Cardiac glycosides suppress the maintenance of stemness and malignancy via inhibiting HIF-1α in human glioma stem cells.

Tissue hypoxia contributes to solid tumor pathogenesis by activating a series of adaptive programs. We previously showed that hypoxia promotes the preferential expansion and maintenance of CD133 positive human glioma stem cells (GSC) in a hypoxia inducible factor 1 alpha (HIF-1α)-dependent mechanism. Here, we examined the activity of digitoxin (DT), a cardiac glycoside and a putative inhibitor of HIF-1α, on human GSC in vitro and in vivo. During hypoxic conditions (1% O2), we observed the effect of DT on the intracellular level of HIF-1α and the extracellular level of vascular endothelial growth factor (VEGF) in human GSC. We found that DT at clinically achievable concentrations, suppressed HIF-1α accumulation during hypoxic conditions in human GSC and established glioma cell lines. DT treatment also significantly attenuated hypoxia-induced expression of VEGF, a downstream target of HIF-1α. Exposure to DT also reduced hypoxia-induced activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. Furthermore, DT potently inhibited neurosphere formation, and decreased CD133 expression even at concentrations that were not overtly cytotoxic. Lastly, treatment with DT reduced GSC engraftment in an in vivo xenograft model of glioblastoma. Intraperitoneal injections of DT significantly inhibited the growth of established glioblastoma xenografts, and suppressed expression of HIF-1α and carbonic anhydrase (CA9), a surrogate marker of hypoxia. Taken together, these results suggest that DT at clinically achievable concentration functions as an inhibitor of HIF-1α, worthy of further investigations in the therapy of glioblastoma.

Purification, enzymatic activity and inhibitor discovery for recombinant human carbonic anhydrase XIV.

Human carbonic anhydrase XIV (CA XIV), a transmembrane protein, highly expressed in the central nervous system, is difficult to recombinantly express and purify in large scale for the measurements of inhibitor binding and drug design. CA XIV belongs to the family of twelve catalytically active CA isoforms in the human body. Disorders in the expression of CA XIV cause serious diseases and CA XIV has been described as a possible drug target for the treatment of epilepsy, some retinopathies, and skin tumors. In this study, the effect of different promoters, E. coli strains, and the length of recombinant CA XIV protein construct were analyzed for the production CA XIV in large scale by using affinity purification. Active site titration by inhibitors and the isothermal titration calorimery revealed over 96% purity of the protein. Enzymatic activity of the purified CA XIV was determined by following the CO2 hydration using the stopped-flow technique. Several inhibitors were discovered that exhibited selectivity towards CA XIV over other CA isoforms and could be developed as drugs.

Loss of carbonic anhydrase XII function in individuals with elevated sweat chloride concentration and pulmonary airway disease.

Elevated sweat chloride levels, failure to thrive (FTT), and lung disease are characteristic features of cystic fibrosis (CF, OMIM #219700). Here we describe variants in CA12 encoding carbonic anhydrase XII in two pedigrees exhibiting CF-like phenotypes. Exome sequencing of a white American adult diagnosed with CF due to elevated sweat chloride, recurrent hyponatremia, infantile FTT and lung disease identified deleterious variants in each CA12 gene: c.908-1 G>A in a splice acceptor and a novel frameshift insertion c.859_860insACCT. In an unrelated consanguineous Omani family, two children with elevated sweat chloride, infantile FTT, and recurrent hyponatremia were homozygous for a novel missense variant (p.His121Gln). Deleterious CFTR variants were absent in both pedigrees. CA XII protein was localized apically in human bronchiolar epithelia and basolaterally in the reabsorptive duct of human sweat glands. Respiratory epithelial cell RNA from the adult proband revealed only aberrant CA12 transcripts and in vitro analysis showed greatly reduced CA XII protein. Studies of ion transport across respiratory epithelial cells in vivo and in culture revealed intact CFTR-mediated chloride transport in the adult proband. CA XII protein bearing either p.His121Gln or a previously identified p.Glu143Lys missense variant localized to the basolateral membranes of polarized Madin-Darby canine kidney (MDCK) cells, but enzyme activity was severely diminished when assayed at physiologic concentrations of extracellular chloride. Our findings indicate that loss of CA XII function should be considered in individuals without CFTR mutations who exhibit CF-like features in the sweat gland and lung.

