Modulation of a Stem Cell Gene: LGR4 Knockout in a Human Cell Line by CRISPR/Cas Method.

The modulation of gene expression is essential for the investigation of function or involved pathway of a single gene of interest, in particular in the developmental/stem cell biology. The temporary knock down of gene expression via siRNA is a well-established but with a residual expression connected modulation method. The chapter describes the complete knockout of a defined target and allows a comprehensive study of different gene like the stem cell gene LGR4 (Leucine-rich repeat-containing G-protein-coupled receptor 4) using the new developed CRISPR/Cas method (clustered regularly interspaced short palindromic repeats).

Prognostic impact of cytoplasmatic EGFR upregulation in patients with oral squamous cell carcinoma: A pilot study.

In various tumors, epidermal growth factor-receptor (EGFR) serves a role in tumorigenesis and has an impact on survival. Usually the EGF-receptor is located on the surface of the cell membrane and is involved in various signaling pathways. The dimerization of EGFR with other ErbB family proteins, such as HER2, is important for the tumor progression. Nevertheless, a second EGFR-associated signaling pathway appears to be important for tumor cells, which is cytoplasmic/nuclear EGFR. The present study examined the influence of membranous or cytoplasmic localized EGFR on the prognosis of patients with oral squamous cell carcinoma (OSCC). Slides from 45 OSCC tumor samples were stained against EGFR using immunohistochemistry and analysed by the Remmele score system. The association with histopathological parameters and survival data was analyzed. Cytoplasmatic EGFR localization was identified as an independent predictive biomarker for overall survival in the examined OSCC cohort according to multivariate Cox regression analysis. Positive cytoplasmatic EGFR staining was correlated with a higher risk of early death (RR=3.0; P=0.035), while membranous EGFR localization did not affect patient survival. To the best of our knowledge, the present study is the first study to demonstrate that cytoplasmatic-localized EGFR is an independent prognostic biomarker for the overall survival of patients with OSCC.

Causes and Consequences of A Glutamine Induced Normoxic HIF1 Activity for the Tumor Metabolism.

The transcription factor hypoxia-inducible factor 1 (HIF1) is the crucial regulator of genes that are involved in metabolism under hypoxic conditions, but information regarding the transcriptional activity of HIF1 in normoxic metabolism is limited. Different tumor cells were treated under normoxic and hypoxic conditions with various drugs that affect cellular metabolism. HIF1α was silenced by siRNA in normoxic/hypoxic tumor cells, before RNA sequencing and bioinformatics analyses were performed while using the breast cancer cell line MDA-MB-231 as a model. Differentially expressed genes were further analyzed and validated by qPCR, while the activity of the metabolites was determined by enzyme assays. Under normoxic conditions, HIF1 activity was significantly increased by (i) glutamine metabolism, which was associated with the release of ammonium, and it was decreased by (ii) acetylation via acetyl CoA synthetase (ACSS2) or ATP citrate lyase (ACLY), respectively, and (iii) the presence of L-ascorbic acid, citrate, or acetyl-CoA. Interestingly, acetylsalicylic acid, ibuprofen, L-ascorbic acid, and citrate each significantly destabilized HIF1α only under normoxia. The results from the deep sequence analyses indicated that, in HIF1-siRNA silenced MDA-MB-231 cells, 231 genes under normoxia and 1384 genes under hypoxia were transcriptionally significant deregulated in a HIF1-dependent manner. Focusing on glycolysis genes, it was confirmed that HIF1 significantly regulated six normoxic and 16 hypoxic glycolysis-associated gene transcripts. However, the results from the targeted metabolome analyses revealed that HIF1 activity affected neither the consumption of glucose nor the release of ammonium or lactate; however, it significantly inhibited the release of the amino acid alanine. This study comprehensively investigated, for the first time, how normoxic HIF1 is stabilized, and it analyzed the possible function of normoxic HIF1 in the transcriptome and metabolic processes of tumor cells in a breast cancer cell model. Furthermore, these data imply that HIF1 compensates for the metabolic outcomes of glutaminolysis and, subsequently, the Warburg effect might be a direct consequence of the altered amino acid metabolism in tumor cells.

