Stromal matrix metalloproteinase-11 is involved in the mammary gland postnatal development.

MMP-11 is a bad prognosis paracrine factor in invasive breast cancers. However, its mammary physiological function remains largely unknown. In the present study we have investigated MMP-11 function during postnatal mammary gland development and function using MMP-11-deficient (MMP-11-/-) mice. Histological and immunohistochemical analyses as well as whole-mount mammary gland staining show alteration of the mammary gland in the absence of MMP-11, where ductal tree, alveolar structures and milk production are reduced. Moreover, a series of transplantation experiments allowed us to demonstrate that MMP-11 exerts an essential local paracrine function that favors mammary gland branching and epithelial cell outgrowth and invasion through adjacent connective tissues. Indeed, MMP-11-/- cleared fat pads are not permissive for wild-type epithelium development, whereas MMP-11-/- epithelium transplants grow normally when implanted in wild-type cleared fat pads. In addition, using primary mammary epithelial organoids, we show in vitro that this MMP-11 pro-branching effect is not direct, suggesting that MMP-11 acts via production/release of stroma-associated soluble factor(s). Finally, the lack of MMP-11 leads to decreased periductal collagen content, suggesting that MMP-11 has a role in collagen homeostasis. Thus, local stromal MMP-11 might also regulate mammary epithelial cell behavior mechanically by promoting extracellular matrix stiffness. Collectively, the present data indicate that MMP-11 is a paracrine factor involved during postnatal mammary gland morphogenesis, and support the concept that the stroma strongly impact epithelial cell behavior. Interestingly, stromal MMP-11 has previously been reported to favor malignant epithelial cell survival and promote cancer aggressiveness. Thus, MMP-11 has a paracrine function during mammary gland development that might be harnessed to promote tumor progression, exposing a new link between development and malignancy.

Deficiency in trefoil factor 1 (TFF1) increases tumorigenicity of human breast cancer cells and mammary tumor development in TFF1-knockout mice.

Although trefoil factor 1 (TFF1; previously named pS2) is abnormally expressed in about 50% of human breast tumors, its physiopathological role in this disease has been poorly studied. Moreover, controversial data have been reported. TFF1 function in the mammary gland therefore needs to be clarified. In this study, using retroviral vectors, we performed TFF1 gain- or loss-of-function experiments in four human mammary epithelial cell lines: normal immortalized TFF1-negative MCF10A, malignant TFF1-negative MDA-MB-231 and malignant TFF1-positive MCF7 and ZR75.1. The expression of TFF1 stimulated the migration and invasion in the four cell lines. Forced TFF1 expression in MCF10A, MDA-MB-231 and MCF7 cells did not modify anchorage-dependent or -independent cell proliferation. By contrast, TFF1 knockdown in MCF7 enhanced soft-agar colony formation. This increased oncogenic potential of MCF7 cells in the absence of TFF1 was confirmed in vivo in nude mice. Moreover, chemically induced tumorigenesis in TFF1-deficient (TFF1-KO) mice led to higher tumor incidence in the mammary gland and larger tumor size compared with wild-type mice. Similarly, tumor development was increased in the TFF1-KO ovary and lung. Collectively, our results clearly show that TFF1 does not exhibit oncogenic properties, but rather reduces tumor development. This beneficial function of TFF1 is in agreement with many clinical studies reporting a better outcome for patients with TFF1-positive breast primary tumors.

MLN64 and MENTHO, two mediators of endosomal cholesterol transport.

MLN64 (metastatic lymph node 64) and MENTHO (MLN64 N-terminal homologue) are two late-endosomal proteins that share a conserved region of four transmembrane helices with three short intervening loops called the MENTAL domain (MLN64 N-terminal domain). This domain mediates MLN64 and MENTHO homo- and hetero-interactions, targets both proteins to late endosomes and binds cholesterol in vivo. In addition to the MENTAL domain, MLN64 contains a cholesterol-specific START domain [StAR (steroidogenic acute regulatory protein)-related lipid transfer domain]. The START domain is a protein module of approx. 210 residues that binds lipids, including sterols, and is present in 15 distinct proteins in mammals. Thus MLN64 and MENTHO define discrete cholesterol-containing subdomains within the membrane of late endosomes where they may function in cholesterol transport. The MENTAL domain might serve to maintain cholesterol at the membrane of late endosomes prior to its shuttle to cytoplasmic acceptor(s) through the START domain.