Hypoxia-Related Marker GLUT-1, CAIX, Proliferative Index and Microvessel Density in Canine Oral Malignant Neoplasia.

For various types of tumor therapy, it is suggested that co-targeting of tumor microenvironment, mainly tumor vasculature, mediates tumor response mechanisms. Immunohistochemistry for glucose transporter-1 (GLUT-1), carbonic anhydrase-IX (CAIX), Ki-67, and von Willebrand factor VIII for microvessel density (MVD) were performed on formalin-fixed paraffin-embedded samples of canine oral malignant neoplasms. Polarographic oxygen measurements (median pO2) and perfusion data via contrast-enhanced power Doppler ultrasound (median vascularity, median blood volume) provided additional information. Ninety-two samples were analyzed: sarcomas (n = 32), carcinomas (n = 30), and malignant melanomas (n = 30). Polarographic oxygen and perfusion data was available in 22.8% (sarcomas n = 9, carcinomas n = 7, melanomas n = 5), and 27.1% (sarcomas n = 10, carcinomas n = 8, melanomas n = 7) of cases, respectively. GLUT-1 expression was detected in 46.7% of all samples, and was generally weak. CAIX expression was found in 34.8% of all samples. Median Ki-67 score and MVD count was 19% and 17, respectively. The evaluation of the GLUT-1 score and continuous data showed significantly lower GLUT-1 levels in sarcomas (mean 5.1%, SD 6.2) versus carcinomas and melanomas (mean 16.5%/ 19.0%, SD 17.3/ 20.9, p = 0.001). The expression of CAIX correlated mildly positively with GLUT-1 (p = 0.018, rho = 0.250) as well as with Ki-67 (p = 0.014, rho = 0.295). MVD showed a significantly lower level in melanomas (mean 12.6, SD 7.7) versus sarcomas and carcinomas (mean 21.8/ 26.9, SD 13.0/20.4, p = 0.001). Median vascularity and blood volume were significantly lower in sarcomas (mean 10.4%, SD 11.0, and mean 6.3%, SD 6.5, respectively) versus carcinomas (mean 39.2%, SD 16.4 and mean 33.0%, SD 25.6, respectively) and melanomas (mean 36.0%, SD 18.3, and 31.5%, SD 24.5). Between the 3 histological groups, there was neither a significant difference in the GLUT-1 and CAIX score and continuous data, nor the Ki67 score, or polarographic oxygen measurements. GLUT-1 continuous data and Ki-67 (p<0.001, rho = 0.403), as well as Ki-67 and MVD (p = 0.029, rho = 0.228) correlated positively and a mild correlation was found between vascularity and GLUT-1 (p = 0.043, rho = 0.408). GLUT-1, CAIX, proliferative index and MVD levels were established as microenvironmental descriptors with the purpose of creating a baseline in order to follow changes seen in the tumor microenvironment after hypofractionated radiation with high doses.

Carbonic Anhydrase 9 mRNA/microRNA34a Interplay in Hypoxic Human Mammospheres.