Prognostic impact of mRNA levels of LGR5 transcript variants in OSCC patients.

BACKGROUND: The human leucine-rich, repeat-containing G protein-coupled receptor 5 (LGR5) is a stem cell marker in numerous adult tissues and is overexpressed in a large number of human carcinoma including colon cancer, breast cancer and oral squamous cell carcinomas (OSCC). The role of the full length transcript (LGR5FL) in progression and prognosis of several cancers was reported. However, the biological function of three splice variants of LGR5 (LGR5Δ5, LGR5Δ8 and LGR5Δ5-8) has yet to be thoroughly investigated. METHODS: Seventy-eight frozen tumor samples from adult OSCC patients were studied using quantitative real-time TaqMan™ PCR analysis. The mRNA levels of full length LGR5, the splice variant of LGR5 lacking exon 5 (LGR5Δ5), the splice variant of LGR5 lacking exon 8 (LGR5Δ8) and the mRNA level of all known transcript variants together (LGR5all) were quantified and correlated to overall and disease-specific survival of OSCC patients, clinical parameters and the mRNA level of different tumor-associated markers. RESULTS: An elevated level of tumoral LGR5Δ5 mRNA, but not LGR5FL, LGR5Δ8 or LGR5all mRNA was significantly associated with a poor prognosis for the overall and disease-specific survival of OSCC patients (hazard ratio (HR) = 2.0; p = 0.02; 95% CI: 1.1-3.7; HR = 3.2; p = 0.01; 95% CI: 1.3-8.0; multivariable Cox regression), respectively. Additionally, a higher tumoral level of LGR5Δ5 mRNA in primary tumors was associated with the occurrence of regional lymph node metastases in OSCC patients (odds ratio (OR) = 3.1; p = 0.022; 95% CI: 1.2-7.9; binary logistic regression). Furthermore, the mRNA levels of all investigated LGR5 transcript variants were significantly correlated with the mRNA expression of Wnt-target genes and markers of epithelial-to-mesenchymal transition (EMT). CONCLUSION: The mRNA level of the LGR5 splice variant LGR5Δ5 is an independent negative prognostic marker for overall and disease-specific survival and metastasis in OSCC patients. Additionally, we suggest, all LGR5 transcript variants are involved in the EMT process mainly through activating the Wnt-signalling pathway.

Investigation of the Prognostic Role of Carbonic Anhydrase 9 (CAIX) of the Cellular mRNA/Protein Level or Soluble CAIX Protein in Patients with Oral Squamous Cell Carcinoma.

s: Carbonic anhydrase 9 (CAIX) is an important protein that stabilizes the extracellular pH value and is transcriptionally regulated by hypoxia-inducible factor 1 (HIF1), but more stable than HIF1α. Here we show a comparative study that examines the prognostic value of CA9 mRNA, CAIX protein of tumor cells and secreted CAIX protein for oral squamous cell carcinoma (OSCC) patients. Tumor samples from 72 OSCC patients and 24 samples of normal tissue were analyzed for CA9 mRNA levels. A total of 158 OSCC samples were stained for CAIX by immunohistochemistry and 89 blood serum samples were analyzed by ELISA for soluble CAIX protein content. Survival analyses were performed by Kaplan⁻Meier and Cox's regression analysis to estimate the prognostic effect of CA9/CAIX in OSCC patients. The CA9 mRNA and CAIX protein levels of tumor cells correlated with each other, but not with those of the secreted CAIX protein level of the blood of patients. ROC curves showed a significant (p < 0.001) higher mRNA-level of CA9 in OSCC samples than in adjacent normal tissue. Cox's regression analysis revealed an increased risk (i) of death for patients with a high CA9 mRNA level (RR = 2.2; p = 0.02), (ii) of locoregional recurrence (RR = 3.2; p = 0.036) at higher CA9 mRNA levels and (iii) of death at high CAIX protein level in their tumors (RR = 1.7; p = 0.066) and especially for patients with advanced T4-tumors (RR = 2.0; p = 0.04). However, the secreted CAIX protein level was only as a trend associated with prognosis in OSCC (RR = 2.2; p = 0.066). CA9/CAIX is an independent prognostic factor for OSCC patients and therefore a potential therapeutic target.

