Perturbation of Rb, p53, and Brca1 or Brca2 Cooperate in Inducing Metastatic Serous Epithelial Ovarian Cancer

The majority of human high grade serous epithelial ovarian cancer (SEOC) is characterized by frequent mutations in p53 and alterations in the RB and FOXM1 pathways. A subset of human SEOC harbors a combination of germline and somatic mutations as well as epigenetic dysfunction for BRCA1/2. Using Cre-conditional alleles and intrabursal induction by Cre-expressing adenovirus in genetically engineered mice, we analyzed the roles of pathway perturbations in epithelial ovarian cancer initiation and progression. Inactivation of RB-mediated tumor suppression induced surface epithelial proliferation with progression to stage I carcinoma. Additional biallelic inactivation and/or missense p53 mutation in the presence or absence of Brca1/2 caused progression to stage IV disease. As in human SEOC, mice developed peritoneal carcinomatosis, ascites, and distant metastases. Unbiased gene expression and metabolomic profiling confirmed that Rb, p53, and Brca1/2-triple mutant tumors aligned with human SEOC, and not with other intraperitoneal cancers. Together, our findings provide a novel resource for evaluating disease etiology and biomarkers, therapeutic evaluation, and improved imaging strategies in epithelial ovarian cancer

Membrane-type MMPs enable extracellular matrix permissiveness and mesenchymal cell proliferation during embryogenesis

AbstractPeri-cellular remodeling of mesenchymal extracellular matrices is considered a prerequisite for cell proliferation, motility and development. Here we demonstrate that membrane-type 3 MMP, MT3-MMP, is expressed in mesenchymal tissues of the skeleton and in peri-skeletal soft connective tissue. Consistent with this localization, MT3-MMP-deficient mice display growth inhibition tied to a decreased viability of mesenchymal cells in skeletal tissues. We document that MT3-MMP works as a major collagenolytic enzyme, enabling cartilage and bone cells to cleave high-density fibrillar collagen and modulate their resident matrix to make it permissive for proliferation and migration. Collectively, these data uncover a novel extracellular matrix remodeling mechanism required for proper function of mesenchymal cells. The physiological significance of MT3-MMP is highlighted in mice double deficient for MT1-MMP and MT3-MMP. Double deficiency transcends the combined effects of the individual single deficiencies and leads to severe embryonic defects in palatogenesis and bone formation incompatible with life. These defects are directly tied to loss of indispensable collagenolytic activities required in collagen-rich mesenchymal tissues for extracellular matrix remodeling and cell proliferation during embryogenesis

MT1-MMP and Type II Collagen Specify Skeletal Stem Cells and Their Bone and Cartilage Progeny

Skeletal formation is dependent on timely recruitment of skeletal stem cells and their ensuing synthesis and remodeling of the major fibrillar collagens, type I collagen and type II collagen, in bone and cartilage tissues during development and postnatal growth. Loss of the major collagenolytic activity associated with the membrane-type 1 matrix metalloproteinase (MT1-MMP) results in disrupted skeletal development and growth in both cartilage and bone, where MT1-MMP is required for pericellular collagen dissolution. We show here that reconstitution of MT1-MMP activity in the type II collagen–expressing cells of the skeleton rescues not only diminished chondrocyte proliferation, but surprisingly, also results in amelioration of the severe skeletal dysplasia associated with MT1-MMP deficiency through enhanced bone formation. Consistent with this increased bone formation, type II collagen was identified in bone cells and skeletal stem/progenitor cells of wildtype mice. Moreover, bone marrow stromal cells isolated from mice expressing MT1-MMP under the control of the type II collagen promoter in an MT1-MMP–deficient background showed enhanced bone formation in vitro and in vivo compared with cells derived from nontransgenic MT1-MMP–deficient littermates. These observations show that type II collagen is not stringently confined to the chondrocyte but is expressed in skeletal stem/progenitor cells (able to regenerate bone, cartilage, myelosupportive stroma, marrow adipocytes) and in the chondrogenic and osteogenic lineage progeny where collagenolytic activity is a requisite for proper cell and tissue function

Membrane-type MMPs are indispensable for placental labyrinth formation and development

