PPARδ is pro-tumorigenic in a mouse model of COX-2-induced mammary cancer

Cyclooxygenase-2 (COX-2), overexpressed in inflammatory conditions and cancer, regulates angiogenesis and tumorigenesis via the production of biologically active prostanoids. Previously, we showed that COX-2 over-expression in the mammary gland of transgenic mice induces an angiogenic switch and transforms the mammary epithelium into invasive mammary carcinoma. Since COX-2-derived prostanoids can activate the nuclear receptor PPARδ, we crossed Pparδ^?/? mice with COX-2 transgenic mice in the FVB/N background. PPARδ was expressed constitutively in the mammary gland of virgin, pregnant and lactating mice. Mammary hyperplasia and tumorigenesis in the COX-2 transgenic mice was markedly reduced in the Pparδ^?/? mice compared to their wild type counterparts. Analysis of the mammary tissues indicated that immunoreactive Ki-67, cyclin D1 and phosphorylated histone 3 (Phospho H3) were reduced in Pparδ^?/? mice, suggesting that PPARδ activation regulates cell proliferation in the mammary gland. We postulate that activation of the nuclear receptor PPARδ by COX-2-derived prostanoids may be involved in the proliferation of mammary epithelial cells and therefore contribute to mammary cancer development.     

Abnormal expression of Muc5b in Cftr-null mice and in mammary tumors of MMTV-ras mice

Gel-forming mucins are large, high molecular weight, and heavily O-glycosylated proteins that are responsible for the rheological properties of mucus gel. Among them, the mucin MUC5B has been implicated in breast cancer and cystic fibrosis. We obtained a new polyclonal serum, named CP1, which was isolated from a rabbit immunized with a mouse Muc5b peptide. The immunoprofile of Muc5b was determined on paraffin-embedded and frozen mouse tissue sections and showed a similar expression pattern in mouse to that in the human. The “nonmammary” mucin Muc5b was detected in all mammary tumors analyzed from MMTV-ras mice, suggesting that the CP1 antibody is a valuable tool for investigating the involvement of this mucin in mammary cancer. We also found that uninfected Cftr ^−/− mice harbored more Clara cells, which were Muc5b-positive, than did their wild-type control littermates. The number of Muc5b-positive cells increased in Cftr ^−/− mice infected experimentally with Pseudomonas aeruginosa, and the mice developed mucus plugs in their bronchi and bronchioles with a high frequency of Muc5b content (87%, Cohen’s kappa = 0.82; p < 0.0001). These findings suggest that mice genetically deficient in the Cftr gene are predisposed to develop mucus plugs and that MUC5B may provide a valuable target for decreasing mucus viscosity in cystic fibrosis.     

Core3 O-Glycan Synthase Suppresses Tumor Formation and Metastasis of Prostate Carcinoma PC3 and LNCaP Cells through Down-regulation of ��2��1 Integrin Complex

