Hyaluronan Binding Motifs of USP17 and SDS3 Exhibit Anti-Tumor Activity

BACKGROUND: We previously reported that the USP17 deubiquitinating enzyme having hyaluronan binding motifs (HABMs) interacts with human SDS3 (suppressor of defective silencing 3) and specifically deubiquitinates Lys-63 branched polyubiquitination of SDS3 resulting in negative regulation of histone deacetylase (HDAC) activity in cancer cells. Furthermore, USP17 and SDS3 mutually interact with each other to block cell proliferation in HeLa cells but the mechanism for this inhibition in cell proliferation is not known. We wished to investigate whether the HABMs of USP17 were responsible for tumor suppression activity. METHODOLOGY/PRINCIPAL FINDINGS: Similarly to USP17, we have identified that SDS3 also has three consecutive HABMs and shows direct binding with hyaluronan (HA) using cetylpyridinium chloride (CPC) assay. Additionally, HA oligosaccharides (6-18 sugar units) competitively block binding of endogenous HA polymer to HA binding proteins. Thus, administration of HA oligosaccharides antagonizes the interaction between HA and USP17 or SDS3. Interestingly, HABMs deleted USP17 showed lesser interaction with SDS3 but retain its deubiquitinating activity towards SDS3. The deletion of HABMs of USP17 could not alter its functional regulation on SDS3-associated HDAC activity. Furthermore, to explore whether HABMs in USP17 and SDS3 are responsible for the inhibition of cell proliferation, we investigated the effect of USP17 and SDS3-lacking HABMs on cell proliferation by soft agar, apoptosis, cell migration and cell proliferation assays. CONCLUSIONS: Our results have demonstrated that these HABMs in USP17 and its substrate SDS3 are mainly involved in the inhibition of anchorage-independent tumor growth

Tissue hyaluronan expression, as reflected in the sputum of lung cancer patients, is an indicator of malignancy

Hyaluronan (HA) shows promise for detecting cancerous change in pleural effusion and urine. However, there is uncertainty about the localization of HA in tumor tissue and its relationship with different histological types and other components of the extracellular matrix, such as angiogenesis. We evaluated the association between HA and degree of malignancy through expression in lung tumor tissue and sputum. Tumoral tissue had significantly increased HA compared to normal tissue. Strong HA staining intensity associated with cancer cells was significant in squamous cell carcinoma compared to adenocarcinoma and large cell carcinoma. A significant direct association was found between tumors with a high percentage of HA and MVD (microvessel density) in tumoral stroma. Similarly significant was the direct association between N1 tumors and high levels of HA in cancer cells. Cox multivariate analysis showed significant association between better survival and low HA. HA increased in sputum from lung cancer patients compared to cancer-free and healthy volunteers and a significant correlation was found between HA in sputum and HA in cancer tissue. Localization of HA in tumor tissue was related to malignancy and reflected in sputum, making this an emerging factor for an important diagnostic procedure in patients suspected to have lung cancer. Further study in additional patients in a randomized prospective trial is required to finalize these results and to validate our quantitative assessment of HA, as well as to couple it to gold standard sputum cytology.Research supported by FAPESP (2010/11005-5 and 2010/04462) and CNPq (#471939/2010-2 and 483005/2012-6

Synthetic emmprin peptides with chitobiose substitution stimulate MMP-2 production by fibroblasts

<p>Abstract</p> <p>Background</p> <p>Emmprin, a glycoprotein containing two Ig domains, is enriched on tumor cell surfaces and stimulates matrix metalloproteinase (MMP) production by adjacent stromal cells. Its first Ig domain (ECI) contains the biologically active site. The dependence of emmprin activity on N-glycosylation is controversial. We investigated whether synthetic ECI with the shortest sugar is functionally active.</p> <p>Methods</p> <p>The whole ECI peptides carrying sugar chains, a chitobiose unit or N-linked core pentasaccharide, were synthesized by the thioester method and added to fibroblasts to examine whether they stimulate MMP-2 production.</p> <p>Results</p> <p>ECI carrying a chitobiose unit, ECI-(GlcNAc) ₂, but not ECI without a chitobiose unit or the chitobiose unit alone, dose-dependently stimulated MMP-2 production by fibroblasts. ECI with longer chitobiose units, ECI-[(Man)₃(GlcNAc)₂], also stimulated MMP-2 production, but the extent of its stimulation was lower than that of ECI-(GlcNAc)₂.</p> <p>Conclusions</p> <p>Our results indicate that ECI can mimic emmprin activity when substituted with chitobiose, the disaccharide with which N-glycosylation starts.</p

EMMPRIN Promotes Melanoma Cells Malignant Properties through a HIF-2alpha Mediated Up-Regulation of VEGF-Receptor-2

