Marisa Mariani

MiR-200c and HuR in ovarian cancer

BackgroundMicroRNAs in solid malignancies can behave as predictors of either good or poor outcome. This is the case with members of the miR-200 family, which are the primary regulators of the epithelial to mesenchymal transition and have been reported to act as both oncogenes and tumor suppressors. This study assessed the role of miR-200c as regulator of class III β-tubulin (TUBB3), a factor associated with drug-resistance and poor prognosis in ovarian cancer.MethodsExpression of miR-200c was assessed in a panel of ovarian cancer cell lines with inherent or acquired drug-resistance. Stable overexpression of miR-200c was obtained in A2780 and Hey cell lines. Crosslinking-coupled affinity purification method and ribonucleic-immunoprecipitation assay were used to characterise the complexes between miR-200c, HuR and 3′UTR region of TUBB3 mRNA. Nanofluidic technology and immunohistochemistry were used to analyze the expression of HuR, TUBB3 and miR-200c in 220 ovarian cancer patients.ResultsIn a panel of ovarian adenocarcinoma cell lines, we observed a direct correlation between miR-200c expression and chemoresistance. In A2780 cells miR-200c targeted TUBB3 3′UTR, while a positive correlation was observed between miR-200c and TUBB3 expression in most of the other cell lines. Through the analysis of 3′UTR-associated complexes, we found that the miR-200c can increase the association of the RNA binding protein HuR with TUBB3 mRNA, whereas HuR binding enhanced TUBB3 mRNA translation. Most importantly, in our analysis on 220 ovarian cancer patients we observed that overexpression of miR-200c correlated with poor or good outcome depending on the cellular localization of HuR.ConclusionThis study suggests a model for the combined regulatory activity of miR-200c and HuR on TUBB3 expression in ovarian cancer. When HuR is nuclear, high expression of miR-200c inhibits TUBB3 expression and results in a good prognosis, whereas when HuR occurs in cytoplasm, the same miRNA enhances TUBB3 expression and produces a poor outcome. These findings reveal the usefulness of multidimensional analysis in the investigation of the prognostic role of miRNA expression.

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.

Class III β-tubulin and the cytoskeletal gateway for drug resistance in ovarian cancer.

The Class III β‐tubulin isotype (βIII‐tubulin) is a predictive biomarker in ovarian cancer and other solid tumor malignancies. We discovered that βIII‐tubulin function is linked to two GTPases: guanylate‐binding protein 1 (GBP1), which activates its function, and GNAI1, which inhibits it. This finding was demonstrated in a panel of ovarian cancer cells resistant to several chemotherapeutic agents. Using a protein microarray, we identified PIM1 as the downstream partner of GBP1, recruited into the cytoskeleton under hypoxic conditions. The clinical value of these observations was tested by performing an archive study of 98 ovarian cancer patients, which demonstrated that the βIII‐tubulin ‐/PIM1‐ cohort responded to treatment, exhibiting long overall survival (OS), while βIII‐tubulin +/PIM+ patients experienced poor outcomes and OS times similar to patients receiving palliation alone. βIII‐tubulin expression is commonly believed responsible for paclitaxel resistance due to its enhancement of the dynamic instability of microtubules, which counteracts the activity of taxanes. In contrast, our research reveals that βIII‐tubulin behaves as a gateway for prosurvival signals, such as PIM1, to move into the cytoskeleton. When cells are exposed to microenvironmental stressors, they activate this pathway by telling the cytoskeleton to incorporate PIM1 through GBP1 and βIII‐tubulin, which ultimately leads to drug resistance. This discovery reveals that βIII‐tubulin does not act alone but requires partners to play its role. The discovery of such protein:protein interactions underlying this prosurvival cascade makes feasible the development of therapeutic approaches using novel compounds that are capable of inhibiting the transmission of prosurvival signals into the cytoskeleton. J. Cell. Physiol. 227: 1034–1041, 2012.

