The prognostic value of the hypoxia markers CA IX and GLUT 1 and the cytokines VEGF and IL 6 in head and neck squamous cell carcinoma treated by radiotherapy ± chemotherapy

BACKGROUND: Several parameters of the tumor microenvironment, such as hypoxia, inflammation and angiogenesis, play a critical role in tumor aggressiveness and treatment response. A major question remains if these markers can be used to stratify patients to certain treatment protocols. The purpose of this study was to investigate the inter-relationship and the prognostic significance of several biological and clinicopathological parameters in patients with head and neck squamous cell carcinoma (HNSCC) treated by radiotherapy ± chemotherapy. METHODS: We used two subgroups of a retrospective series for which CT-determined tumoral perfusion correlated with local control. In the first subgroup (n = 67), immunohistochemistry for carbonic anhydrase IX (CA IX) and glucose transporter-1 (GLUT-1) was performed on the pretreatment tumor biopsy. In the second subgroup (n = 34), enzyme linked immunosorbent assay (ELISA) was used to determine pretreatment levels of the cytokines vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) in serum. Correlation was investigated between tumoral perfusion and each of these biological markers, as well as between the markers mutually. The prognostic value of these microenvironmental parameters was also evaluated. RESULTS: For CA IX and GLUT-1, the combined assessment of patients with both markers expressed above the median showed an independent correlation with local control (p = 0.02) and disease-free survival (p = 0.04) with a trend for regional control (p = 0.06). In the second subgroup, IL-6 pretreatment serum level above the median was the only independent predictor of local control (p = 0.009), disease-free survival (p = 0.02) and overall survival (p = 0.005). CONCLUSION: To our knowledge, we are the first to report a link in HNSCC between IL-6 pretreatment serum levels and radioresistance in vivo. This link is supported by the strong prognostic association of pretreatment IL-6 with local control, known to be the most important parameter to judge radiotherapy responses. Furthermore, the combined assessment of CA IX and GLUT-1 correlated independently with prognosis. This is a valuable indication that a combined approach is important in the investigation of prognostic markers

Carbonic anhydrase IX induction defines a heterogeneous cancer cell response to hypoxia and mediates stem cell-like properties and sensitivity to HDAC inhibition.

Carbonic anhydrase IX (CAIX) is strongly induced by hypoxia and its overexpression is associated with poor therapeutic outcome in cancer. Here, we report that hypoxia promotes tumour heterogeneity through the epigenetic regulation of CAIX. Based on hypoxic CAIX expression we identify and characterize two distinct populations of tumour cells, one that has inducible expression of CAIX and one that does not. The CAIX+ve population is enriched with cells expressing cancer stem cell markers and which have high self-renewal capacity. We show that differential CAIX expression is due to differences in chromatin structure. To further investigate the relationship between chromatin organization and hypoxic induction of CAIX expression we investigated the effect of JQ1 an inhibitor of BET bromodomain proteins and A366 a selective inhibitor of the H3K9 methyltransferase G9a/GLP. We identified that these drugs were able to modulate hypoxic CAIX expression induction. This further highlights the role of epigenetic modification in adaption to hypoxia and also in regulation of heterogeneity of cells within tumours. Interestingly, we identified that the two subpopulations show a differential sensitivity to HDAC inhibitors, NaBu or SAHA, with the CAIX positive showing greater sensitivity to treatment. We propose that drugs modulating chromatin regulation of expression may be used to reduce heterogeneity induced by hypoxia and could in combination have significant clinical consequences

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Hypoxia-mediated upregulation of MCT1 expression supports the glycolytic phenotype of glioblastomas

