Role and prognostic significance of the epithelial-mesenchymal transition factor ZEB2 in ovarian cancer.

ZEB2 is a key factor in epithelial-mesenchymal transition (EMT), a program controlling cell migration in embryonic development and adult tissue homeostasis. We demonstrated a role of ZEB2 in migration and anchorage-independent cell growth in ovarian cancer, as shown by ZEB2 silencing. We found that the RNA-binding protein HuR bound the 3'UTR of ZEB2 mRNA, acting as a positive regulator of ZEB2 protein expression. In Hey ovarian cell line, HuR silencing decreased ZEB2 and ZEB1 nuclear expression and impaired migration. In hypoglycemic conditions ZEB2 expression decreased, along with ZEB1, vimentin and cytoplasmic HuR, and a reduced cellular migration ability was observed. Analysis of ZEB2 and HuR expression in ovarian cancers revealed that nuclear ZEB2 is localized in tumor leading edge and co-localizes with cytoplasmic HuR. In a series of 143 ovarian cancer patients high expression of ZEB2 mRNA significantly correlated with a poor prognosis in term of both overall survival and progression- free survival. Moreover, at immunohistochemical evaluation, we found that prognostic significance of ZEB2 protein relies on its nuclear expression and co-localization with cytoplasmic HuR. In conclusion our findings indicated that nuclear ZEB2 may enhance progression of EMT transition and acquisition of an aggressive phenotype in ovarian cancer.

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 ßIII-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 ßIII-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 ßIII-tubulin and nuclear Sox9. CONCLUSIONS: Sox9 allows the survival of OC cells upon hypoxic condition, through the activation of ßIII-tubulin expression and its aberrant activation in OC is prominent in patients with aggressive OC.

MiR-200c and HuR in ovarian cancer.

BACKGROUND: MicroRNAs 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 ß-tubulin (TUBB3), a factor associated with drug-resistance and poor prognosis in ovarian cancer. METHODS: Expression 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. RESULTS: In 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. CONCLUSION: This 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.

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.

RON and cisplatin resistance in ovarian cancer cell lines.

RON (recepteur d'origine nantais) tyrosine kinase receptor has revealed its tumorigenic potential in recent studies. RON was reported to be overexpressed in 55% of primary ovarian carcinoma samples and furthermore its activation increases cell motility and invasiveness. In this study, we investigated the correlation between RON expression and chemoresistance in ovarian cancer cells. In A2780 cells, a model featured by high chemosensitivity to cisplatin, stable overexpression of RON was able to reduce sensitivity to this agent, while incubation with a blocking anti-RON antibody (ID1) increased the cisplatin-induced growth inhibition effect. Moreover, we observed an increased RON expression both at the mRNA and protein level in A2780 cells made resistant to doxorubicin and paclitaxel (A2780ADR and TC 1, respectively), two cell lines exhibiting a collateral resistance to cisplatin. OVCAR-3 cells, showing high levels of RON expression, also displayed inherent cisplatin resistance. The morphology observed in these resistant cells is consistent with a scattering phenotype and a RON-activated state. RON expression levels were monitored upon hypoxia. A 2.5-fold increase of RON expression was noticed in response to hypoxia in OVCAR-3 cells, in parallel with a decrease of E-cadherin mRNA. Altogether these results suggest an involvement of RON in the acquisition of cisplatin resistance and highlight the importance of this factor as a promising target for combination with cisplatin-based chemotherapy in ovarian cancer.

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.

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.

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

Class III beta-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 beta-actin. These findings suggest for SEMA6A a novel function in the cytoskeleton and a role in modulating tubulin isotype composition and microtubule dynamics.

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.

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.

Lack of stimulatory activity of a phytoestrogen-containing soy extract on the growth of breast cancer tumors in mice.