The hypoxic environment is a crucial component of the cancer stem cell niche and it is capable of eliciting stem cell features in cancer cells. We previously reported that SNAI2 up-regulates the expression of Carbonic Anhydrase iso-enzyme 9 (CA9) in hypoxic MCF7 cells. Here we show that SNAI2 down-regulates miR34a expression in hypoxic MCF7 cell-derived mammospheres. Next, we report on the capability of miR34a to decrease CA9 mRNA stability and CA9 protein expression. We also convey that the over-expression of cloned CA9-mRNA-3'UTR increases the mRNA half-life and protein levels of two miR34a targets JAGGED1 and NOTCH3. The data here reported shows that the SNAI2-dependent down-regulation of miR34a substantially contributes to the post-transcriptional up-regulation of CA9, and that CA9-mRNA-3'UTR acts as an endogenous microRNA sponge. We conclude that CA9/miR34 interplay shares in the hypoxic regulation of mammospheres and therefore, may play a relevant role in the hypoxic breast cancer stem cell niche.

The Expression of Carbonic Anhydrase (CA) IX/XII and Lymph Node Metastasis in Early Breast Cancer.

PURPOSE: The aim of study was to test by immunohistochemical (IHC) staining whether carbonic anhydrase (CA) 9 and 12 have an effect on sentinel lymph node (SLN) metastasis in early breast cancer and to find clinicopathologic factors associated with SLN metastasis. MATERIALS AND METHODS: Between June 2003 and June 2011, medical records of 470 patients diagnosed with breast cancer with pT1-2, pN0-2, and M0 were reviewed. Of these 470, 314 patients who underwent SLN biopsy±axillary dissection were subjects of this study. Using tissue microarray, IHC staining for CA9 and CA12 was performed. Clinicopathologic factors such as patient age, tumour size, lymphatic invasion, hormone receptor status, and the Ki-67 labeling index were analysed together. RESULTS: The mean age of all patients was 51.7 years. The mean number of harvested SLN was 3.62, and 212 patients (67.5%) had negative SLN. Lymphatic invasion, the Ki-67 labelling index of primary tumours, and CA9 staining of stromal cells, were independent risk factors for SLN metastasis in the multivariate analysis. In 33 patients (10.5%) without the three risk factors, no patient had SLN metastasis. In 80 patients without lymphatic invasion of primary tumours or CA9 staining of stromal cells, only four patients (5%) had positive SLN. CONCLUSION: CA9 staining of stromal cells is an independent risk factor for SLN metastasis as well as lymphatic invasion and a low Ki-67 labelling index of primary tumours in patients with early breast cancer. IHC staining of primary tumours for CA12was not associated with SLN metastasis.

Characteristics of carbonic anhydrase 9 expressing cells in human intestinal crypt base.

Though recent studies have revealed that stem cells of many tissues are harbored in hypoxic microenvironment, little is known about the relationship between hypoxia and intestinal crypt base, where intestinal stem cells are supposed to exist. In this study, we focused on carbonic anhydrase IX (CA9), a hypoxia-inducible membrane-tethered protein, in normal intestinal crypt base, adenoma and early colorectal cancer. Using surgically resected human colorectal cancer specimen, we searched for the expression pattern and functional association of CA9 in human adult normal intestinal epithelia, adenoma and early colorectal cancer by immunofluorescent and immunohistochemical staining, flow cytometry, and quantitative real-time-polymerase chain reaction. We demonstrated that almost all crypt base slender cells in ileum and crypt base cells with eosinophilic structure in their basal cytoplasm in right and left colon were CA9+ with the ratio of 25 to 40%, and that adenoma and T1 colorectal cancer showed broad expression of CA9. Flow cytometrically sorted CA9+ population showed increased mRNA level of a Wnt signaling factor AXIN2. In conclusion, these observations indicate that CA9 expression in normal crypt base cells has association with intestinal epithelial stemness and CA9 may be involved in the carcinogenesis of colorectal cancer.

CA IX is upregulated in CoCl2-induced hypoxia and associated with cell invasive potential and a poor prognosis of breast cancer.