Background: Glioblastomas (GBM) present a high cellular heterogeneity with conspicuous necrotic regions associated with hypoxia, which is related to tumor aggressiveness. GBM tumors exhibit high glycolytic metabolism with increased lactate production that is extruded to the tumor microenvironment through monocarboxylate transporters (MCTs). While hypoxia-mediated regulation of MCT4 has been characterized, the role of MCT1 is still controversial. Thus, we aimed to understand the role of hypoxia in the regulation of MCT expression and function in GBM, MCT1 in particular. Methods: Expression of hypoxia-and glycolytic-related markers, as well as MCT1 and MCT4 isoforms was assessed in in vitro and in vivo orthotopic glioma models, and also in human GBM tissues by immunofluorescence/immunohistochemistry and Western blot. Following MCT1 inhibition, either pharmacologically with CHC (a-cyano-4-hydroxynnamic acid) or genetically with siRNAs, we assessed GBM cell viability, proliferation, metabolism, migration and invasion, under normoxia and hypoxia conditions. Results: Hypoxia induced an increase in MCT1 plasma membrane expression in glioma cells, both in in vitro and in vivo models. Additionally, treatment with CHC and downregulation of MCT1 in glioma cells decreased lactate production, cell proliferation and invasion under hypoxia. Moreover, in the in vivo orthotopic model and in human GBM tissues, there was extensive co-expression of MCT1, but not MCT4, with the GBM hypoxia marker CAIX. Conclusion: Hypoxia-induced MCT1 supports GBM glycolytic phenotype, being responsible for lactate efflux and an important mediator of cell survival and aggressiveness. Therefore, MCT1 constitutes a promising therapeutic target in GBM.This study was supported by Projecto Estratégico- LA 26 – 2013-2014 (PEst-C/SAL/LA0026/2013) and ON.2 SR&TD Integrated Program (NORTE-07-0124FEDER-000017)” co-funded by Programa Operacional Regional do Norte (ON.2- O Novo Norte), Quadro de Referência Estratégico Nacional (QREN), through Fundo Europeu de Desenvolvimento Regional (FEDER), as well as MCTI/CNPq Nº 73/2013 (Brazil). VMG received a fellowship from Fundação para a Ciência e Tecnologia (FCT) ref. SFRH/BD/51997/2012.info:eu-repo/semantics/publishedVersio

‘With Friends Like These’: Human Rights, Neoconservatism and U.S. Foreign Policy from Carter to Reagan.

This thesis engages with two emerging bodies of scholarship: the history of human rights and the history of U.S. neoconservatism. It begins with an exploration of the genesis of the contemporary international human rights movement, arguing that human rights as we know and understand them today were a product of the latter half of the twentieth century. Their path, however, was not a clear one. The emergence of neoconservative ideology in U.S. domestic politics would greatly impact upon the trajectory of the human rights movement under the presidencies of Jimmy Carter and Ronald Reagan. The latter period witnessed a conflict between America’s Watch and the Reagan administration over human rights as an ‘idea’ and as praxis, with U.S. policy towards Latin America as the primary battle field

Studies on breeding dwarf poinsettias (Euphorbia pulcherrima Willd.) and the influence of infective agents

BACKGROUND: Hypoxia, metabolism, and growth factor signaling are important prognostic features in most solid tumors. The purpose of this study was to determine whether head and neck squamous cell carcinoma (HNSCC) xenografts show similar biological and molecular characteristics as the primary tumor they originate from. METHODS: Eighteen HNSCC primary tumor-xenograft pairs were immunofluorescently stained for pimonidazole (hypoxia), carbonic anhydrase IX (CAIX), glucose transporter-1 (GLUT-1), monocarboxylate transporter-1 (MCT-1), monocarboxylate transporter-4 (MCT-4), epidermal growth factor receptor (EGFR), and phosphorylated protein kinase B (pAKT). RESULTS: Although no correlation was found for the amount of hypoxia, significant correlations between primary tumors and xenografts were observed for both the percentage of cells positive for expression and the hypoxia-related expression pattern of CAIX, GLUT-1, and MCT-1. For EGFR and MCT-4, the intensity of expression was correlated. No correlation was observed for pAKT. CONCLUSION: Xenografts did not always resemble the primary tumor they originate from, but the xenografts did represent the variability in expression levels and patterns observed in the primary tumors

Multiparameter analysis of vasculature, perfusion and proliferation in human tumour xenografts.

A method is presented in this report for concurrent analysis of vascular architecture, blood perfusion and proliferation characteristics in whole-tumour cross-sections of human larynx carcinoma and glioblastoma xenografts. Tumours were implanted subcutaneously in nude mice. After i.v. injection with Hoechst 33342 and bromodeoxyuridine (BrdUrd) as perfusion and proliferation markers, animals were killed. An antiendothelial antibody (9F1) was used to delineate vascular structures. Cross-sections were analysed by a multistep immune staining and a computer-controlled microscope scanning method. Each tumour section was stained and scanned four times (Hoechst, 9F1, BrdUrd and Fast Blue for all nuclei). When these images were combined, vasculature, perfusion and proliferation parameters were analysed. The labelling index (LI) was defined as the ratio of the BrdUrd-labelled area to the total nuclear area. The LI based on manual counting and the LI calculated by flow cytometry (FCM) were in good agreement with the LI based on surface analysis. LI decreased at increasing distance from its nearest vessel. In the vicinity of perfused vessels, the LI was 30-70% higher than near non-perfused vessels. This method shows that both vasculature/perfusion and proliferation characteristics can be measured in the same whole-tumour section in a semiautomatic way. This could be applied in clinical practice to identify combined human tumour characteristics that predict for a favourable response to treatment modifications