Shohreh Shahabi

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.

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.

Urinary screening tests for fetal Down syndrome: II. Hyperglycosylated hCG

Hyperglycosylated hCG is a form of hCG with more complex oligosaccharide side chains. A specific immunoassay was developed to measure hyperglycosylated hCG. Levels were measured in urine samples from 1157 women between 11 to 22 weeks of gestation, undergoing genetic analysis because of advanced maternal age. Values were normalized to urine creatinine concentration and plotted against gestational age, median values were determined and multiples of the control median (MoM) calculated. The median MoM and log standard deviation (log SD) of the 1134 control samples was 1.0 and 0.47, and of the 23 Down syndrome cases was 7.8 and 0.48, respectively. This indicated a 7.8‐fold increase in hyperglycosylated hCG levels in Down syndrome cases.

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.

Urinary screening tests for fetal Down syndrome: I. Fresh β‐core fragment

Variable results have been reported using urine β‐core fragment as a marker for fetal Down syndrome. Initial studies by Cuckle et al. (1994) and Canick et al. (1995) indicated that β‐core fragment was an outstanding marker, detecting >80 per cent of Down syndrome cases. Since these reports, widely varying results have been published, indicating between 20 per cent and 66 per cent detection of cases at 5 per cent false‐positive rate. The wide variation in the reported data has led to a loss of enthusiasm for this marker as a useful test for Down syndrome screening.

Prenatal diagnosis of an epidermal scalp cyst simulating an encephalocoele

The majority of extracranial masses detected by prenatal ultrasound screening are encephalocoeles. Several recent publications have drawn attention to extracranial masses which resemble encephalocoeles. The uncertainty in these situations can result in the unnecessary termination of a pregnancy. We report a case of an epidermal cyst of the scalp presenting as an encephalocoele at a routine second‐trimester ultrasound scan.

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.

A phase II study of weekly topotecan and docetaxel in heavily treated patients with recurrent uterine and ovarian cancers

OBJECTIVES A phase II trial designed to evaluate the safety and efficacy of weekly topotecan and docetaxel in heavily treated patients with recurrent uterine or epithelial ovarian cancers. METHODS Eligible patients with recurrent epithelial ovarian or uterine cancers were treated with weekly topotecan 3.5 mg/m(2) and docetaxel 30 mg/m(2) for 3 consecutive weeks. Cycles were repeated every 4 weeks for 6 cycles or until evidence of disease progression, unacceptable toxicity, or death. Response was assessed as per RECIST or Rustins criteria. Time to best response and overall survival were calculated using Kaplan-Meier statistical methods. RESULTS Twenty-seven patients registered, of which 24 were evaluable for response. The majority of patients had received 2 prior chemotherapy regimens. Of the total 86 cycles of chemotherapy that were administered, there were three grade 4 (all neutropenia) and ten grade 3 toxicities. Six of the grade 3 non-hematologic toxicities were unrelated to treatment. There were 8 dose delays and 4 dose reductions. The overall response rate was 25% (95% CI: 7.7%-42.3%, 8% CR, 17% PR), and 38% of the patients had clinical benefit (95% CI: 18.1%-56.9%; CR+PR+13% SD). The median duration of response was 8.5 months (range 3-19 months). The median overall survival was 18.5 months (range 1.8-50.7 months). CONCLUSION The combination of weekly topotecan and docetaxel has clinical benefit and is well tolerated in this heavily treated patient population. Patients with platinum-resistant tumors had clinical benefit and should be considered for further study with this regimen.