Expression of claudin-11 by tumor cells in cutaneous squamous cell carcinoma is dependent on the activity of p38δ

The incidence of cutaneous squamous cell carcinoma (cSCC) is rapidly increasing, and the prognosis of patients with metastatic disease is poor. There is an emerging need to identify molecular markers for predicting aggressive behaviour of cSCC. Here, we have examined the role of tight junction (TJ) components in the progression of cSCC. The expression pattern of mRNAs for TJ components was determined with RNA sequencing and oligonucleotide array-based expression analysis from cSCC cell lines (n=8) and normal human epidermal keratinocytes (NHEK, n=5). The expression of CLDN11 was specifically elevated in primary cSCC cell lines (n=5), but low or absent in metastatic cSCC cell lines (n=3) and NHEKs. Claudin-11 was detected in cell-cell contacts of primary cSCC cells in culture by indirect immunofluorescence analysis. Analysis of a large panel of tissue samples from sporadic UV-induced cSCC (n=65), cSCC in situ (n=56), actinic keratoses (n=31), seborrhoeic keratoses (n=7) and normal skin (n=16) by immunohistochemistry showed specific staining for claudin-11 in intercellular junctions of keratinizing tumor cells in well and moderately differentiated cSCCs, whereas no staining for claudin-11 was detected in poorly differentiated tumors. The expression of claudin-11 in cSCC cells was dependent on the activity of p38 delta MAPK and knock-down of claudin-11 enhanced cSCC cell invasion. These findings provide evidence for the role of claudin-11 in regulation of cSCC invasion and suggest loss of claudin-11 expression in tumor cells as a biomarker for advanced stage of cSCC

Loss of the laminin subunit alpha-3 induces cell invasion and macrophage infiltration in cutaneous squamous cell carcinoma

Background Cutaneous squamous cell carcinoma (cSCC) is a common cancer that invades the dermis through the basement membrane. The role of the basement membrane in poorly differentiated cSCC is not well understood.Objectives To study the effect that loss of the laminin subunit alpha-3 (alpha 3) chain from the tumour microenvironment has on tumour invasion and inflammatory cell recruitment.Methods We examined the role of the basement membrane proteins laminin subunits alpha 3, beta 3 and gamma 2 in SCC invasion and inflammatory cell recruitment using immunohistochemistry, short hairpin RNA knockdown, RNA-Seq, mouse xenograft models and patient tumour samples.Results Analysis of SCC tumours and cell lines using antibodies specific to laminin chains alpha 3, beta 3 and gamma 2 identified a link between poorly differentiated SCC and reduced expression of laminin alpha 3 but not the other laminin subunits investigated. Knockdown of laminin alpha 3 increased tumour invasion both in vitro and in vivo. Western blot and immunohistochemical staining identified increased phosphorylated myosin light chain with loss of laminin alpha 3. Inhibition of ROCK (rho-associated protein kinase) but not Rac1 significantly reduced the invasive potential of laminin alpha 3 knockdown cells. Knockdown of laminin subunits alpha 3 and gamma 2 increased monocyte recruitment to the tumour microenvironment. However, only the loss of laminin alpha 3 correlated with increased tumour-associated macrophages both in xenografted tumours and in patient tumour samples.Conclusions These data provide evidence that loss of the laminin alpha 3 chain in cSCC has an effect on both the epithelial and immune components of cSCC, resulting in an aggressive tumour microenvironment

Significant Role of Collagen XVII And Integrin β4 in Migration and Invasion of The Less Aggressive Squamous Cell Carcinoma Cells

Collagen XVII and integrin alpha 6 beta 4 have well-established roles as epithelial adhesion molecules. Their binding partner laminin 332 as well as integrin alpha 6 beta 4 are largely recognized to promote invasion and metastasis in various cancers, and collagen XVII is essential for the survival of colon and lung cancer stem cells. We have studied the expression of laminin.2, collagen XVII and integrin beta 4 in tissue microarray samples of squamous cell carcinoma (SCC) and its precursors, actinic keratosis and Bowen's disease. The expression of laminin.2 was highest in SCC samples, whereas the expression of collagen XVII and integrin beta 4 varied greatly in SCC and its precursors. Collagen XVII and integrin beta 4 were also expressed in SCC cell lines. Virus-mediated RNAi knockdown of collagen XVII and integrin beta 4 reduced the migration of less aggressive SCC-25 cells in horizontal scratch wound healing assay. Additionally, in a 3D organotypic myoma invasion assay the loss of collagen XVII or integrin beta 4 suppressed equally the migration and invasion of SCC-25 cells whereas there was no effect on the most aggressive HSC-3 cells. Variable expression patterns and results in migration and invasion assays suggest that collagen XVII and integrin beta 4 contribute to SCC tumorigenesis

