Do ultrastructural changes in aged peritoneum contribute to ovarian cancer metastasis? [abstract]

Epithelial ovarian cancer (EOC) will affect 1 in 69 women born in the United States today. Currently, 80% of women newly diagnosed with EOC already have metastatic disease, thus early intervention during the metastatic process will improve the long-term survival rates of women with EOC. Metastasis in EOC occurs through a unique process where cells are shed from a primary tumor and form multicellular aggregates (MCA) that disseminate intraperitoneally in the ascites fluid

Fluorescence In Situ Hybridization for MicroRNA Detection in Archived Oral Cancer Tissues

The noncoding RNA designated as microRNA (miRNA) is a large group of small single-stranded regulatory RNA and has generated wide-spread interest in human disease studies. To facilitate delineating the role of microRNAs in cancer pathology, we sought to explore the feasibility of detecting microRNA expression in formalin-fixed paraffin-embedded (FFPE) tissues. Using FFPE materials, we have compared fluorescent in situ hybridization (FISH) procedures to detect miR-146a with (a) different synthetic probes: regular custom DNA oligonucleotides versus locked nucleic acid (LNA) incorporated DNA oligonucleotides; (b) different reporters for the probes: biotin versus digoxigenin (DIG); (c) different visualization: traditional versus tyramide signal amplification (TSA) system; (d) different blocking reagents for endogenous peroxidase. Finally, we performed miR-146a FISH on a commercially available oral cancer tissue microarray, which contains 40 cases of oral squamous cell carcinoma (OSCC) and 10 cases of normal epithelia from the human oral cavity. A sample FISH protocol for detecting miR-146a is provided. In summary, we have established reliable in situ hybridization procedures for detecting the expression of microRNA in FFPE oral cancer tissues. This method is an important tool for studies on the involvement of microRNA in oral cancer pathology and may have potential prognostic or diagnostic value

Lysophoshatidic acid regulation of cell surface-associated proteases

Abstract only availableLysophosphatidic acid (LPA) is a potential biomarker of ovarian cancer and is thought to promote early stages of cancer progression through the stimulation of two cell surface associated proteases. The affects of LPA on the expression and cell surface association of two proteolytic enzymes associated with ovarian cancer progression, matrix metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator (uPA), were analyzed. Both MMP-9 and uPA have been linked with cancer cell invasion due to their proteolytic activity. The cell surface association and activation of MMP-9 is a chief mechanism by which cells invade collagen rich barriers, whereas the increased binding of uPA to its cell surface receptor promotes the conversion of plasminogen to plasmin which also promotes cell invasion. LPA was shown to increase the expression of the MMP-9 protease in a concentration dependent manner in both OVCA 429 and OVCA 433 ovarian cancer cell cultures at concentrations well below those normally found in ascites fluids ( 1 M). LPA treatment (80 M) showed as much as a 3.5 fold increase in MMP-9 expression. Further, LPA treatment increased the expression of MMP-9 over MMP-2 in conditioned media of both OVCA 429 and OVCA 433 cells. Stimulation of uPA activity was also shown in culture medium but required the elevated concentrations ( 20 M) often found in the ascites of ovarian cancer patients. Inhibitor studies showed that inhibition of PI-3K signaling (most evidently in OVCA 433 cells) and p38 MAPK (namely in OVCA 429 cells) repressed LPA stimulation of MMP-9 expression in a dose-dependent fashion. Future studies involving matrigel invasion assays will evaluate the functional consequence of LPA-stimulated MMP-9 expression and enhanced cell surface proteolysis on ovarian cancer cell invasive activity.NIH grant to M.S Stac

The Tumor Microenvironment of High Grade Serous Ovarian Cancer

The Special Issue on high grade serous ovarian cancer (HGSOC) and the contribution of the tumor micro-environment (TME) consisted of reviews contributed by leaders in the ovarian cancer (OC) field. [...]

Lysophosphatidic Acid Disrupts Junctional Integrity and Epithelial Cohesion in Ovarian Cancer Cells

Ovarian cancer metastasizes via exfoliation of free-floating cells and multicellular aggregates from the primary tumor to the peritoneal cavity. A key event in EOC metastasis is disruption of cell-cell contacts via modulation of intercellular junctional components including cadherins. Ascites is rich in lysophosphatidic acid (LPA), a bioactive lipid that may promote early events in ovarian cancer dissemination. The objective of this paper was to assess the effect of LPA on E-cadherin junctional integrity. We report a loss of junctional E-cadherin in OVCAR3, OVCA429, and OVCA433 cells exposed to LPA. LPA-induced loss of E-cadherin was concentration and time dependent. LPA increased MMP-9 expression and promoted MMP-9-catalyzed E-cadherin ectodomain shedding. Blocking LPA receptor signaling inhibited MMP-9 expression and restored junctional E-cadherin staining. LPA-treated cells demonstrated a significant decrease in epithelial cohesion. Together these data support a model wherein LPA induces MMP-9 expression and MMP-9-catalyzed E-cadherin ectodomain shedding, resulting in loss of E-cadherin junctional integrity and epithelial cohesion, facilitating metastatic dissemination of ovarian cancer cells

Masticatory loading and soft-tissue plasticity in the mammalian circumorbital region

