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Despite major progress in breast cancer research, the functional contribution of distinct cancer cell clones to malignant tumor progression and metastasis remains largely elusive. We have assessed clonal heterogeneity within individual primary tumors and metastases and also during the distinct stages of malignant tumor progression using clonal tracking of cancer cells in the MMTV-PyMT mouse model of metastatic breast cancer. Comparative gene expression analysis of clonal subpopulations reveals a substantial level of heterogeneity across and also within the various stages of breast carcinogenesis. The intra-stage heterogeneity is primarily manifested by differences in cell proliferation, also found within invasive carcinomas of luminal A-, luminal B-, and HER2-enriched human breast cancer. Surprisingly, in the mouse model of clonal tracing of cancer cells, chemotherapy mainly targets the slow-proliferative clonal populations and fails to efficiently repress the fast-proliferative populations. These insights may have considerable impact on therapy selection and response in breast cancer patients.

Although it has been demonstrated that transformed progenitor cell population can contribute to tumor initiation, factors contributing to this malignant transformation are poorly known. Using in vitro and xenograft based models, previous studies demonstrated that miR-489 acts as a tumor suppressor miRNA by targeting various oncogenic pathways. It has been demonstrated that miR-489 directly targets HER2 and inhibits the HER2 signaling pathway; however, its role in mammary gland development and HER2 induced tumor initiation hasn’t been studied. To dissect the role of miR-489, we sorted different populations of mammary epithelial cells and determined that miR-489 was highly expressed in mammary stem cells. MMTV-miR-489 mice that overexpressed miR-489 in mammary epithelial cells were developed and these mice exhibited an inhibition of mammary gland development in early ages with a specific impact on highly proliferative cells. Double transgenic MMTV-Her2-miR489 mice were then generated to observe how miR-489 overexpression affects HER2 induced tumorigenesis. miR-489 overexpression delayed HER2 induced tumor initiation significantly. Moreover, miR-489 overexpression inhibited tumor growth and lung metastasis. miR-489 overexpression reduced mammary progenitor cell population significantly in preneoplastic mammary glands of MMTV-Her2 mice which showed a putative transformed population in HER2 induced tumorigenesis. The miR-489 overexpression reduced CD49fhiCD61hi populations in tumors that have stem- like properties, and miR-489 overexpression altered the HER2 signaling pathway in mammary tumors. Altogether, these data indicate that the inhibition of HER2 induced tumorigenesis by miR-489 overexpression was due to altering progenitor cell populations while decreasing tumor growth and metastasis via influencing tumor promoting genes DEK and SHP2.

The remodeling of the stromal extracellular matrix (ECM) plays a crucial, but incompletely understood role during tumor progression and metastasis. Hic-5, a focal adhesion scaffold protein, has previously been implicated in tumor cell invasion, proliferation and metastasis. To investigate the role of Hic-5 in breast tumor progression in vivo, Hic-5−/− mice were generated and crossed with the Mouse Mammary Tumor Virus-Polyoma Middle T Antigen (MMTV-PyMT) mouse. Tumors from the Hic-5−/−;PyMT mice exhibited increased latency and reduced growth, with fewer lung metastases, as compared to Hic-5+/−;PyMT mice. Immunohistochemical analysis showed that Hic-5 is primarily expressed in the cancer associated fibroblasts (CAFs). Further analysis revealed that the Hic-5−/−;PyMT tumor stroma contains fewer CAFs and exhibits reduced ECM deposition. The remodeling of the stromal matrix by CAFs has been shown to increase tumor rigidity to indirectly regulate FAK Y397 phosphorylation in tumor cells to promote their growth and invasion. Accordingly, the Hic-5−/−;PyMT tumor cells exhibited a reduction in FAK Y397 phosphorylation. Isolated Hic-5−/−;PyMT CAFs were defective in stress fiber organization and exhibited reduced contractility. These cells also failed to efficiently deposit and organize the ECM in two and three dimensions. This, in turn, impacted three dimensional MDA-MB-231 tumor cell migration behavior. Thus, using a new knockout mouse model, we have identified Hic-5 expression in CAFs as a key requirement for deposition and remodeling of the stromal ECM to promote non-cell autonomous breast tumor progression.

