient to induce aneuploidy and tumorigenesis in a p53 mutant background.

Immortalized, pre-malignant epithelial cell populations contain long-lived, label-retaining cells that asymmetrically divide and retain their template DNA.

INTRODUCTION: During selective segregation of DNA, a cell asymmetrically divides and retains its template DNA. Asymmetric division yields daughter cells whose genome reflects that of the parents', simultaneously protecting the parental cell from genetic errors that may occur during DNA replication. We hypothesized that long-lived epithelial cells are present in immortal, premalignant cell populations, undergo asymmetric division, retain their template DNA strands, and cycle both during allometric growth and during pregnancy. METHODS: The glands of 3-week old immune competent Balb/C female mice were utilized intact or cleared of host epithelium and implanted with ductal-limited, lobule-limited, or alveolar-ductal progenitor cells derived from COMMA-D1 pre-malignant epithelial cells. 5-bromo-2-deoxyuridine (5-BrdU) was administered to identify those cells which retain their template DNA. Nulliparous mice were then either injected with [(3)H]-thymidine ((3)H-TdR) to distinguish 5-BrdU-label retaining cells that enter the cell cycle and euthanized, or mated, injected with (3)H-TdR, and euthanized at various days post-coitus. Sections were stained for estrogen receptor-α(ER-α) or progesterone receptor (PR) via immunohistochemistry. Cells labelled with both 5-BrdU and (3)H-TdR were indicative of label-retaining epithelial cells (LREC). RESULTS: Cells that retained a 5-BrdU label and cells labelled with [(3)H]-thymidine were found in all mice and were typically detected along the branching epithelium of mature mouse mammary glands. Cells containing double-labelled nuclei (LREC) were found in the intact mammary gland of both pregnant and nulliparous mice, and in mammary glands implanted with pre-malignant cells. Double-labelled cells ((3)H-TdR/5-BrdU) represent a small portion of cells in the mammary gland that cycle and retain their template DNA (5-BrdU). Some label-retaining cells were also ER-α or PR positive. LRECs distributed their second label ((3)H-TdR) to daughter cells; and this effect persisted during pregnancy. LRECs, and small focal hyperplasia, were found in all immortalized premalignant mammary implant groups. CONCLUSIONS: The results indicate that a subpopulation of long-lived, label-retaining epithelial cells (LRECs) is present in immortal premalignant cell populations. These LRECs persist during pregnancy, retain their original DNA, and a small percentage express ER-α and PR. We speculate that LRECs in premalignant hyperplasia represent the long-lived (memory) cells that maintain these populations indefinitely.

An intraductal human-in-mouse transplantation model mimics the subtypes of ductal carcinoma in situ.

INTRODUCTION: Human models of noninvasive breast tumors are limited, and the existing in vivo models do not mimic inter- and intratumoral heterogeneity. Ductal carcinoma in situ (DCIS) is the most common type (80%) of noninvasive breast lesions. The aim of this study was to develop an in vivo model whereby the natural progression of human DCIS might be reproduced and studied. To accomplish this goal, the intraductal human-in-mouse (HIM) transplantation model was developed. The resulting models, which mimicked some of the diversity of human noninvasive breast cancers in vivo, were used to show whether subtypes of human DCIS might contain distinct subpopulations of tumor-initiating cells. METHODS: The intraductal models were established by injection of human DCIS cell lines (MCF10DCIS.COM and SUM-225), as well as cells derived from a primary human DCIS (FSK-H7), directly into the primary mouse mammary ducts via cleaved nipple. Six to eight weeks after injections, whole-mount, hematoxylin and eosin, and immunofluorescence staining were performed to evaluate the type and extent of growth of the DCIS-like lesions. To identify tumor-initiating cells, putative human breast stem/progenitor subpopulations were sorted from MCF10DCIS.COM and SUM-225 with flow cytometry, and their in vivo growth fractions were compared with the Fisher's Exact test. RESULTS: Human DCIS cells initially grew within the mammary ducts, followed by progression to invasion in some cases into the stroma. The lesions were histologically almost identical to those of clinical human DCIS. This method was successful for growing DCIS cell lines (MCF10DCIS.COM and SUM-225) as well as a primary human DCIS (FSK-H7). MCF10DCIS.COM represented a basal-like DCIS model, whereas SUM-225 and FSK-H7 cells were models for HER-2+ DCIS. With this approach, we showed that various subtypes of human DCIS appeared to contain distinct subpopulations of tumor-initiating cells. CONCLUSIONS: The intraductal HIM transplantation model provides an invaluable tool that mimics human breast heterogeneity at the noninvasive stages and allows the study of the distinct molecular and cellular mechanisms of breast cancer progression.