Low HIF-1α and low EGFR mRNA Expression Significantly Associate with Poor Survival in Soft Tissue Sarcoma Patients; the Proteins React Differently.

In various tumors, the hypoxia inducible factor-1α (HIF-1α) and the epidermal growth factor-receptor (EGFR) have an impact on survival. Nevertheless, the prognostic impact of both markers for soft tissue sarcoma (STS) is not well studied. We examined 114 frozen tumor samples from adult soft tissue sarcoma patients and 19 frozen normal tissue samples. The mRNA levels of HIF-1α, EGFR, and the reference gene hypoxanthine phosphoribosyltransferase (HPRT) were quantified using a multiplex qPCR technique. In addition, levels of EGFR or HIF-1α protein were determined from 74 corresponding protein samples using ELISA techniques. Our analysis showed that a low level of HIF-1α or EGFR mRNA (respectively, relative risk (RR) = 2.8; p = 0.001 and RR = 1.9; p = 0.04; multivariate Cox´s regression analysis) is significantly associated with a poor prognosis in STS patients. The combination of both mRNAs in a multivariate Cox's regression analysis resulted in an increased risk of early tumor-specific death of patients (RR = 3.1, p = 0.003) when both mRNA levels in the tumors were low. The EGFR protein level had no association with the survival of the patient's cohort studied, and a higher level of HIF-1α protein associated only with a trend to significance (multivariate Cox's regression analysis) to a poor prognosis in STS patients (RR = 1.9, p = 0.09). However, patients with low levels of HIF-1α protein and a high content of EGFR protein in the tumor had a three-fold better survival compared to patients without such constellation regarding the protein level of HIF-1α and EGFR. In a bivariate two-sided Spearman's rank correlation, a significant correlation between the expression of HIF-1α mRNA and expression of EGFR mRNA (p < 0.001) or EGFR protein (p = 0.001) was found, additionally, EGFR mRNA correlated with EGFR protein level (p < 0.001). Our results show that low levels of HIF-1α mRNA or EGFR mRNA are negative independent prognostic markers for STS patients, especially after combination of both parameters. The protein levels showed a different effect on the prognosis. In addition, our analysis suggests a possible association between HIF-1α and EGFR expression in STS.

CMG2 Expression Is an Independent Prognostic Factor for Soft Tissue Sarcoma Patients.

The capillary morphogenesis gene 2 (CMG2), also known as the anthrax toxin receptor 2 (ANTXR2), is a transmembrane protein putatively involved in extracellular matrix (ECM) adhesion and tissue remodeling. CMG2 promotes endothelial cell proliferation and exhibits angiogenic properties. Its downregulation is associated with a worsened survival of breast carcinoma patients. Aim of this study was to analyze the CMG2 mRNA and protein expression in soft tissue sarcoma and their association with patient outcome. CMG2 mRNA was measured in 121 tumor samples of soft tissue sarcoma patients using quantitative real-time PCR. CMG2 protein was evaluated in 52 tumor samples by ELISA. CMG2 mRNA was significantly correlated with the corresponding CMG2 protein expression (rs = 0.31; p = 0.027). CMG2 mRNA expression was associated with the mRNA expressions of several ECM and tissue remodeling enzymes, among them CD26 and components of the uPA system. Low CMG2 mRNA expression was correlated with a worsened patients' disease-specific survival in Kaplan-Meier analyses (mean patient survival was 25 vs. 96 months; p = 0.013), especially in high-stage tumors. A decreased CMG2 expression is a negative prognostic factor for soft tissue sarcoma patients. CMG2 may be an interesting candidate gene for the further exploration of soft tissue sarcoma genesis and progression.