Separase, a protease encoded by the ESPL1 gene, cleaves the chromosomal cohesin during mitosis. Separase protein and transcripts are overexpressed in a wide range of human cancers (Meyer et al., Clin Cancer Res 2009; 15: 2703-2710). To investigate the physiological consequence of Separase overexpression in animals, we have generated a transgenic MMTVEspl1 mouse model that overexpresses Separase protein in the mammary glands. MMTV-Espl1 mice in a C57BL/6 genetic background develop aggressive, highly aneuploid, and estrogen receptor alpha positive (ERα+) mammary adenocarcinomas with an 80% penetrance. The mammary tumors caused by overexpression of Separase, alone or combined with p53 heterozygosity, in mammary epithelium mimic several aspects of the most aggressive forms of human breast cancer, including high levels of genetic instability, cell cycle defects, poor differentiation, distant metastasis, and metaplasia. Histopathologically, MMTV-Espl1 tumors are highly heterogeneous showing features of both luminal as well as basal subtypes of breast cancers, with aggressive disease phenotype. In addition to aneuploidy, Separase overexpression results in chromosomal instability (CIN) including premature chromatid separation (PCS), lagging chromosomes, anaphase bridges, micronuclei, centrosome amplification, multi nucleated cells, gradual accumulation of DNA damage, and progressive loss of tumor suppressors p53 and cadherin gene loci. These results suggest that Separase overexpressing mammary cells are not only susceptible to chromosomal missegregation-induced aneuploidy but also other genetic instabilities including DNA damage and loss of key tumor suppressor gene loci, which in combination can initiate tumorigenesis and disease progression.

The microenvironment of the mammary gland has been shown to exert a deterministic control over cells from different normal organs during murine mammary gland regeneration in transplantation studies. When mouse mammary tumor virus (MMTV)-neu-induced tumor cells were mixed with normal mammary epithelial cells (MECs) in a dilution series and inoculated into epithelium-free mammary fat pads, they were redirected to non-carcinogenic cell fates by interaction with untransformed MECs during regenerative growth. In the presence of non-transformed MECs (50:1), tumor cells interacted with MECs to generate functional chimeric outgrowths. When injected alone, tumor cells invariably produced tumors. Here, the normal microenvironment redirects MMTV-neu-transformed tumorigenic cells to participate in the regeneration of a normal, functional mammary gland. In addition, the redirected tumor cells show the capacity to differentiate into normal mammary cell types, including luminal, myoepithelial and secretory. The results indicate that signals emanating from a normal mammary microenvironment, comprised of stromal, epithelial and host-mediated signals, combine to suppress the cancer phenotype during glandular regeneration. Clarification of these signals offers improved therapeutic possibilities for the control of mammary cancer growth.

Although Wnt signaling activation is frequently observed in human breast cancer, mutations in genes encoding intracellular components of the Wnt signaling pathway are rare. We found that the expression of Wnt signaling co-receptor, LRP6, is upregulated in a subset of human breast cancer tissues and cell lines. To examine whether the overexpression of LRP6 in mammary epithelial cells is sufficient to activate Wnt signaling and promote cell proliferation, we generated transgenic mice overexpressing LRP6 in mammary epithelial cells driven by the mouse mammary tumor virus (MMTV) promoter. We found that mammary glands from MMTV-LRP6 mice exhibit significant Wnt activation evidenced by the translocation of beta-catenin from membrane to cytoplasmic/nuclear fractions. The expression of several Wnt target genes including Axin2, Cyclin D1 and c-Myc was also increased in MMTV-LRP6 mice. More importantly, mammary glands from virgin MMTV-LRP6 mice exhibit significant hyperplasia, a precursor to breast cancer, when compared with wild-type littermate controls. Several matrix metalloproteinases are upregulated in MMTV-LRP6 mice that could contribute to the hyperplasia phenotype. Our results suggest that Wnt signaling activation at the cell-surface receptor level can contribute to breast cancer tumorigenesis.

