Giuseppina Raspaglio

Paclitaxel Directly Binds to Bcl-2 and Functionally Mimics Activity of Nur77

We reported previously that Bcl-2 is paradoxically down-regulated in paclitaxel-resistant cancer cells. We reveal here that paclitaxel directly targets Bcl-2 in the loop domain, thereby facilitating the initiation of apoptosis. Molecular modeling revealed an extraordinary similarity between the paclitaxel binding sites in Bcl-2 and beta-tubulin, leading us to speculate that paclitaxel could be mimetic of an endogenous peptide ligand, which binds both proteins. We tested the hypothesis that paclitaxel mimics Nur77, which, like paclitaxel, changes the function of Bcl-2. This premise was confirmed by Nur77 interacting with both paclitaxel targets (Bcl-2 and beta-tubulin) and a peptide sequence mimicking the Nur77 structural region, thus reproducing the paclitaxel-like effects of tubulin polymerization and opening the permeability transition pore channel in mitochondria. This discovery could help in the development of novel anticancer agents with nontaxane skeleton as well as in identifying the clinical subsets responsive to paclitaxel-based therapy.

The seco-taxane IDN5390 is able to target class III beta-tubulin and to overcome paclitaxel resistance.

A prominent mechanism of drug resistance to taxanes is the overexpression of class III β-tubulin. The seco-taxane IDN5390 was chosen for its selective activity in paclitaxel-resistant cells with an overexpression of class III β-tubulin. Moreover, the combined treatment paclitaxel/IDN5390 yielded a strong synergism, which was also evident in cell-free tubulin polymerization assays. In the presence of an anti-class III β-tubulin as a blocking antibody, tubulin polymerization induced by paclitaxel and IDN5390 was enhanced and not affected, respectively, whereas synergism was abolished, thereby indicating that IDN5390 activity is not modulated by class III β-tubulin levels. Such properties can be explained by taking into consideration the composition of class III β-tubulin paclitaxel binding site; in fact, Ser277 interacting with paclitaxel C group in class I is replaced by an Arginine in class III. IDN5390 that has an open and flexible C ring and an acidic α-unsaturated enol-keton moiety better fits with class III β-tubulin than paclitaxel at the binding site. Taking altogether, these findings indicate that the concomitant treatment IDN5390/paclitaxel is able to successfully target class I and III β-tubulin and the combined use of two taxanes with diverse spectrum activity against tubulin isotypes could represent a novel approach to overcome paclitaxel resistance.

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.

Microsatellite Instability Is an Independent Indicator of Recurrence in Sporadic Stage I-II Endometrial Adenocarcinoma

PURPOSE The aim of this study was to define the prognostic role of microsatellite status in 65 stage I-II primary sporadic endometrioid endometrial adenocarcinoma (EEA) patients. PATIENTS AND METHODS Familiarity for neoplasia was ascertained in all patients on the basis of a questionnaire. Microsatellite status was assessed by matching normal and tumoral DNA probed for five dinucleotide repeats and one mononucleotide repeat marker. Microsatellite status was analyzed in relation to clinicopathologic characteristics of the patients and length of disease-free survival (DFS). RESULTS Eleven tumors (17%) of 65 had instability at two or more loci and were considered as unstable or microsatellite instability (MI). Tumors with no instability or instability at one locus were classified as microsatellite stable (MS). The percentage of MI was significantly higher in poorly than in well to moderately differentiated tumors (50% v 9%; P =.003). The 5-year DFS rate of MI patients was 63% (95% confidence interval [CI], 35% to 91%) versus 96% (95% CI, 91% to 101%) of MS patients (P =.0004). In multivariate analysis, only the presence of MI, stage II of disease, and depth of myometrial invasion greater than 50% retained independent prognostic roles. CONCLUSION The assessment of microsatellite status may provide useful information for preoperative prognostic characterization of stage I-II primary sporadic EEA patients in which more individualized treatment options can be attempted.

Antiproliferative and antiangiogenic effects of the benzophenanthridine alkaloid sanguinarine in melanoma

This study was aimed at evaluating the potential application of benzophenanthridine alkaloids, sanguinarine and cheleritrine, in the therapy of melanoma cancer. In vitro antiproliferative activity of sanguinarine was higher than that of cheleritrine against the B16 melanoma 4A5 cells. Both agents were able to produce DNA breaks, and the DNA unwinding assay showed that they act as DNA intercalating agents. Sanguinarine was selected for determination of its in vivo preclinical efficacy. Oral treatment with sanguinarine reduced the tumor burden in a transplantable murine tumor grown in a syngeneic host (B16 melanoma 4A5 in C57BL/6 mice), and in a human tumor xenograft grown in immunodeficient mice (A375 human melanoma in athymic nude mice). In A375 tumors a significant decrease in the proliferation marker Ki67, and a reduction in the activated mitogen-activated protein kinases (p-p44/42 MAPK), and in protein kinase B (pAKT) were also observed. Three out of eleven A375-bearing treated mice were tumor-free at the end of treatment, and did not develop any tumor after a further, treatment-free, observation period of 60 days. Sanguinarine also showed a striking antiangiogenic activity in mice. Data from the present study support the concept that sanguinarine can be effective in melanoma skin cancer.

Looking at Drug Resistance Mechanisms for Microtubule Interacting Drugs: Does TUBB3 Work?

