Search

Overexpression of βIII-tubulin is associated with resistance to tubulin-binding agents (TBA) in a range of tumor types. We previously showed that small interfering RNA silencing of βIII-tubulin expression hypersensitized non–small cell lung cancer cells to TBAs. To determine whether βIII-tubulin mediates its effect on drug-induced mitotic arrest and cell death by differentially regulating microtubule behavior, the effects of βIII-tubulin knockdown on microtubule dynamics were analyzed in H460 non–small cell lung cancer cells stably expressing green fluorescent protein-βI-tubulin. Interphase cells were examined at three vincristine and paclitaxel concentrations that (a) inhibited cell proliferation, (b) induced 5% to 10% mitotic arrest, and (c) induced 30% to 40% mitotic arrest. In the absence of either drug, βIII-tubulin knockdown caused no significant change in microtubule dynamic instability. At 2 nmol/L vincristine (IC50), overall microtubule dynamicity was significantly suppressed in βIII-tubulin knockdowns (−31.2%) compared with controls (−6.5%). Similar results were obtained with paclitaxel, suggesting that knockdown of βIII-tubulin induces hypersensitivity by enhancing stabilization of microtubule dynamics at low drug concentrations. At higher drug concentrations (≥40 nmol/L vincristine; ≥20 nmol/L paclitaxel), βIII-tubulin knockdown resulted in significantly reduced suppressive effects on microtubule dynamicity with little or no further increase in mitotic arrest, compared with control cells. Importantly, apoptosis was markedly increased by βIII-tubulin knockdown independent of further suppression of microtubule dynamics and mitotic arrest. These results show that βIII-tubulin knockdown enhances the effectiveness of TBAs through two mechanisms: suppression of microtubule dynamics at low drug concentrations and a mitosis-independent mechanism of cell death at higher drug concentrations. Mol Cancer Ther; 9(5); 1339–48. ©2010 AACR.

Supplementary Data from Microtubule Dynamics, Mitotic Arrest, and Apoptosis: Drug-Induced Differential Effects of βIII-Tubulin

Individual differences in cognitive processing have been the subject of intensive research. One important type of such individual differences is the tendency for global versus local processing, which was shown to correlate with a wide range of processing differences in fields such as decision making, social judgments and creativity. Yet, whether these global/local processing tendencies are correlated within a subject across different domains is still an open question. To address this question, we develop and test a novel method to quantify global/local processing tendencies, in which we directly set in opposition the local and global information instead of instructing subjects to specifically attend to one processing level. We apply our novel method to two different domains: (1) a numerical cognition task, and (2) a preference task. Using computational modeling, we accounted for classical effects in choice and numerical-cognition. Global/local tendencies in both tasks were quantified using a salience parameter. Critically, the salience parameters extracted from the numerical cognition and preference tasks were highly correlated, providing support for robust perceptual organization tendencies within an individual.

Objective: Tumor Treating Fields (TTFields) are alternating electrical fields that are approved and effective for glioblastoma therapy with a frequency of 200 kHz. At a frequency of 100 kHz, we have previously shown that TTFields can transiently open the blood-brain barrier (BBB) in vitro and in vivo [for full text, please go to the a.m. URL]

Targeting cell division by chemotherapy is a highly effective strategy to treat a wide range of cancers. However, there are limitations of many standard-of-care chemotherapies: undesirable drug toxicity, side-effects, resistance and high cost. New small molecules which kill a wide range of cancer subtypes, with good therapeutic window in vivo, have the potential to complement the current arsenal of anti-cancer agents and deliver improved safety profiles for cancer patients. We describe results with a new anti-cancer small molecule, WEHI-7326, which causes cell cycle arrest in G2/M, cell death in vitro, and displays efficacious anti-tumor activity in vivo. WEHI-7326 induces cell death in a broad range of cancer cell lines, including taxane-resistant cells, and inhibits growth of human colon, brain, lung, prostate and breast tumors in mice xenografts. Importantly, the compound elicits tumor responses as a single agent in patient-derived xenografts of clinically aggressive, treatment-refractory neuroblastoma, breast, lung and ovarian cancer. In combination with standard-of-care, WEHI-7326 induces a remarkable complete response in a mouse model of high-risk neuroblastoma. WEHI-7326 is mechanistically distinct from known microtubule-targeting agents and blocks cells early in mitosis to inhibit cell division, ultimately leading to apoptotic cell death. The compound is simple to produce and possesses favorable pharmacokinetic and toxicity profiles in rodents. It represents a novel class of anti-cancer therapeutics with excellent potential for further development due to the ease of synthesis, simple formulation, moderate side effects and potent in vivo activity. WEHI-7326 has the potential to complement current frontline anti-cancer drugs and to overcome drug resistance in a wide range of cancers.

