The accumulation of mutations is a contributing factor in the initiation of premalignant mammary lesions and their progression to malignancy and metastasis. We have used a mouse model in which the carcinogen is the mouse mammary tumor virus (MMTV) which induces clonal premalignant mammary lesions and malignant mammary tumors by insertional mutagenesis. Identification of the genes and signaling pathways affected in MMTV-induced mouse mammary lesions provides a rationale for determining whether genetic alteration of the human orthologues of these genes/pathways may contribute to human breast carcinogenesis. A high- throughput platform for inverse PCR to identify MMTV-host junction fragments and their nucleotide sequences in a large panel of MMTV-induced lesions was developed. Validation of the genes affected by MMTV-insertion was carried out by microarray analysis. Common integration site (CIS) means that the gene was altered by an MMTV proviral insertion in at least two independent lesions arising in different hosts. Three of the new genes identified as CIS for MMTV were assayed for their capability to confer on HC11 mouse mammary epithelial cells the ability for invasion, anchorage independent growth and tumor development in nude mice. Analysis of MMTV induced mammary premalignant hyperplastic outgrowth (HOG) lines and mammary tumors led to the identification of CIS restricted to 35 loci. Within these loci members of the Wnt, Fgf and Rspo gene families plus two linked genes (Npm3 and Ddn) were frequently activated in tumors induced by MMTV. A second group of 15 CIS occur at a low frequency (2-5 observations) in mammary HOGs or tumors. In this latter group the expression of either Phf19 or Sdc2 was shown to increase HC11 cells invasion capability. Foxl1 expression conferred on HC11 cells the capability for anchorage- independent colony formation in soft agar and tumor development in nude mice. The published transcriptome and nucleotide sequence analysis of gene expression in primary human breast tumors was interrogated. Twenty of the human orthologues of MMTV CIS associated genes are deregulated and/or mutated in human breast tumors. We are currently testing the hypothesis that non-cellular extracts from normal mammary tissue can reprogram non-mammary and cancer cell in the epithelium-divested mammary fat pad. Breast cancer is the second leading cause of cancer deaths in the United States. At present, the etiology of breast cancer is unknown;however the possibility of a distinct cell of origin, i.e. a cancer stem cell, is a heavily investigated area of research. Influencing signals from the tissue niche are known to affect stem cells. Literature has shown that cancer cells lose their tumorigenic potential and display 'normal'behavior when placed into 'normal'ontogenic environments. Therefore, it may be the case that the tissue microenvironment is able to generate signals to redirect cancer cell fate. Previously, we showed that pluripotent human embryonal carcinoma cells could be redirected by the regenerating mammary gland microenvironment to contribute epithelial progeny for 'normal'gland development in-vivo. We showed that that human metastatic, non-metastatic, and metastasis-suppressed breast cancer cells proliferate and contribute to normal mammary gland development in-vivo without tumor formation. Immunochemistry for human-specific mitochondria, keratin 8 and 14, as well as human-specific milk proteins (alpha-lactalbumin, impregnated transplant hosts) confirmed the presence of human cell progeny. Features consistent with normal mammary gland development as seen in intact hosts (duct, lumen formation, development of secretory acini) were recapitulated in both primary and secondary outgrowths from chimeric implants. These results suggest the dominance of the tissue microenvironment over cancer cell fate. This work demonstrates that cultured human breast cancer cells (metastatic and non-metastatic) respond developmentally to signals generated by the mouse mammary gland microenvironment during gland regeneration in-vivo. Experiments were conducted to redirect mouse Embryonic Stem (ES) cells from a tumorigenic phenotype to a normal mammary epithelial phenotype in vivo. Mixing LacZ-labeled ES cells with normal mouse mammary epithelial cells at ratios of 1:5 and 1:50 in phosphate buffered saline and immediately inoculating them into epithelium-divested mammary fat pads of immune-compromised mice accomplished this. Our results indicate that