P4HA1: A single-gene surrogate of hypoxia signatures in oral squamous cell carcinoma patients.

BACKGROUND AND PURPOSE: Hypoxia gene expression signatures are of high prognostic value for head and neck cancer patients. Recently, the prognostic information of a multiple-gene hypoxia signature was found to be provided by the mRNA level of P4HA1 alone (Tawk et al., 2016). Therefore, we studied the prognostic value of P4HA1 in an independent cohort of oral squamous cell carcinoma (OSCC) patients. MATERIAL AND METHODS: Frozen tumor samples of 118 adult OSCC patients were analysed for P4HA1 mRNA level by quantitative real-time TaqMan™ PCR analysis. Kaplan-Meier analysis and Cox's regression analysis were performed to characterize the prognostic impact of P4HA1 mRNA level in OSCC patients. RESULTS: The analyzed patient cohort was divided into four subgroups according to the quartiles of the P4HA1 mRNA levels. The highest intratumoral P4HA1 mRNA level was significantly correlated with a poor overall survival (RR = 2.2; P = 0.04) and an increased risk of locoregional recurrence (RR = 4.8; P = 0.02). In patients who received radiotherapy (n = 82) highest intratumoral P4HA1 mRNA level was significantly correlated with a poor overall survival (RR = 3.4; P = 0.01) and an increased risk of locoregional recurrence (RR = 10.3; P = 0.005). Moreover, significant correlations between the P4HA1 mRNA level and the mRNA level of several EMT and stem cell markers were found. CONCLUSIONS: A high P4HA1 mRNA level, as a single-gene surrogate of hypoxia, is an independent prognostic marker for the overall survival and locoregional recurrence of OSCC patients.

Normoxic accumulation of HIF1α is associated with glutaminolysis.

OBJECTIVES: The stabilization of the transcription factor and prognostic tumor marker hypoxia-inducible factor 1α (HIF1α) is considered to be crucial for cellular metabolic adaptations to hypoxia. However, HIF1α has also been shown to accumulate under normoxic conditions, although this phenomenon is poorly understood. METHODS: We investigated the conditions for normoxic HIF1α stabilization in different tumor cell lines (e.g., two mammary carcinoma cell lines and three oral squamous cell carcinoma cell lines) via Western blot analysis or immunohistochemical staining. The transcriptional activity of HIF1 was demonstrated by analyzing the messenger RNA (mRNA) expression of the HIF1 target carbonic anhydrase 9 (CA9) via PCR. RESULTS: Our data demonstrate that the combined incubation of tumor cells with glutamine and growth factors (e.g., EGF, insulin, and serum) mediates the normoxic accumulation of HIF1α in vitro. Consequently, the inhibition of glutaminolysis by a glutaminase inhibitor blocked the normoxic accumulation of HIF1α. Additionally, the normoxic HIF1α protein displayed nuclear translocation and transcriptional activity, which was confirmed by the induction of CA9 mRNA expression. Furthermore, the normoxic accumulation of HIF1α was associated with impaired proliferation of tumor cells. Finally, ammonia, the toxic waste product of glutaminolysis, induced a normoxic accumulation of HIF1α to the same extent as glutamine. CONCLUSION: Our study suggests that HIF1α is involved in the regulation of glutamine metabolism and the cellular levels of the toxic metabolic waste product ammonia under normoxia. Hence, our results, together with data presented in the literature, support the hypothesis that HIF1α and its target genes play a crucial role in metabolic pathways, such as glutaminolysis and glycolysis, under both hypoxic and normoxic conditions. CLINICAL RELEVANCE: Therefore, the inhibition of HIF1α (and/or HIF1α target genes) could emerge as a promising therapeutic approach that would result in the accumulation of toxic metabolic waste products in tumor cells as well as the reduction of their nutrition and energy supply.

miR-199a-5p regulates HIF-1α and OSGIN2 and its expression is correlated to soft-tissue sarcoma patients' outcome.