We identified a somatic mutation in estrogen receptor-alpha (ERalpha) in breast cancer causing a lysine to arginine transition (K303R) resulting in hypersensitivity to estrogen, altered associations with coactivators and corepressors and altered posttranslational modifications of ERalpha. We have developed a transgenic mouse expressing the K303R mutant ERalpha under control of the mouse mammary tumor virus (MMTV) promoter. At 4 months of age, K303R ERalpha transgenic animals demonstrate precocious alveolar budding compared with wild-type ERalpha transgenic mice or nontransgenic littermates. Despite these morphologic differences, K303R ERalpha transgenic mice displayed no differences in levels of ERalpha, progesterone receptor or proliferation at this time-point. Pregnancy or chronic estrogen plus progesterone exposure in K303R ERalpha transgenic mice also resulted in significantly more alveolar budding, increased beta-casein production and dilated ducts when compared with nontransgenic littermates. To examine the effects of mutant expression on tumorigenesis, mutant ERalpha mice were crossed with FVB-MMTVneu mice and significantly delayed time to neu-mediated tumorigenesis in bigenic animals. In contrast, mutant expression did not affect carcinogen-induced tumorigenesis. Collectively, these data demonstrate that aberrant estrogenic signaling through the K303R ERalpha mutation may lead to precocious alveolar budding in virgin mice, and to an expedited maturation and differentiation phenotype in the mammary glands of hormonally stimulated animals.

Transgenic mice expressing the Notch 4 intracellular domain (ICD) (Int3) in the mammary gland have two phenotypes: arrest of mammary alveolar/lobular development and mammary tumorigenesis. Notch4 signaling is mediated primarily through the interaction of Int3 with the transcription repressor/activator Rbpj. We have conditionally ablated the Rbpj gene in the mammary glands of mice expressing whey acidic protein (Wap)-Int3. Interestingly, Rbpj knockout mice (Wap-Cre(+)/Rbpj(-/-)/Wap-Int3) have normal mammary gland development, suggesting that the effect of endogenous Notch signaling on mammary gland development is complete by day 15 of pregnancy. RBP-J heterozygous (Wap-Cre(+)/Rbpj(-/+)/Wap-Int3) and Rbpj control (Rbpj(flox/flox)/Wap-Int3) mice are phenotypically the same as Wap-Int3 mice with respect to mammary gland development and tumorigenesis. In addition, the Wap-Cre(+)/Rbpj(-/-)/Wap-Int3-knockout mice also developed mammary tumors at a frequency similar to Rbpj heterozygous and Wap-Int3 control mice but with a slightly longer latency. Thus, the effect on mammary gland development is dependent on the interaction of the Notch ICD with the transcription repressor/activator Rbpj, and Notch-induced mammary tumor development is independent of this interaction.

Cyclin D1 levels are maintained at steady state by phosphorylation-dependent nuclear export and polyubiquitination by the SCFFBX4-αB crystallin E3 ligase. Inhibition of cyclin D1 proteolysis has been implicated as a causative factor leading to its overexpression in carcinomas of the breast and esophagus; however evaluation of the contribution of stable cyclin D1 to the genesis of such carcinomas has not been performed. We therefore generated transgenic mice wherein expression of either wild-type or a stable cyclin D1 allele (D1T286A) is regulated by MMTV-LTR. MMTV-D1T286A mice developed mammary adenocarcinomas at an increased rate relative to MMTV-D1 mice. Similar to human cancers that overexpress cyclin D1, D1T286A tumors were estrogen receptor positive and exhibited estrogen-dependent growth. MMTV-D1T286A tumors specifically overexpressed genes involved in DNA replication and DNA damage checkpoints suggesting that stabilization and nuclear accumulation of cyclin D1-dependent kinase contributes to genomic instability through perturbations in DNA replication. Collectively, these results suggest that temporal control of cyclin D1 subcellular localization and proteolysis is critical for the maintenance of homeostasis within the mammary epithelium.