Identification of novel amplification gene targets in mouse and human breast cancer at a syntenic cluster mapping to mouse ch8A1 and human ch13q34.

Serial analysis of gene expression from aggressive mammary tumors derived from transplantable p53 null mouse mammary outgrowth lines revealed significant up-regulation of Tfdp1 (transcription factor Dp1), Lamp1 (lysosomal membrane glycoprotein 1) and Gas6 (growth arrest specific 6) transcripts. All of these genes belong to the same linkage cluster, mapping to mouse chromosome band 8A1. BAC-array comparative genomic hybridization and fluorescence in situ hybridization analyses revealed genomic amplification at mouse region ch8A1.1. The minimal region of amplification contained genes Cul4a, Lamp1, Tfdp1, and Gas6, highly overexpressed in the p53 null mammary outgrowth lines at preneoplastic stages, and in all its derived tumors. The same amplification was also observed in spontaneous p53 null mammary tumors. Interestingly, this region is homologous to human chromosome 13q34, and some of the same genes were previously observed amplified in human carcinomas. Thus, we further investigated the occurrence and frequency of gene amplification affecting genes mapping to ch13q34 in human breast cancer. TFDP1 showed the highest frequency of amplification affecting 31% of 74 breast carcinomas analyzed. Statistically significant positive correlation was observed for the amplification of CUL4A, LAMP1, TFDP1, and GAS6 genes (P < 0.001). Meta-analysis of publicly available gene expression data sets showed a strong association between the high expression of TFDP1 and decreased overall survival (P = 0.00004), relapse-free survival (P = 0.0119), and metastasis-free interval (P = 0.0064). In conclusion, our findings suggest that CUL4A, LAMP1, TFDP1, and GAS6 are targets for overexpression and amplification in breast cancers. Therefore, overexpression of these genes and, in particular, TFDP1 might be of relevance in the development and/or progression in a significant subset of human breast carcinomas.

Hormone-induced protection of mammary tumorigenesis in genetically engineered mouse models.

INTRODUCTION: The experiments reported here address the question of whether a short-term hormone treatment can prevent mammary tumorigenesis in two different genetically engineered mouse models. METHODS: Two mouse models, the p53-null mammary epithelial transplant and the c-neu mouse, were exposed to estrogen and progesterone for 2 and 3 weeks, respectively, and followed for development of mammary tumors. RESULTS: In the p53-null mammary transplant model, a 2-week exposure to estrogen and progesterone during the immediate post-pubertal stage (2 to 4 weeks after transplantation) of mammary development decreased mammary tumorigenesis by 70 to 88%. At 45 weeks after transplantation, analysis of whole mounts of the mammary outgrowths demonstrated the presence of premalignant hyperplasias in both control and hormone-treated glands, indicating that the hormone treatment strongly affects the rate of premalignant progression. One possible mechanism for the decrease in mammary tumorigenesis may be an altered proliferation activity as the bromodeoxyuridine labeling index was decreased by 85% in the mammary glands of hormone-treated mice. The same short-term exposure administered to mature mice at a time of premalignant development also decreased mammary tumorigenesis by 60%. A role for stroma and/or systemic mediated changes induced by the short-term hormone (estrogen/progesterone) treatment was demonstrated by an experiment in which the p53-null mammary epithelial cells were transplanted into the cleared mammary fat pads of previously treated mice. In such mice, the tumor-producing capabilities of the mammary cells were also decreased by 60% compared with the same cells transplanted into unexposed mice. In the second set of experiments using the activated Her-2/neu transgenic mouse model, short-term estradiol or estradiol plus progesterone treatment decreased mammary tumor incidence by 67% and 63%, and tumor multiplicity by 91% and 88%, respectively. The growth rate of tumors arising in the hormone-treated activated Her-2/neu mice was significantly lower than tumors arising in non-hormone treated mice. CONCLUSION: Because these experiments were performed in model systems that mimic many essential elements of human breast cancer, the results strengthen the rationale for translating this prevention strategy to humans at high risk for developing breast cancer.

