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.

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.

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.

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.

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.

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.

p19ARF determines the balance between normal cell proliferation rate and apoptosis during mammary gland development.

This study demonstrated, for the first time, the following events related to p19(ARF) involvement in mammary gland development: 1) Progesterone appears to regulate p19(ARF) in normal mammary gland during pregnancy. 2) p19(ARF) expression levels increased sixfold during pregnancy, and the protein level plateaus during lactation. 3) During involution, p19(ARF) protein level remained at high levels at 2 and 8 days of involution and then, declined sharply at day 15. Absence of p19(ARF) in mammary epithelial cells leads to two major changes, 1) a delay in the early phase of involution concomitant with downregulation of p21(Cip1) and decrease in apoptosis, and 2) p19(ARF) null cells are immortal in vivo measured by serial transplantion, which is partly attributed to complete absence of p21(Cip1) compared with WT cells. Although, p19(ARF) is dispensable in mammary alveologenesis, as evidenced by normal differentiation in the mammary gland of pregnant p19(ARF) null mice, the upregulation of p19(ARF) by progesterone in the WT cells and the weakness of p21(Cip1) in mammary epithelial cells lacking p19(ARF) strongly suggest that the functional role(s) of p19(ARF) in mammary gland development is critical to sustain normal cell proliferation rate during pregnancy and normal apoptosis in involution possibly through the p53-dependent pathway.

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.

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.

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.

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.

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.

From mice to humans: identification of commonly deregulated genes in mammary cancer via comparative SAGE studies.

Genetically engineered mouse mammary cancer models have been used over the years as systems to study human breast cancer. However, much controversy exists on the utility of such models as valid equivalents to the human cancer condition. To perform an interspecies gene expression comparative study in breast cancer we used a mouse model that most closely resembles human breast carcinogenesis. This system relies on the transplant of p53 null mouse mammary epithelial cells into the cleared mammary fat pads of syngeneic hosts. Serial analysis of gene expression (SAGE) was used to obtain gene expression profiles of normal and tumor samples from this mouse mammary cancer model (>300,000 mouse mammary-specific tags). The resulting mouse data were compared with 25 of our human breast cancer SAGE libraries (>2.5 million human breast-specific tags). We observed significant similarities in the deregulation of specific genes and gene families when comparing mouse with human breast cancer SAGE data. A total of 72 transcripts were identified as commonly deregulated in both species. We observed a systematic and significant down-regulation in all of the tumors from both species of various cytokines, including CXCL1 (GRO1), LIF, interleukin 6, and CCL2. All of the mouse and most human mammary tumors also displayed decreased expression of genes known to inhibit cell proliferation, including NFKBIA (IKBalpha), GADD45B, and CDKN1A (p21); transcription-related genes such as CEBP, JUN, JUNB, and ELF1; and apoptosis-related transcripts such as IER3 and GADD34/PPP1R15A. Examples of overexpressed transcripts in tumors from both species include proliferation-related genes such as CCND1, CKS1B, and STMN1 (oncoprotein 18); and genes related to other functions such as SEPW1, SDFR1, DNCI2, and SP110. Importantly, abnormal expression of several of these genes has not been associated previously with breast cancer. The consistency of these observations was validated in independent mouse and human mammary cancer sets. This is the first interspecies comparison of mammary cancer gene expression profiles. The comparative analysis of mouse and human SAGE mammary cancer data validates this p53 null mouse tumor model as a useful system closely resembling human breast cancer development and progression. More importantly, these studies are allowing us to identify relevant biomarkers of potential use in human studies while leading to a better understanding of specific mechanisms of human breast carcinogenesis.

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.

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).