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The E3 ubiquitin ligase RNF8 plays critical roles in maintaining genomic stability by promoting the repair of DNA double-strand breaks (DSBs) through ubiquitin signaling. Abnormal activation of Notch signaling and defective repair of DSBs promote breast cancer risk. Here, we found that low expression of the full- length RNF8 correlated with poor prognosis for breast cancer patients. Our data revealed that in addition to its role in the repair of DSBs, RNF8 regulated Notch1 signaling and cell-fate determination of mammary luminal progenitors. Mechanistically, RNF8 acted as a negative regulator of Notch signaling by ubiquitylating the active NOTCH1 protein (N1ICD), leading to its degradation. Consistent with abnormal activation of Notch signaling and impaired repair of DSBs in Rn/8-mutant mammary epithelial cells, we observed increased risk of mammary tumorigenesis in mouse models for RNF8 deficiency. Notably, deficiency of RNF8 sensitized breast cancer cells to combination of pharmacological inhibitors of Notch signaling and poly(ADP-ribose) polymerase (PARP), suggesting implications for treatment of breast cancer associated with impaired RNF8 expression or function., Introduction Regulation of Notch signaling and repair of double-strand breaks (DSBs) is critical for maintaining tissue homeostasis and suppression of cancer. Notch signaling is important for the control of cell-fate [...]

Breast cancer is one of the most common cancers in humans and will on average affect up to one in eight women in their lifetime in the United States and Europe (1). The Women's Health Initiative and the Million Women Study have shown that hormone replacement therapy is associated with an increased risk of incident and fatal breast cancer (2,3). In particular, synthetic progesterone derivatives (progestins) such as medroxyprogesterone acetate (MPA), used in millions of women for hormone replacement therapy and contraceptives, markedly increase the risk of developing breast cancer. Here we show that the invivoadministration of MPA triggers massive induction of the key osteoclast differentiation factor RANKL (receptor activator of NF-κB ligand) in mammary-gland epithelial cells. Genetic inactivation of the RANKL receptor RANK in mammary-gland epithelial cells prevents MPA-induced epithelial proliferation, impairs expansion of the [CD49f.sup.hi] stem-cell-enriched population, and sensitizes these cells to DNA-damage-induced cell death. Deletion of RANK from the mammary epithelium results in a markedly decreased incidence and delayed onset of MPA-driven mammary cancer. These data show that the RANKL/RANK system controls the incidence and onset of progestin-driven breast cancer., RANKL (also known as ODF, TRANCE, OPGL and TNFSF11) and its receptor RANK (also known as TRANCE-R and TNFRSF11A) are essential for the development and activation of osteoclasts (4,5). The [...]

Some cancers have been stratified into subclasses based on their unique involvement of specific signaling pathways. The mapping of human cancer genomes is revealing a vast number of somatic alterations; however, the identification of clinically relevant molecular tumor subclasses and their respective driver genes presents challenges. This information is key to developing more targeted and personalized cancer therapies. Here, we generate a new mouse model of genomically unstable osteosarcoma (OSA) that phenocopies the human disease. Integrative oncogenomics pinpointed cAMP-dependent protein kinase type I, α regulatory subunit (Prkar1a) gene deletions at 11qE1 as a recurrent genetic trait for a molecularly distinct subclass of mouse OSA featuring RANKL overexpression. Using mouse genetics, we established that Prkar1a is a bone tumor suppressor gene capable of directing subclass development and driving RANKL overexpression during OSA tumorigenesis. Finally, we uncovered evidence for a PRKAR1A-low subset of human OSA with distinct clinical behavior. Thus, tumor subclasses develop in mice and can potentially provide information toward the molecular stratification of human cancers., Introduction Common mutational processes underpin the development of diverse cancers by perturbing key regulatory networks that have evolved to resist the emergence of neoplastic cells in normal tissues. Some human [...]

