Biosynthesis of Promatrix Metalloproteinase-9/Chondroitin Sulphate Proteoglycan Heteromer Involves a Rottlerin-Sensitive Pathway

BACKGROUND: Previously we have shown that a fraction of the matrix metalloproteinase-9 (MMP-9) synthesized by the macrophage cell line THP-1 was bound to a chondroitin sulphate proteoglycan (CSPG) core protein as a reduction sensitive heteromer. Several biochemical properties of the enzyme were changed when it was bound to the CSPG. METHODOLOGY/PRINCIPAL FINDINGS: By use of affinity chromatography, zymography, and radioactive labelling, various macrophage stimulators were tested for their effect on the synthesis of the proMMP-9/CSPG heteromer and its components by THP-1 cells. Of the stimulators, only PMA largely increased the biosynthesis of the heteromer. As PMA is an activator of PKC, we determined which PKC isoenzymes were expressed by performing RT-PCR and Western Blotting. Subsequently specific inhibitors were used to investigate their involvement in the biosynthesis of the heteromer. Of the inhibitors, only Rottlerin repressed the biosynthesis of proMMP-9/CSPG and its two components. Much lower concentrations of Rottlerin were needed to reduce the amount of CSPG than what was needed to repress the synthesis of the heteromer and MMP-9. Furthermore, Rottlerin caused a minor reduction in the activation of the PKC isoenzymes δ, ε, θ and υ (PKD3) in both control and PMA exposed cells. CONCLUSIONS/SIGNIFICANCE: The biosynthesis of the proMMP-9/CSPG heteromer and proMMP-9 in THP-1 cells involves a Rottlerin-sensitive pathway that is different from the Rottlerin sensitive pathway involved in the CSPG biosynthesis. MMP-9 and CSPGs are known to be involved in various physiological and pathological processes. Formation of complexes may influence both the specificity and localization of the enzyme. Therefore, knowledge about biosynthetic pathways and factors involved in the formation of the MMP-9/CSPG heteromer may contribute to insight in the heteromers biological function as well as pointing to future targets for therapeutic agents

Characterisation and prognostic value of tertiary lymphoid structures in oral squamous cell carcinoma

BACKGROUND: Oral squamous cell carcinomas are often heavily infiltrated by immune cells. The organization of B-cells, follicular dendritic cells, T-cells and high-endothelial venules into structures termed tertiary lymphoid structures have been detected in various types of cancer, where their presence is found to predict favourable outcome. The purpose of the present study was to evaluate the incidence of tertiary lymphoid structures in oral squamous cell carcinomas, and if present, analyse whether they were associated with clinical outcome. METHODS: Tumour samples from 80 patients with oral squamous cell carcinoma were immunohistochemically stained for B-cells, follicular dendritic cells, T-cells, germinal centre B-cells and high-endothelial venules. Some samples were sectioned at multiple levels to assess whether the presence of tertiary lymphoid structures varied within the tumour. RESULTS: Tumour-associated tertiary lymphoid structures were detected in 21 % of the tumours and were associated with lower disease-specific death. The presence of tertiary lymphoid structures varied within different levels of a tissue block. CONCLUSIONS: Tertiary lymphoid structure formation was found to be a positive prognostic factor for patients with oral squamous cell carcinoma. Increased knowledge about tertiary lymphoid structure formation in oral squamous cell carcinoma might help to develop and guide immune-modulatory cancer treatments

Presence of tumour high-endothelial venules is an independent positive prognostic factor and stratifies patients with advanced-stage oral squamous cell carcinoma