Tamoxifen inhibition of estrogen receptor-alpha-negative mouse mammary tumorigenesis.

Tamoxifen reduces the relative risk of breast cancer developing from specific premalignant lesions. Many breast cancers that arise after tamoxifen treatment are estrogen receptor-alpha (ER-alpha)-negative, although premalignant lesions such as atypical ductal hyperplasia are highly ER-alpha-positive. The p53 null mouse mammary epithelial transplant model is characterized by ER-alpha-positive premalignant lesions that give rise to both ER-alpha-positive and ER-alpha-negative tumors. Given this progression from ER-alpha-positive to ER-alpha-negative lesions, we tested the ability of tamoxifen to block or delay mammary tumorigenesis in several versions of this model. In groups 1 and 2, p53 null normal mammary epithelial transplants were maintained in virgin mice. In groups 3 to 5, the p53 null and mammary transplants were maintained in mice continuously exposed to high levels of progesterone. In groups 6 and 7, transplants of the premalignant outgrowth line PN8a were maintained in virgin mice. Tamoxifen blocked estrogen signaling in these mice as evidenced by decreases in progesterone-induced lateral branching and epithelial proliferation in the mammary epithelium. Tamoxifen did not alter the elevated levels of progesterone in the blood while significantly reducing the circulating level of prolactin. Tamoxifen reduced tumor incidence in p53 null normal mammary epithelial transplants maintained in virgin mice from 55% to 5% and in progesterone-stimulated mice from 81% to 21%. The majority of the resultant tumors were ER-alpha-negative. Tamoxifen also significantly delayed tumorigenesis in the ER-alpha-positive high premalignant line PN8a from 100% to 75%. These results show that tamoxifen delays the emergence of ER-alpha-negative tumors if given early in premalignant progression.

p53 function is required for hormone-mediated protection of mouse mammary tumorigenesis.

Limited hormonal stimulation of the mammary gland during a critical window in postpubertal development imparts a long-lasting protective effect against breast cancer in humans and in rodent models. The hormonal stimulation can be achieved by full-term pregnancy or low doses of estradiol-17beta and progesterone administered for 21 days. The mechanism(s) behind this effect of hormones is not understood at the molecular level. The experiments reported here demonstrate that the absence of p53 tumor suppressor gene function abrogates the protective effect of hormones against carcinogen-induced mammary carcinogenesis in BALB/c mice. This is the first identification of a specific gene product that mediates the protective effect of hormones. Additionally, the experiments highlight the usefulness of transgenic mouse models in the testing of hypotheses derived from the classic rat mammary models.

Hormone dependence in premalignant mammary progression.

Human breast cancers that are estrogen receptor (ER) negative convey a poor prognosis for patient survival. A mouse model that mimics essential biological and genetic attributes of a subset of human breast cancer is the BALB/c p53-null mammary epithelium, in which deletion of the tumor suppressor gene p53 results in enhanced tumorigenic risk. The experiments reported herein examine the hormone dependence of premalignant mammary progression in this model. The p53-null normal mammary epithelium exhibits the same dependence as p53 wild-type mammary epithelium on ovarian hormones for growth. However, in contrast to p53 wild-type epithelium, estrogen and progesterone, singly or in combination, strongly enhance tumorigenesis in p53-null mammary epithelium. The removal of progesterone signaling by deletion of the progesterone receptor eliminates progesterone enhancement of tumorigenesis. The immortalized premalignant outgrowth lines, termed PN, possess different tumorigenic capabilities, but the majority of these lines showed a strong dependence on ovarian hormones for growth and tumorigenesis. Although these lines are highly ER positive, a large number of tumors arising from these lines were ER negative and grew when implanted in ovariectomized mice. As was the case for p53-null normal mammary cells, hormonal stimulation was a strong promoter for tumorigenesis in the premalignant outgrowth lines and, surprisingly, was much stronger than the chemical carcinogen 7,12-dimethylbenzanthracene. In summary, these results demonstrate that p53-null mammary cells, which generate a significant percentage of ER-negative tumors, are highly responsive to the absence or presence of ovarian hormones during the normal and premalignant stages. This model would appear an excellent one to test the effects of chemopreventive agents on the development of both ER-negative and ER-positive mammary tumors.