BackgroundStathmin mediates cell migration and invasion in vitro, and metastasis in vivo. To investigate stathmin’s role on the metastatic process, we performed integrated mRNA–miRNA expression analysis to identify pathways regulated by stathmin.MethodsMiRNA and gene arrays followed by miRNA-target-gene integration were performed on stathmin-depleted neuroblastoma cells (CtrlshRNAvs. Stmn Seq2shRNA). The expression of the predicted target PTPN14 was evaluated by RT-qPCR, western blot and immunohistochemistry. Gene-silencing technology was used to assess the role of PTPN14 on proliferation, migration, invasion and signalling pathway.ResultsStathmin levels modulated the expression of genes and miRNA in neuroblastoma cells, leading to a deregulation of migration and invasion pathways. Consistent with gene array data, PTPN14 mRNA and protein expression were downregulated in stathmin- depleted neuroblastoma cells and xenografts. In two independent neuroblastoma cells, suppression of PTPN14 expression led to an increase in cell migration and invasion. PTPN14 and stathmin expression did not act in a feedback regulatory loop in PTPN14- depleted cells, suggesting a complex interplay of signalling pathways. The effect of PTPN14 on YAP pathway activation was cell-type dependent.ConclusionsOur findings demonstrate that stathmin levels can regulate PTPN14 expression, which can modulate neuroblastoma cell migration and invasion.

Purpose Adolescents' mental well-being has become a growing public health concern. Adolescents' daily lives and their engagement in risks have changed dramatically in the course of the 21st century, leading to a need to update traditional models of risk to include new exposures and behaviors. To date, studies have examined the relationship between (mainly traditional) risk behaviors and adolescent mental well-being or looked at risk factors that jeopardize mental well-being such as lack of social support but have not combined them together to highlight the most significant risks for adolescent mental well-being today. The present study included new and traditional risk behaviors and risk factors, robustly derived an empirically based model of clusters of risk, and examined the relative association of these clusters to adolescent mental well-being. Methods Data from the 2017–2018 Health Behaviours in School-aged Children study were used. The sample included 32,884 adolescents (51.7% girls) aged 15 years from 37 countries and regions. The principal component analysis was used to determine the existence of clusters of risk, using 21 items related to adolescent mental well-being that included both risk behaviors (e.g., substance use) and risk factors (e.g., peer support). Analysis was conducted in both a randomly split training and test set and in gender separate models. Mixed-effects logistic regressions examined the association between clusters of risk and mental well-being indices (low life satisfaction and psychosomatic complaints). Results Seven clusters of risk were identified: substance use and early sex, low social support, insufficient nutrition, bullying, sugary foods and drinks, physical health risk, and problematic social media use (SMU). Low social support and SMU were the strongest predictors of low life satisfaction (odds ratios = 2.167 and 1.330, respectively) and psychosomatic complaints (odds ratio = 1.687 and 1.386, respectively). Few gender differences in predictors were found. Exposure to bullying was somewhat more associated with psychosomatic complaints for girls, whereas physical health risk was associated with reduced relative odds of low life satisfaction among boys. Split-sample validation and out-of-sample prediction confirmed the robustness of the results. Conclusions The results highlight the importance of contemporary clusters of risk, such as low social support and SMU in the mental well-being of young people and the need to focus on these as targets for prevention. We propose that future studies should use composite risk measures that take into account both risk behaviors and risk factors to explain adolescents' mental well-being.