One common characteristic of breast cancers arising in carriers of the predisposition gene BRCA1 is a loss of expression of the CDK inhibitor p27(Kip1) (p27), suggesting that p27 interacts epistatically with BRCA1. To investigate this relationship, we examined expression of p27 in mice expressing a dominant negative allele of Brca1 (MMTV-trBr) in the mammary gland. While these mice rarely develop tumors, they showed a 50% increase in p27 protein and a delay in mammary gland development associated with reduced proliferation. In contrast, on a p27 heterozygote background, MMTV-trBrca1 mice showed an increase in S phase cells, and normal mammary development. p27 was the only protein in the cyclin-cyclin-dependent kinase network to show altered expression, suggesting that it may be a central mediator of cell cycle arrest in response to loss of function of BRCA1. Furthermore, in human mammary epithelial MCF7 cells expressing BRCA1-specific RNAi and in the BRCA1-deficient human tumor cell line HCC1937, p27 is elevated at the mRNA level compared to cells expressing wild-type BRCA1. We hypothesize that disruption of BRCA1 induces an increase in p27 that inhibits proliferation. Accordingly, reduction in p27 expression leads to enhancement of cellular proliferation in the absence of BRCA1.

MUC1 is a tumor antigen, overexpressed in approximately 90% of human breast cancers. In normal glandular epithelia, MUC1 is expressed at the apical surface; however, in carcinomas an aberrantly glycosylated form of MUC1 is upregulated and expressed around the entire surface of the cell. Previously, we have shown that a lack of Muc1 significantly delays tumor progression and/or onset in MMTV-PyV-mT and MMTV-Wnt-1 transgenic mice. Here we show that, unlike the models mentioned above, a loss of Muc1 in MMTV-c-Neu mice (MMTV-c-Neu/Muc1(-/-)) altered neither mammary tumor onset nor progression. Moreover, characterization of MMTV-c-Neu/Muc1(+/+) tumors revealed that Muc1 expression was repressed at the level of transcription. In contrast, normal mammary gland tissue adjacent to tumor tissue expressed Muc1 and pregnant mammary glands from c-Neu transgenic animals expressed high levels of Muc1. We found that transient transfection of activated ErbB2 into human embryonic kidney 293/MUC1 cells resulted in the repression of MUC1 expression. Further, transient transfection of activated ErbB2 resulted in the inhibition of Muc1 transcriptional activation in luciferase reporter assays. These data suggest that the activation of ErbB2, which only occurs in c-Neu tumors, selectively inhibits Muc1 expression.

The MMTV/neu transgenic (Tg) mice spontaneously develop mammary tumors stochastically after a long latent period, suggesting that the c-neu/erbB2 oncogene is not sufficient for tumor formation. To identify putative collaborator(s) of the c-neu/erbB2, we used the provirus insertional mutagenesis approach with mammary tumors arising in MMTV/neu Tg mice infected with the mouse mammary tumor virus (MMTV). The Notch1 gene was identified as a novel target for MMTV provirus insertional activation. In Notch1-rearranged tumors, the Notch1 gene was interrupted by the MMTV provirus insertion upstream of the exons coding for the TM domain. These insertions led to overexpression of novel 5' truncated approximately 7 kb RNA coding for 280 kDa mutant protein harboring only the Notch1 ectodomain, N(EC)mut. These may be involved in tumor formation. Another consequence of these insertions was the expression of truncated 3' Notch1 transcripts (3.5 - 4.5 kb) and proteins (86 - 110 kDa) deleted of most of the extracellular sequences (Notch1intra). We found that 3' truncated Notch1intra can transform HC11 mouse mammary epithelial cells in vitro. Deletion analysis revealed that the ankyrin-repeats and the domain 1 (aa 1751 - 1821) are required, while a signal peptide, the two conserved cysteines (C1652 and C1685) and the OPA and PEST sequences are dispensable for transformation. These results indicate that the N-terminally truncated Notch1intra protein behaves as an oncogene in this system.