Hormone-induced chromosomal instability in p53-null mammary epithelium.

The absence of p53 function increases risk for spontaneous tumorigenesis in the mammary gland. Hormonal stimulation enhances tumor risk in p53-null mammary epithelial cells as well as the incidence of aneuploidy. Aneuploidy appears in normal p53-null mammary epithelial cells within 5 weeks of hormone stimulation. Experiments reported herein assessed a possible mechanism of hormone-induced aneuploidy. Hormones increased DNA synthesis equally between wild-type (WT) and p53-null mammary epithelial cells. There were two distinct responses in p53-null cells to hormone exposure. First, Western blot analysis demonstrated that the levels of two proteins involved in regulating sister chromatid separation and the spindle checkpoint, Mad2 and separase (ESPL1) were increased in null compared with WT cells. In contrast, the levels of securin and Rad21 proteins were not increased in hormone-stimulated p53-null compared with WT cells. ESPL1 RNA was also increased in p53-null mouse mammary cells in vivo by 18 h of hormone stimulation and in human breast MCF7 cells in monolayer culture by 8 h of hormone stimulation. Furthermore, both promoters contained p53 and steroid hormone response elements. Mad2 protein was increased as a consequence of the absence of p53 function. The increase in Mad2 protein was observed also at the cellular level by immunohistochemistry. Second, hormones increased gene amplication in the distal arm of chromosome 2, as shown by comparative genomic hybridization. These results support the hypothesis that hormone stimulation acts to increase aneuploidy by several mechanisms. First, by increasing mitogenesis in the absence of the p53 checkpoint in G2, hormones allow the accumulation of cells that have experienced chromosome missegregation. Second, the absolute rate of chromosome missegregation may be increased by alterations in the levels of two proteins, separase and Mad2, which are important for maintaining chromosomal segregation and the normal spindle checkpoint during mitosis.

Biological and genetic properties of the p53 null preneoplastic mammary epithelium.

The absence of the tumor suppressor gene p53 confers an increased tumorigenic risk for mammary epithelial cells. In this report, we describe the biological and genetic properties of the p53 null preneoplastic mouse mammary epithelium in a p53 wild-type environment. Mammary epithelium from p53 null mice was transplanted serially into the cleared mammary fat pads of p53 wild-type BALB/c female to develop stable outgrowth lines. The outgrowth lines were transplanted for 10 generations. The outgrowths were ductal in morphology and progressed through ductal hyperplasia and ductal carcinoma in situ before invasive cancer. The preneoplastic outgrowth lines were immortal and exhibited activated telomerase activity. They are estrogen and progesterone receptor-positive, and aneuploid, and had various levels of tumorigenic potential. The biological and genetic properties of these lines are distinct from those found in most hyperplastic alveolar outgrowth lines, the form of mammary preneoplasia occurring in most traditional models of murine mammary tumorigenesis. These results indicate that the preneoplastic cell populations found in this genetically engineered model are similar in biological properties to a subset of precurser lesions found in human breast cancer and provide a unique model to identify secondary events critical for tumorigenicity and invasiveness.

Osteopontin is a potential target gene in mouse mammary cancer chemoprevention by Se-methylselenocysteine.

