Expression of the Laminin-5γ2 Chain Is Associated With Acquisition of Malignant Traits in Ovarian Serous and Mucinous Tumors.

BackgroundOvarian tumors are predominantly of epithelial origin and are currently divided into three groups: Adenoma, borderline tumor, and adenocarcinoma by the presence or absence of invasion and cellular atypia. However, it is difficult to assess invasion, so a more objective biomarker would be desirable. Laminin-5 (Lam5) is a protein that constitutes the extracellular matrix of the basement membrane, and it is composed of three short-chain subunits. One of them, the Lam5γ2 chain (Lam5γ2), has been reported to be expressed in some malignant tumors and is suggested to be related to tumor cell invasion. Against this background, we investigated immunohistologically whether the Lam5γ2 would be useful as a marker to predict invasion in ovarian serous and mucinous tumors.MethodsImmunohistochemistry for Lam5γ2 was performed on a total of 80 cases of serous and mucinous tumor adenomas, borderline tumors, and adenocarcinomas, and the differences in the localization of Lam5γ2 expression were observed.ResultsThe basement membrane expression of Lam5γ2 tended to be preserved or had disappeared in half of the adenomas and borderline serous tumors. In all cases of adenocarcinoma, the expression on the basement membrane had disappeared. The frequency of expression in tumor cells increased in the order of adenoma, borderline tumor, adenocarcinoma. In particular, in most adenocarcinoma cases a cytoplasmic expression was observed. The same tendency was noted in mucinous tumors, and the basement membrane expression tended to disappear in the order of adenoma, borderline tumor and adenocarcinoma. Many cases of adenocarcinoma were observed among the tumor cells. In adenocarcinoma cases of both types of tumor, many tumor cells showed prominent cytoplasmic expression particularly in the microinvasive foci and in the invasive front.ConclusionsThe disappearance of Lam5γ2 from the basement membrane and its aberrant expression in serous and mucinous tumor cells may serve as a phenotype for invasiveness.

In vitro beneficial effects of a flax extract on papillary fibroblasts define it as an anti-aging candidate

Objective: During aging, skin undergoes structural, cellular and molecular changes, which not only alter skin mechanical properties but also biological and physiological functions. Structurally the epidermis becomes thinner, the dermal epidermal junction flattens and the extra-cellular matrix component of the dermis is disorganized and degraded. The dermis is composed of two compartments: The Reticular dermis is the deepest and thickest part while the upper layer, the papillary dermis, which is much thinner and is in close contact with epidermis, plays an important role in the structure and function of the skin. We have recently shown that the papillary dermis was preferentially affected by skin aging because the activity of fibroblasts in this region was especially altered as a function of age. The purpose of this study was to investigate the capacity of a flax extract as anti-aging component. Method: We investigated the capacity of a flax extract to stimulate or restore the activity of papillary fibroblasts from young and old donors in cultured monolayers and in reconstructed skin. Several biological markers of extracellular matrix homeostasis and mechanical properties were investigated. Results: The tested flax extract seemed to improve parameters known to change with age: I/ In monolayers after treatment the number of aged fibroblasts increased II/ In reconstructed skin the flax extract appears to positively regulate some biological activities; particularly in aged fibroblasts where the deposition of laminin 5, fibrillin 1, procollagen I were increased in the dermis and the secretion of specific soluble factors like MMP1, MMP3 and KGF were regulated to levels similar to those observed in young fibroblasts III/ Mechanical properties were improved particularly for elastics parameters (R5, R2 and R7). Conclusion: The flax extract is a promising anti-aging compound. The treatment of aged papillary fibroblasts resulted in a return to a younger-like profile for some of the studied parameters.