Soft tissue sarcomas are a heterogeneous group of malignant neoplasms of mesenchymal origin. Partly due to hypoxia, an aggressive and radioresistant phenotype frequently develops, resulting in poorer patient outcome. microRNAs (miRNAs) are tiny, non-coding regulators of gene expression and in situations of cellular stress situations may predict clinical progression and patient outcome. In the present study, hypoxia-associated miR-199a-5p expression in 96 soft tissue sarcoma samples was analysed by reverse transcription-quantitative polymerase chain reaction and associations between miR-199a-5p expression and patient clinicopathological characteristics and survival were measured. Additionally, luciferase reporter assays analyzed the post-transcriptional regulation of hypoxia-associated genes hypoxia-inducible factor 1α (HIF-1α), oxidative stress induced growth inhibitor 2 (OSGIN2) and vascular endothelial growth factor (VEGF) by miR-199a-5p. Survival analyses indicated that low expression of miR-199a-5p was significantly correlated with poorer tumor-specific survival (univariate Cox's-Regression analyses; relative risk=1.92, P=0.029). Furthermore, it was demonstrated that the 3'UTR of HIF-1α and OSGIN2 genes were regulated by miR-199a-5p in-vitro, although the 3'UTR of VEGF was not. To the best of our knowledge, this is the first report demonstrating the regulation of the 3'untranslated region of the OSGIN2 gene by miR-199a-5p and a significant correlation between low miR-199a-5p expression and a poor outcome of patients with soft tissue sarcoma.

IDH1(R132H) mutation causes a less aggressive phenotype and radiosensitizes human malignant glioma cells independent of the oxygenation status.

BACKGROUND AND PURPOSE: In malignant glioma the presence of the IDH1 mutation (IDH1(R132H)) is associated with better clinical outcome. However, it is unclear whether IDH1 mutation is associated with a less aggressive phenotype or directly linked to increased sensitivity to radiotherapy. MATERIAL AND METHODS: We determined the influence of IDH1(R132H) mutant protein on proliferation and growth in 3D culture, migration, cell survival and radiosensitivity in vitro under normoxia (21% O2) and hypoxia (<1% O2) in a panel of human malignant glioma cell lines (U-251MG, U-343MG, LN-229) with stable overexpression of wild-type (IDH1(wt)) and mutated IDH1 (IDH1(R132H)). RESULTS: Overexpression of IDH1(R132H) in glioma cells resulted in slightly decreased cell proliferation, considerably reduced cell migration and caused differences in growth properties in 3D spheroid cultures. Furthermore, IDH1(R132H)-positive cells consistently demonstrated an increased radiosensitivity in human malignant glioma cells U-251MG (DMF10: 1.52, p<0.01 and 1.42, p<0.01), U-343MG (DMF10: 1.78, p<0.01 and 1.75, p<0.01) and LN-229 (DMF10: 1.41, p<0.05 and 1.68, p<0.01) under normoxia and hypoxia, respectively. CONCLUSION: Our data indicate that IDH1(R132H) mutation causes both a less aggressive biological behavior and direct radiosensitization of human malignant glioma cells. Targeting IDH1 appears to be an attractive approach in combination with radiotherapy.

mRNA expression levels of hypoxia-induced and stem cell-associated genes in human glioblastoma.