Although there are numerous reports of carbohydrates enriched in cancer cells, very few studies have addressed the functions of carbohydrates present in normal cells that decrease in cancer cells. It has been reported that core3 O-glycans are synthesized in normal gastrointestinal cells but are down-regulated in cancer cells. To determine the roles of core3 O-glycans, we transfected PC3 and LNCaP prostate cancer cells with β3-N-acetylglucosaminyltransferase-6 (core3 synthase) required to synthesize core3 O-glycans. Both engineered cell lines exhibited reduced migration and invasion through extracellular matrix components compared with mock-transfected cells. Moreover we found that α2β1 integrin acquired core3 O-glycans in cells expressing core3 synthase with decreased maturation of β1 integrin, leading to decreased levels of the α2β1 integrin complex, decreased activation of focal adhesion kinase, and reduced lamellipodia formation. Upon inoculation into the prostate of nude mice, PC3 cells expressing core3 O-glycans produced much smaller tumors without metastasis to the surrounding lymph nodes in contrast to robust tumor formation and metastasis seen in mock-transfected PC3 cells. Similarly LNCaP cells expressing core3 O-glycans barely produced subcutaneous tumors in contrast to robust tumor formation by mock-transfected LNCaP cells. These findings indicate that addition of core3 O-glycans to β1 and α2 integrin subunits in prostate cancer cells suppresses tumor formation and tumor metastasis.Cancer cells often express surface carbohydrates different from normal cells (1). One such change is expression of sialyl Lewis X and Lewis B blood group antigens in cancer cells (2, 3). These structural elements are seen as capping oligosaccharides attached to the underlying glycan backbone where they likely function as ligands for cell adhesion molecules.The structure of underlying glycans also changes during malignant transformation and differentiation. In particular, there are several reports that an increase in the β1,6-N-acetylglucosaminyl branch in N-glycans synthesized by β1,6-N-acetylglucosaminyltransferase-V is associated with oncogenic transformation (4–7). Similar structural changes are seen in mucin-type O-glycans, which have N-acetylgalactosamine at the reducing end linked to polypeptide threonine or serine residues. Addition of different carbohydrate residues to N-acetylgalactosamine confers a variety of backbone structures on mucin-type O-glycans; the most abundant of those are classified as core1, core2, core3, and core4 O-glycans (8) (Fig. 1). Among these O-glycans, the synthesis of the core2 branch has been extensively studied particularly because conversion of core1 to core2 O-glycans was observed in T cell activation (9). Expression of core2 branch apparently represents an oncodifferentiation antigen because core2 branched O-glycans are synthesized in early stages of T cell differentiation, down-regulated in mature T cells, and reappear in T cell leukemia and immune deficiencies such as AIDS and Wiskott-Aldrich syndrome (for a review, see Ref. 10). In addition, overexpression of core2 O-glycans is seen in many cancers, including lung and breast carcinoma cells (11, 12).Open in a separate windowFIGURE 1.Biosynthetic pathways of mucin-type O-glycans. N-Acetylgalactosamine is transferred to a serine or threonine residue in a polypeptide. Resultant GalNAcα1→Ser/Thr is converted by core3 synthase (β3GnT-6) to GlcNAcβ1→3GalNAcα1→Ser/Thr (core3). Core3 is then converted to core4 by C2GnT-2 (C2GnT-M). GalNAcα1→Ser/Thr is also converted to core1, Galβ1→3GalNAcα1→Ser/Thr, by core1 synthase. Core1 is then converted to core2 by C2GnT-1, C2GnT-2, and C2GnT-3.By contrast, core3 and core4 O-glycans are synthesized in normal cells but apparently down-regulated in gastric and colorectal carcinoma (13, 14). Core3 O-glycans are synthesized by core3 synthase (β3GnT-6),² which adds β1,3-linked N-acetylglucosamine to N-acetylgalactosamine at the reducing terminus (15) (Fig. 1). Iwai et al. (16) showed that forced expression of core3 synthase in human fibrosarcoma HT1080 FP-10 cells resulted in significant reduction in the formation of lung tumor foci in mice after intravenous injection of tumor cells through a tail vein. However, the same study did not address whether the expression of core3 influences tumor metastasis because the cancer cells were intravenously injected and no primary tumor was formed to spread into the lung as metastasis in contrast to the other studies (17, 18). Core4 O-glycan is synthesized by addition of β1,6-linked N-acetylglucosamine to a core3 acceptor by core2 β1,6-N-acetylglucosamine M type (C2GnT-M) or C2GnT-2 (19, 20) (Fig. 1). Huang et al. (21) reported that C2GnT-M is down-regulated in colonic carcinoma cells and that forced expression of C2GnT-M in HCT116 colonic carcinoma cells significantly decreased cell invasion and subcutaneous tumor formation. How up-regulation of core3 and core4 O-glycans influences the pathophysiology of cells expressing core3 and core4 O-glycans has not been addressed.Cell-extracellular matrix interaction plays an essential role during acquisition of migration and invasive behavior of cancer cells. For example, α2β1 integrin is the major receptor for collagen (22) and most abundantly expressed in prostate cancer cells (23). Glycosylation on integrin is one of the important modulators of integrin functions, and many glycan structures, mainly N-glycans, have been studied. An increase of bisecting GlcNAc structure on α5β1 integrin inhibits the cell spreading and migration (24), and induced β1,6-GlcNAc sugar chains on N-glycans of β1 integrin result in stimulation of cell migration (25). However, it has not been addressed whether changes in O-glycans affect integrin maturation and functions.To determine the role of core3 O-glycans in tumor formation and metastasis, we analyzed PC3 and LNCaP human prostate cancer cells. We found that these cell lines express only small amounts of detectable core3 synthase; thus we transfected the cell lines with core3 synthase. Core3 synthase-transfected PC3 and LNCaP cells expressed increased amounts of core3 O-glycans in α2β1 integrin, showed the reduced maturation of β1 integrin and low levels of α2β1 integrin formation, migrated less efficiently through collagen and other extracellular matrix components, and were less invasive than mock-transfected cells. Moreover those cells exhibited decreased activation of focal adhesion kinase (FAK) compared with mock-transfected cells. Significantly PC3 cells expressing core3 O-glycans produced almost no primary tumors in the prostate and formed much fewer metastases in the draining lymph nodes than mock-transfected cells. Similarly LNCaP cells expressing core3 O-glycans produced much smaller subcutaneous tumors than mock-transfected LNCaP cells. These findings indicate that addition of core3 O-glycans to the α2β1 integrin leads to decreased cell migration and invasion, resulting in decreased prostate tumor formation and metastasis.     