Wnt-10b is expressed during the formation of the mammary rudiment in mouse embryos and its expression continues through puberty when the mammary ductal pattern is established under control of ovarian steroids. Recently, viral activation of the Wnt-10b locus has linked its overexpression to mammary tumor formation, suggesting a role for Wnt-10b in patterning and growth-regulation of the mammary gland. To test this notion, we created lines of transgenic mice that express elevated levels of Wnt-10b under the control of the MMTV promoter/enhancer. Overexpression of this gene resulted in profound developmental alterations in the mammary gland, including expanded glandular development and the precocious appearance of alveoli in virgin females. Moreover, transgenic male mice also exhibited dramatic mammary development involving highly branched mammary ducts and gynecomastia. Aberrant expression of Wnt-10b in the mammary rudiments of males evidently bypasses the normal requirement for ovarian hormonal control in stimulating mammary ductal growth and the repressive effects of androgens. In addition to these developmental effects, transgenic mice of both sexes were highly susceptible to the development of mammary adenocarcinomas. Such tumors arose in a solitary manner indicating that Wnt-10b is a proto-oncogene which provides a necessary, but insufficient signal for oncogenesis. Relevant to this, there was no evidence of amplified expression of FGF mRNAs in these tumors though the Fgf's are a class of genes often implicated as collaborators in Wnt-mediated tumor formation. Indeed, co-expression of MMTV-Wnt-10b and MMTV-FGF-3/int-2 resulted in sterile offspring with highly disorganized mammary epithelium, demonstrating a potent interaction between their respective developmental pathways. These results suggest that Wnt-10b, or other Wnt genes expressed early in mammary development, play a role in regulating sexual dimorphism and show potent transforming activity when overexpressed.

The role of the canonical NF-kappaB pathway in mammary tumorigenesis was investigated using a transgenic (TG) mouse expressing a dominant-negative inhibitor of kappaB (IkappaBalpha(SR (S32A/S36A))) in the mammary gland under the control of the mouse mammary tumor virus promoter (MMTV). TG and control mice were subjected to a chemical carcinogenesis protocol. Hyperkeratinized squamous metaplasias (cytokeratin-6+/p63+) sometimes with a basaloid island component, were found in both TG and control mice whereas luminal (cytokeratin-19+/MUC1+) ErbB2+ papillary and adenomatous lesions developed almost exclusively in control mice. p65/RelA- and NF-kappaB DNA-binding activity were detected in mammary luminal lesions, but rarely in squamous metaplasias. Analysis of NF-kappaB family proteins and target genes using microarray data from a cohort of human mammary tumors revealed the expression of a canonical NF-kappaB pathway, but not non-canonical pathway proteins in HER2+ luminal cancers. HER2+ tumors also showed differential regulation of specific NF-kappaB target genes relative to basal and ER+ luminal cancers. Isolation of mammary cell populations enriched for stem and progenitor cell characteristics from an NF-kappaB-EGFP reporter mouse by fluorescence-activated cell sorting demonstrated that luminal progenitors contain activated NF-kappaB whereas the mammary stem cell-enriched population, does not. Together these data suggest that the canonical NF-kappaB pathway is active in normal luminal progenitor cells before transformation and is required for the formation of mammary luminal-type epithelial neoplasias.

Transgenic mice overexpressing Notch4 intracellular domain (Int3) under the control of the whey acidic protein (WAP) or mouse mammary tumor virus-long terminal repeat promoters, develop mammary tumors. Microarray analysis of these tumors revealed high levels of c-Kit expression. Gleevec is a tyrosine kinase inhibitor that targets c-Kit, platelet-derived growth factor receptors (PDGFRs) and c-Abl. This led us to speculate that tyrosine kinase receptor activity might be a driving force in the development of Int3 mammary tumors. WAP-Int3 tumor-bearing mice were treated with continuous release of Gleevec using subcutaneously implanted Alzet pumps. Phosphorylation of c-Kit, PDGFRs and c-Abl is inhibited in Int3 transgenic mammary tumors by Gleevec. Inhibition of these enzymes is associated with a decrease in cell proliferation and angiogenesis, and an induction of apoptosis. To examine the signaling mechanisms underlying Notch4/Int3 tumorigenesis, we employed small interfering RNA (siRNA) to knock down c-Kit, PDGFRs and c-Abl alone or in combination and observed the effects on soft agar growth of HC11 cells overexpressing Int3. Only siRNA constructs for c-Kit and/or PDGFR-alpha were able to inhibit HC11-Int3 colony formation in soft agar. Our data demonstrate an inhibitory effect of Gleevec on Int3-induced transformation of HC11 cells and mammary tumors and indicate an oncogenic role for c-Kit and PDGFR-alpha tyrosine kinases in the context of Int3 signaling.