BACKGROUND: Se-methylselenocysteine (MSC) is a naturally occurring organoselenium compound that inhibits mammary tumorigenesis in laboratory animals and in cell culture models. Previously we have documented that MSC inhibits DNA synthesis, total protein kinase C and cyclin-dependent kinase 2 kinase activities, leading to prolonged S-phase arrest and elevation of growth-arrested DNA damage genes, followed by caspase activation and apoptosis in a synchronized TM6 mouse mammary tumor model. The aim of the present study was to examine the efficacy of MSC against TM6 mouse mammary hyperplastic outgrowth (TM6-HOG) and to determine in vivo targets of MSC in this model system. METHODS: Twenty mammary fat pads each from female Balb/c mice transplanted with TM6-HOG and fed with 0.1 ppm selenium and with 3 ppm selenium respectively, were evaluated at 4 and 12 weeks after transplantation for growth spread, proliferative index and caspase-3 activity. Thirteen mice transplanted with TM6-HOG in each selenium group were observed for tumor formation over 23 weeks. Tumors from mice in both groups were compared by cDNA array analysis and data were confirmed by reverse transcription-polymerase chain reaction. To determine the effect of MSC on the expression of the novel target gene and on cell migration, experiments were performed in triplicate. RESULTS: A dietary dose of 3 ppm selenium significantly reduced the growth spread and induced caspase-3 activity in mammary fat pads in comparison with mice fed with the basal diet (0.1 ppm selenium). The extended administration (23 weeks) of 3 ppm selenium in the diet resulted in a tumor incidence of 77% in comparison with 100% tumor incidence in 0.1 ppm selenium-fed animals. The size of TM6 tumors in the supplemented group was smaller (mean 0.69 cm2) than in the mice fed with the basal diet (mean 0.93 cm2). cDNA array analysis showed a reduced expression of osteopontin (OPN) in mammary tumors of mice fed with the 3 ppm selenium diet in comparison with OPN expression in tumors arising in 0.1 ppm selenium-fed mice. A 24-hour treatment of TM6 cells with MSC significantly inhibited their migration and also reduced their OPN expression in comparison with untreated cells. CONCLUSIONS: OPN is a potential target gene in the inhibition of mammary tumorigenesis by selenium.

Separation by cell size enriches for mammary stem cell repopulation activity.

Mammary gland reconstitution experiments, as well as lineage tracing experiments, have provided evidence for the existence of adult mammary stem cells (MaSCs). In addition, cell sorting techniques for specific cell surface markers (CD24(+)CD29(H)CD49f(H)Sca1(-)) have been used to prospectively isolate MaSC-enriched populations. Although these markers enrich for cell subpopulations that harbor MaSCs, they do not identify regenerative stem cells uniquely. Here, we report that MaSCs can be further defined by the property of cell size. Fluorescence-activated cell sorting was used to analyze sizing beads and further separate populations of cells with varying degrees of forward scatter (FSC). Cells with a low FSC that were approximately <10 µm in size lacked outgrowth potential and failed to reconstitute the mammary gland when transplanted into the cleared fat pads of syngeneic mice. In contrast, cells >10 µm in size with a higher FSC had increased outgrowth potential as compared with lineage-negative (LIN(-)) control cells. Limiting dilution transplantation assays indicated that the repopulating ability of LIN(-)CD24(+)CD29(H) cells that were >10 µm in size was significantly increased as compared with cells marked by CD24 and CD29 alone. These results suggest that MaSCs can be further isolated by sorting based on size/FSC. These findings have critical implications for understanding mammary gland stem cell biology, an important requisite step for understanding the etiology of breast cancer.

Identification of modulated genes by three classes of chemopreventive agents at preneoplastic stages in a p53-null mouse mammary tumor model.

Genetically engineered mouse cancer models are among the most useful tools for testing the in vivo effectiveness of the various chemopreventive approaches. The p53-null mouse model of mammary carcinogenesis was previously characterized by us at the cellular, molecular, and pathologic levels. In a companion article, Medina et al. analyzed the efficacy of bexarotene, gefitinib, and celecoxib as chemopreventive agents in the same model. Here we report the global gene expression effects on mammary epithelium of such compounds, analyzing the data in light of their effectiveness as chemopreventive agents. SAGE was used to profile the transcriptome of p53-null mammary epithelium obtained from mice treated with each compound versus controls. This information was also compared with SAGE data from p53-null mouse mammary tumors. Gene expression changes induced by the chemopreventive treatments revealed a common core of 87 affected genes across treatments (P < 0.05). The effective compounds, bexarotene and gefitinib, may exert their chemopreventive activity, at least in part, by affecting a set of 34 genes related to specific cellular pathways. The gene expression signature revealed various genes previously described to be associated with breast cancer, such as the activator protein-1 complex member Fos-like antigen 2 (Fosl2), early growth response 1 (Egr1), gelsolin (Gsn), and tumor protein translationally controlled 1 (Tpt1), among others. The concerted modulation of many of these transcripts before malignant transformation seems to be conducive to predominantly decrease cell proliferation. This study has revealed candidate key pathways that can be experimentally tested in the same model system and may constitute novel targets for future translational research.