Complex <Emphasis Type="Italic">N</Emphasis>-glycans or core 1-derived <Emphasis Type="Italic">O</Emphasis>-glycans are not required for the expression of stage-specific antigens SSEA-1, SSEA-3, SSEA-4, or Le<Superscript>Y</Superscript> in the preimplantation mouse embryo

The glycan epitopes termed stage-specific embryonic antigens (SSEA) occur on glycoproteins and glycolipids in mammals. However, it is not known whether these epitopes are attached to N- or O-glycans on glycoproteins and/or on glycolipids in the developing mouse embryo. In this paper the expression of the antigens SSEA-1, SSEA-3, SSEA-4 and Le^Y was examined on ovulated eggs, early embryos and blastocysts lacking either complex and hybrid N-glycans or core-1 derived O-glycans. In all cases, antigen expression determined by fluorescence microscopy of bound monoclonal antibodies to embryos at the stage of development of maximal expression was similar in mutant and control embryos. Thus, none of these developmental antigens are expressed solely on either complex N- or core 1-derived O-glycans attached to glycoproteins in the preimplantation mouse embryo. Furthermore, neither of these classes of glycan is essential for the expression of SSEA-1, SSEA-3, SSEA-4 or Le^Y on mouse embryos.     

Comparative Oncogenomics Implicates the Neurofibromin 1 Gene (NF1) as a Breast Cancer Driver

Identifying genomic alterations driving breast cancer is complicated by tumor diversity and genetic heterogeneity. Relevant mouse models are powerful for untangling this problem because such heterogeneity can be controlled. Inbred Chaos3 mice exhibit high levels of genomic instability leading to mammary tumors that have tumor gene expression profiles closely resembling mature human mammary luminal cell signatures. We genomically characterized mammary adenocarcinomas from these mice to identify cancer-causing genomic events that overlap common alterations in human breast cancer. Chaos3 tumors underwent recurrent copy number alterations (CNAs), particularly deletion of the RAS inhibitor Neurofibromin 1 (Nf1) in nearly all cases. These overlap with human CNAs including NF1, which is deleted or mutated in 27.7% of all breast carcinomas. Chaos3 mammary tumor cells exhibit RAS hyperactivation and increased sensitivity to RAS pathway inhibitors. These results indicate that spontaneous NF1 loss can drive breast cancer. This should be informative for treatment of the significant fraction of patients whose tumors bear NF1 mutations.     

Loss of Panx1 Impairs Mammary Gland Development at Lactation: Implications for Breast Tumorigenesis

Pannexin1 (Panx1) subunits oligomerize to form large-pore channels between the intracellular and extracellular milieu that have been shown to regulate proliferation, differentiation and cell death mechanisms. These key cellular responses are ultimately necessary for normal tissue development and function but the role of Panx1 in development, differentiation and function in many tissues remains unexplored, including that of the breast. Panx1 was identified to be expressed in the mammary gland through western blot and immunofluorescent analysis and is dynamically upregulated during pregnancy and lactation. In order to evaluate the role of Panx1 in the context of mammary gland development and function, Panx1^-/- mice were evaluated in comparison to wild-type mice in the mammary glands of virgin, lactating and involuting mice. Our results revealed that Panx1 ablation did not affect virgin or involuting mammary glands following histological and whole mount analysis. Panx1 was necessary for timely alveolar development during early lactation based on a decreased number of alveolar lumen following histological analysis and reduced proliferation following Ki67 immunofluorescent labelling. Importantly, the loss of Panx1 in lactating mammary glands did not overtly affect epithelial or secretory differentiation of the mammary gland suggesting that Panx1 is not critical in normal mammary gland function. In addition, PANX1 mRNA expression was correlated with negative clinical outcomes in patients with breast cancer using in silico arrays. Together, our results suggest that Panx1 is necessary for timely alveolar development following the transition from pregnancy to lactation, which may have implications extending to patients with breast cancer.     