The cell death receptor Fas plays a role in the establishment of fulminant hepatitis, a major cause of druginduced liver failure. Fas activation elicits extrinsic apoptotic and hepatoprotective signals; however, the mechanisms by which these signals are integrated during disease are unknown. Tissue inhibitor of metalloproteinases 3 (TIMP3) controls the critical sheddase a disintegrin and metalloproteinase 17 (ADAM17) and may dictate stress signaling. Using mice and cells lacking TIMP3, ADAM17, and ADAM17-regulated cell surface molecules, we have found that ADAM17-mediated ectodomain shedding of TNF receptors and EGF family ligands controls activation of multiple signaling cascades in Fas-induced hepatitis. We demonstrated that TNF signaling promoted hepatotoxicity, while excessive TNF receptor 1 (TNFR1) shedding in [Timp3.sup.-/-] mice was protective. Compound [Timp3.sup.-/-][Tnf.sup.-/-] and [Timp3.sup.-/-] [Tnfr1.sup.-/-] knockout conferred complete resistance to Fas-induced toxicity. Loss of Timp3 enhanced metalloproteinase-dependent EGFR signaling due to increased release of the EGFR ligands TGF-α, amphiregulin, and HB-EGF, while depletion of shed amphiregulin resensitized [Timp3.sup.-/-] hepatocytes to apoptosis. Finally, adenoviral delivery of Adam17 prevented acetaminophen-induced liver failure in a clinically relevant model of Fas-dependent fulminant hepatitis. These findings demonstrate that TIMP3 and ADAM17 cooperatively dictate cytokine signaling during death receptor activation and indicate that regulated metalloproteinase activity integrates survival and death signals during acute hepatotoxic stress., Introduction Hepatocytes are highly sensitive to death receptor-mediated apoptosis. Engagement of this pathway elicits multiple and complex extrinsic signals, some apoptotic and others hepatoprotective. The cumulative tissue response to these [...]

Reproductive history is the strongest risk factor for breast cancer after age, genetics and breast density (1,2). Increased breast cancer risk is entwined with a greater number of ovarian hormone-dependent reproductive cycles, yet the basis for this predisposition is unknown (3-5). Mammary stem cells (MaSCs) are located within a specialized niche in the basal epithelial compartment that is under local and systemic regulation (6). The emerging role of MaSCs in cancer initiation warrants the study of ovarian hormones in MaSC homeostasis. Here we show that the MaSC pool increases 14-fold during maximal progesterone levels at the luteal dioestrus phase of the mouse. Stem-cell-enriched [CD49f.sup.hi] cells amplify at dioestrus, or with exogenous progesterone, demonstrating a key role for progesterone in propelling this expansion. In aged mice, [CD49f.sup.hi] cells display stasis upon cessation of the reproductive cycle. Progesterone drives a series of events where luminal cells probably provide Wnt4 and RANKL signals to basal cells which in turn respond by upregulating their cognate receptors, transcriptional targets and cell cycle markers. Our findings uncover a dynamic role for progesterone in activating adult MaSCs within the mammary stem cell niche during the reproductive cycle, where MaSCs are putative targets for cell transformation events leading to breast cancer., Mammary stem cells (MaSCs) are widely considered to be constant and quiescent unless activated at puberty and pregnancy, which are brief and isolated developmental periods (7-9). In contrast, the reproductive [...]

Author(s): D. Holstead Jones [1, 2, 3, 11, 10]; Tomoki Nakashima [1, 11]; Otto H. Sanchez [4, 10]; Ivona Kozieradzki [1, 2, 3]; Svetlana V. Komarova [5]; Ildiko Sarosi [6]; [...]

Mus81-Eme1 endonuclease has been implicated in the rescue of stalled replication forks and the resolution of meiotic recombination intermediates in yeast. We used gene targeting to study the physiological requirements of Mus81 in mammals. Mus[81.sup.-/-] mice are viable and fertile, which indicates that mammalian Mus81 is not essential for recombination processes associated with meiosis. Mus81-deficient mice and cells were hypersensitive to the DNA cross-linking agent mitomycin C but not to γ-irradiation. Remarkably, both homozygous Mus[81.sup.-/-] and heterozygous Mus[81.sup.+/-] mice exhibited a similar susceptibility to spontaneous chromosomal damage and a profound and equivalent predisposition to lymphomas and other cancers. These studies demonstrate a critical role for the proper biallelic expression of the mammalian Mus81 in the maintenance of genomic integrity and tumor suppression., Homology-directed DNA repair is a major pathway that facilitates the accurate removal of chromosomal damage resulting from exogenous stimuli, stalled replication forks (RFs), or genetically programmed processes (1-3). Homologous recombination [...]

