Stromal Gas6 promotes the progression of premalignant mammary cells.

Tumor progression is regulated by a complex interplay between neoplastic cells and the tumor microenvironment. Tumor-associated macrophages have been shown to promote breast cancer progression in advanced disease and more recently, in early stage cancers. However, little is known about the macrophage-derived factors that promote tumor progression in early stage lesions. Using a p53-null model of early stage mammary tumor progression, we found that Gas6 is highly expressed in pre-invasive lesions associated with increased infiltrating macrophages, as compared with those with few recruited macrophages. We show that F4/80+CD11b+ macrophages produce Gas6 in premalignant lesions in vivo, and that macrophage-derived Gas6 induces a tumor-like phenotype ex vivo. Using a 3-D co-culture system, we show that macrophage-derived Gas6 activates its receptor Axl and downstream survival signals including Akt and STAT3, which was accompanied by altered E-cadherin expression to induce a malignant morphology. In vivo studies demonstrated that deletion of stromal Gas6 delays early stage progression and decreases tumor formation, while tumor growth in established tumors remains unaffected. These studies suggest that macrophage-derived Gas6 is a critical regulator of the transition from premalignant to invasive cancer, and may lead to the development of unique biomarkers of neoplastic progression for patients with early stage breast cancer, including ductal carcinoma in situ.

Whole Mammary Gland Transplantation in Mice Protocol.

Whole Mammary Gland Transplantation involves transplanting an excised mammary gland into another, more suitable host. This method can be used to extend the life of a mammary gland past the mouse's life span by transplanting the mammary gland of an older mouse into a young healthy mouse. As you can see in the video below (Video 1), by attaching it to the abdomen of the mouse, the gland will receive a steady blood supply and both epithelial and stromal cells will remain viable for up to one year. Although this method is not used often, it has been part of several experiments including determining whether the stroma or epithelium is the primary target in chemically induced mouse mammary tumorigenesis (Medina and Kittrell, 2005). To monitor transplants, palpate every week for tumor formation. The transplanted mammary gland may also be passaged serially every 8-10 weeks. Keep transplanted gland in the same mouse for no longer than one year.

Macrophages promote the progression of premalignant mammary lesions to invasive cancer.

Breast cancer initiation, progression and metastasis rely on a complex interplay between tumor cells and their surrounding microenvironment. Infiltrating immune cells, including macrophages, promote mammary tumor progression and metastasis; however, less is known about the role of macrophages in early stage lesions. In this study, we utilized a transplantable p53-null model of early progression to characterize the immune cell components of early stage lesions. We show that macrophages are recruited to ductal hyperplasias with a high tumor-forming potential where they are differentiated and polarized toward a tumor-promoting phenotype. These macrophages are a unique subset of macrophages, characterized by pro-inflammatory, anti-inflammatory and immunosuppressive factors. Macrophage ablation studies showed that macrophages are required for both early stage progression and primary tumor formation. These studies suggest that therapeutic targeting of tumor-promoting macrophages may not only be an effective strategy to block tumor progression and metastasis, but may also have critical implications for breast cancer prevention.

Pituitary Isograft Transplantation in Mice.

The mouse pituitary isograft is a technique developed to administer persistent hormone stimulation, thereby increasing cellular proliferation in the mammary tissue ( Christov et al., 1993 ). The pituitary isograft procedure was first described in 'Induction of Mammary Cancer in Mice without the Mammary Tumor Agent by Isografts of Hypophyses' by O. Mühlbock and L. M. Boot in 1959 (Muhlbock and Boot, 1959). Since then, the procedure has seen wide use. A pituitary gland is harvested posthumously from a donor mouse and implanted under the renal capsule of the recipient mouse through a small abdominal incision just below the last rib. Once the pituitary gland is implanted, it begins releasing hormones. These secretions increase serum levels of multiple hormones including prolactin, progesterone and 17ß-estradiol ( Christov et al., 1993 ). Although the effects of these hormones on cancer cell proliferation, growth, differentiation, and longevity are not well characterized, and, in some cases, controversial, the net effect of a pituitary isograft is to increase the proliferation of murine breast tissue depending upon strain specific characteristics ( Lydon et al., 1999 ). Below is a protocol describing how to perform the pituitary isograft procedure. After many of the steps, a time reference is listed in parentheses. Each reference corresponds to a time point in the embedded video of the procedure. (Video 1) Video 1.Pituitary isograft transplantation in mice. Video portraying pituitary isograft transplantation procedure in donor and recipient mice.

Mouse Mammary Intraductal (MIND) Method for Transplantation of Patient Derived Primary DCIS Cells and Cell Lines.

