HuR regulates beta-tubulin isotype expression in ovarian cancer.

The supply of oxygen and nutrients to solid tumors is inefficient because cancer tissues have an inadequate number of microvessels, thus inducing the selective growth of the most aggressive cancer cells. This explains why many of the factors underlying a poor prognosis are induced in hypoxic/hypoglycemic conditions. Among these factors, a prominent role in several solid tumors is played by the class III beta-tubulin gene (TUBB3). The study described here reveals that glucose deprivation enhances TUBB3 expression at both the gene and protein levels in A2780 ovarian cancer cells. In silico analysis of TUBB3 mRNA sequence predicted a putative binding site for the RNA-binding protein Hu antigen (HuR) in the 3' flanking untranslated region. A hypoglycemic-dependent engagement of this site was shown using RNA pull-down and ribonucleoimmunoprecipitation techniques. Thereafter, HuR gene silencing revealed that TUBB3 translation is HuR dependent in hypoglycemia because HuR silencing inhibited the entry of TUBB3 mRNA into cytoskeletal and free polysomes. Finally, the clinical value of this finding was assessed in a clinical cohort of 46 ovarian cancer patients in whom it was found that HuR cytoplasmic staining was associated with high levels of TUBB3 and poor survival.

IgG autoantibody to brain beta tubulin III associated with cytokine cluster-II discriminate cerebral malaria in central India.

BACKGROUND: The main processes in the pathogenesis of cerebral malaria caused by Plasmodium falciparum involved sequestration of parasitized red blood cells and immunopathological responses. Among immune factors, IgG autoantibodies to brain antigens are increased in P. falciparum infected patients and correlate with disease severity in African children. Nevertheless, their role in the pathophysiology of cerebral malaria (CM) is not fully defined. We extended our analysis to an Indian population with genetic backgrounds and endemic and environmental status different from Africa to determine if these autoantibodies could be either a biomarker or a risk factor of developing CM. METHODS/PRINCIPAL FINDINGS: We investigated the significance of these self-reactive antibodies in clinically well-defined groups of P. falciparum infected patients manifesting mild malaria (MM), severe non-cerebral malaria (SM), or cerebral malaria (CM) and in control subjects from Gondia, a malaria epidemic site in central India using quantitative immunoprinting and multivariate statistical analyses. A two-fold complete-linkage hierarchical clustering allows classifying the different patient groups and to distinguish the CM from the others on the basis of their profile of IgG reactivity to brain proteins defined by PANAMA Blot. We identified beta tubulin III (TBB3) as a novel discriminant brain antigen in the prevalence of CM. In addition, circulating IgG from CM patients highly react with recombinant TBB3. Overall, correspondence analyses based on singular value decomposition show a strong correlation between IgG anti-TBB3 and elevated concentration of cluster-II cytokine (IFNgamma, IL1beta, TNFalpha, TGFbeta) previously demonstrated to be a predictor of CM in the same population. CONCLUSIONS/SIGNIFICANCE: Collectively, these findings validate the relationship between antibody response to brain induced by P. falciparum infection and plasma cytokine patterns with clinical outcome of malaria. They also provide significant insight into the immune mechanisms associated to CM by the identification of TBB3 as a new disease-specific marker and potential therapeutic target.

Molecular mechanisms of patupilone resistance.

Patupilone is an epothilone in advanced clinical development that has shown promising efficacy in heavily pretreated patients. This study aimed at characterizing the mechanisms of patupilone activity in resistant patients. To this end, we generated patupilone-resistant cells using two cellular models, the first characterized by high chemosensitivity and low class III beta-tubulin (TUBB3) expression (A2780), and the second by low chemosensitivity and high TUBB3 expression (OVCAR-3). The obtained cell lines were named EPO3 and OVCAR-EPO, respectively. The same selection procedure was done in A2780 cells to generate a paclitaxel-resistant cell line (TAX50). Factors of resistance are expected to increase in the drug-resistant cell lines, whereas factors of drug sensitivity will be down-regulated. Using this approach, we found up-regulation of TUBB3 in TAX50, but not EPO3, cells, showing that TUBB3 mediates the resistance to paclitaxel but not to patupilone. Moreover, TUBB3 was a factor of patupilone sensitivity because OVCAR-EPO cells exhibited a dramatic reduction of TUBB3 and a concomitant sensitization to hypoxia and cisplatin-based chemotherapy. To identify the mechanisms underlying patupilone resistance, tubulin genes were sequenced, thereby revealing that a prominent mechanism of drug resistance is represented by point mutations in class I beta-tubulin. Overall, these results suggest that paclitaxel and patupilone have nonoverlapping mechanisms of resistance, thus allowing the use of patupilone for those patients relapsing after paclitaxel-based chemotherapy. Furthermore, patupilone represents a promising first-line option for the treatment of high-risk ovarian cancer patients, who exhibit high TUBB3 levels and poor response to standard paclitaxel-platin chemotherapy.

Hypoxia induces class III beta-tubulin gene expression by HIF-1alpha binding to its 3' flanking region.