A role for the collagen I/III and MMP-1/-13 genes in primary inguinal hernia?

BACKGROUND: Abnormal collagen metabolism is thought to play an important role in the development of primary inguinal hernia. This is underlined by detection of altered collagen metabolism and structural changes of the tissue in patients with primary inguinal hernia. However, it is still unknown whether these alterations reflect a basic dysfunction of the collagen synthesis, or of collagen degradation. METHODS: In the present study, we analysed type I and type III procollagen messenger ribonucleic acid (mRNA) and MMP-1 and MMP-13 mRNA in cultured fibroblasts from the skin of patients with primary inguinal hernia, and from patients without hernia (controls) by reverse transcription polymerase chain reaction (RT-PCR) and Northern Blot. RESULTS: The results indicated that the ratio of type I to type III procollagen mRNA was decreased in patients with primary hernia, showing significant differences as compared to controls (p = 0.01). This decrease was mainly due to the increase of type III procollagen mRNA. Furthermore, RT-PCR analysis revealed that the expression of MMP-1 mRNA in patients with primary hernia is equivalent to that of controls (p > 0.05). In addition, MMP-13 mRNA is expressed neither in patients with primary hernia nor in controls. CONCLUSION: We concluded that abnormal change of type I and type III collagen mRNAs contribute to the development of primary inguinal hernia, whereas the expressions of MMP-1 and MMP-13 mRNA appears not to be involved in the development of primary inguinal hernia. Thus, the knowledge on the transcriptional regulation of collagen in patients with primary inguinal hernia may help to understand the pathogenesis of primary inguinal hernia, and implies new therapeutic strategies for this disease

Synergistic effect of stromelysin-1 (matrix metalloproteinase-3) promoter (-1171 5A->6A) polymorphism in oral submucous fibrosis and head and neck lesions

<p>Abstract</p> <p>Background</p> <p>Matrix metalloproteinases (MMPs) are enzymes that degrade all the components of extra cellular matrix and collagen. Various types of MMPs are known to be expressed and activated in patients with oral submucous fibrosis (OSMF) as well as head and neck squamous cell carcinoma (HNSCC). The purpose of this study was to asses the association of the single nucleotide polymorphism (SNP) adenosine insertion/deletion polymorphism (-1171 5A->6A) in the MMP-3 promoter region in these lesions.</p> <p>Methods</p> <p>MMP-3 SNP was genotyped by polymerase chain reaction-restriction fragment polymorphism (PCR-RFLP) analysis in a case control study consisting of 362 participants; 101 cases of OSMF, 135 of HNSCC and 126 controls, compared for age, sex and habits. ROC distribution was plotted to assess the contributions of genetic variation in MMP-3 genotypes with relation to age.</p> <p>Results</p> <p>Analysis of MMP 3 (-1171 5A->6A) polymorphism revealed the frequency of 5A allele in OSMF, HNSCC and controls to be 0.15, 0.13 and 0.07, respectively. A significant difference was found in 5A genotype frequency between OSMF (5A genotype frequency = 0.15, p = 0.01, OR = 2.26, 95% CI = 1.22-4.20) and in controls (5A genotype frequency 0.07) as well as HNSCC (5A genotype frequency 0.13, p = 0.03,95%CI = 1.06-3.51) and controls (5A genotype frequency = 0.07) In this study, 5A genotype had greater than two fold risk for developing OSMF (OR = 2.26) and nearly the same in case of HNSCC (OR = 1.94) as compared to controls. In patients with OSMF as well as HNSCC, the ROC analysis between the MMP-3 genotype and age, 6A/6A allele was found to be significant in patients both over and under 45 years of age; while the 5A/5A carrier alleles showed an association only in patients less than 45 years of age.</p> <p>Conclusions</p> <p>This study concluded that the expression of MMP-3 genotype associated with the 5A alleles, it may have an important role in the susceptibility of the patients to develop OSMF and HNSCC.</p