Major Reservoir for Heparin-Releasable TFPIα (Tissue Factor Pathway Inhibitor α) Is Extracellular Matrix

Objective: Human endothelial cells produce 2 alternatively spliced TFPI (tissue factor pathway inhibitor) isoforms that maintain anticoagulant properties of the vasculature. TFPIβ is glycosylphosphatidylinositol anchored on the cell surface. TFPIα has a basic C terminus sharing homology with VEGF (vascular endothelial growth factor) and is a heparin-releasable protein, suggesting it binds glycosaminoglycans on the endothelium surface. However, this is unclear because TFPIα is not on the surface of cultured endothelial cells. This study identifies the source of heparin-releasable TFPIα. Approach and Results: ELISA assays localized heparin-releasable TFPIα to the extracellular matrix (ECM) of Ea.hy926 cells and human umbilical vein endothelial cells. Immunofluorescence microscopy for TFPIα showed punctate intracytoplasmic staining and ECM staining beneath individual cells. Flow cytometry identified TFPIβ but not TFPIα on the cell surface. TFPIα localization to ECM was confirmed with ELISA and immunohistochemistry studies of umbilical cord veins. The TFPIα C terminus interacted with Ea.hy926 ECM glycosaminoglycans, and a homologous VEGF peptide competed for this binding, suggesting these interactions modulate VEGF responses. Immobilized TFPIα C-terminal peptide bound to several ECM proteoglycans in Ea.hy926 conditioned media. Immunofluorescence studies of human kidney colocalized TFPIα with 4 of these proteoglycans surrounding the microvasculature: glypican-1, syndecan-4, thrombospondin, and laminin-5. The absence of TFPIα on the surface of endothelial cells and its co-localization with specific ECM proteins suggests TFPIα binds to unique proteoglycan structures. Conclusions: ECM contained the primary vascular pool of heparin-releasable TFPIα. By localizing to ECM, TFPIα is positioned to inhibit the procoagulant activity of tissue factor surrounding the vasculature.

LAMA3 is a differentially expressed gene in both lymph node and brain metastases in human breast cancer.

Metastasis to the brain is a clinical problem in patients with breast cancer (1-3). We mined published microarray data (4, 5) to compare primary and metastatic tumor transcriptomes for the discovery of genes associated with brain metastasis in humans with metastatic breast cancer. We found that the alpha 3 subunit of laminin 5, encoded by LAMA3, was among the genes whose expression was most quantitatively different in the brain metastases of patients with metastatic breast cancer. LAMA3 mRNA was present at decreased quantities in brain metastatic tissues as compared to primary tumors of the breast. Importantly, expression of LAMA3 in primary tumors was significantly correlated with patient post-progression survival in patients with breast cancer. Modulation of LAMA3 expression may be relevant to the biology by which tumor cells metastasize from the breast to the brain.

NR2F1 is a barrier to dissemination of early-evolved mammary cancer cells

SummaryCancer cells disseminate during very early and sometimes asymptomatic stages of tumor progression. Granted that biological barriers to tumorigenesis exist, there must also be limiting steps to early dissemination, all of which remain largely unknown. We report that the orphan nuclear receptor NR2F1/COUP-TF1 serves as a barrier to early dissemination. High-resolution intravital imaging revealed that loss of function of NR2F1 in HER2+ early cancer cells increased in vivo dissemination without accelerating mammary tumor formation. NR2F1 expression was positively regulated by the tumor suppressive MMK3/6-p38-MAPK pathway and downregulated by HER2 and Wnt4 oncogenic signaling. NR2F1 downregulation by HER2 in early cancer cells led to decreased E-cadherin expression and β-catenin membrane localization, disorganized laminin 5 deposition, and increased expression of CK14, TWIST1, ZEB1 and PRRX1. Our findings reveal the existence of an inhibitory mechanism of dissemination regulated by NR2F1 downstream of HER2 signaling.

Ultraviolet Light Treatment of Titanium Enhances Attachment, Adhesion, and Retention of Human Oral Epithelial Cells via Decarbonization

Early establishment of soft-tissue adhesion and seal at the transmucosal and transcutaneous surface of implants is crucial to prevent infection and ensure the long-term stability and function of implants. Herein, we tested the hypothesis that treatment of titanium with ultraviolet (UV) light would enhance its interaction with epithelial cells. X-ray spectroscopy showed that UV treatment significantly reduced the atomic percentage of surface carbon on titanium from 46.1% to 28.6%. Peak fitting analysis revealed that, among the known adventitious carbon contaminants, C–C and C=O groups were significantly reduced after UV treatment, while other groups were increased or unchanged in percentage. UV-treated titanium attracted higher numbers of human epithelial cells than untreated titanium and allowed more rapid cell spread. Hemi-desmosome-related molecules, integrin β4 and laminin-5, were upregulated at the gene and protein levels in the cells on UV-treated surfaces. The result of the detachment test revealed twice as many cells remaining adherent on UV-treated than untreated titanium. The enhanced cellular affinity of UV-treated titanium was equivalent to laminin-5 coating of titanium. These data indicated that UV treatment of titanium enhanced the attachment, adhesion, and retention of human epithelial cells associated with disproportional removal of adventitious carbon contamination, providing a new strategy to improve soft-tissue integration with implant devices.