Class III beta-tubulin (TUBB3) overexpression represents a major mechanism of drug resistance to microtubule interacting agents such as taxanes and Vinca alkaloids. Here, we tested hypoxia as a possible inducer of TUBB3. The effects of hypoxia on TUBB3 expression were monitored at mRNA and protein level in A2780, in its paclitaxel-resistant counterpart (TC1) and in HeLa cells. Hypoxia was a strong inducer of TUBB3 in A2780, but not in TC1 and HeLa cells. In A2780 HIF-1alpha was knocked down using RNA interference and TUBB3 expression was assessed in normoxia and hypoxia. The silencing abolished the hypoxia-dependent increase of TUBB3, thereby demonstrating that HIF-1alpha mediates TUBB3 induction in hypoxia. To investigate this phenomenon, the 5' flanking region of human TUBB3 was cloned upstream GFP as a reporter. This region contained the promoter gene, but activity of the reporter was unaffected by hypoxia. Thus, we looked at the 3' flanking region and, at +168 nucleotides from the stop codon, an HIF-1alpha binding site was proven to be active in hypoxia, using a construct in which the site was cloned downstream GFP as reporter gene. Deletion of the site in the construct abolished GFP enhancement upon hypoxia. Chromatin immunoprecipitation revealed the engagement by HIF-1alpha of this site in hypoxia. Methylation analysis of this 3' enhancer showed that it was free of methylation in 70% of cells in A2780, while in less than 16% in both TC1 and HeLa cells, thereby suggesting that TUBB3 increase upon hypoxia is abolished through methylation of the 3' enhancer.

Deletion of the glucose-6-phosphate dehydrogenase gene KlZWF1 affects both fermentative and respiratory metabolism in Kluyveromyces lactis.

In Kluyveromyces lactis, the pentose phosphate pathway is an alternative route for the dissimilation of glucose. The first enzyme of the pathway is the glucose-6-phosphate dehydrogenase (G6PDH), encoded by KlZWF1. We isolated this gene and examined its role. Like ZWF1 of Saccharomyces cerevisiae, KlZWF1 was constitutively expressed, and its deletion led to increased sensitivity to hydrogen peroxide on glucose, but unlike the case for S. cerevisiae, the Klzwf1Delta strain had a reduced biomass yield on fermentative carbon sources as well as on lactate and glycerol. In addition, the reduced yield on glucose was associated with low ethanol production and decreased oxygen consumption, indicating that this gene is required for both fermentation and respiration. On ethanol, however, the mutant showed an increased biomass yield. Moreover, on this substrate, wild-type cells showed an additional band of activity that might correspond to a dimeric form of G6PDH. The partial dimerization of the G6PDH tetramer on ethanol suggested the production of an NADPH excess that was negative for biomass yield.

KlADH3, a gene encoding a mitochondrial alcohol dehydrogenase, affects respiratory metabolism and cytochrome content in Kluyveromyces lactis.

A Kluyveromyces lactis strain, harbouring KlADH3 as the unique alcohol dehydrogenase (ADH) gene, was used in a genetic screen on allyl alcohol to isolate mutants deregulated in the expression of this gene. Here we report the characterization of some mutants that lacked or had highly reduced amounts of KlAdh3p activity; in addition, these mutants showed alterations in glucose metabolism, reduced respiration and reduced cytochrome content. Our results confirm that the KlAdh3p activity contributes to the reoxidation of cytosolic NAD(P)H feeding the respiratory chain through KlNdi1p, the mitochondrial internal transdehydrogenase. The low levels of KlAdh3p in two of the mutants were associated with mutations in KlSDH1, one of the genes of complex II, suggesting signalling between the respiratory chain and expression of the KlADH3 gene.

The deletion of the succinate dehydrogenase gene KlSDH1 in Kluyveromyces lactis does not lead to respiratory deficiency.

We have isolated a Kluyveromyces lactis mutant unable to grow on all respiratory carbon sources with the exception of lactate. Functional complementation of this mutant led to the isolation of KlSDH1, the gene encoding the flavoprotein subunit of the succinate dehydrogenase (SDH) complex, which is essential for the aerobic utilization of carbon sources. Despite the high sequence conservation of the SDH genes in Saccharomyces cerevisiae and K. lactis, they do not have the same relevance in the metabolism of the two yeasts. In fact, unlike SDH1, KlSDH1 was highly expressed under both fermentative and nonfermentative conditions. In addition to this, but in contrast with S. cerevisiae, K. lactis strains lacking KlSDH1 were still able to grow in the presence of lactate. In these mutants, oxygen consumption was one-eighth that of the wild type in the presence of lactate and was normal with glucose and ethanol, indicating that the respiratory chain was fully functional. Northern analysis suggested that alternative pathway(s), which involves pyruvate decarboxylase and the glyoxylate cycle, could overcome the absence of SDH and allow (i) lactate utilization and (ii) the accumulation of succinate instead of ethanol during growth on glucose.