Hypoxia, a common phenomenon during the development of malignant solid tumors including breast cancer, serves to propagate a cascade of molecular pathways triggered by hypoxia-inducible factor-1α (HIF-1α). Carbonic anhydrase IX (CA IX), one of the target genes of HIF-1α, has been reported to be involved in progression of malignant tumors. The objective of this study was to investigate the expression of HIF-1α and CA IX in hypoxia, involvement of CA IX in the regulation of migration and invasion/metastasis and its prognostic significance in breast cancer. We used cobalt chloride (CoCl2) as a hypoxia-mimetic agent and found that the expression of HIF-1α protein, CA IX mRNA and protein, is effectively upregulated, except for HIF-1α mRNA. Data showed that the elevated CA IX expression is closely related to in vitro cell migration and invasion, and the underlying mechanism of this process may be associated with epithelial-mesenchymal transition (EMT). The study of clinical tissue samples also demonstrated that CA IX is an independent prognostic marker that may serve as a useful clinical biomarker for predicting tumor progression and the invasion/metastasis of breast cancer. These results provide further insight into the role of CA IX in tumor progression and put forward further strong evidence as well as new consideration for CA IX target therapy.

Circulating protein synthesis rates reveal skeletal muscle proteome dynamics.

Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders.

Efficient Expression and Crystallization System of Cancer-Associated Carbonic Anhydrase Isoform IX.

Human carbonic anhydrase IX (CA IX) is overexpressed in a number of solid tumors and is considered to be a marker for cellular hypoxia that it is not produced in most normal tissues. CA IX contributes to the acidification of the extracellular matrix, which, in turn, favors tumor growth and metastasis. Therefore, CA IX is considered to be a promising anti-cancer drug target. However, the ability to specifically target CA IX is challenging due to the fact that the human genome encodes 15 different carbonic anhydrase isoforms that have a high degree of homology. Furthermore, structure-based drug design of CA IX inhibitors so far has been largely unsuccessful due to technical difficulties regarding the expression and crystallization of the enzyme. Currently, only one baculovirus-produced CA IX structure in complex with a nonspecific CA inhibitor, acetazolamide, is available in Protein Data Bank. We have developed an efficient system for the production of the catalytic domain of CA IX in methylotrophic yeast Pichia pastoris. The produced protein can be easily crystallized in the presence of inhibitors, as we have demonstrated for several 2-thiophene-sulfonamide compounds. We have also observed significant differences in the binding mode of chemically identical compounds to CA IX and CA II, which can be further exploited in the design of CA IX-specific inhibitors.

Hypoxia biomarkers in squamous cell carcinoma of the uterine cervix.

BACKGROUND: There is significant evidence that severe tumor hypoxia may cause resistance to chemoradiotherapy and promote metastatic spread in locally advanced carcinoma of the uterine cervix. Some clinical investigations have suggested that high expression of hypoxia-inducible factor-1α (HIF-1α) and/or its target gene carbonic anhydrase IX (CAIX) may be useful biomarkers of tumor hypoxia and poor outcome in cervical cancer. Here, we challenged this view by investigating possible associations between HIF-1α expression, CAIX expression, fraction of hypoxic tissue, and lymph node metastasis in experimental human tumors. METHODS: Tumors of two cervical carcinoma xenograft lines (CK-160 and TS-415) were included in the study. Pimonidazole was used as a hypoxia marker, and tumor hypoxia, HIF-1α expression, and CAIX expression were detected by immunohistochemistry. Metastatic status was assessed by examining external lymph nodes in the inguinal, axillary, interscapular, and submandibular regions and internal lymph nodes in the abdomen and mediastinum. RESULTS: Tissue regions staining positive for pimonidazole, HIF-1α, or CAIX were poorly colocalized, both in CK-160 and TS-415 tumors. The expression of HIF-1α or CAIX did not correlate with the fraction of hypoxic tissue in any of the two tumor lines. Furthermore, clinically relevant associations between HIF-1α or CAIX expression and lymph node metastasis were not found. CONCLUSION: Because significant associations between HIF-1α expression, CAIX expression, fraction of hypoxic tissue, and incidence of lymph node metastases could not be detected in any of two preclinical models of human cervical cancer, it is not realistic to believe that high expression of HIF-1α or CAIX can be useful biomarkers of tumor hypoxia and poor outcome in a highly heterogeneous disease like cervical carcinoma.