Regulation of epithelial cell turnover and macrophage phenotype by epithelial cell-derived transforming growth factor beta1 in the mammary gland

Transforming growth factor beta1 (TGFB1) is a multi-functional cytokine that regulates cell proliferation, apoptosis and immune system responses. In the breast, the mammary epithelium is the primary source of TGFB1 and increased expression is associated with increased breast cancer risk. This study was conducted to investigate the roles of epithelial cell-derived TGFB1 in regulation of epithelial cell activity and macrophage phenotype in the mammary gland. Tgfb1 null mutant and wildtype mammary epithelium was transplanted into contra-lateral sides of the cleared mammary gland of TGFB1 replete scid mice. Transplanted tissue was analysed for markers of proliferation and apoptosis to determine the effect of Tgfb1 null mutation on epithelial cell turnover, and was analysed by immunohistochemistry to investigate the location, abundance and phenotype of macrophages. The number of proliferating and dying ductal epithelial cells, determined by BrdU and TUNEL, was increased by 35% and 3.3-fold respectively in mammary gland transplanted with Tgfb1 null epithelium compared to wildtype epithelium (p < 0.05). Abundance of F4/80+ macrophages in between Tgfb1 null epithelial cells compared to wildtype epithelial cells was increased by 50%. The number of iNOS+ and CCR7+ cells in the stroma surrounding Tgfb1 null alveolar epithelium was increased by 78% and 2-fold respectively, and dendriform MHC class II+ cells within ductal epithelium were decreased by 30%. We conclude that epithelial cell-derived TGFB1 in the mammary gland has two functions: (1) regulation of cellular turnover of epithelial cells, and (2) regulation of local macrophage phenotype. These findings shed new light on the diversity of roles of TGFB1 in the mammary gland which are likely to impact on breast cancer risk.     

RUNX2 correlates with subtype-specific breast cancer in a human tissue microarray,and ectopic expression of Runx2 perturbs differentiation in the mouse mammary gland

RUNX2, a master regulator of osteogenesis, is oncogenic in the lymphoid lineage; however, little is known about its role in epithelial cancers. Upregulation of RUNX2 in cell lines correlates with increased invasiveness and the capacity to form osteolytic disease in models of breast and prostate cancer. However, most studies have analysed the effects of this gene in a limited number of cell lines and its role in primary breast cancer has not been resolved. Using a human tumour tissue microarray, we show that high RUNX2 expression is significantly associated with oestrogen receptor (ER)/progesterone receptor (PR)/HER2-negative breast cancers and that patients with high RUNX2 expression have a poorer survival rate than those with negative or low expression. We confirm RUNX2 as a gene that has a potentially important functional role in triple-negative breast cancer. To investigate the role of this gene in breast cancer, we made a transgenic model in which Runx2 is specifically expressed in murine mammary epithelium under the control of the mouse mammary tumour virus (MMTV) promoter. We show that ectopic Runx2 perturbs normal development in pubertal and lactating animals, delaying ductal elongation and inhibiting lobular alveolar differentiation. We also show that the Runx2 transgene elicits age-related, pre-neoplastic changes in the mammary epithelium of older transgenic animals, suggesting that elevated RUNX2 expression renders such tissue more susceptible to oncogenic changes and providing further evidence that this gene might have an important, context-dependent role in breast cancer.KEY WORDS: RUNX2, Breast cancer, Transgenic model, Mammary development     

Reduced Androgen Receptor Expression Accelerates the Onset of ERBB2 Induced Breast Tumors in Female Mice