Purpose Adolescents' mental well-being has become a growing public health concern. Adolescents' daily lives and their engagement in risks have changed dramatically in the course of the 21st century, leading to a need to update traditional models of risk to include new exposures and behaviors. To date, studies have examined the relationship between (mainly traditional) risk behaviors and adolescent mental well-being or looked at risk factors that jeopardize mental well-being such as lack of social support but have not combined them together to highlight the most significant risks for adolescent mental well-being today. The present study included new and traditional risk behaviors and risk factors, robustly derived an empirically based model of clusters of risk, and examined the relative association of these clusters to adolescent mental well-being. Methods Data from the 2017–2018 Health Behaviours in School-aged Children study were used. The sample included 32,884 adolescents (51.7% girls) aged 15 years from 37 countries and regions. The principal component analysis was used to determine the existence of clusters of risk, using 21 items related to adolescent mental well-being that included both risk behaviors (e.g., substance use) and risk factors (e.g., peer support). Analysis was conducted in both a randomly split training and test set and in gender separate models. Mixed-effects logistic regressions examined the association between clusters of risk and mental well-being indices (low life satisfaction and psychosomatic complaints). Results Seven clusters of risk were identified: substance use and early sex, low social support, insufficient nutrition, bullying, sugary foods and drinks, physical health risk, and problematic social media use (SMU). Low social support and SMU were the strongest predictors of low life satisfaction (odds ratios = 2.167 and 1.330, respectively) and psychosomatic complaints (odds ratio = 1.687 and 1.386, respectively). Few gender differences in predictors were found. Exposure to bullying was somewhat more associated with psychosomatic complaints for girls, whereas physical health risk was associated with reduced relative odds of low life satisfaction among boys. Split-sample validation and out-of-sample prediction confirmed the robustness of the results. Conclusions The results highlight the importance of contemporary clusters of risk, such as low social support and SMU in the mental well-being of young people and the need to focus on these as targets for prevention. We propose that future studies should use composite risk measures that take into account both risk behaviors and risk factors to explain adolescents' mental well-being.

Reorganization of the actin cytoskeleton during mitosis is crucial for regulating cell division. A functional role for γ-actin in mitotic arrest induced by the microtubule-targeted agent, paclitaxel, has recently been demonstrated. We hypothesized that γ-actin plays a role in mitosis. Herein, we investigated the effect of γ-actin in mitosis and demonstrated that γ-actin is important in the distribution of β-actin and formation of actin-rich retraction fibers during mitosis. The reduced ability of paclitaxel to induce mitotic arrest as a result of γ-actin depletion was replicated with a range of mitotic inhibitors, suggesting that γ-actin loss reduces the ability of broad classes of anti-mitotic agents to induce mitotic arrest. In addition, partial depletion of γ-actin enhanced centrosome amplification in cancer cells and caused a significant delay in prometaphase/metaphase. This prolonged prometaphase/metaphase arrest was due to mitotic defects such as uncongressed and missegregated chromosomes, and correlated with an increased presence of mitotic spindle abnormalities in the γ-actin depleted cells. Collectively, these results demonstrate a previously unknown role for γ-actin in regulating centrosome function, chromosome alignment and maintenance of mitotic spindle integrity.