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.

Inhibition of carbonic anhydrase IX (CA9) sensitizes renal cell carcinoma to ionizing radiation.

While normal kidneys are relatively sensitive to ionizing radiation (IR), renal cell carcinoma (RCC) is considered radioresistant. Carbonic anhydrase IX (CA9), an enzyme that maintains intracellular pH by carbon dioxide dissolution, is upregulated in the majority of RCC, but not in normal kidneys. Since regulation of intracellular pH may enhance radiation effects, we hypothesized that inhibition of CA9 may radiosensitize RCC. Clonogenic survival assay of human clear cell RCC 786-O and murine RCC RAG cells in the presence of a pharmacological CA9 inhibitor or with shRNA-mediated knockdown of CA9 was performed to investigate the response to IR in vitro (single dose or fractionated) and in vivo. Extracellular pH changes were measured in vitro. Treatment with AEBS [4-(2-aminoethyl)benzene sulfonamide], a sulfonamide, was used as a pharmacological inhibitor of the enzymatic activity of CA9. Nude mice bearing subcutaneous xenografts of 786-O cells stably expressing CA9 shRNA or scrambled control were irradiated (6 Gy). Tumor growth was followed longitudinally in the 786-O-bearing mice receiving AEBS (50-200 µg/ml drinking water) or control (vehicle only) which were irradiated (6 Gy) and compared with mice receiving either IR or AEBS alone. In vitro inhibition of CA9 activity or expression significantly sensitized RCC cells to the effects of IR (p<0.05), an effect even more significant when hypofractionated IR was applied. In vivo irradiated xenografts from RCC cells transfected with CA9 shRNA were significantly smaller compared to irradiated xenografts from the scrambled shRNA controls (p<0.05). RCC xenografts from mice treated with AEBS in combination with IR grew significantly slower than all controls (p<0.05). Inhibition of CA9 expression or activity resulted in radiation sensitization of RCC in a preclinical mouse model.

Evidence of Placental Hypoxia in Maternal Sleep Disordered Breathing.

Sleep disordered breathing (SDB) represents a spectrum of disorders, including habitual snoring and obstructive sleep apnea (OSA). Sleep disordered breathing is characterized by chronic intermittent hypoxia, airflow limitation, and recurrent arousals, which may lead to tissue hypoperfusion, hypoxia, and inflammation. In this study, we aimed to examine whether SDB during pregnancy was associated with histopathologic evidence of chronic placental hypoxia and/or uteroplacental underperfusion. The placentas of women with OSA (n  =  23) and habitual snoring (n  =  78) as well as nonsnorer controls (n  =  47) were assessed for histopathologic and immunohistochemical markers of chronic hypoxia and uteroplacental underperfusion. Fetal normoblastemia was significantly more prevalent in SDB placentas than in those of nonsnorer controls (34.6% and 56.5% in snorers and OSA, respectively, versus 6.4% in controls). Expression of the tissue hypoxia marker carbonic anhydrase IX (CAIX) was more common in OSA placentas than controls (81.5% and 91.3% in snorers and OSA, respectively, versus 57.5% in controls). Adjusting for confounders such as body mass index, diabetes mellitus, or chronic hypertension did not alter the results. The uteroplacental underperfusion scores were similar among the 3 groups. Our findings suggest that SDB during pregnancy is associated with fetoplacental hypoxia, as manifested by fetal normoblastemia and increased placental carbonic anhydrase IX immunoreactivity. The clinical implications and underlying mechanisms remain to be determined.

Carbonic anhydrase IX overexpression regulates the migration and progression in oral squamous cell carcinoma.