Accepted manuscript version. The final publication is available at Springer via http://dx.doi.org/10.1007/s13277-015-4036-4Background: Staging of oral squamous cell carcinoma is based on the TNM system, which has been deemed insufficient for prognostic purposes. Hence, better prognostic tools are needed to reflect the biological diversity of these cancers. Previously, high numbers of specialized blood vessels called high-endothelial venules have been reported to be associated with prolonged survival in patients with breast cancer. In this study, we analysed the prognostic value and morphological characteristics of tumour-associated high-endothelial venules in oral cancer. Methods: The presence of tumour-associated high-endothelial venules was evaluated by immunohistochemistry in 75 patients with oral squamous cell carcinoma and analysed with correlation to clinicopathological parameters, patients’ survival, and vessel morphology. Ten of the samples were analysed at multiple levels to evaluate intratumoural heterogeneity. Results: The presence of tumour-associated high-endothelial venules was found to be associated with lower disease-specific death in multivariate regression analyses (P=0.002). High-endothelial venules were present in all (n=53) T1-T2 tumours, but only in two-thirds (n=14) of the T3-T4 tumours. The morphology of high-endothelial venules was heterogeneous and correlated with lymphocyte density. High-endothelial venules were found to be distributed homogeneously within the tumours. Conclusion: We found the presence of tumour-associated high-endothelial venules to be an easy-to-use, robust, and independent positive prognostic factor for patients with oral cancer. Absence of these vessels in advancedstage tumours might identify patients with more aggressive disease. Evaluating the presence of tumour-associated high-endothelial venules might help to tailor the treatment of oral cancer patients to their individual need

Intracellular MMP-2 Activity in Skeletal Muscle is Associated with Type II Fibers

Matrix metalloproteinase 2 (MMP-2) is a proteolytic enzyme implicated in motility, differentiation, and regeneration of skeletal muscle fibers through processing of extracellular substrates. Although MMP-2 has been found to be localized intracellularly in cardiomyocytes where the enzyme is thought to contribute to post-ischemic loss of contractility, little is known about intracellular MMP-2 activity in skeletal muscle fibers. In the present study we demonstrate intracellular MMP-2 in normal skeletal muscle by immunohistochemical staining. Immunogold electron microscopic analyses indicated that the enzyme was concentrated in Z-lines of the sarcomers, in the nuclear membrane, and in mitochondria. By use of in situ zymography, we found that gelatinolytic activity in muscle fibers was co-localized with immunofluorecent staining for MMP-2. Staining for MMP-9, the other member of the gelatinase group of the MMPs, was negative. The broad-spectrum metalloprotease inhibitor EDTA and the selective gelatinase inhibitor CTT2, but not the cysteine inhibitor E64, strongly reduced the gelatinolytic activity. The intracellular gelatinolytic activity was much more prominent in fast twitch type II fibers than in slow twitch type I fibers, and there was a decrease in intracellular gelatinolytic activity and MMP-2 expression in muscles from mice exposed to high intensity interval training. Together our results indicate that MMP-2 is part of the intracellular proteolytic network in normal skeletal muscle, especially in fast twitch type II fibers. Further, the results suggest that intracellular MMP-2 in skeletal muscle fibers is active during normal homeostasis, and affected by the level of physical activity

Presence of high-endothelial venules correlates with a favorable immune microenvironment in oral squamous cell carsinoma

Oral squamous cell carcinomas (OSCC) are associated with a poor prognosis, which may be partly due to functional impairment of the immune response. Lymphocyte recruitment to the tumor site is facilitated by high-endothelial venules (HEV), whereas expression of programmed-death ligand 1 (PD-L1) can impair T cell function. Thus, we hypothesize that these factors are important in shaping the immune response in OSCC. In the present study, we characterized the immune infiltrate in formalin-fixed, paraffin-embedded tumor samples from 75 OSCC patients. We used immunohistochemistry to determine the distribution of immune cell subsets, HEV and PD-L1, as well as quantitative real-time polymerase chain reaction to assess the expression of inflammatory cytokines and chemokines associated with lymphocyte trafficking. Finally, we calculated correlations between the presence of immune cell subsets, the gene expression patterns, HEV, PD-L1 and the clinicopathological parameters including patient survival. The presence of HEV correlated with increased number of CD3+ T cells and CD20+ B cells, higher levels of the chemokines CXCL12 and CCL21, and lower levels of CCL20, irrespective of the tumors’ T-stage. In univariate analysis, high levels of CD20+ B cells and CD68+ macrophages, positive HEV-status, and low T- and N-stages predicted longer patient survival. However, only the presence of HEV and a low T-stage were independent positive prognosticators. This indicates that HEV are important mediators and a convenient marker of an antitumor immune response in OSCC. Our findings support HEV as a potential immunomodulatory target in OSCC. PD-L1 staining in tumor cells correlated with lower T-stage, increased infiltration of CD4+ cells and higher expression of several inflammation-related cytokines. Thus, OSCC tumors rich in CD4+ cells may preferentially respond to PD-1/PD-L1 blockade therapy

