Int6 has been shown to be an interactive participant with the protein translation initiation complex eIF3, the COP9 signalosome and the regulatory lid of the 26S proteasome. Insertion of mouse mammary tumor virus into the Int6 locus creates a C-terminally truncated form of the protein. Expression of the truncated form of Int6 (Int6sh) in stably transfected human and mouse mammary epithelial cell lines leads to cellular transformation. In addition, decreased expression of Int6/eIF3e is observed in approximately one third of all human breast carcinomas. To validate that Int6sh has transforming activity in vivo, a transgenic mouse model was designed using the whey acidic protein (Wap) promoter to target expression of truncated Int6 to differentiating alveolar epithelial cells in the mammary gland. Microarray analyses were performed on normal, premalignant and malignant WapInt6sh expressing tissues. Mammary tumors developed in 40% of WapInt6sh heterozygous parous females at an average age of 18 months. Contralateral mammary glands from WapInt6sh mice with tumors and glands from non-tumor bearing mice contained widespread focal alveolar hyperplasia. Only 4% of WapInt6sh non-breeding females developed tumors by two years of age. The Wap promoter is active only during estrus in the mammary tissue of cycling non-pregnant mice. Microarray analyses of mammary tissues demonstrated that Int6sh expression in the alveolar tissue altered the mammary transcriptome in a specific manner that was detectable even in the first pregnancy. These observations provide the first in vivo evidence that mammary-specific expression of the Int6sh mutation leads to persistence of alveolar hyperplasia with the accompanying increased predisposition to mammary tumorigenesis. We investigated the co-localization of [3H]-thymidine and ERα or PR in mammary tissue from mice that had received treatment of estrogen, progesterone and prolactin subsequent to a long chase period to identify label-retaining cells. Label retaining epithelial cells (LREC) comprised approximately 2.0% of the entire mammary epithelium. ERα-positive (ERα+) and PR-positive (PR+) cells represented roughly 30-40% of the LREC subpopulation. Administration of estrogen, progesterone and prolactin altered the percent of LREC expressing ERα. The results presented here support the premise that a subpopulation of LREC in the murine mammary gland is ERα and/or PR-positive. This suggests that certain mammary LREC (sic stem cells) remain stably positive for these receptors, raising the possibility, that LREC comprise a hierarchy of asymmetrically cycling mammary stem/progenitor cells distinguished one from another by the presence or absence of nuclear steroid receptor expression. Growth factors and hormones are responsible for development of the mammary gland and can contribute to mammary carcinogenesis. The transforming growth factors (TGF) α and β1 demonstrate opposing effects on the mammary epithelium. TGFα is a mitogen and survival factor for mammary secretory cells and is often upregulated in cancer while TGFβ1 may act as a growth suppressor and has been shown to inhibit alveolar development and lactogenesis. To examine the contradistinct effects of TGFα and TGFβ1 on mammary epithelium, we crossed MT-TGFα mice with WAP-TGFβ1 transgenic mice. The newly generated bitransgenic mice failed to nurse their pups and were resistant to mammary tumorigenesis (0% at 12 months of age), compared to single transgenic MT-TGFα in which the majority (65% at 12 months of age) of the mice developed hyperplastic alveolar mammary lesions. Transplantation studies showed that bitransgenic tissue was highly resistant to tumor formation even after multiple pregnancies. WAP-TGFβ1 mammary transplants often failed to grow and fully fill cleared mammary fat pads upon transplantation. This phenotype was completely reversed in the bitransgenic implants, which grew as well as normal epithelium upon transplantation. In addition TGFα and bitransgenic TGFα/TGFβ1 mice had reduced rates of apoptosis during involution as compared to wild type and TGFβ1. These data demonstrate that TGFβ1 and TGFα exhibit opposing effects upon mammary epithelium when expressed alone but when expressed together result in reciprocally suppressive effects upon one another in the context of mammary development and tumorigenesis. We have previously described pluripotent, parity-induced mammary epithelial cells (PI-MEC) marked by Rosa26-lacZ expression in the mammary glands of parous females, PI-MEC act as lobule-limited epithelial stem/progenitor cells. To determine whether parity is necessary to generate PI-MEC, we incubated mammary explant cultures from virgin mice in vitro with insulin alone (I), hydrocortisone alone (H), prolactin alone (Prl), or a combination of these lactogenic hormones (IHPrl). Insulin alone activated the WAP-Cre gene. Hydrocortisone and prolactin alone did not. Any combination of hormones that included insulin was effective. Only I, H and Prl together were able to induce secretory differentiation and milk protein synthesis. In addition, EGF, IGF-2 and IGF-1 added individually produced activated (lacZ+) PI-MEC in explant cultures. Neither estrogen nor progesterone induced WAP-Cre expression in the explants. None of these positive initiators of WAP-Cre expression in PI-MEC were effective in mammospheres or two-dimensional cultures of mammary epithelium, indicating the indispensability of epithelial-stromal interaction in PI-MEC activation. Like PI-MEC, lacZ+ cells from virgin explants proliferated and contributed progeny to mammospheres in vitro and to epithelial outgrowths in vivo after transplantation. lacZ+ cells induced in virgin mouse mammary explants were multipotent (like PI-MEC) in impregnated hosts producing lacZ+ mammary alveolar structures comprised of both myoepithelial and luminal progeny. These data demonstrate that PI-MEC, a mammary epithelial sub-population of lobule-limited progenitor cells, are present in nulliparous female mice before parity and, like the PI-MEC observed following parity, are capable of proliferation, self-renewal and the capacity to produce progeny of diverse cell fates. Transgenic mice expressing the wildtype Her2/neu (ErbB2) oncogene under transcriptional regulation of the MMTV-LTR seem to be suitable for studying the involvement of PI-MECs in pregnancy-associated mammary tumorigenesis since this animal model exhibits a relatively long latency of tumorigenesis (T50 of 205 days). Using this animal model, we demonstrated that a) multiparous females consistently exhibited accelerated tumorigenesis compared to their nulliparous littermate controls in a mixed genetic background, and b) PI-MECs were indeed primary targets of neoplastic transformation. To further substantiate that PI-MECs are primary targets for neoplastic transformation in MMTV-neu transgenic mice, we eliminated or greatly impaired the growth of PI-MECs by deleting the Tsg101 gene in cells that transiently activated WAP-Cre (i.e. females that carry two transgenes, MMTV-neu and WAP-Cre, in a homozygous Tsg101 conditional knockout background). In multiparous MMTV-neu females, impaired genesis or elimination of PI-MECs resulted in a significantly reduced tumor onset suggesting that restraining the growth and survival of differentiating alveolar cells during pregnancy eliminates the cellular basis for transformation in this model. This assumption is currently being verified again by deleting the Jak2 gene from the entire mammary ductal tree in MMTV-neu transgenic mice
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