Tissue inhibitors of metalloproteinases (TIMPs) regulate extracellular matrix (ECM) degradation by matrix metalloproteinases (MMPs). We have examined the role of TIMP-3 on ECM homeostasis and bronchiole branching morphogenesis during murine embryogenesis. Employing an in vitro organ culture system, we found decreased bronchiolar branching in null lungs when compared with wild type (WT) counterparts after 2 days in culture. When a synthetic inhibitor of MMPs at low dose was added to the culture system, branching was augmented regardless of genotype. Gelatin and in situ zymography revealed that null lungs exhibited enhanced activation of MMPs throughout lung development. We analysed the impact of increased MMP activity on a number of ECM molecules by Western blot analysis, but found that only fibronectin abundance was consistently reduced in the null lungs throughout development. To confirm that our observed defect in culture was not simply a developmental delay in the null lung, we examined null and WT lungs from newborn pups. Here, we found not only a reduced number of bronchioles in the null, but also that the bronchiole tubes were dilated compared with controls and that alveologenesis was attenuated. We propose that the deletion of TIMP-3 disrupts the exquisite TIMP/MMP balance required for proper focal ECM proteolysis, which leads to correct bronchiole branching morphogenesis in the developing mouse lung. Keywords: TIMP; MMP; ECM; Lung development; Epithelial-mesenchyme interactions

Extracellular matrix (ECM) fragments or cryptic sites unmasked by proteinases have been postulated to affect tissue remodeling and cancer progression. Therefore, the elucidation of their identities and functions is of great interest. Here, we show that matrix metalloproteinases (MMPs) generate a domain (Dill) from the ECM macromolecule laminin-5. Binding of a recombinant Dill fragment to epidermal growth factor receptor stimulates downstream signaling (mitogen-activated protein kinase), MMP-2 gene expression, and cell migration. Appearance of this cryptic ECM ligand in remodeling mammary gland coincides with MMP-mediated involution in wild-type mice, but not in tissue inhibitor of metalloproteinase 3 (TIMP-3)-deficient mice, supporting physiological regulation of Dill liberation. These findings indicate that ECM cues may operate via direct stimulation of receptor tyrosine kinases in tissue remodeling, and possibly cancer invasion.

The osteoclast differentiation factor osteoprotegerin-ligand (OPGL), essential for bone remodeling, has been found to be required for mammary gland development. Mice that have no OPGL or lack its receptor RANK fail to form lobulo-alveolar mammary structures in pregnancy, resulting in death of newborns. The data give a novel paradigm in mammary gland development and give a rationale related to evolution for hormonal regulation and gender bias for female osteoporosis.

Author(s): Takehiko Sasaki [1]; Junko Irie-Sasaki [1]; Yasuo Horie; Kurt Bachmaier [1]; Jimmie E. Fata [2]; Martin Li [3]; Akira Suzuki [1]; Dennis Bouchard [1]; Alexandra Ho [1]; Mark Redston [...]

Golgi [beta]1,6N-acetylglucosaminyltransferase V (MGAT5) is required in the biosynthesis of [beta]1,6GlcNAc-branched N-linked glycans attached to cell surface and secreted glycoproteins. Amounts of MGAT5 glycan products are commonly increased in malignancies, and correlate with disease progression. To study the functions of these N-glycans in development and disease, we generated mice deficient in Mgat5 by targeted gene mutation. These Mgat5[sup.-/-] mice lacked Mgat5 products and appeared normal, but differed in their responses to certain extrinsic conditions. Mammary tumor growth and metastases induced by the polyomavirus middle T oncogene was considerably less in Mgat5[sup.-/-] mice than in transgenic littermates expressing Mgat5. Furthermore, Mgat5 glycan products stimulated membrane ruffling and phosphatidylinositol 3 kinase-protein kinase B activation, fueling a positive feedback loop that amplified oncogene signaling and tumor growth in vivo. Our results indicate that inhibitors of MGAT5 might be useful in the treatment of malignancies by targeting their dependency on focal adhesion signaling for growth and metastasis., Author(s): Maria Granovsky [1, 2]; Jimmie Fata [3]; Judy Pawling [1]; William J. Muller [4]; Rama Khokha [3]; James W. Dennis (corresponding author) [1, 2] Malignant transformation is accompanied by [...]