Androgen receptor (AR) is commonly expressed in both the epithelium of normal mammary glands and in breast cancers. AR expression in breast cancers is independent of estrogen receptor alpha (ERα) status and is frequently associated with overexpression of the ERBB2 oncogene. AR signaling effects on breast cancer progression may depend on ERα and ERBB2 status. Up to 30% of human breast cancers are driven by overactive ERBB2 signaling and it is not clear whether AR expression affects any steps of tumor progression in this cohort of patients. To test this, we generated mammary specific Ar depleted mice (MARKO) by combining the floxed allele of Ar with the MMTV-cre transgene on an MMTV-NeuNT background and compared them to littermate MMTV-NeuNT, Ar^fl/+ control females. Heterozygous MARKO females displayed reduced levels of AR in mammary glands with mosaic AR expression in ductal epithelium. The loss of AR dramatically accelerated the onset of MMTV-NeuNT tumors in female MARKO mice. In this report we show that accelerated MMTV-NeuNT-dependent tumorigenesis is due specifically to the loss of AR, as hormonal levels, estrogen and progesterone receptors expression, and MMTV-NeuNT expression were similar between MARKO and control groups. MMTV-NeuNT induced tumors in both cohorts displayed distinct loss of AR in addition to ERα, PR, and the pioneer factor FOXA1. Erbb3 mRNA levels were significantly elevated in tumors in comparison to normal mammary glands. Thus the loss of AR in mouse mammary epithelium accelerates malignant transformation rather than the rate of tumorigenesis.     

Changes in gene expression during the development of mammary tumors in MMTV-Wnt-1 transgenic mice

Background

In human breast cancer normal mammary cells typically develop into hyperplasia, ductal carcinoma in situ, invasive cancer, and metastasis. The changes in gene expression associated with this stepwise progression are unclear. Mice transgenic for mouse mammary tumor virus (MMTV)-Wnt-1 exhibit discrete steps of mammary tumorigenesis, including hyperplasia, invasive ductal carcinoma, and distant metastasis. These mice might therefore be useful models for discovering changes in gene expression during cancer development.

Results

We used cDNA microarrays to determine the expression profiles of five normal mammary glands, seven hyperplastic mammary glands and 23 mammary tumors from MMTV-Wnt-1 transgenic mice, and 12 mammary tumors from MMTV-Neu transgenic mice. Adipose tissues were used to control for fat cells in the vicinity of the mammary glands. In these analyses, we found that the progression of normal virgin mammary glands to hyperplastic tissues and to mammary tumors is accompanied by differences in the expression of several hundred genes at each step. Some of these differences appear to be unique to the effects of Wnt signaling; others seem to be common to tumors induced by both Neu and Wnt-1 oncogenes.

Conclusion

We described gene-expression patterns associated with breast-cancer development in mice, and identified genes that may be significant targets for oncogenic events. The expression data developed provide a resource for illuminating the molecular mechanisms involved in breast cancer development, especially through the identification of genes that are critical in cancer initiation and progression.     

ERBB2 overexpression suppresses stress-induced autophagy and renders ERBB2-induced mammary tumorigenesis independent of monoallelic Becn1 loss

Defective autophagy has been implicated in mammary tumorigenesis, as the gene encoding the essential autophagy regulator BECN1 is deleted in human breast cancers and Becn1^+/− mice develop mammary hyperplasias. In agreement with a recent study, which reports concurrent allelic BECN1 loss and ERBB2 amplification in a small number of human breast tumors, we found that low BECN1 mRNA correlates with ERBB2-overexpression in breast cancers, suggesting that BECN1 loss and ERBB2 overexpression may functionally interact in mammary tumorigenesis. We now report that ERBB2 overexpression suppressed autophagic response to stress in mouse mammary and human breast cancer cells. ERBB2-overexpressing Becn1^+/+ and Becn1^+/− immortalized mouse mammary epithelial cells (iMMECs) formed mammary tumors in nude mice with similar kinetics, and monoallelic Becn1 loss did not alter ERBB2- and PyMT-driven mammary tumorigenesis. In human breast cancer databases, ERBB2-expressing tumors exhibit a low autophagy gene signature, independent of BECN1 mRNA expression, and have similar gene expression profiles with non-ERBB2-expressing breast tumors with low BECN1 levels. We also found that ERBB2-expressing BT474 breast cancer cells, despite being partially autophagy-deficient under stress, can be sensitized to the anti-ERBB2 antibody trastuzumab (tzb) by further pharmacological or genetic autophagy inhibition. Our results indicate that ERBB2-driven mammary tumorigenesis is associated with functional autophagy suppression and ERBB2-positive breast cancers are partially autophagy-deficient even in a wild-type BECN1 background. Furthermore and extending earlier findings using tzb-resistant cells, exogenously imposed autophagy inhibition increases the anticancer effect of trastuzumab on tzb-sensitive ERBB2-expressing breast tumor cells, indicating that pharmacological autophagy suppression has a wider role in the treatment of ERBB2-positive breast cancer.     