The Reconstructed Human Epidermis in vitro — a Model for Basic and Applied Research of Human Skin

Background. The reconstructed human epidermis (RE) is an in vitro tissue-engineering construct similar to the native epidermis. Objective. To develop a full-layer RE. Describe its structure: determine the presence of all layers of the epidermal component, including basal, spinous and granular layers and stratum corneum of the epidermis; detect the basement membrane, the border between the epidermal and mesenchymal component. Materials and methods. Isolation of keratinocytes and fibroblasts from human donor skin. Cultivation of keratinocytes and fibroblasts in vitro under 2D conditions, cell subculturing and 3D modeling of RE, obtaining cryosections, histological staining, immunohistochemical (IHC) study with antibodies to cytokeratins 14 and 10, Ki67 protein, loricrin, laminin 5 and plectin. Results. A technique was developed for the formation of RE. Histological examination showed that the stratification of keratinocyte layers occurs during the formation of RE. Layers are formed including basal, spinous and granular layers and stratum corneum. The IHC study has shown the proliferative activity of keratinocytes of the basal layer and has detected the presence of marker proteins of keratinocytes at different stages of differentiation. RE basal keratinocytes, like native ones, form hemidesmosomes and synthesize basement membrane proteins. Conclusions. A full-layer human RE was obtained in vitro. RE meets all the characteristics of the native epidermis and it is suitable for basic and practical research in the field of skin biology, dermatology, and cosmetology.

IMMUNOHISTOCHEMICAL EVALUATION OF LAMININ-5 AND MYOFIBROBLASTS IN EPITHELIAL DYSPLASIA AND ORAL SQUAMOUS CELL CARCINOMA

Background: Oral squamous cell carcinoma is one of the most prevalent cancers worldwide. The transformation of the normal epithelial cell into a tumor cell bestows upon them certain features at the cellular and molecular level which aids in its survival and proliferation. Invasion of the altered tumor cells through the basement membrane into the connective tissue stroma and their subsequent spread and metastasis is an important prognostic indicator. Laminin-5 is a protein associated with a migratory phenotype in epithelial neoplastic cells. Along with laminin, the stromal myofibroblasts play a significant role in tumor invasion, due to its ability to modify the extracellular matrix. Aim: To evaluate the role of laminin 5 and stromal myofibroblasts in oral epithelial dysplasia and oral squamous cell carcinoma. Methods: Paraffin-embedded archival samples of 25 normal, 30 oral epithelial dysplasia and 30 oral squamous cell carcinoma (OSCC) were evaluated for laminin-5 and α- smooth muscle actin (SMA) using standard immunohistochemistry. Semi-quantitative assessment of the expression of laminin and alpha SMA was done in all the study samples. The area of staining and the staining intensity was evaluated in order to determine the staining index which were then statistically analyzed between the three groups. Results: All the cases of laminin showed cytoplasmic staining in the basal cell layer and basement membrane. Expression of laminin was observed in the basal cell layer of normal and epithelial dysplasia study group and mainly around the tumor islands in OSCC group. α- SMA was seen with increasing intensity with increasing grade of the disease. Comparison of laminin expression between the three groups showed a statistically significant decrease in the staining index from normal to epithelial dysplasia to OSCC (p < 0.01). Statistical comparison of α-SMA in between the three groups using Kruskal- Wallis test showed a significant increase in the expression of α-SMA from normal to epithelial dysplasia to OSCC (p < 0.01) Conclusion: Decreased laminin expression in the basement membrane and increased expression of α-SMA favors tumor invasion, establishment of an invasive phenotype of neoplastic cells and a permissive environment for tumor invasion. Key words: Epithelial dysplasia, Oral cancer, Laminin, Alpha- Smooth muscle actin, Immunohistochemistry, Myofibroblasts