Aurora-A/STK15/BTAK, which encodes a centrosome-associated kinase, is amplified and overexpressed in multiple types of human tumors, including breast cancer. However, the causal relationship between overexpression of Aurora-A and tumorigenesis has not been fully established due to contradictory data obtained from different experimental systems. To investigate this, we generated a mouse strain that carries an MMTV-Aurora-A transgene. We showed that all the MMTV-Aurora-A mice displayed enhanced branch morphogenesis in the mammary gland and about 40% developed mammary tumors at 20 months of age. The tumor incidence was significantly increased in a p53(+/-) mutation background with about 70% MMTV-Aurora-A;p53(+/-) animals developed tumors at 18 months of age. Of note, overexpression of Aurora-A led to genetic instability, characterized by centrosome amplification, chromosome tetraploidization and premature sister chromatid segregation, at stages prior to tumor formation. Most notably, the severe chromosomal abnormality did not cause cell death owing to the activation of AKT pathway, including elevated levels of phosphorylated AKT and mammalian target of rapamycin, and nuclear accumulation of cyclin D1, which enabled continuous proliferation of the tetraploid cells. These data establish Aurora-A as an oncogene that causes malignant transformation through inducing genetic instability and activating oncogenic pathways such as AKT and its downstream signaling.

MUC1 is an integral membrane mucin glycoprotein that is normally expressed on the apical surface of most simple, secretory epithelia and hematopoietic cells. Overexpression of aberrantly glycosylated MUC1 is a hallmark of many carcinomas including 90% of breast carcinomas. MUC1 has been shown to bind to c-Src tyrosine kinase in vitro, whereby c-Src phosphorylates the MUC1 cytoplasmic domain at a YEKV motif. c-Src is an extensively studied nonreceptor tyrosine kinase implicated in mammary tumorigenesis. Previously, mouse mammary tumor virus-driven polyoma middle T-antigen (MMTV-PyV MT) transgenic mice crossed onto a Muc1 null background exhibited a significant delay in tumor progression. c-Src has been shown to interact with PyV MT, and to play an integral and indispensable role in MMTV-PyV MT-induced mammary tumorigenesis. Here, we determine the effect of Muc1 expression on c-Src activation and signaling. Examination of MMTV-PyV MT glands on a wild-type or Muc1 null background demonstrates that Muc1 expression promotes c-Src signaling by influencing its association with known substrates such as the p85 subunit of phosphatidylinositol 3-kinase and beta-catenin. These findings may provide a mechanism for the delay in tumor progression that is observed in the absence of Muc1.

The three human D-type cyclins, cyclin D1, D2 and D3 share the ability to bind to and activate cdk4 and 6. MMTV-cyclin D1 transgenic mice develop mainly adenocarcinoma, while MMTV-cyclin D2 mice show a lack of alveologenesis during pregnancy and only develop carcinoma at low frequency. The effect of cyclin D3 overexpression in mammary glands remains hitherto unknown. We generated MMTV-cyclin D3 transgenic mice and report here that they develop exclusively squamous cell carcinoma. We show that although cyclin D3 transgene expression was detected early in puberty, postnatal development and mammary gland proliferation were normal in virgin animals. In contrast, multiparous mice develop multiple foci of abnormal growth that correspond to various stages of squamous metaplasia. Therefore, our results support a role for cyclin D3 in squamous differentiation. In addition, we found that p16 expression during involution is abolished, while p27 expression increased in MMTV-cyclin D3 mice, two modifications that have been reported in the other MMTV-D-type cyclin transgenic models. Our observations indicate that despite biochemical redundancy in vitro and in vivo, D-type cyclins promote distinct oncogenic pathways.

The Wnt/beta-catenin signaling pathway is critical for normal mammalian development, the specification of epidermal cells and neoplastic transformation of intestinal epithelium. However, precise molecular information regarding cell-specific responses to beta-catenin signaling has been limited. This question was addressed using a mouse model in which exon 3 of the beta-catenin gene was deleted in several cell types with loxP-mediated recombination utilizing a Cre transgene under control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR). The stabilization of beta-catenin in prostate epithelium resulted in hyperplasias and extensive transdifferentiation into epidermal-like structures, which expressed keratins 1 and 6, filaggrin, loricrin and involucrin. The cell-specific loss of NKCC1 protein and reduced nuclear Stat5a is further suggestive of a loss of prostate epithelial characteristics. In addition to the prostate, hyperplasias and squamous metaplasias were detected in epithelia of the epididymis, vas deferens, coagulating gland, preputial gland and salivary gland. However, and in contrast to a recent study, no lesions reminiscent of high-grade prostate intraepithelial neoplasia were detected. Since beta-catenin was activated in several cell types and impinged upon the viability of these mice, it was not possible to evaluate the cumulative effect over more than 3 months. To assess long-term consequences of beta-catenin activation, mutant and control prostate tissues were transplanted into the mammary fat pads of wild-type males. Notably, squamous metaplasias, intra-acinous hyperplasia and possible neoplastic transformation were observed after a total of 18 weeks of beta-catenin stimulation. This suggests that the transdifferentiation into squamous metaplasias is an early response of endoderm-derived cells to beta-catenin, and that the development of intra-acinous hyperplasias or neoplastic foci is a later event.