Vinca alkaloids and taxanes represent the mainstay of medical treatment of hematological and solid tumors. Unfortunately, a major clinical problem with these agents is drug resistance. Although a plethora of mechanisms of drug resistance have been described, only a few of them have been validated in clinical trials. Among these, the one involving the protein TUBB3 seems to represent a promising target for studying drug resistance. In fact, it seems that this protein is a factor promoting cell survival and represents an endogenous element of an inherent drug-resistance program built into cells to counteract the activity of microtubule-interacting drugs. Its pivotal role has been ascertained in clinical trials in lung, breast, and ovarian cancer, three diseases that can be successfully treated with microtubule-interacting drugs. Although TUBB3 is probably not a unique factor in drug resistance, the hope is that direct targeting of this protein will increase the response to microtubule-interacting drugs, thereby overcoming an important element in the growth of drug resistance.

Bcl-2 down-regulation and tubulin subtype composition are involved in resistance of ovarian cancer cells to vinflunine

Vinflunine, a new microtubule-targeting drug, has a marked antitumor activity in vitro and in vivo. Here, we studied the mechanisms mediating resistance to vinflunine. We investigated the response to vinflunine of ovarian cancer cells initially selected as paclitaxel-resistant cells (A2780-TC1 cells). By comparison with A2780-wild-type (wt) cells, we showed that A2780-TC1 cells were highly resistant to vinflunine, with resistance factors reaching 800 and 1,830 for IC50 and IC70, respectively. We showed that P-glycoprotein minimally participated in this cell resistance. The examination of tubulin composition revealed increased levels of acetylated α-tubulin, βII-tubulin, and βIII-tubulin in A2780-TC1 cells before vinflunine treatment. As a consequence, vinflunine unequally affected microtubule network organization and function in A2780-wt and A2780-TC1 cells. Whereas the drug depolymerized microtubules and induced a mitotic block in A2780-wt cells, it did not depolymerize microtubules and induced a G2 block in A2780-TC1 cells. Elsewhere, the mitochondrial protein Bcl-2 was down-regulated in A2780-TC1 cells. This down-regulation was related to resistance, as A2780-TC1 cells stably transfected with a Bcl-2 construct recovered a partial sensitivity to vinflunine. Lastly, we confirmed the role played by Bcl-2 by showing that the mitochondrial membrane potential was only disrupted by vinflunine in cells expressing Bcl-2. Altogether, our results indicate that modifications acquired during treatment (i.e., paclitaxel) have significant consequences on cell response to the following drug (i.e., vinflunine). Especially, this study shows that a specific pool of tubulin subtypes and a down-regulation of Bcl-2 are associated with resistance of ovarian cancer cells to vinflunine. [Mol Cancer Ther 2006;5(11):2824–33]

From plasma membrane to cytoskeleton: a novel function for semaphorin 6A

Class III β-tubulin (TUBB3) overexpression has been reported in ovary, lung, breast, and gastric cancer patients. Currently, no clinical drugs are available for a specific targeting of TUBB3, whereas the investigational drug IDN5390 specifically interacts with TUBB3. To gain insight into the pathways leading to TUBB3 up-regulation, we did a human genome microarray analysis in A2780 cells made resistant to IDN5390 to identify selected pathways specifically disrupted in resistant cells. Using this approach, we discovered that semaphorin 6A (SEMA6A) is down-regulated not only in IDN5390-resistant cells but also in cells made resistant to cisplatin, topotecan, and doxorubicin, whereas no changes were noticed in paclitaxel- and gemcitabine-resistant cells. Acute treatment with IDN5390 was able to down-regulate SEMA6A in cells unselected for drug resistance. TUBB3 expression was assessed in A2780 clones with stable overexpression of SEMA6A and in a panel of clones in which silencing of the protein was obtained. Quantitative PCR was then used to check the modulation of SEMA6A as well as to assess the expression of TUBB3. TUBB3 was increased (median value, 5.4) and reduced (median value, 0.47) in cells with overexpression and silencing of SEMA6A, respectively. Thus, the findings indicate a correlation between the expression of SEMA6A and TUBB3. Then, we found that a form of 83 kDa of SEMA6A is expressed in the cytoskeleton in association with β-actin. These findings suggest for SEMA6A a novel function in the cytoskeleton and a role in modulating tubulin isotype composition and microtubule dynamics. [Mol Cancer Ther 2008;7(1):233–41]

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.

Gli family transcription factors are drivers of patupilone resistance in ovarian cancer

Epothilones constitute a novel class of antitubulin agents that are active in patients who relapse after treatment with other chemotherapeutics. This study investigated the molecular mechanisms leading to the onset of epothilone-B (patupilone) resistance in ovarian cancer. Results demonstrated that the Gli family of transcription factors was overexpressed in resistant cells and that treatment with a specific Gli1 inhibitor (GANT58) made cells more susceptible to treatment, partially reversing drug resistance. We also demonstrated that Gli1 knockdown halted growth in resistant cells that were exposed to patupilone, confirming that Gli1 is capable of directly mediating epothilone-B resistance. Another observation from our research was that patupilone-resistant cells produced HGF and acquired characteristics of a mesenchymal phenotype. However, HGF silencing alone was not capable of converting the drug-resistant phenotype to a susceptible one, and in this case we demonstrated that Gli1 overexpression led to an increase in HGF, establishing a functional link between Gli1 and HGF. These results demonstrated that Gli1 played a key role in driving resistance to patupilone, suggesting that the combination of epothilones and Gli1-targeted agents could be exploited to improve outcomes in ovarian cancer patients resistant to standard treatments.