Carbonic anhydrase IX (CAIX) is reportedly overexpressed in several types of carcinomas and is generally considered a marker of malignancy. The current study investigated the association between membrane expression of CAIX and the clinicopathological characteristics in oral squamous cell carcinoma (OSCC) patients. The study used immunohistochemistry to examine CAIX expression in 271 OSCC specimens by tissue microarray (TMA) and assessed the effect of CAIX overexpression and knockdown on migration of oral cancer cells in vitro. We found that CAIX expression was associated with more advanced clinical stages (p = 0.030) and positive lymph node metastasis (p = 0.026). Importantly, CAIX expression was correlated with a poorer patient prognosis in a univariate survival analysis (p = 0.025). Moreover, CAIX suppression by small interfering RNA (siRNA) significantly reduced cellular migration in OECM-1 oral cancer cell. In conclusion, our study showed that the expression of CAIX in OSCC samples can predict the progression of OSCC and survival of OSCC patients in Taiwan.

The impact of immunohistochemical staining with ezrin-carbonic anhydrase IX and neuropilin-2 on prognosis in patients with metastatic renal cell cancer receiving tyrosine kinase inhibitors.

The identification of prognostic factors in patients with renal cell carcinoma (RCC) represents an area of increasing interest. In this retrospective study, we evaluated the prognostic role of carbonic anhydrase-IX, ezrin, and neuropilin in metastatic RCC patients. The expression of several biomarkers were measured by immunohistochemistry (IHC) in 45 patients with advanced stage RCC treated with second-line tyrosine kinase inhibitors (TKIs) targeting vascular endothelial growth factor (VEGF) after failure of interferon-alpha between January 2007 and June 2012. Kaplan-Meier curves and log-rank tests were used for analysis of progression-free survival (PFS) and overall survival (OS), and a multivariate Cox proportional hazard model was employed to identify factors with an independent effect on the survival. Age, ezrin and neuropilin-2 overexpression were found to be statistically significant factors (P < 0.05) for PFS in the univariate analysis. Ezrin and neuropilin-2 overexpression, hemoglobin and albumin level were statistically significant factors (P < 0.05) for OS in the univariate analysis. Multivariate analysis revealed that low expression of ezrin and neuropilin-2 was an independent prognostic factor for PFS and OS. The median PFS was 4 months for patients overexpressing neuropilin-2 versus 11 months for those with lower expression of neuropilin-2 (p = 0.033). The median OS was longer in patients with low levels of neuropilin-2 expression (26 months) compared to patients overexpressing neuropilin-2 (13 months) (p = 0.023). Increased expression of ezrin was associated with poor prognosis in patients treated with TKIs targeting VEGF (PFS, 3 vs 7 months; p = 0.012). High ezrin expression was associated with shorter OS (p = 0.009). This is the first study in the literature showing that neuropilin-2 and ezrin are related with prognosis in patients with advanced RCC.

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.

Carbonic anhydrase-IX score is a novel biomarker that predicts recurrence and survival for high-risk, nonmetastatic renal cell carcinoma: Data from the phase III ARISER clinical trial.

INTRODUCTION AND OBJECTIVE: With a limited number of prognostic and predictive biomarkers available, carbonic anhydrase-IX (CAIX) has served as an important prognostic biomarker for patients with clear cell renal cell carcinoma (ccRCC). However, studies have recently called into question the role of CAIX as a biomarker for ccRCC. To investigate this uncertainty, we quantified the association of CAIX with lymphatic involvement and survival using data from ARISER study (WX-2007-03-HR)--a prospective trial involving subjects with high-risk nonmetastatic ccRCC. METHODS AND MATERIALS: We reviewed the records of 813 patients enrolled in the ARISER study. Central review of histology, grade, and CAIX staining (frequency and intensity) was performed. CAIX score was derived by multiplying the staining intensity (1-3) by percent positive cells (0%-100%), yielding a range of 0 to 300. We quantified the association of CAIX expression and score with lymphatic spread and survival (disease-free survival [DFS] and overall survival [OS]) using Kaplan-Meier and multivariable propensity score adjusted Cox regression analyses. RESULTS: Median follow-up of the cohort was 54.2 months. Although 56% of subjects with lymphatic involvement had CAIX>85%, only 33% had CAIX score ≥ 200. On multivariable analysis, CAIX>85% was not a statistically significant predictor of DFS and OS (P = 0.06 and P = 0.15, respectively). However, CAIX score ≥ 200, when compared with CAIX score ≤ 100, was associated with improved DFS and OS (P = 0.01 and P = 0.01, respectively) on multivariable analysis. CONCLUSIONS: The largest, multicenter, prospective analysis of patients with high-risk nonmetastatic ccRCC demonstrates the utility of CAIX score as a statistically significant prognostic biomarker for survival. We recommend that CAIX score be quantified for all patients with high-risk disease after nephrectomy.