Characterization of the putative cholesterol transport protein metastatic lymph node 64 in the brain.

Intracellular management of cholesterol is a critical process in the brain. Deficits with cholesterol transport and storage are linked to neurodegenerative disorders such as Neimann-Pick disease type C and Alzheimer's disease. One protein putatively involved in cholesterol transport is metastatic lymph node 64 (MLN64). MLN64 localizes to late endosomes which are part of the cholesterol internalization pathway. However, a detailed pattern of MLN64 expression in the brain is unclear. Using immunocytochemical and immunoblot analyses, we demonstrated the presence of MLN64 in several tissue types and various regions within the brain. MLN64 immunostaining in the CNS was heterogeneous, indicating selective expression in discrete specific cell populations and regions. MLN64 immunoreactivity was detected in glia and neurons, which displayed intracellular labeling consistent with an endosomal localization. Although previous studies suggested that MLN64 may promote steroid production in the brain, MLN64 immunoreactivity did not colocalize with steroidogenic cells in the CNS. These results demonstrate that MLN64 is produced in the mouse and human CNS in a restricted pattern of expression, suggesting that MLN64 serves a cell-specific function in cholesterol transport.

Generation of a conditionally null allele of the laminin alpha1 gene.

Laminins are heterotrimeric glycoproteins of the basement membranes. Laminin 1 (alpha1, beta1, gamma1) is the major laminin expressed during early mouse embryogenesis. To gain access to the physiological function of laminin alpha1 chain, we developed a conditionally null allele of its encoding gene (Lama1) using the cre/loxP system. Floxed-allele-carrying mice (Lama1(flox/flox)) display no overt phenotype. Lama1(flox/flox) mice were crossed with transgenic deleter mice (CMV-Cre) to generate Lama1-deficient mice (Lama1(Delta/Delta)). Lama1(Delta/Delta) embryos die during the early postimplantation period after embryonic day 6.5. They lack Reichert's membrane, an extraembryonic basement membrane in which laminin alpha1 is normally highly expressed. In parallel, Lama1(Delta/Delta) embryos display 1) parietal and visceral endoderm differentiation defects with altered expression of cytokeratin 19 and GATA4, respectively, and 2) an induction of apoptosis. This new mouse model is of particular interest as it will allow time- and tissue-specific inactivation of the Lama1 gene in various organs.

The steroidogenic acute regulatory protein homolog MLN64, a late endosomal cholesterol-binding protein.

MLN64 is a transmembrane protein that shares homology with the cholesterol binding domain (START domain) of the steroidogenic acute regulatory protein. The steroidogenic acute regulatory protein is located in the inner membrane of mitochondria, where it facilitates cholesterol import into the mitochondria. Crystallographic analysis showed that the START domain of MLN64 is a cholesterol-binding domain. The present work was undertaken to determine which step of the intracellular cholesterol pathway MLN64 participates in. Using immunocytofluorescence, MLN64 colocalizes with LBPA, a lipid found specifically in late endosomes. Electron microscopy indicates that MLN64 is restricted to the limiting membrane of late endosomes. Microinjection or endocytosis of specific antibodies shows that the START domain of MLN64 is cytoplasmic. Deletion and mutagenesis experiments demonstrate that the amino-terminal part of MLN64 is responsible for its addressing. Although this domain does not contain conventional dileucine- or tyrosine-based targeting signals, we show that a dileucine motif (Leu(66)-Leu(67)) and a tyrosine residue (Tyr(89)) are critical for the targeting or the proper folding of the molecule. Finally, MLN64 colocalizes with cholesterol and Niemann Pick C1 protein in late endosomes. However, complementation assays show that MLN64 is not involved in the Niemann Pick C2 disease which, results in cholesterol lysosomal accumulation. Together, our results show that MLN64 plays a role at the surface of the late endosomes, where it might shuttle cholesterol from the limiting membrane to cytoplasmic acceptor(s).