Stromal impact on tumor growth and lymphangiogenesis in human carcinoma xenografts

Squamous cell carcinomas (SCCs) arising in the oral cavity are associated with poor survival, mainly due to metastatic disease. In contrast, skin SCCs rarely metastasize and are usually curable. To study influence of tongue and skin stroma on cancer growth and induction of lymphangiogenesis, xenograft tumors of human carcinoma cells were established either in tongue or skin of BALB/c nude mice. Two oral and two skin SCC cell lines were used, as well as an endometrial adenocarcinoma cell line. Tongue tumors established from all cell lines were larger than corresponding skin tumors. Peritumoral lymphatic vessel density was up to five times higher in tongue than in corresponding skin tumors, and mRNA level of the lymphangiogenic growth factor vascular endothelial growth factor (VEGF)-C was twice as high in tongue tumors compared with corresponding skin tumors. Contrary to lymphatic vessel density, blood vessel density was higher in skin tumors than in tongue tumors. In a cohort of patient samples, lymphatic vessel density was found to be higher in tongue SCCs compared with skin SCCs, supporting a clinical relevance of our findings. Our results show that the tumor stroma has a profound impact on cancer growth and induction of lymphangiogenesis and angiogenesis. The difference in lymphatic vessel density between tongue and skin tumors may be important in directing metastatic potential of tumors arising in these organs

Tumor budding score predicts lymph node status in oral tongue squamous cell carcinoma and should be included in the pathology report

Background - The majority of oral cavity cancers arise in the oral tongue. The aim of this study was to evaluate the prognostic value of tumor budding in oral tongue squamous cell carcinoma, both as a separate variable and in combination with depth of invasion. We also assessed the prognostic impact of the 8th edition of the American Joint Committee on Cancer’s TNM classification (TNM8), where depth of invasion (DOI) supplements diameter in the tumor size (T) categorization. Methods - Patients diagnosed with primary oral tongue squamous cell carcinoma were evaluated retrospectively. Spearman bivariate correlation analyses with bootstrapping were used to identify correlation between variables. Prognostic value of clinical and histopathological variables was assessed by Log rank and Cox regression analyses with bootstrapping using 5-year disease specific survival as outcome. The significance level for the hypothesis test was 0.05. Results - One-hundred and fifty patients had available material for microscopic evaluation on Hematoxylin and Eosin-stained slides and were included in the analyses. Reclassification of tumors according to TNM8 caused a shift towards a higher T status compared to the previous classification. The tumor budding score was associated with lymph node metastases where 23% of the patients with low-budding tumors had lymph node metastases, compared with 43% of those with high-budding tumors. T-status, lymph node status, tumor budding, depth of invasion, and the combined tumor budding/depth of invasion score were all significantly associated with survival in univariate analyses. In multivariate analyses only N-status was an independent prognosticator of survival. Conclusion - Reclassification according to TNM8 shifted many tumors to a higher T-status, and also increased the prognostic value of the T-status. This supports the implementation of depth of invasion to the T-categorization in TNM8. Tumor budding correlated with lymph node metastases and survival. Therefore, information on tumor budding can aid clinicians in treatment planning and should be included in pathology reports of oral tongue squamous cell carcinomas

Cleavage of the urokinase receptor (uPAR) on oral cancer cells: Regulation by transforming growth factor - beta1 (TGF-beta1) and potential effects on migration and invasion