Prognostic value of carbonic anhydrase VII expression in colorectal carcinoma.

BACKGROUND: Carbonic anhydrases (CAs) have been implicated in the pathogenesis of human cancers. Carbonic anhydrase VII (CA7), a member of the CA gene family, was recently demonstrated to be expressed in several human tissues including colon. Nevertheless, the expression and clinical relevance of CA7 in colorectal carcinoma (CRC) has not been investigated. METHODS: Real-time PCR, western blot, and immunohistochemistry analyses were used to determine CA7 expression in CRC clinical samples. The correlation of CA7 expression with clinicopathologic features was assessed in 228 patients from Luoyang, China (training cohort) and validated in 151 patients from Shanghai, China (validation cohort). Kaplan-Meier and Cox proportional regression analyses were used to estimate the association between CA7 expression and patients' survival. RESULTS: CA7 expression was frequently downregulated in CRC tissues at both the mRNA and protein levels. Reduced expression of CA7 was significantly correlated with poor differentiation, positive lymph node metastasis, advanced TNM stage and unfavorable clinical outcome not only in the training cohort but also in the validation set. Survival analysis indicated that patients with lower CA7 expression had a significantly shorter disease-specific survival (DSS) than those with higher CA7 expression. Importantly, further stage-based analyses revealed that decreased CA7 expression significantly predicted poor DSS and was an independent adverse prognostic indicator for patients with early stage tumors in both cohorts. CONCLUSIONS: Our results indicate that decreased expression of CA7 correlates with disease progression and predicts poor prognosis in CRC, especially for patients with early stage tumors.

Specific immunohistochemical pattern of carbonic anhydrase IX is helpful for the diagnosis of CNS hemangioblastoma.

Hemangioblastomas are rare capillary-rich tumors predominantly found in the CNS. The histological appearance of these tumors varies across a broad spectrum. Several entities show considerable histomorphological similarities to hemangioblastomas. Therefore, morphological evaluation can be challenging. In this study, we evaluated the diagnostic utility of immunohistochemistry using antibodies against carbonic anhydrase IX and cytokeratin staining. Within our files, we identified 20 hemangioblastomas. A collection of 46 other tumors relevant to the differential diagnosis (12 pilocytic astrocytomas, 11 meningiomas, one pleomorphic xanthoastrocytoma, one angiomatous fibrous histiocytoma, 14 carcinoma metastases and seven gliomas grades II-IX) served as control. The pattern of strong, diffuse expression of carbonic anhydrase IX with membranous accentuation in combination with keratin negativity was considered diagnostic for hemangioblastomas. It was found in 18 out of 20 (90%) hemangioblastomas and in none of the control cases (P < 0.001). This resulted in a sensitivity of 90% and a specificity of 100%. The positive and negative predictive values were 100% and 96%, respectively. Carbonic anhydrase IX with cytokeratin is thus a highly sensitive and specific marker combination for hemangioblastomas. It is therefore very helpful in the diagnosis of these tumors and in their discrimination from other entities.