Localization of uPAR and MMP-9 in lipid rafts is critical for migration, invasion and angiogenesis in human breast cancer cells

<p>Abstract</p> <p>Background</p> <p>uPAR and MMP-9, which play critical roles in tumor cell invasion, migration and angiogenesis, have been shown to be associated with lipid rafts.</p> <p>Methods</p> <p>To investigate whether cholesterol could regulate uPAR and MMP-9 in breast carcinoma, we used MβCD (methyl beta cyclodextrin, which extracts cholesterol from lipid rafts) to disrupt lipid rafts and studied its effect on breast cancer cell migration, invasion, angiogenesis and signaling.</p> <p>Results</p> <p>Morphological evidence showed the association of uPAR with lipid rafts in breast carcinoma cells. MβCD treatment significantly reduced the colocalization of uPAR and MMP-9 with lipid raft markers and also significantly reduced uPAR and MMP-9 at both the protein and mRNA levels. Spheroid migration and invasion assays showed inhibition of breast carcinoma cell migration and invasion after MβCD treatment. <it>In vitro </it>angiogenesis studies showed a significant decrease in the angiogenic potential of cells pretreated with MβCD. MβCD treatment significantly reduced the levels of MMP-9 and uPAR in raft fractions of MDA-MB-231 and ZR 751 cells. Phosphorylated forms of Src, FAK, Cav, Akt and ERK were significantly inhibited upon MβCD treatment. Increased levels of soluble uPAR were observed upon MβCD treatment. Cholesterol supplementation restored uPAR expression to basal levels in breast carcinoma cell lines. Increased colocalization of uPAR with the lysosomal marker LAMP1 was observed in MβCD-treated cells when compared with untreated cells.</p> <p>Conclusion</p> <p>Taken together, our results suggest that cholesterol levels in lipid rafts are critical for the migration, invasion, and angiogenesis of breast carcinoma cells and could be a critical regulatory factor in these cancer cell processes mediated by uPAR and MMP-9.</p

Differentiation of embryonic stem cells into fibroblast-like cells in three-dimensional type I collagen gel cultures

Fibroblasts are heterogeneous mesenchymal cells that play important roles in the production and maintenance of extracellular matrix. Although their heterogeneity is recognized, progenitor progeny relationships among fibroblasts and the factors that control fibroblast differentiation are poorly defined. The current study was designed to develop a reliable method that would permit in vitro differentiation of fibroblast-like cells from human and murine embryonic stem cells (ESCs). Undifferentiated ESCs were differentiated into embryoid bodies (EBs) with differentiation media. EBs were then cast into type I collagen gels and cultured for 21 d with basal media. The spindle-shaped cells that subsequently grew from the EBs were released from the gels and subsequently cultured as monolayers in basal media supplemented with serum. Differentiated cells showed a characteristic spindle-shaped morphology and had ultrastructural features consistent with fibroblasts. Immunocytochemistry showed positive staining for vimentin and alpha-smooth muscle actin but was negative for stage-specific embryonic antigens and cytokeratins. Assays of fibroblast function, including proliferation, chemotaxis, and contraction of collagen gels demonstrated that the differentiated cells, derived from both human and murine ESCs, responded to transforming growth factor-β1 and prostaglandin E2 as would be expected of fibroblasts, functions not expected of endothelial or epithelial cells. The current study demonstrates that cells with the morphologic and functional features of fibroblasts can be reliably derived from human and murine ESCs. This methodology provides a means to investigate and define the mechanisms that regulate fibroblast differentiation

Role of zinc and α2macroglobulin on thymic endocrine activity and on peripheral immune efficiency (natural killer activity and interleukin 2) in cervical carcinoma