Escape of cancer cells from the circulation (extravasation) is thought to be a major rate-limiting step in metastasis, with few cells being able to extravasate. Furthermore, highly metastatic cells are believed to extravasate more readily than poorly metastatic cells. We assessed in vivo the extravasation ability of highly metastatic ras-transformed NIH 3T3 cells (PAP2) versus control nontumorigenic non-transformed NIH 3T3 cells and primary mouse embryo fibroblasts. Fluorescently labeled cells were injected intravenously into chicken embryo chorioallantoic membrane and analyzed by intravital videomicroscopy. The chorioallantoic membrane is an appropriate model for studying extravasation, since, at the embryonic stage used, the microvasculature exhibits a continuous basement membrane and adult permeability properties. The kinetics of extravasation were assessed by determining whether individual cells (n = 1481) were intravascular, extravascular, or in the process of extravasation, at 3, 6, and 24 h after injection. Contrary to expectations, our results showed that all three cell types extravasated with the same kinetics. By 24 h after injection >89% of observed cells had completed extravasation from the capillary plexus. After extravasation, individual fibroblasts of all cell types demonstrated preferential migration within the mesenchymal layer toward arterioles, not to venules or lymphatics. Thus in this model and for these cells, extravasation is independent of metastatic ability. This suggests that the ability to extravasate in vivo is not necessarily predictive of subsequent metastasis formation, and that postextravasation events may be key determinants in metastasis.

A study is conducted on Rhoc deficient mice to show that RhoC is dispensable for embryonic and post-natal development. It is demonstrated that loss of RhoC does not affect tumor development but decreases tumor cell motility and metastatic cell survival leading to a drastic inhibition of metastasis.

Author(s): D. Holstead Jones [1, 2, 3, 4]; Tomoki Nakashima [2]; Otto H. Sanchez [6]; Ivona Kozieradzki [2, 3, 4]; Svetlana V. Komarova [5]; Ildiko Sarosi [7]; Sean Morony [7]; [...]

Antisense RNA--Induced Reduction in Murine TIMP Levels Confers Oncogenicity on Swiss 3T3 Cells AMURINE CDNA CLONE CALLED 16C8 was identified initially by differential screening of a cDNA library with radiolabeled [...]

Author(s): Takehiko Sasaki; Junko Irie-Sasaki; Yasuo Horie; Kurt Bachmaier; Jimmie E. Fata; Martin Li; Akira Suzuki; Dennis Bouchard; Alexandra Ho; Mark Redston; Steven Gallinger; Rama Khokha; Tak W. Mak; Phillip [...]

Research was conducted to examine insulin-like growth factor II (IGF-II) signaling in neoplastic proliferation. The objective was to show that tissue inhibitor of metalloproteinase 1 blocks liver hyperplasia during tumor development despite TAg-mediated reactivation of IGF-II. Results indicate that TIMP-1 inhibits liver hyperplasia by reducing the activity of the tumor-inducing mitogen, IGF-II.

The dynamic process of mammary ductal morphogenesis depends on regulated epithelial proliferation and extracellular matrix (ECM) turnover. Epithelial cell-matrix contact closely dictates epithelial proliferation, differentiation, and survival. Despite the fact that tissue inhibitors of metalloproteinases (Timps) regulate ECM turnover, their function in mammary morphogenesis is unknown. We have delineated the spatiotemporal expression of all Timps (Timp-1 to Timp-4) during discrete phases of murine mammary development. Timp mRNAs were abundant in mammary tissue, each displaying differential expression patterns with predominant localization in luminal epithelial cells. Timp-1 mRNA was unique in that its expression was limited to the stage at which epithelial proliferation was high. To assess whether Timp-1 promotes or inhibits epithelial cell proliferation we manipulated mammary Timp-1 levels, genetically and biochemically. Down-regulation of epithelial-derived Timp-1 in transgenic mice, by mouse mammary tumor virus promoter-directed Timp-1 antisense RNA expression, led to augmented ductal expansion and increased number of ducts (P < 0.004). In these transgenics the integrity of basement membrane surrounding epithelial ducts, as visualized by laminin-specific immunostaining, was breached. In contrast to these mice, ductal expansion was markedly attenuated in the proximity of implanted recombinant Timp-1-releasing pellets (rTIMP-1), without an increase in basement membrane deposition around migrating terminal end buds. Epithelial proliferation and apoptosis were measured to determine the basis of altered ductal expansion. Luminal epithelial proliferation was increased by 55% (P < 0.02) in Timp-1-reduced transgenic mammary tissue and, conversely, decreased by 38% (P < 0.02) in terminal end buds by implanted rTIMP-1. Epithelial apoptosis was minimal and remained unaffected by Timp-1 manipulations. We conclude that Timps have an integral function in mammary morphogenesis and that Timp-1 regulates mammary epithelial proliferation in vivo, at least in part by maintaining basement membrane integrity. Key Words: Timps; mammary morphogenesis; transgenic mice; epithelial proliferation.