The roles of hypoxia-induced and stem cell-associated genes in the development of malignancy and tumour progression are well known. However, there are a limited number of studies analysing the impact of mRNA expression levels of hypoxia-induced and stem cell-associated genes in the tissues of brain tumours and glioblastoma patients. In this study, tumour tissues from patients with glioblastoma multiforme and tumour adjacent tissues were analysed. We investigated mRNA expression levels of hypoxia-inducible factor-1α (HIF-1α), hypoxia-inducible factor-2α (HIF-2α), carbonic anhydrase 9 (CA9), vascular endothelial growth factor (VEGF), glucose transporter-1 (GLUT-1) and osteopontin (OPN), and stem cell-associated genes survivin, epidermal growth factor receptor (EGFR), human telomerase reverse transcriptase (hTERT), Nanog and octamer binding transcription factor 4 (OCT4) using quantitative real-time polymerase chain reaction (qRT-PCR). Our data revealed higher mRNA expression levels of hypoxia-induced and stem cell-associated genes in tumour tissue than levels in the tumour adjacent tissues in patients with glioblastoma multiforme. A strong positive correlation between the mRNA expression levels of HIF-2α, CA9, VEGF, GLUT-1 and OPN suggests a specific hypoxia-associated profile of mRNA expression in glioblastoma multiforme. Additionally, the results indicate the role of stem-cell-related genes in tumour hypoxia. Kaplan-Maier analysis revealed that high mRNA expression levels of hypoxia-induced markers showed a trend towards shorter overall survival in glioblastoma patients (P=0.061). Our data suggest that mRNA expression levels of hypoxia-induced genes are important tumour markers in patients with glioblastoma multiforme.

Targeting of EGFR and HER2 with therapeutic antibodies and siRNA: a comparative study in glioblastoma cells.

BACKGROUND: The epidermal growth factor receptors, EGFR (HER1) and HER2, have proven prognostic relevance in a variety of human malignancies and both are functionally involved in the molecular pathogenesis of malignant gliomas. MATERIAL AND METHODS: We selectively inhibited EGFR and HER2 in glioblastoma cell lines via EGFR- and HER2-specific siRNAs and through the binding of the therapeutic antibodies cetuximab and trastuzumab. The expression of EGFR and HER2 was verified by real-time PCR and western blot analyses. We examined the growth rate, cell cycle distribution, cell migration, clonogenic survival, and radiosensitivity of U251MG and LN-229 glioblastoma cell lines to determine the physiological and cell biological effects of EGFR and HER2 targeting. RESULTS: EGFR and HER2 targeting using the therapeutic antibodies cetuximab and trastuzumab had no effect on cellular growth rate, cell cycle distribution, cell migration, clonogenic survival, and radiosensitivity in the cell lines U251 and LN-229. In contrast, siRNA knock-down of EGFR and HER2, reduced the growth rate by 40-65 %. The knock-down of EGFR did not change the cell migration rate in the cell lines U251 and LN-229. However, knock-down of HER2 reduced the cell migration rate by 50 %. Radiobiological analysis revealed that EGFR knock-down induced no radiosensitization in U251MG and LN-229 cells. However, the knock-down of HER2 induced radiosensitization in U251MG cells. CONCLUSION: The epidermal growth factor receptor HER2 is a promising anti-tumor target for the therapy of glioblastoma. HER2 targeting may represent a promising strategy to induce cell physiological and radiobiological anti-tumor effects in glioblastoma.

A novel splice variant of the stem cell marker LGR5/GPR49 is correlated with the risk of tumor-related death in soft-tissue sarcoma patients.

BACKGROUND: The human leucine-rich, repeat-containing G protein-coupled receptor (LGR) 5, also called GPR49, is a marker of stem cells in adult intestinal epithelium, stomach and hair follicles. LGR5/GPR49 is overexpressed in tumors of the colon, ovary and liver and in basal cell carcinomas. Moreover, an expression in skeletal muscle tissues was also detected. However, there has been no investigation regarding the expression and function of LGR5/GPR49 in soft-tissue sarcomas (STS) yet. METHODS: Seventy-seven frozen tumor samples from adult STS patients were studied using quantitative real-time TaqMan™ PCR analysis. The mRNA levels of wild type LGR5/GPR49 and a newly identified splice variant of LGR5/GPR49 lacking exon 5 (that we called GPR49Δ5) were quantified. RESULTS: A low mRNA expression level of GPR49Δ5, but not wild type LGR5/GPR49, was significantly correlated with a poor prognosis for the disease-associated survival of STS patients (RR = 2.6; P = 0.026; multivariate Cox's regression hazard analysis). Furthermore, a low mRNA expression level of GPR49Δ5 was associated with a shorter recurrence-free survival (P = 0.043). However, tumor onset in patients with a lower expression level of GPR49Δ5 mRNA occurred 7.5 years later (P = 0.04) than in patients with a higher tumor level of GPR49Δ5 mRNA. CONCLUSION: An attenuated mRNA level of the newly identified transcript variant GPR49Δ5 is a negative prognostic marker for disease-associated and recurrence-free survival in STS patients. Additionally, a lower GPR49Δ5 mRNA level is associated with a later age of tumor onset. A putative role of GPR49Δ5 expression in tumorigenesis and tumor progression of soft tissue sarcomas is suggested.