Decreased natural killer (NK) activity as well as interleukin 2 (IL-2) are risk factors for the progression of cervical carcinoma. NK activity and IL-2 may be thymus controlled. Plasma levels of active thymulin, a zinc-dependent thymic hormone (ZnFTS), are reduced in cancer because of the low peripheral zinc bioavailability. Zinc and thymulin are relevant for normal immune functions. α2-Macroglobulin is an inhibitor of matrix metalloproteases (MMPs) against invasive tumour proliferation. Because α2-macroglobulin has a binding affinity (Kd) for zinc that is higher than does thymulin, it may play a key role in immune efficiency in cancer. Plasma samples of 22 patients (age range 35–60 years) with locally advanced squamous cervical carcinoma and with FIGO stage Ib2–IIb were examined. They showed reduced active thymulin, decreased NK activity and IL-2 production, increased soluble IL-2 receptor (sIL-2R) and augmented α2-macroglobulin in the circulation, whereas plasma zinc levels were within the normal range for age. Significant positive correlations were found between zinc or active thymulin and α2-macroglobulin (r = 0.75, P< 0.01, r = 0.78, P< 0.01, respectively) in cancer patients. In vitro zinc increases IL-2 production from peripheral blood mononuclear cells (PBMCs) of cancer patients. These data suggest that an increase in α2-macroglobulin, which competes with thymulin for zinc binding, may be involved in causing a thymulin deficit with a consequent decrease of IL-2 and NK cytotoxicity. Thus, physiological zinc treatment in cervical carcinoma maybe restores impaired central and peripheral immune efficiency. © 1999 Cancer Research Campaig

The role of versican isoforms V0/V1 in glioma migration mediated by transforming growth factor-β2

Versican is a large chondroitin sulphate proteoglycan produced by several tumour cell types, including high-grade glioma. The increased expression of certain versican isoforms in the extracellular matrix (ECM) plays a role in tumour cell growth, adhesion and migration. Transforming growth factor-β2 (TGF-β2) is an important modulator of glioma invasion, partially by remodeling the ECM. However, it is unknown whether it interacts with versican during malignant progression of glioma cells. Here, we analysed the effect of TGF-β2 on the expression of versican isoforms. The expression of versican V0/V1 was upregulated by TGF-β2 detected by quantitative polymerase chain reaction and immunoprecipitation, whereas V2 was not induced. Using time-lapse scratch and spheroid migration assays, we observed that the glioma migration rate is significantly increased by exogenous TGF-β2 and inhibited by TGF-β2-specific antisense oligonucleotides. Interestingly, an antibody specific for the DPEAAE region of glycosaminoglycan-β domain of versican was able to reverse the effect of TGF-β2 on glioma migration in a dose-dependent manner. Taken together, we report here that TGF-β2 triggers the malignant phenotype of high-grade gliomas by induction of migration, and that this effect is, at least in part, mediated by versican V0/V1

Hydrolyzed eggshell membrane immobilized on phosphorylcholine polymer supplies extracellular matrix environment for human dermal fibroblasts

We have found that a water-soluble alkaline-digested form of eggshell membrane (ASESM) can provide an extracellular matrix (ECM) environment for human dermal fibroblast cells (HDF) in vitro. Avian eggshell membrane (ESM) has a fibrous-meshwork structure and has long been utilized as a Chinese medicine for recovery from burn injuries and wounds in Asian countries. Therefore, ESM is expected to provide an excellent natural material for biomedical use. However, such applications have been hampered by the insolubility of ESM proteins. We have used a recently developed artificial cell membrane biointerface, 2-methacryloyloxyethyl phosphorylcholine polymer (PMBN) to immobilize ASESM proteins. The surface shows a fibrous structure under the atomic force microscope, and adhesion of HDF to ASESM is ASESM-dose-dependent. Quantitative mRNA analysis has revealed that the expression of type III collagen, matrix metalloproteinase-2, and decorin mRNAs is more than two-fold higher when HDF come into contact with a lower dose ASESM proteins immobilized on PMBN surface. A particle-exclusion assay with fixed erythrocytes has visualized secreted water-binding molecules around the cells. Thus, HDF seems to possess an ECM environment on the newly designed PMBN-ASESM surface, and future applications of the ASESM-PMBN system for biomedical use should be of great interest