EMMPRIN's expression in melanoma tissue was reported to be predictive of poor prognosis. Here we demonstrate that EMMPRIN up-regulated VEGF receptor-2 (VEGFR-2) in two different primary melanoma cell lines and consequently increased migration and proliferation of these cells while inhibiting their apoptosis. SiRNA inhibition of VEGFR-2 expression abrogated these EMMPRIN effects. EMMPRIN regulation of VEGFR-2 was mediated through the over-expression of HIF-2α and its translocation to the nucleus where it forms heterodimers with HIF-1β. These results were supported by an in vivo correlation between the expression of EMMPRIN with that of VEGFR-2 in human melanoma tissues as well as with the extent of HIF-2α localization in the nucleus. They demonstrate a novel mechanism by which EMMPRIN promotes tumor progression through HIF-2α/VEGFR-2 mediated mechanism, with an autocrine role in melanoma cell malignancy. The inhibition of EMMPRIN in cancer may thus simultaneously target both the VEGFR-2/VEGF system and the matrix degrading proteases to block tumor cell growth and invasion

Upregulated EMMPRIN/CD147 might contribute to growth and angiogenesis of gastric carcinoma: a good marker for local invasion and prognosis

Tumour growth depends on angiogenesis, which is closely associated with vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs). Extracellular MMP inducer (EMMPRIN) was reported to involve in the progression of malignancies by regulating expression of VEGF and MMPs in stromal cells. To clarify the role of EMMPRIN in progression and angiogenesis of gastric carcinoma, expression of EMMPRIN, ki-67, MMP-2, MMP-9 and VEGF was examined on tissue microarray containing gastric carcinomas (n=234) and non-cancerous mucosa adjacent to carcinoma (n=85) by immunohistochemistry. Additionally, microvessel density (MVD) was assessed after labelling with anti-CD34 antibody. Extracellular MMP inducer expression was compared with clinicopathological parameters of tumours, including levels of ki-67, MMP-2, MMP-9 and vascular endothelial growth factor (VEGF), MVD as well as survival time of carcinoma patients. Gastric carcinoma cell lines (HGC-27, MKN28 and MKN45) were studied for EMMPRIN expression by immunohistochemistry and Western blot. Extracellular MMP inducer expression was gradually increased from normal mucosa to carcinomas through hyperplastic or metaplastic mucosa of the stomach (P<0.05). There was strong EMMPRIN expression in all gastric carcinoma cell lines despite different levels of glycosylation. Extracellular MMP inducer expression was positively correlated with tumour size, depth of invasion, lymphatic invasion, expression of ki-67, MMP-2, MMP-9 and VEGF of tumours (P<0.05), but not with lymph node metastasis, UICC staging or differentiation (P>0.05). Interestingly, there was a significantly positive relationship between EMMPRIN expression and MVD in gastric carcinomas (P<0.05). Survival analysis indicated EMMPRIN expression to be negatively linked to favourable prognosis (P<0.05), but not be independent factor for prognosis (P>0.05). Further analysis showed three independent prognostic factors, depth of invasion, lymphatic and venous invasion, to influence the relationship between EMMPRIN expression and prognosis. Upregulated expression of EMMPRIN possibly contributes to genesis, growth and local invasion of gastric carcinomas. Altered EMMPRIN expression might enhance growth, invasion and angiogenesis of gastric carcinoma via upregulating MMP expression of both stromal fibroblasts and gastric cancer cells and could be considered as an objective and effective marker to predict invasion and prognosis

Identification of uPAR-positive Chemoresistant Cells in Small Cell Lung Cancer

BACKGROUND: The urokinase plasminogen activator (uPA) and its receptor (uPAR/CD87) are major regulators of extracellular matrix degradation and are involved in cell migration and invasion under physiological and pathological conditions. The uPA/uPAR system has been of great interest in cancer research because it is involved in the development of most invasive cancer phenotypes and is a strong predictor of poor patient survival. However, little is known about the role of uPA/uPAR in small cell lung cancer (SCLC), the most aggressive type of lung cancer. We therefore determined whether uPA and uPAR are involved in generation of drug resistant SCLC cell phenotype. METHODS AND FINDINGS: We screened six human SCLC cell lines for surface markers for putative stem and cancer cells. We used fluorescence-activated cell sorting (FACS), fluorescence microscopy and clonogenic assays to demonstrate uPAR expression in a subpopulation of cells derived from primary and metastatic SCLC cell lines. Cytotoxic assays were used to determine the sensitivity of uPAR-positive and uPAR-negative cells to chemotherapeutic agents. The uPAR-positive cells in all SCLC lines demonstrated multi-drug resistance, high clonogenic activity and co-expression of CD44 and MDR1, putative cancer stem cell markers. CONCLUSIONS: These data suggest that uPAR-positive cells may define a functionally important population of cancer cells in SCLC, which are resistant to traditional chemotherapies, and could serve as critical targets for more effective therapeutic interventions in SCLC