Abstract only availableMasticatory loading is the key to understanding the plasticity or epigenetic responses of many of the soft and hard tissues of the mammalian skull. Diet-induced variation in the magnitude and/or frequency of masticatory loads influences the organization, functional adaptation and postnatal development of craniofacial systems. While experimental work exists concerning masticatory stress as a determinant of maxillomandibular form, similar research concerning the mammalian circumorbital region of soft-tissue structures remains sparse. Indeed, controversy remains over the function of the circumorbital region, specifically whether the postorbital region in primates and other mammals is responsive to biomechanical loading. This hinders our understanding of evolutionary transformations during primate origins, where the earliest primates evolved a bony postorbital bar from an ancestor with a soft-tissue structure along the lateral orbital margin. To fill this gap, we examined the postorbital microanatomy of white rabbits (Oryctolagus cuniculus). Rabbits exhibit a masticatory complex and feeding behaviors like primates, yet retain a primitive circumorbital region similar to their common ancestor. To address the plasticity and function of soft tissues of the lateral orbital wall, three cohorts of 10 rabbits each were raised from weaning (1 month old) to adulthood (6 months old) on diets of different mechanical properties (under-use, control, over-use). Once sacrificed, tissues were collected from the left lateral orbital wall. Dissections revealed that, rather than the anticipated postorbital ligament, rabbits instead exhibit fibrocartilage. Samples were analyzed histologically and immunohistochemically for general anatomy, fibril architecture, collagen expression/organization, and protein abundance using Western Blot analysis. Preliminary data suggests that collagen fibers are aligned differently, with an overexpression of collagen in the over-use dietary cohort versus both the control and under-use groups. In sum, these experimental findings suggest that the postorbital fibrocartilage is mechanically responsive, which contrasts with the non-masticatory nature of bony elements in the circumorbital region.Life Sciences Undergraduate Research Opportunity Progra

The Tumor Microenvironment of High Grade Serous Ovarian Cancer

The Special Issue on high grade serous ovarian cancer (HGSOC) and the contribution of the tumor microenviroment (TME) consists of reviews contributed by leaders in the OC field. As HGSOC metastases have a highly complex TME, there is an urgent need to better understand the TME in general, its distinct components in particular, and the role of the TME in the context of disease recurrence and development of chemoresistance. The Special Issue incorporates the current understanding of the different parts of thd TME components, including the cancer cells themselves, the cells surrounding the cancer cells or stromal cells, and the cells of the immune system, which are attracted to the site of metastases. In addition to these cells of the TME, the role of various cellular factors made by the cells of the TME are also the subject of the reviews. In addition, reviews in this Special Issue cover the complex relationships between the molecular mechanisms of HGSOC progression, including genomic, epigenomic and transcriptomic changes and changes in the immune cell landscape, as these may provide attractive new molecular targets for HGSOC therapy

Lysophosphatidic acid modulates ovarian cancer multicellular aggregate assembly and metastatic dissemination

Epithelial ovarian cancer (EOC) metastasis occurs by exfoliation of cells and multicellular aggregates (MCAs) from the tumor into the peritoneal cavity, adhesion to and retraction of peritoneal mesothelial cells and subsequent anchoring. Elevated levels of lysophosphatidic acid (LPA) have been linked to aberrant cell proliferation, oncogenesis, and metastasis. LPA disrupts junctional integrity and epithelial cohesion in vitro however, the fate of free-floating cells/MCAs and the response of host peritoneal tissues to LPA remain unclear. EOC MCAs displayed significant LPA-induced changes in surface ultrastructure with the loss of cell surface protrusions and poor aggregation, resulting in increased dissemination of small clusters compared to untreated control MCAs. LPA also diminished the adhesive capacity of EOC single cells and MCAs to murine peritoneal explants and impaired MCA survival and mesothelial clearance competence. Peritoneal tissues from healthy mice injected with LPA exhibited enhanced mesothelial surface microvilli. Ultrastructural alterations were associated with restricted peritoneal susceptibility to metastatic colonization by single cells as well as epithelial-type MCAs. The functional consequence is an LPA-induced dissemination of small mesenchymal-type clusters, promoting a miliary mode of peritoneal seeding that complicates surgical removal and is associated with worse prognosis

Epidermal Growth Factor Receptor-Mediated Membrane Type 1 Matrix Metalloproteinase Endocytosis Regulates the Transition between Invasive versus Expansive Growth of Ovarian Carcinoma Cells in Three-Dimensional Collagen

Abstract The epidermal growth factor receptor (EGFR) is overexpressed in ovarian carcinomas and promotes cellular responses that contribute to ovarian cancer pathobiology. In addition to modulation of mitogenic and motogenic behavior, emerging data identify EGFR activation as a novel mechanism for rapid modification of the cell surface proteome. The transmembrane collagenase membrane type 1 matrix metalloproteinase (MT1-MMP, MMP-14) is a major contributor to pericelluar proteolysis in the ovarian carcinoma microenvironment and is subjected to extensive posttranslational regulation. In the present study, the contribution of EGFR activation to control of MT1-MMP cell surface dynamics was investigated. Unstimulated ovarian cancer cells display caveolar colocalization of EGFR and MT1-MMP, whereas EGFR activation prompts internalization via distinct endocytic pathways. EGF treatment results in phosphorylation of the MT1-MMP cytoplasmic tail, and cells expressing a tyrosine mutated form of MT1-MMP (MT1-MMP-Y 573 F) exhibit defective MT1-MMP internalization. As a result of sustained cell surface MT1-MMP activity, a phenotypic epithelial-mesenchymal transition is observed, characterized by enhanced migration and collagen invasion, whereas growth within three-dimensional collagen gels is inhibited. These data support an EGFR-dependent mechanism for regulation of the transition between invasive and expansive growth of ovarian carcinoma cells via modulation of MT1-MMP cell surface dynamics