Design and Synthesis of 2-Heterocyclyl-3-arylthio-1H-indoles as Potent Tubulin Polymerization and Cell Growth Inhibitors with Improved Metabolic Stability

New arylthioindoles (ATIs) were obtained by replacing the 2-alkoxycarbonyl group with a bioisosteric 5-membered heterocycle nucleus. The new ATIs 5, 8, and 10 inhibited tubulin polymerization, reduced cell growth of a panel of human transformed cell lines, and showed higher metabolic stability than the reference ester 3. These compounds induced mitotic arrest and apoptosis at a similar level as combretastatin A-4 and vinblastine and triggered caspase-3 expression in a significant fraction of cells in both p53-proficient and p53-defective cell lines. Importantly, ATIs 5, 8, and 10 were more effective than vinorelbine, vinblastine, and paclitaxel as growth inhibitors of the P-glycoprotein-overexpressing cell line NCI/ADR-RES. Compound 5 was shown to have medium metabolic stability in both human and mouse liver microsomes, in contrast to the rapidly degraded reference ester 3, and a pharmacokinetic profile in the mouse characterized by a low systemic clearance and excellent oral bioavailability.

Gender Influences the Class III and V β-Tubulin Ability to Predict Poor Outcome in Colorectal Cancer

Purpose: Colorectal cancer is one of the deadliest diseases in Western countries. To predict the outcome of therapy, we assessed the role of class III (TUBB3) and class V β-tubulin (TUBB6) as predictive biomarkers. Experimental Design: Using immunohistochemistry and nanofluidics, the expression of TUBB3 and TUBB6 was assessed in two cohorts of 180 and 134 patients, respectively. The CYP17A1 RS743572 was genotyped to identify GG carriers with enhanced androgen levels. TUBB3 and TUBB6 were investigated in 22 colorectal cancer cell lines in basal conditions and after serum starvation, the latter serving as activator of this prosurvival pathway. To ascertain the role of androgen receptor (AR) in such regulation, we silenced AR and checked TUBB3 and TUBB6 expression and sensitivity to chemotherapy. Results: There was a link between poor survival, the expression of TUBB3/TUBB6, and AR only in females. Conversely, only in males carriers of the GG phenotype exhibited the worst outcome. Importantly, male cell lines were resistant to serum starvation and exhibited higher levels of TUBB6, thereby suggesting that the pathway is activated by androgens. In female cells this phenomenon was absent. In both genders, AR was the main driver of TUBB3/TUBB6 expression, as constitutive silencing of AR was associated with downregulation of TUBB3/TUBB6 expression and increased sensitivity to oxaliplatin and SN-38. Conclusions: The involvement of androgens in the TUBB3 pathway opens the way for clinical trials to assess the efficacy of antiandrogens for increasing the efficacy of chemotherapy in male colorectal cancer patients. Clin Cancer Res; 18(10); 2964–75. ©2012 AACR.

βIII-Tubulin: biomarker of taxane resistance or drug target?

Introduction: βIII-Tubulin (TUBB3) is predominantly expressed in neurons of the central and peripheral nervous systems, while in normal non-neoplastic tissues it is barely detectable. By contrast, this cytoskeletal protein is abundant in a wide range of tumors. βIII-Tubulin is linked to dynamic instability of microtubules (MTs), weakening the effects of agents interfering with MT polymerization. Based on this principle, early studies introduced the classical theory linking βIII-tubulin with a mechanism of counteracting taxane activity and accordingly, prompted its investigation as a predictive biomarker of taxane resistance. Areas covered: We reviewed 59 translational studies, including cohorts from lung, ovarian, breast, gastric, colorectal and various miscellaneous cancers subject to different chemotherapy regimens. Expert opinion: βIII-Tubulin functions more as a prognostic factor than as a predictor of response to chemotherapy. We believe this view can be explained by βIII-tubulins association with prosurvival pathways in the early steps of the metastatic process. Its prognostic response increases if combined with additional biomarkers that regulate its expression, since βIII-tubulin can be expressed in conditions, such as estrogen exposure, unrelated to survival mechanisms and without any predictive activity. Additional avenues for therapeutic intervention could emerge if drugs are designed to directly target βIII-tubulin and its mechanism of regulation.

Novel Drugs Targeting Microtubules: the Role of Epothilones

Among the drugs targeting microtubule functions by interfering with tubulin subunits, epothilones represent a class of anticancer agents which recently entered clinical development. Although epothilones share mechanisms of action similar to taxanes, they have non-overlapping mechanisms of resistance; in particular, while overexpression of class III β-tubulin plays a major role in taxane resistance, epothilones display their highest efficacy in class III β-tubulin overexpressing malignancies. Three compounds belonging to this family (patupilone, ixabepilone and sagopilone), have been actively investigated in clinical trials, and some of them are at an advanced stage of development. This review provides a comprehensive analysis of the available literature on epothilones, focusing on their clinical development and potential as an additional weapon in the arsenal against tumors.