The membrane-type matrix metalloproteinases (MT-MMPs) are essential for pericellular matrix remodeling in late stages of development, as well as in growth and tissue homeostasis in postnatal life. Although early morphogenesis is perceived to involve substantial tissue remodeling, the roles of MT-MMPs in these processes are only partially characterized. Here we explore the functions of 2 prominently expressed MT-MMPs, MT1-MMP and MT2-MMP, and describe their roles in the process of placental morphogenesis. The fetal portion of the placenta, in particular the labyrinth (LA), displays strong overlapping expression of MT1-MMP and MT2-MMP, which is critical for syncytiotrophoblast formation and in turn for fetal vessels. Disruption of trophoblast syncytium formation consequently leads to developmental arrest with only a few poorly branched fetal vessels entering the LA causing embryonic death at embryonic day 11.5. Through knockdown of MMP expression, we demonstrate that either MT1-MMP or MT2-MMP is crucial specifically during development of the LA. In contrast, knockdown of MT-MMP activity after LA formation is compatible with development to term and postnatal life. Taken together these data identify essential but interchangeable roles for MT1-MMP or MT2-MMP in placental vasculogenesis and provide the first example of selective temporal and spatial MMP activity required for development of the mouse embryo

MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis

SummaryProteolysis is essential during branching morphogenesis, but the roles of MT-MMPs and their proteolytic products are not clearly understood. Here, we discover that decreasing MT-MMP activity during submandibular gland branching morphogenesis decreases proliferation and increases collagen IV and MT-MMP expression. Specifically, reducing epithelial MT2-MMP profoundly decreases proliferation and morphogenesis, increases Col4a2 and intracellular accumulation of collagen IV, and decreases the proteolytic release of collagen IV NC1 domains. Importantly, we demonstrate the presence of collagen IV NC1 domains in developing tissue. Furthermore, recombinant collagen IV NC1 domains rescue branching morphogenesis after MT2-siRNA treatment, increasing MT-MMP and proproliferative gene expression via β1 integrin and PI3K-AKT signaling. Additionally, HBEGF also rescues MT2-siRNA treatment, increasing NC1 domain release, proliferation, and MT2-MMP and Hbegf expression. Our studies provide mechanistic insight into how MT2-MMP-dependent release of bioactive NC1 domains from collagen IV is critical for integrating collagen IV synthesis and proteolysis with epithelial proliferation during branching morphogenesis

Perturbation of Rb, p53, and Brca1 or Brca2 Cooperate in Inducing Metastatic Serous Epithelial Ovarian Cancer

The majority of human high grade serous epithelial ovarian cancer (SEOC) is characterized by frequent mutations in p53 and alterations in the RB and FOXM1 pathways. A subset of human SEOC harbors a combination of germline and somatic mutations as well as epigenetic dysfunction for BRCA1/2. Using Cre-conditional alleles and intrabursal induction by Cre-expressing adenovirus in genetically engineered mice, we analyzed the roles of pathway perturbations in epithelial ovarian cancer initiation and progression. Inactivation of RB-mediated tumor suppression induced surface epithelial proliferation with progression to stage I carcinoma. Additional biallelic inactivation and/or missense p53 mutation in the presence or absence of Brca1/2 caused progression to stage IV disease. As in human SEOC, mice developed peritoneal carcinomatosis, ascites, and distant metastases. Unbiased gene expression and metabolomic profiling confirmed that Rb, p53, and Brca1/2-triple mutant tumors aligned with human SEOC, and not with other intraperitoneal cancers. Together, our findings provide a novel resource for evaluating disease etiology and biomarkers, therapeutic evaluation, and improved imaging strategies in epithelial ovarian cancer

Pathway-Specific Engineered Mouse Allograft Models Functionally Recapitulate Human Serous Epithelial Ovarian Cancer

<div><p>The high mortality rate from ovarian cancers can be attributed to late-stage diagnosis and lack of effective treatment. Despite enormous effort to develop better targeted therapies, platinum-based chemotherapy still remains the standard of care for ovarian cancer patients, and resistance occurs at a high rate. One of the rate limiting factors for translation of new drug discoveries into clinical treatments has been the lack of suitable preclinical cancer models with high predictive value. We previously generated genetically engineered mouse (GEM) models based on perturbation of <i>Tp53</i> and <i>Rb</i> with or without <i>Brca1</i> or <i>Brca2</i> that develop serous epithelial ovarian cancer (SEOC) closely resembling the human disease on histologic and molecular levels. Here, we describe an adaptation of these GEM models to orthotopic allografts that uniformly develop tumors with short latency and are ideally suited for routine preclinical studies. Ovarian tumors deficient in <i>Brca1</i> respond to treatment with cisplatin and olaparib, a PARP inhibitor, whereas <i>Brca1</i>-wild type tumors are non-responsive to treatment, recapitulating the relative sensitivities observed in patients. These mouse models provide the opportunity for evaluation of effective therapeutics, including prediction of differential responses in <i>Brca1</i>-wild type and <i>Brca1</i>–deficient tumors and development of relevant biomarkers.</p></div