Transcriptomic signature of bexarotene (rexinoid LGD1069) on mammary gland from three transgenic mouse mammary cancer models.

BACKGROUND: The rexinoid bexarotene (LGD1069, Targretin) is a highly selective retinoid x receptor (RXR) agonist that inhibits the growth of pre-malignant and malignant breast cells. Bexarotene was shown to suppress the development of breast cancer in transgenic mice models without side effects. The chemopreventive effects of bexarotene are due to transcriptional modulation of cell proliferation, differentiation and apoptosis. Our goal in the present study was to obtain a profile of the genes modulated by bexarotene on mammary gland from three transgenic mouse mammary cancer models in an effort to elucidate its molecular mechanism of action and for the identification of biomarkers of effectiveness. METHODS: Serial analysis of gene expression (SAGE) was employed to profile the transcriptome of p53-null, MMTV-ErbB2, and C3(1)-SV40 mammary cells obtained from mice treated with bexarotene and their corresponding controls. RESULTS: This resulted in a dataset of approximately 360,000 transcript tags representing over 20,000 mRNAs from a total of 6 different SAGE libraries. Analysis of gene expression changes induced by bexarotene in mammary gland revealed that 89 genes were dysregulated among the three transgenic mouse mammary models. From these, 9 genes were common to the three models studied. CONCLUSION: Analysis of the indicated core of transcripts and protein-protein interactions of this commonly modulated genes indicate two functional modules significantly affected by rexinoid bexarotene related to protein biosynthesis and bioenergetics signatures, in addition to the targeting of cancer-causing genes related with cell proliferation, differentiation and apoptosis.

Ataxia telangiectasia-mutated and p53 are potential mediators of chloroquine-induced resistance to mammary carcinogenesis.

The use of agents to prevent the onset of and/or the progression to breast cancer has the potential to lower breast cancer risk. We have previously shown that the tumor-suppressor gene p53 is a potential mediator of hormone (estrogen/progesterone)-induced protection against chemical carcinogen-induced mammary carcinogenesis in animal models. Here, we show for the first time a breast cancer-protective effect of chloroquine in an animal model. Chloroquine significantly reduced the incidence of N-methyl-N-nitrosourea-induced mammary tumors in our animal model similar to estrogen/progesterone treatment. No protection was seen in our BALB/c p53-null mammary epithelium model, indicating a p53 dependency for the chloroquine effect. Using a human nontumorigenic mammary gland epithelial cell line, MCF10A, we confirm that in the absence of detectable DNA damage, chloroquine activates the tumor-suppressor p53 and the p53 downstream target gene p21, resulting in G(1) cell cycle arrest. p53 activation occurs at a posttranslational level via chloroquine-dependent phosphorylation of the checkpoint protein kinase, ataxia telangiectasia-mutated (ATM), leading to ATM-dependent phosphorylation of p53. In primary mammary gland epithelial cells isolated from p53-null mice, chloroquine does not induce G(1) cell cycle arrest compared with cells isolated from wild-type mice, also indicating a p53 dependency. Our results indicate that a short prior exposure to chloroquine may have a preventative application for mammary carcinogenesis.

A mechanism of cyclin D1 action encoded in the patterns of gene expression in human cancer.

Here we describe how patterns of gene expression in human tumors have been deconvoluted to reveal a mechanism of action for the cyclin D1 oncogene. Computational analysis of the expression patterns of thousands of genes across hundreds of tumor specimens suggested that a transcription factor, C/EBPbeta/Nf-Il6, participates in the consequences of cyclin D1 overexpression. Functional analyses confirmed the involvement of C/EBPbeta in the regulation of genes affected by cyclin D1 and established this protein as an indispensable effector of a potentially important facet of cyclin D1 biology. This work demonstrates that tumor gene expression databases can be used to study the function of a human oncogene in situ.