MUC1 is aberrantly expressed in greater than 90% of all breast carcinomas, yet its function as a tumor antigen is not fully understood. Recently, studies have shown that MUC1 interacts with beta-catenin, erbB receptors, src, GSK-3beta and protein kinase Cdelta, possibly in a complex that promotes the disassembly of adherens junctions and the invasion of cells. Here we show that the deletion of Muc1 expression from MMTV-Wnt-1 transgenic mice results in a significant increase in the time to mammary gland tumor onset. Analysis of MMTV-Wnt-1 tumors on a wild-type Muc1 background shows a tumor-specific complex formation between Muc1 and beta-catenin that can be observed in both the membrane and the cytoplasm of transformed epithelium. Analysis of primary human adenocarcinomas revealed that this MUC1/beta-catenin interaction occurs in both primary and metastatic tumors, but is dramatically increased in metastatic lesions. Addition of MUC1-cytoplasmic domain peptides to the invasive MDA-MB-468 and MDA-MB-231 cell lines increases their invasive capability, and these peptides colocalize with both beta-catenin and the focal adhesion protein vinculin, primarily at sites of membrane invasion into a collagen matrix. These data indicate a potential mechanism for MUC1 promotion of invasive tumorigenesis in the breast through the modulation of beta-catenin localization and subsequent cytoskeletal dynamics.

Protein kinase CK2 is a ubiquitous and evolutionarily conserved serine/threonine kinase that is upregulated in many human cancers and can serve as an oncogene in lymphocytes. Recently, we have demonstrated that CK2 potentiates Wnt/beta-catenin signaling in mammary epithelial cells. To determine whether CK2 overexpression contributes to mammary tumorigenesis, we have performed comparative studies of human and rat breast cancer specimens and we have engineered transgenic mice with dysregulated expression of CK2alpha in the mammary gland. We find that CK2 is highly expressed in human breast tumor specimens and in carcinogen-induced rat mammary tumors. Overexpression of CK2alpha in the mammary gland of transgenic mice, under control of the MMTV-LTR, causes hyperplasia and dysplasia of the female mammary gland. Thirty per cent of the female MMTV-CK2alpha transgenic mice develop mammary adenocarcinomas at a median of 23 months of age, often associated with Wnt pathway activation, as evidenced by upregulation of beta-catenin protein. NF-kappaB activation and upregulation of c-Myc also occur frequently. Thus, in mice, rats, and humans, dysregulated expression of CK2 is associated with and is capable of contributing to mammary tumorigenesis. Targeted inhibition of CK2 could be useful in the treatment of breast cancer.

Wnt-1 was first identified as a protooncogene activated by viral insertion in mouse mammary tumors. Transgenic expression of this gene using a mouse mammary tumor virus LTR enhancer causes extensive ductal hyperplasia early in life and mammary adenocarcinomas in approximately 50% of the female transgenic (TG) mice by 6 months of age. Metastasis to the lung and proximal lymph nodes is rare at the time tumors are detected but frequent after the removal of the primary neoplasm. The potent mitogenic effect mediated by Wnt-1 expression does not require estrogen stimulation; tumors form after an increased latency in estrogen receptor alpha-null mice. Several genetic lesions, including inactivation of p53 and over-expression of Fgf-3, collaborate with Wnt-1 in leading to mammary tumors, but loss of Sky and inactivation of one allele of Rb do not affect the rate of tumor formation in Wnt-1 TG mice.