tumorigenesis occurs only when normal mammary ductal growth is not achieved in the inoculated fat pads. When normal mammary gland growth occurs, we find ES cells (LacZ+) progeny interspersed with normal mammary cell progeny in the mammary epithelial structures. We demonstrate that these progeny, marked by LacZ expression, differentiate into multiple epithelial subtypes including steroid receptor positive luminal cells and myoepithelial cells indicating that the ES cells are capable of epithelial multipotency in this context but do not form teratomas. In addition, in secondary transplants, ES cell progeny proliferate, contribute apparently normal mammary progeny, maintain their multipotency and do not produce teratomas. The mouse mammary epithelial cell hierarchy contains both multipotent stem cell as well as lineage limited duct and lobular progenitor cell functions. The latter-also termed parity-identified mammary epithelial cells (PI-MEC)-are marked by beta-galactosidase (beta-Gal) expression following pregnancy and involution in WAP-Cre/Rosa26-flox-stop-flox-lacZ (WC/R26) mice and are the targets of tumorigenic transformation in MMTV-erbB2 transgenic mice. Here we demonstrate that an epithelial population distinct from PI-MEC is transformed during WAP-Int3 tumorigenesis. As expected, WAP-Int3/WC/R26 triple-transgenic mice failed to undergo secretory alveolar development, failed to lactate and developed mammary tumors. Following pregnancy and involution, Beta-gal+ mammary epithelial cells were found in the normal mammary tissue, but the resulting mammary tumors were all beta-gal-. WAP-Int3/WC/R26 mammary glands contained ample ERa+ mammary epithelial cells (MEC), but only rare (1%) PR+ and RANKL+ cells. In addition, dissociated MEC from WAP-Int3/WC/R26 glands failed to regenerate a mammary tree upon transplantation into a cleared fat-pad of a nu/nu recipient mouse. However, when mixed with normal MEC, PI-MEC from WAP-Int3/WC/R26 mice contributed progeny to the resulting functional outgrowth. The WAP-Int3/WC/R26 derived PI-MEC displayed all of the properties of fully functional lobular progenitors including giving rise to ERa+, PR+, SMA+, and RANKL+ epithelial progeny. These results demonstrate that WAP-Int3 has no oncogenic effect upon PI-MEC and that the expansion of functional lobular progenitors is required for secretory alveolar development and lactation. Furthermore, lobular progenitor function is ultimately controlled by signals within its microenvironment.

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Bruno, Robert D; Boulanger, Corinne A; Rosenfield, Sonia M et al. (2014) Paracrine-rescued lobulogenesis in chimeric outgrowths comprising progesterone-receptor-null mammary epithelium and redirected wild-type testicular cells. J Cell Sci 127:27-32
Boulanger, Corinne A; Bruno, Robert D; Mack, David L et al. (2013) Embryonic stem cells are redirected to non-tumorigenic epithelial cell fate by interaction with the mammary microenvironment. PLoS One 8:e62019
Wagner, K-U; Booth, B W; Boulanger, C A et al. (2013) Multipotent PI-MECs are the true targets of MMTV-neu tumorigenesis. Oncogene 32:1338
Bruno, Robert D; Rosenfield, Sonia M; Smith, Gilbert H (2013) Late developing mammary tumors and hyperplasia induced by a low-oncogenic variant of mouse mammary tumor virus (MMTV) express genes identical to those induced by canonical MMTV. Mol Cancer 12:79
Boulanger, Corinne A; Smith, Gilbert H (2009) Reprogramming cell fates in the mammary microenvironment. Cell Cycle 8:1127-32
Smith, Gilbert H; Salomon, David S; Vonderhar, Barbara K (2009) Re: "Evidence that an early pregnancy causes a persistent decrease in the number of functional mammary epithelial stem cells-implications for pregnancy-induced protection against breast cancer" by Siwko et al. Stem Cells 27:1223;author reply 1224-5
Smith, Gilbert H (2008) Stem cells, hormones, and mammary cancer. Adv Exp Med Biol 617:69-78
Booth, Brian W; Mack, David L; Androutsellis-Theotokis, Andreas et al. (2008) The mammary microenvironment alters the differentiation repertoire of neural stem cells. Proc Natl Acad Sci U S A 105:14891-6
Booth, Brian W; Boulanger, Corinne A; Smith, Gilbert H (2008) Selective segregation of DNA strands persists in long-label-retaining mammary cells during pregnancy. Breast Cancer Res 10:R90
Booth, Brian W; Boulanger, Corinne A; Smith, Gilbert H (2008) Stem cells and the mammary microenvironment. Breast Dis 29:57-67

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