Histone Demethylase Jumonji AT-rich Interactive Domain 1B (JARID1B) Controls Mammary Gland Development by Regulating Key Developmental and Lineage Specification Genes

The JmjC domain-containing H3K4 histone demethylase jumonji AT-rich interactive domain 1B (JARID1B) (also known as KDM5B and PLU1) is overexpressed in breast cancer and is a potential target for breast cancer treatment. To investigate the in vivo function of JARID1B, we developed Jarid1b^−/− mice and characterized their phenotypes in detail. Unlike previously reported Jarid1b^−/− strains, the majority of these Jarid1b^−/− mice were viable beyond embryonic and neonatal stages. This allowed us to further examine phenotypes associated with the loss of JARID1B in pubertal development and pregnancy. These Jarid1b^−/− mice exhibited decreased body weight, premature mortality, decreased female fertility, and delayed mammary gland development. Related to these phenotypes, JARID1B loss decreased serum estrogen level and reduced mammary epithelial cell proliferation in early puberty. In mammary epithelial cells, JARID1B loss diminished the expression of key regulators for mammary morphogenesis and luminal lineage specification, including FOXA1 and estrogen receptor α. Mechanistically, JARID1B was required for GATA3 recruitment to the Foxa1 promoter to activate Foxa1 expression. These results indicate that JARID1B positively regulates mammary ductal development through both extrinsic and cell-autonomous mechanisms.     

Highly sialylated mucin-type glycopeptide from porcine intestinal mucosa after heparin extraction: O-glycan profiling and immunological activity evaluation

Mucins are the major proteins that distributed on the intestinal mucosa layer and protect the intestine from pathogens infection. The composition of intestinal mucin O-glycans can affect the health of the gastrointestinal tract in pigs. Porcine intestinal mucosa is widely used as the main raw material of heparin extraction. The heparin extraction residues rich in mucins were usually wasted. The structure of mucin derived O-glycans in porcine intestinal mucosa are currently unknown. In this study, we isolated the mucins from the heparin extraction residues and profiled the O-glycans. After heparin extraction, mucin was digested with trypsin, and separated by strong anion exchange chromatography. The mucin derived O-glycans were release by alkaline β elimination, and analyzed by ultra high performance liquid chromatography-porous graphitized carbon-Fourier transform mass spectrometry (UPLC-PGC-FTMS/MS). Thirty five kinds of O-glycans were identified, most of which were Core 3-derived glycans. In particular, the O-glycans containing sialic acid Neu5Ac accounted for 71.93% of the total O-glycans, which were different from that of other species, including mouse intestine, fish intestine, and porcine colon. The high content sialylated mucin may explain its effect in biological processes. Furthermore, the immunological activity results indicated that the porcine intestinal mucin could promote phagocytosis and proliferation without any cytotoxic effects, which may aid in the development of immunomodulators.

     

Enpp1: A Potential Facilitator of Breast Cancer Bone Metastasis

Bone is the most common site of breast cancer metastasis and once established, it is frequently incurable. Critical to our ability to prevent and treat bone metastasis is the identification of the key factors mediating its establishment and understanding their biological function. To address this issue we previously carried out an in vivo selection process to isolate murine mammary tumor sublines possessing an enhanced ability to colonize the bone. A comparison of gene expression between parental cells and sublines by genome-wide cDNA microarray analysis revealed several potential mediators of bone metastasis, including the pyrophosphate-generating ectoenzyme Enpp1. By qRT-PCR and Western analysis we found that expression of Enpp1 was elevated in human breast cancer cell lines known to produce bone metastasis in animal models compared to non-metastatic and normal mammary epithelial cell lines. Further, in clinical specimens, levels of Enpp1 were significantly elevated in human primary breast tumors relative to normal mammary epithelium, with highest levels observed in breast-bone metastasis as determined by qRT-PCR and immunohistochemical analysis. To examine the potential role of Enpp1 in the development of bone metastasis, Enpp1 expression was stably increased in the breast cancer cell line MDA-MB-231 and the ability to colonize the bone following intracardiac and direct intratibial injection of athymic nude mice was determined. By both routes of administration, increased expression of Enpp1 enhanced the ability of MDA-MB-231 cells to form tumors in the bone relative to cells expressing vector alone, as determined by digital radiography and histological analysis. Taken together, these data suggest a potential role for Enpp1 in the development of breast cancer bone metastasis.