The MIND method involves intraductal injection of patient derived ductal carcinoma in situ (DCIS) cells and DCIS cell lines (MCF10DCIS.COM and SUM225) inside the mouse mammary ducts [Video 1 and Figure 1 in Behbod et al. (2009)]. This method mimics the normal environment of DCIS and facilitates study of the natural progression of human DCIS, i.e., their initial growth as carcinoma in situ within the ducts, followed by invasion into the stroma through the myoepithelial cell layer and basement membrane (Behbod et al., 2009; Valdez et al., 2011). In order to demonstrate that transplantation procedure is successful, the transplanted mammary glands may be excised as early as two weeks following intraductal injection of cells followed by Hematoxylin and Eosin (H&E) staining and/or immunofluorescence staining using human specific cytokeratin 5 and/or 19 [please see Figures 2-4 in Behbod et al. (2009)]. Additionally, the presence of trypan blue inside the mouse mammary ducts immediately following intraductal injection is the best indicator that the injection was successful (Video 1 starting at 4:33 sec).

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.

Intra-mammary ductal transplantation: a tool to study premalignant progression.

Ductal carcinoma in situ (DCIS) is a non-obligate precursor to invasive breast cancer. Although there is extensive information on the cellular and molecular changes in DCIS, there is limited ability to functionally test. The critical changes in premalignant progression. This review summarizes our experience with a recently developed method which provides. The opportunity to functionally test the molecular events occuring to functionally test the molecular events occurring in the initial changes in premaligant progression; i.e., the step from non-invasive to invasive behavior.

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.

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.

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.

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.

Prevention of tumorigenesis in p53-null mammary epithelium by rexinoid bexarotene, tyrosine kinase inhibitor gefitinib, and celecoxib.

The chemopreventive effects of three agents, rexinoid bexarotene, tyrosine kinase inhibitor gefitinib, and celecoxib, were tested on mammary tumor development arising in p53-null mammary epithelium. The rexinoid bexarotene was the most efficacious inhibitor as it reduced mammary tumor development by 75% in virgin mice and significantly delayed mean tumor development by 98 days in hormone-stimulated mice. The tyrosine kinase inhibitor gefitinib reduced mammary tumor incidence by 50% in virgin mice but did not significantly delay mean tumor latency in hormone-stimulated mice. Celecoxib did not reduce tumor incidence or mean tumor latency in either of the two models. The high doses of the rexinoid and the tyrosine kinase inhibitor did not affect the progression of tumors arising from the premalignant mammary outgrowth line, PN8a. A comparison of these agents with tamoxifen shows the superiority of tamoxifen in preventing tumor development in p53-null mammary cells. Similarly, a comparison of the results of the p53 model with other transgenic models in their response to the chemopreventive agents showed that mammary tumors arising from different oncogenic events will respond differently to the different agents.

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.

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.

Identification of tumor-initiating cells in a p53-null mouse model of breast cancer.

Using a syngeneic p53-null mouse mammary gland tumor model that closely mimics human breast cancer, we have identified, by limiting dilution transplantation and in vitro mammosphere assay, a Lin(-)CD29(H)CD24(H) subpopulation of tumor-initiating cells. Upon subsequent transplantation, this subpopulation generated heterogeneous tumors that displayed properties similar to the primary tumor. Analysis of biomarkers suggests the Lin(-)CD29(H)CD24(H) subpopulation may have arisen from a bipotent mammary progenitor. Differentially expressed genes in the Lin(-)CD29(H)CD24(H) mouse mammary gland tumor-initiating cell population include those involved in DNA damage response and repair, as well as genes involved in epigenetic regulation previously shown to be critical for stem cell self-renewal. These studies provide in vitro and in vivo data that support the cancer stem cell (CSC) hypothesis. Furthermore, this p53-null mouse mammary tumor model may allow us to identify new CSC markers and to test the functional importance of these markers.

Altered mammary gland development in the p53+/m mouse, a model of accelerated aging.

The tumor suppressor p53 is important for inhibiting the development of breast carcinomas. However, little is known about the effects of increased p53 activity on mammary gland development. Therefore, the effect of p53 dosage on mammary gland development was examined by utilizing the p53+/m mouse, a p53 mutant which exhibits increased wild-type p53 activity, increased tumor resistance, a shortened longevity, and a variety of accelerated aging phenotypes. Here we report that p53+/m virgin mice exhibit a defect in mammary gland ductal morphogenesis. Transplants of mammary epithelium into p53+/m recipient mice demonstrate decreased outgrowth of wild-type and p53+/m donor epithelium, suggesting systemic or stromal alterations in the p53+/m mouse. Supporting these data, p53+/m mice display decreased levels of serum IGF-1 and reduced IGF-1 signaling in virgin glands. The induction of pregnancy or treatment of p53+/m mice with estrogen, progesterone, estrogen and progesterone in combination, or IGF-1 stimulates ductal outgrowth, rescuing the p53+/m mammary phenotype. Serial mammary epithelium transplants demonstrate that p53+/m epithelium exhibits decreased transplant capabilities, suggesting early stem cell exhaustion. These data indicate that appropriate levels of p53 activity are important in regulating mammary gland ductal morphogenesis, in part through regulation of the IGF-1 pathway.

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.

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.