We have examined defects in mammary development and tumorigenesis in a transgenic model expressing the c-myc gene under the MMTV-LTR promoter. The stochastic tumors which arise from hyperplastic ductal and lobular lesions in this model are characterized by high rates both of apoptosis and of chromosomal instability. Since the p53 gene product is thought to be central in the maintenance of genomic integrity, in part due to its ability to induce apoptosis in cells harboring DNA damage, we examined its expression and possible mutation. Initially, we observed that unmutated p53 is strongly expressed in premalignant mammary glands and in mammary tumors derived from the MMTV-c-myc strain. We then mated the MMTV-myc strain to a p53-deficient strain as a means of examining the effect of this lesion on mammary development and tumorigenesis in the context of c-myc overexpression. A lack of both p53 alleles in the presence of c-myc overexpression resulted in a dramatic hyerplastic alteration in mammary gland development. Specifically, in female bitransgenic MMTV-c-myc/p53 null mice (MMTV-myc/p53(-/-)), lobular hyperplasias were observed at almost every ductal end bud as early as 32 days of age. In contrast, only mild ductal and lobular hyperplasias were seen in MMTV-myc mice that contained both p53 alleles (MMTV-myc/p53(+/+)); an intermediate phenotype occurred in mice with a single intact (MMTV-myc/p53(+/-)) p53 allele. Mammary carcinomas arose with a high frequency in MMTV-myc/p53(+/-) mice; the tumors were comparable in frequency, histology and apoptotic index to the tumors in MMTV-myc/p53(+/+) mice. Also, as previously observed (Elson et al., 1995), lymphomas arose with extremely short latency in MMTV-myc/p53(-/-) mice, precluding study of the fate of their hyperplastic mammary lesions in situ. The frequency of p53 mutations in MMTV-myc/p53(+/+) and MMTV-myc/p53(+/-) mammary tumors and in cell lines derived from these tumors was examined by direct sequencing. No point mutations or deletions in p53 were observed in mammary tumors or cell lines from either genotype. Finally, a detailed chromosomal analysis using multicolor spectral karyotyping (SKY) revealed that there were multiple chromosomal alterations in the c-myc-overexpressing cells that contained either one or two unmutated p53 alleles. Variable ploidy changes, a common translocation of chromosome 11, and other chromosomal aberrations were observed. Our data thus support an interaction between c-Myc and p53 in mammary development, but suggest that loss of p53 is required neither for c-myc-dependent tumorigenesis nor for c-myc-dependent chromosomal instability.

Mouse mammary tumor virus (MMTV) is an insertional mutagen that has been demonstrated to transcriptionally activate flanking cellular proto-oncogenes. Previously we have used MMTV infection to accelerate mammary tumorigenesis in Wnt1 transgenic mice in order to identify genes that cooperate with the Wnt1 oncogene. Initial investigations into the resulting tumor collection, screened primarily by Southern analysis, showed that three fibroblast growth factor genes, Fgf8, Fgf3 and Fgf4, sustain activating insertion mutations in 10%, 42% and 6% of the tumors, respectively. Here, in an examination of the tumors from MMTV-infected Wnt1 transgenic mice that emphasizes Northern analysis, we report transcriptional activation of Fgf8 in 30 additional tumors (increasing the percentage of activations to 50%), while no significant changes in the activation frequency of Fgf3 or Fgf4 were found. To determine the frequency of insertional activation in normal mice, we examined tumors from MMTV-infected nontransgenic littermates of the Wnt1 transgenics and from MMTV-infected BALB/c mice. Fgf8, Fgf3 and Fgf4 were found to be activated in 11%, 80% and 5%, respectively, of the tumors in the combined nontransgenic groups. Thus, there appears to be an increased predisposition for Fgf8 activations in Wnt1 transgenic mice versus normal mice, suggesting that cells expressing Wnt1 are especially sensitized to stimulation by FGF8 compared with FGF3 or FGF4. In contrast, the activation frequency of Fgf3 in tumors from MMTV-infected Wnt1 transgenic mice was approximately one-half that of normal mice. Our results show that this in vivo model of multistep tumorigenesis reveals significant differences in the activation rates of Fgf3 and Fgf8 depending upon the status of Wnt1 expression in the mammary gland. The differential activation of these Fgfs may relate to differences in their signaling pathways.