Retinoids are an important component of neuroblastoma therapy at the stage of minimal residual disease, yet 40–50% of patients treated with 13-cis-retinoic acid (13-cis-RA) still relapse, indicating the need for more effective retinoid therapy. Vorinostat, or Suberoylanilide hydroxamic acid (SAHA), is a potent inhibitor of histone deacetylase (HDAC) classes I & II and has antitumor activity in vitro and in vivo. Fenretinide (4-HPR) is a synthetic retinoid which acts on cancer cells through both nuclear retinoid receptor and non-receptor mechanisms. In this study, we found that the combination of 4-HPR + SAHA exhibited potent cytotoxic effects on neuroblastoma cells, much more effective than 13-cis-RA + SAHA. The 4-HPR + SAHA combination induced caspase-dependent apoptosis through activation of caspase 3, reduced colony formation and cell migration in vitro, and tumorigenicity in vivo. The 4-HPR and SAHA combination significantly increased mRNA expression of thymosin-beta-4 (Tβ4) and decreased mRNA expression of retinoic acid receptor α (RARα). Importantly, the up-regulation of Tβ4 and down-regulation of RARα were both necessary for the 4-HPR + SAHA cytotoxic effect on neuroblastoma cells. Moreover, Tβ4 knockdown in neuroblastoma cells increased cell migration and blocked the effect of 4-HPR + SAHA on cell migration and focal adhesion formation. In primary human neuroblastoma tumor tissues, low expression of Tβ4 was associated with metastatic disease and predicted poor patient prognosis. Our findings demonstrate that Tβ4 is a novel therapeutic target in neuroblastoma, and that 4-HPR + SAHA is a potential therapy for the disease.

Actin and microtubule interactions are important for many cellular events, however these interactions are poorly described. Alterations in γ-actin are associated with diseases such as hearing loss and cancer. Functional investigations demonstrated that partial depletion of γ-actin affects cell polarity and induces resistance to microtubule-targeted agents. To determine whether γ-actin alterations directly affect microtubule dynamics, microtubule dynamic instability was analyzed in living cells following partial siRNA depletion of γ-actin. Partial depletion of γ-actin suppresses interphase microtubule dynamics by 17.5% due to a decrease in microtubule shortening rates and an increase in microtubule attenuation. γ-Actin partial depletion also increased distance-based microtubule catastrophe and rescue frequencies. In addition, knockdown of γ-actin delayed mitotic progression, partially blocking metaphase–anaphase transition and inhibiting cell proliferation. Interestingly, in the presence of paclitaxel, interphase microtubule dynamics were further suppressed by 24.4% in the γ-actin knockdown cells, which is comparable to 28.8% suppression observed in the control siRNA treated cells. Paclitaxel blocked metaphase–anaphase transition in both the γ-actin knockdown cells and the control siRNA cells. However, the extent of mitotic arrest was much higher in the control cells (28.4%), compared to the γ-actin depleted cells (8.5%). Therefore, suppression of microtubule dynamics by partial depletion of γ-actin is associated with marked delays in metaphase-anaphase transition and not mitotic arrest. This is the first demonstration that γ-actin can modulate microtubule dynamics by reducing the microtubule shortening rate, promoting paused/attenuated microtubules, and increasing transition frequencies suggesting a mechanistic link between γ-actin and microtubules. © 2013 Wiley Periodicals, Inc

Elad Moisseiev,1,3 Tzahi Sela,2 Liza Minkev,2 David Varssano1,31Department of Ophthalmology, Tel-Aviv Medical Center, Tel-Aviv, Israel; 2Care Vision, Tel Aviv, Israel; 3Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, IsraelPurpose: To evaluate the trends in corneal refractive procedure selection for the correction of myopia, focusing on the relative proportions of laser in situ keratomileusis (LASIK) and surface ablation procedures.Methods: Only eyes that underwent LASIK or surface ablation for the correction of myopia between 2008–2011 were included in this retrospective study. Additional recorded parameters included patient age, preoperative manifest refraction, corneal thickness, and calculated residual corneal bed thickness. A risk score was given to each eye, based on these parameters, according to the Ectasia Risk Factor Score System (ERFSS), without the preoperative corneal topography.Results: This study included 16,163 eyes, of which 38.4% underwent LASIK and 61.6% underwent surface ablation. The risk score correlated with procedure selection, with LASIK being preferred in eyes with a score of 0 and surface ablation in eyes with a score of 2 or higher. When controlling for age, preoperative manifest refraction, corneal thickness, and all parameters, the relative proportion of surface ablation compared with LASIK was found to have grown significantly during the study period.Conclusions: Our results indicate that with time, surface ablation tended to be performed more often than LASIK for the correction of myopia in our cohort. Increased awareness of risk factors and preoperative risk assessment tools, such as the ERFSS, have shifted the current practice of refractive surgery from LASIK towards surface ablation despite the former's advantages, especially in cases in which the risk for ectasia is more than minimal (risk score 2 and higher).Keywords: surface ablation, LASIK, PRK, myopia correction, ectasia