The present study was designed to investigate the effects of a phytoestrogens-containing soy extract (SOYSELECT, SSE) on the growth of estrogen-dependent (MCF-7) and estrogen-unresponsive (MDA-MB-231) human breast cancer xenografts in athymic mice. Results obtained provided evidence that MCF-7 tumors did not grow over the treatment period (5 weeks) in ovariectomized females receiving 50 or 100 mg/kg/day SSE (oral route); administration of SSE also did not affect the estradiol-sustained growth of MCF-7 tumors in mice. Similarly, no effects on tumor growth were observed in SSE-treated mice bearing MDA-MB-231 xenografts. Data from pS2, progesterone receptor and cyclin D1 mRNA expression in tumors showed that, although SSE was able to induce a moderate estrogenic effect in MCF-7 cells, it did not increase cellular proliferation and tumor growth, in our experimental conditions. Besides, when used in association with 17beta-estradiol, it displayed antiestrogenic activity. The expression of other genes involved in tumor progression and angiogenesis, such as Thrombospondin 1, Transforming Growth Factor beta2 and Kallikrein 6 was also evaluated in tumor samples, results showing a decrease in mRNA expression upon SSE treatment. The effect of SSE on angiogenesis in vivo was also evaluated in the Matrigel plug assay; results obtained showed a striking anti-angiogenic activity in mice receiving 100 mg/kg/day SSE, thereby confirming that this extract may interfere with angiogenesis. Collectively, these experimental data suggest that SSE could be not harmful for women with a history of or at high risk for breast cancer, at least for short treatment periods; however, further studies are needed to thoroughly characterize the activity profile of the extract in this specific setting of patients.

Class III beta-tubulin overexpression is a prominent mechanism of paclitaxel resistance in ovarian cancer patients.

The vast majority of women with advanced ovarian cancer will ultimately relapse and develop a drug-resistant disease with an overall 5-year survival of <50%. Unfortunately, the mechanisms of drug resistance actually operating in patients are still unknown. To address this issue, in 41 patients affected by advanced ovarian cancer the three main mechanisms of paclitaxel resistance were investigated: overexpression of MDR-1 gene, point mutations at prominently expressed alpha-tubulin and beta-tubulin genes and selective alterations in the expression of beta-tubulin isotypes. MDR-1 and the beta-tubulin isotypes expression were evaluated by semiquantitative and real-time PCR. On the same specimens, quantitative immunohistochemistry was also done in the tumor area. No statistically significant changes of MDR-1 expression were noticed between the sensitive and resistant patients either at the mRNA or protein level. The tubulin mutations for the ubiquitous alpha-tubulin and beta-tubulin genes were evaluated by automated DNA sequencing, and in all patients, no mutations were detected in both resistant and sensitive cases. With regard to the expression of tubulin isoforms, a statistically significant up-regulation of class III beta-tubulin was found in the resistant subset. It is worth noting that this statistically significant increase of the expression of class III beta-tubulin was detectable at the mRNA and protein level. By a direct comparison of the three main known mechanisms of paclitaxel resistance, this study indicates that overexpression of class III beta-tubulin is the most prominent mechanism of paclitaxel resistance in ovarian cancer.

Thiocolchicine dimers: a novel class of topoisomerase-I inhibitors.

During a cellular screening of thiocolchicine analogs, thiocolchicine dimers resulted particularly active in cisplatin-resistant A2780-CIS cells. In order to discover by which mechanism(s) thiocolchicine dimers overcame cisplatin resistance, p53, p21waf1 and MLH1 were assessed by Western blot. Results pointed out that, when combined with cisplatin, dimers increased the amount of all the three proteins with respect to the levels obtained by single drug exposure, thereby suggesting an interference in the process of repair of the cisplatin-induced DNA lesions. Moreover, in isolated nuclei drugs were able to produce DNA breaks, as demonstrated by Comet assay, thereby proving that the compounds were able to target cell nucleus independently from microtubules. Since Topo-I (topoisomerase I) is directly involved in the DNA repair and such activity is overexpressed in cisplatin-resistant cells, Topo-I was investigated as a potential target. Using DNA relaxation assay, thiocolchicine dimers inhibited Topo-I, a property not shared by thiocolchicine. At variance with camptothecin, dimers did not produce cleavable complexes, thereby indicating that Topo-I inhibition occurs upstream of the religation step. To assess the mechanism of inhibition, an electrophoretic mobility shift assay between DNA and Topo-I was performed and revealed that thiocolchicine dimers specifically interfere with binding of Topo-I to DNA. The interference is specific since the same compounds did not modulate DNase activity and did not act as intercalating agents in the DNA unwinding assay. Finally, behaviour of dimers as spindle poisons was investigated and no relevant changes with respect to thiocolchicine in terms of interaction with microtubules were found.