Source at https://doi.org/10.1186/s12885-017-3349-7 Background: Urokinase plasminogen activator (uPA) receptor (uPAR) is up-regulated at the invasive tumour front of human oral squamous cell carcinoma (OSCC), indicating a role for uPAR in tumour progression. We previously observed elevated expression of uPAR at the tumour-stroma interface in a mouse model for OSCC, which was associated with increased proteolytic activity. The tumour microenvironment regulated uPAR expression, as well as its glycosylation and cleavage. Both full-length- and cleaved uPAR (uPAR (II-III)) are involved in highly regulated processes such as cell signalling, proliferation, migration, stem cell mobilization and invasion. The aim of the current study was to analyse tumour associated factors and their effect on uPAR cleavage, and the potential implications for cell proliferation, migration and invasion. Methods: Mouse uPAR was stably overexpressed in the mouse OSCC cell line AT84. The ratio of full-length versus cleaved uPAR as analysed by Western blotting and its regulation was assessed by addition of different protease inhibitors and transforming growth factor - β 1(TGF- β 1). The role of uPAR cleavage in cell proliferation and migration was analysed using real-time cell analysis and invasion was assessed using the myoma invasion model. Results: We found that when uPAR was overexpressed a proportion of the receptor was cleaved, thus the cells presented both full-length uPAR and uPAR (II-III). Cleavage was mainly performed by serine proteases and urokinase plasminogen activator (uPA) in particular. When the OSCC cells were stimulated with TGF- β 1, the production of the uPA inhibitor PAI-1 was increased, resulting in a reduction of uPAR cleavage. By inhibiting cleavage of uPAR, cell migration was reduced, and by inhibiting uPA activity, invasion was reduced. We could also show that medium containing soluble uPAR (suPAR), and cleaved soluble uPAR (suPAR (II-III)), induced migration in OSCC cells with low endogenous levels of uPAR. Conclusions: These results show that soluble factors in the tumour microenvironment, such as TGF- β 1, PAI-1 and uPA, can influence the ratio of full length and uPAR (II-III) and thereby potentially effect cell migration and invasion. Resolving how uPAR cleavage is controlled is therefore vital for understanding how OSCC progresses and potentially provides new targets for therapy

Tumour Microenvironments Induce Expression of Urokinase Plasminogen Activator Receptor (uPAR) and Concomitant Activation of Gelatinolytic Enzymes

Peer reviewe

Small lytic peptides escape the inhibitory effect of heparan sulfate on the surface of cancer cells

Several naturally occurring cationic antimicrobial peptides (CAPs), including bovine lactoferricin (LfcinB), display promising anticancer activities. These peptides are unaffected by multidrug resistance mechanisms and have been shown to induce a protective immune response against solid tumors, thus making them interesting candidates for developing novel lead structures for anticancer treatment. Recently, we showed that the anticancer activity by LfcinB was inhibited by the presence of heparan sulfate (HS) on the surface of tumor cells. Based on extensive structure-activity relationship studies performed on LfcinB, shorter and more potent peptides have been constructed. In the present study, we have investigated the anticancer activity of three chemically modified 9-mer peptides and the influence of HS and chondroitin sulfate (CS) on their cytotoxic activity. Various cell lines and red blood cells were used to investigate the anticancer activity and selectivity of the peptides. The cytotoxic effect of the peptides against the different cell lines was measured by use of a colorimetric MTT viability assay. The influence of HS and CS on their cytotoxic activity was evaluated by using HS/CS expressing and HS/CS deficient cell lines. The ability of soluble HS and CS to inhibit the cytotoxic activity of the peptides and the peptides’ affinity for HS and CS were also investigated. The 9-mer peptides displayed selective anticancer activity. Cells expressing HS/CS were equally or more susceptible to the peptides than cells not expressing HS/CS. The peptides displayed a higher affinity for HS compared to CS, and exogenously added HS inhibited the cytotoxic effect of the peptides. In contrast to the previously reported inhibitory effect of HS on LfcinB, the present study shows that the cytotoxic activity of small lytic peptides was increased or not affected by cell surface HS