HIF-1α inhibition by siRNA or chetomin in human malignant glioma cells: effects on hypoxic radioresistance and monitoring via CA9 expression.

BACKGROUND: Hypoxia induces activation of the HIF-1 pathway and is an essential characteristic of malignant gliomas. Hypoxia has been linked to tumor progression, therapy resistance and poor prognosis. However, little is known about the impact of HIF-1α inhibition on radioresistance of malignant glioma. METHODS: In this study, we investigated the effects of the inhibition of HIF-1α on cell survival and radiosensitivity in U251MG and U343MG glioma cells, using two different strategies. HIF-1α inhibition was achieved by siRNA targeting of HIF-1α or via chetomin, a disruptor of interactions between HIF-1α and p300. The inhibition of the HIF-1 pathway was monitored by quantitative real-time PCR and Western blot analyses of the expression levels of HIF-1α and CA9. CA9 expression was investigated as a potential indicator of the efficacy of HIF-1 inhibition and the resulting radiosensitivity of malignant glioma cell lines was determined by clonogenic assay after irradiation under normoxic (2-10 Gy) or hypoxic (2-15 Gy) conditions. RESULTS: Although siRNA and chetomin show distinct modes of action, both attenuated the hypoxia-induced radioresistance of malignant glioma cell lines U251MG (DMF10: 1.35 and 1.18) and U343MG (DMF10: 1.78 and 1.48). However, siRNA and chetomin showed diverse effects on radiosensitivity under normoxic conditions in U251MG (DMF10: 0.86 and 1.35) and U343MG (DMF10: 1.33 and 1.02) cells. CONCLUSIONS: Results from this in vitro study suggest that inhibition of HIF-1α is a promising strategy to sensitize human malignant gliomas to radiotherapy and that CA9 could serve as an indicator of effective HIF-1-related radiosensitization.

Elevated tumor and serum levels of the hypoxia-associated protein osteopontin are associated with prognosis for soft tissue sarcoma patients.

BACKGROUND: Osteopontin (OPN) overexpression is correlated with a poor prognosis for tumor patients. However, only a few studies investigated the prognostic impact of expression of OPN in soft tissue sarcomas (STS) yet. METHODS: This study is based on tumor and serum samples from 93 adult STS patients. We investigated OPN protein levels in serum (n = 86) and tumor tissue (n = 80) by ELISA and OPN mRNA levels in tumor tissue (n = 68) by quantitative real-time PCR. RESULTS: No correlation was found between OPN levels in serum and tumor tissue. Moreover, an elevated OPN protein level in the serum was significantly associated with clinical parameters such as higher stage (p = 0.004), higher grade (p = 0.003), subtype (p = 0.002) and larger tumor size (p = 0.03). OPN protein levels in the tumor tissue were associated with higher stage (p = 0.06), higher grade (p = 0.003), subtype (p = 0.07) and an increased rate of relapse (p = 0.02). In addition, using a Cox's proportional hazards regression model, we found that an elevated OPN protein level in the serum and tumor tissue extracts is a significant negative prognostic factor for patients with STS. The relative risks of tumor-related death were 2.2 (p < 0.05) and 3.7 (p = 0.01), respectively. CONCLUSION: Our data suggest OPN protein in serum as well as in tumor tissue extracts is an important prognostic factor for soft tissue sarcoma patients.