Overexpression of βIII-tubulin is associated with resistance to tubulin-binding agents (TBA) in a range of tumor types. We previously showed that small interfering RNA silencing of βIII-tubulin expression hypersensitized non–small cell lung cancer cells to TBAs. To determine whether βIII-tubulin mediates its effect on drug-induced mitotic arrest and cell death by differentially regulating microtubule behavior, the effects of βIII-tubulin knockdown on microtubule dynamics were analyzed in H460 non–small cell lung cancer cells stably expressing green fluorescent protein-βI-tubulin. Interphase cells were examined at three vincristine and paclitaxel concentrations that (a) inhibited cell proliferation, (b) induced 5% to 10% mitotic arrest, and (c) induced 30% to 40% mitotic arrest. In the absence of either drug, βIII-tubulin knockdown caused no significant change in microtubule dynamic instability. At 2 nmol/L vincristine (IC50), overall microtubule dynamicity was significantly suppressed in βIII-tubulin knockdowns (−31.2%) compared with controls (−6.5%). Similar results were obtained with paclitaxel, suggesting that knockdown of βIII-tubulin induces hypersensitivity by enhancing stabilization of microtubule dynamics at low drug concentrations. At higher drug concentrations (≥40 nmol/L vincristine; ≥20 nmol/L paclitaxel), βIII-tubulin knockdown resulted in significantly reduced suppressive effects on microtubule dynamicity with little or no further increase in mitotic arrest, compared with control cells. Importantly, apoptosis was markedly increased by βIII-tubulin knockdown independent of further suppression of microtubule dynamics and mitotic arrest. These results show that βIII-tubulin knockdown enhances the effectiveness of TBAs through two mechanisms: suppression of microtubule dynamics at low drug concentrations and a mitosis-independent mechanism of cell death at higher drug concentrations. Mol Cancer Ther; 9(5); 1339–48. ©2010 AACR.

Cell migration plays a crucial role in numerous cellular functions, and alterations in the regulation of cell migration are required for invasive transformation of a tumor cell. While the mechanistic process of actin-based migration has been well documented, little is known as to the specific function of the nonmuscle actin isoforms in mammalian cells. Here, we present a comprehensive examination of γ-actin's role in cell migration using an RNAi approach. The partial suppression of γ-actin expression in SH-EP neuroblastoma cells resulted in a significant decrease in wound healing and transwell migration. Similarly, the knockdown of γ-actin significantly reduced speed of motility and severely affected the cell's ability to explore, which was, in part, due to a loss of cell polarity. Moreover, there was a significant increase in the size and number of paxillin-containing focal adhesions, coupled with a significant decrease in phosphorylated paxillin in γ-actin-knockdown cells. In addition, there was a significant increase in the phosphorylation of cofilin and myosin regulatory light chain, suggesting an overactivated Rho-associated kinase (ROCK) signaling pathway in γ-actin-knockdown cells. The alterations in the phosphorylation of paxillin and myosin regulatory light chain were unique to γ-actin and not β-actin knockdown. Inhibition of the ROCK pathway with the inhibitor Y-27632 restored the ability of γ-actin-knockdown cells to migrate. This study demonstrates γ-actin as a potential upstream regulator of ROCK mediated cell migration.