Sox9 and Hif-2α regulate TUBB3 gene expression and affect ovarian cancer aggressiveness.

UNLABELLED SOX9 [(sex determining region Y)-box9] gene has been implicated in the development and progression of different neoplasms. This study investigated the role of Sox9 in the expression of TUBB3 gene, a marker of aggressiveness in ovarian cancer (OC), encoding βIII-tubulin protein. Gene expression was assessed by quantitative polymerase chain reaction (qPCR) in OC models. Using chromatin immunoprecipitation (ChIP) we found that Sox9 engages TUBB3 promoter at minus 980 base pairs from the transcriptional start site with transcriptional enhancing effects. Furthermore we found that Sox9 is a downstream target of Hif-2α, a transcription factor encoded by endothelial PAS domain protein-1 (EPAS1). Hypoxic microenvironment is a common feature of solid tumors associated with cancer aggressiveness. In the present work we found that knockdown of either SOX9 or EPAS1 abolished TUBB3 gene induction in hypoxia. This phenomenon was associated with a decrease in the number of cell colonies capable of growing in an anchorage-independent way. Using a nanofluidic genetic analyzer, the expression of SOX9, TUBB3 and EPAS1 was evaluated in 182 OC specimens. Double staining immunohistochemistry was employed to evaluate the expression and prognostic role of both Sox9 and βIII-tubulin. Results obtained in cellular models matched the pattern of clinical specimens. We documented a direct correlation among the expression of EPAS1, SOX9 and TUBB3 at mRNA level. Patients displaying no expression for the three genes had the best outcome. A poor prognosis significant in multivariate analysis was visible in patients featuring high expression of βIII-tubulin and nuclear Sox9. CONCLUSIONS Sox9 allows the survival of OC cells upon hypoxic condition, through the activation of βIII-tubulin expression and its aberrant activation in OC is prominent in patients with aggressive OC.

Class III β-tubulin in normal and cancer tissues

Microtubules are polymeric structures composed of tubulin subunits. Each subunit consists of a heterodimer of α- and β-tubulin. At least seven β-tubulin isotypes, or classes, have been identified in human cells, and constitutive isotype expression appears to be tissue specific. Class III β-tubulin (βIII-tubulin) expression is normally confined to testes and tissues derived from neural cristae. However, its expression can be induced in other tissues, both normal and neoplastic, subjected to a toxic microenvironment characterized by hypoxia and poor nutrient supply. In this review, we will summarize the mechanisms underlying βIII-tubulin constitutive and induced expression. We will also illustrate its capacity to serve as a biomarker of neural commitment in normal tissues and as a pure prognostic biomarker in cancer patients.

Identification of the first inhibitor of the GBP1:PIM1 interaction. Implications for the development of a new class of anticancer agents against paclitaxel resistant cancer cells.

Class III β-tubulin plays a prominent role in the development of drug resistance to paclitaxel by allowing the incorporation of the GBP1 GTPase into microtubules. Once in the cytoskeleton, GBP1 binds to prosurvival kinases such as PIM1 and initiates a signaling pathway that induces resistance to paclitaxel. Therefore, the inhibition of the GBP1:PIM1 interaction could potentially revert resistance to paclitaxel. A panel of 44 4-azapodophyllotoxin derivatives was screened in the NCI-60 cell panel. The result is that 31 are active and the comparative analysis demonstrated specific activity in paclitaxel-resistant cells. Using surface plasmon resonance, we were able to prove that NSC756093 is a potent in vitro inhibitor of the GBP1:PIM1 interaction and that this property is maintained in vivo in ovarian cancer cells resistant to paclitaxel. Through bioinformatics, molecular modeling, and mutagenesis studies, we identified the putative NSC756093 binding site at the interface between the helical and the LG domain of GBP1. According to our results by binding to this site, the NSC756093 compound is able to stabilize a conformation of GBP1 not suitable for binding to PIM1.