LIM-kinase 2 (LIMK2) belongs to the LIMK family of proteins, which comprises LIMK1 and LIMK2. Both proteins regulate actin polymerization through phosphorylation and inactivation of the actin depolymerizing factor cofilin. In this study, we show that the level of LIMK2 protein is increased in neuroblastoma, BE(2)-C cells, selected for resistance to microtubule-destabilizing agents, vincristine and colchicine. However, the level of phosphorylated LIMK1 and LIMK2 was similar in the resistant and parental BE(2)-C cells. In contrast, the level of phospho-cofilin was greatly increased in the drug-resistant cells. Downregulation of LIMK2 expression increases sensitivity of neuroblastoma SH-EP cells to vincristine and vinblastine but not to microtubule-stabilizing agents, while it's overexpression increased its resistance to vincristine. Its vincristine-induced mitotic arrest was moderately inhibited in the LIMK2 knockdown cells, suggesting that the increased drug sensitivity is through an alternative mechanism other then mitotic arrest and apoptosis. Moreover, downregulation of LIMK2 expression induces formation of abnormal mitotic spindles, an effect enhanced in the presence of microtubule-destabilizing agents. LIMK2 is important for normal mitotic spindle formation and altered LIMK2 expression mediates sensitivity to microtubule destabilizing agents. These findings suggest that inhibition of LIMK2 activity may be used for the treatment of tumors resistant to microtubule-destabilizing drugs.

Background: Proteomic investigations have revealed alterations in cytoskeletal proteins expressed in human acute lymphoblastic leukemia cells that are resistant to microtubuledisrupting agents. We characterized γ -actin expression in antimicrotubule drug – resistant leukemia and examined the effect of altered γ -actin in resistance of acute lymphoblastic leukemia to antimicrotubule agents. Methods: Twodimensional polyacrylamide gel electrophoresis and mass spectrometry were used to identify actin proteins in human acute lymphoblastic leukemia cell lines resistant to vinblastine (CCRF-CEM/VLB100 cells) and desoxyepothilone B (CCRF-CEM/dEpoB140 cells). Fluorescence-based cycle sequencing was used to detect gene mutations. Site-directed mutagenesis was used to generate mutant γ -actin expression plasmids, which were used to transfect mouse NIH/3T3 cells. Clonogenic analysis was used for drug sensitivity studies. A small interfering RNA (siRNA) was used to block γ -actin gene expression in human neuroblastoma SH-EP cells. Expression of γ -actin (normalized to that of β 2 -microglobulin [ β 2 M]) in primary leukemia cells obtained from patients at diagnosis (n = 44) and relapse (n = 25) was examined using semiquantitative reverse transcription – polymerase chain reaction. Statistical signifi cance of changes in the ratio of γ -actin to β 2 M expression between diagnosis and relapse samples was determined by two-sided unpaired Student’s t tests. Results: We identifi ed novel mutant forms of γ -actin and the concomitant loss of wild-type γ -actin in CCRF-CEM/VLB100 cells and CCRFCEM/dEpoB140 cells. Mouse NIH/3T3 cells that expressed the mutant γ -actin proteins were more resistant to antimicrotubule agents than cells transfected with empty plasmid. Human neuroblastoma SH-EP cells transfected with γ -actin siRNA displayed higher relative resistance to paclitaxel ( P

Aurora kinase inhibitors are new mitosis-targeting drugs currently in clinical trials for the treatment of haematological and solid malignancies. However, knowledge of the molecular factors that influence sensitivity and resistance remains limited. Herein, we developed and characterised an in vitro leukaemia model of resistance to the Aurora B inhibitor ZM447439. Human T-cell acute lymphoblastic leukaemia cells, CCRF-CEM, were selected for resistance in 4 µM ZM447439. CEM/AKB4 cells showed no cross-resistance to tubulin-targeted and DNA-damaging agents, but were hypersensitive to an Aurora kinase A inhibitor. Sequencing revealed a mutation in the Aurora B kinase domain corresponding to a G160E amino acid substitution. Molecular modelling of drug binding in Aurora B containing this mutation suggested that resistance is mediated by the glutamate substitution preventing formation of an active drug-binding motif. Progression of resistance in the more highly selected CEM/AKB8 and CEM/AKB16 cells, derived sequentially from CEM/AKB4 in 8 and 16 µM ZM447439 respectively, was mediated by additional defects. These defects were independent of Aurora B and multi-drug resistance pathways and are associated with reduced apoptosis mostly likely due to reduced inhibition of the catalytic activity of aurora kinase B in the presence of drug. Our findings are important in the context of the use of these new targeted agents in treatment regimes against leukaemia and suggest resistance to therapy may arise through multiple independent mechanisms.