Stathmin levels alter PTPN14 expression and impact neuroblastoma cell migration.

BACKGROUND: Stathmin 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. METHODS: MiRNA and gene arrays followed by miRNA-target-gene integration were performed on stathmin-depleted neuroblastoma cells (CtrlshRNA vs. 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. RESULTS: Stathmin 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. CONCLUSIONS: Our findings demonstrate that stathmin levels can regulate PTPN14 expression, which can modulate neuroblastoma cell migration and invasion.

γ-Actin regulates cell migration and modulates the ROCK signaling pathway.

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 ß-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.

Preclinical small molecule WEHI-7326 overcomes drug resistance and elicits response in patient-derived xenograft models of human treatment-refractory tumors.

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.

Gamma-actin is involved in regulating centrosome function and mitotic progression in cancer cells.

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 ß-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.

Thymosin-ß4 is a determinant of drug sensitivity for Fenretinide and Vorinostat combination therapy in neuroblastoma.

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 (Tß4) and decreased mRNA expression of retinoic acid receptor α (RARα). Importantly, the up-regulation of Tß4 and down-regulation of RARα were both necessary for the 4-HPR + SAHA cytotoxic effect on neuroblastoma cells. Moreover, Tß4 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 Tß4 was associated with metastatic disease and predicted poor patient prognosis. Our findings demonstrate that Tß4 is a novel therapeutic target in neuroblastoma, and that 4-HPR + SAHA is a potential therapy for the disease.

Partial depletion of gamma-actin suppresses microtubule dynamics.

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.

Evolution of resistance to Aurora kinase B inhibitors in leukaemia cells.

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.

Microtubule dynamics, mitotic arrest, and apoptosis: drug-induced differential effects of betaIII-tubulin.

Overexpression of betaIII-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 betaIII-tubulin expression hypersensitized non-small cell lung cancer cells to TBAs. To determine whether betaIII-tubulin mediates its effect on drug-induced mitotic arrest and cell death by differentially regulating microtubule behavior, the effects of betaIII-tubulin knockdown on microtubule dynamics were analyzed in H460 non-small cell lung cancer cells stably expressing green fluorescent protein-betaI-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, betaIII-tubulin knockdown caused no significant change in microtubule dynamic instability. At 2 nmol/L vincristine (IC(50)), overall microtubule dynamicity was significantly suppressed in betaIII-tubulin knockdowns (-31.2%) compared with controls (-6.5%). Similar results were obtained with paclitaxel, suggesting that knockdown of betaIII-tubulin induces hypersensitivity by enhancing stabilization of microtubule dynamics at low drug concentrations. At higher drug concentrations (> or =40 nmol/L vincristine; > or =20 nmol/L paclitaxel), betaIII-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 betaIII-tubulin knockdown independent of further suppression of microtubule dynamics and mitotic arrest. These results show that betaIII-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.

Alterations in gamma-actin and tubulin-targeted drug resistance in childhood leukemia.

BACKGROUND: Proteomic investigations have revealed alterations in cytoskeletal proteins expressed in human acute lymphoblastic leukemia cells that are resistant to microtubule-disrupting agents. We characterized gamma-actin expression in antimicrotubule drug-resistant leukemia and examined the effect of altered gamma-actin in resistance of acute lymphoblastic leukemia to antimicrotubule agents. METHODS: Two-dimensional 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 gamma-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 gamma-actin gene expression in human neuroblastoma SH-EP cells. Expression of gamma-actin (normalized to that of beta2-microglobulin [beta2M]) 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 significance of changes in the ratio of gamma-actin to beta2M expression between diagnosis and relapse samples was determined by two-sided unpaired Student's t tests. RESULTS: We identified novel mutant forms of gamma-actin and the concomitant loss of wild-type gamma-actin in CCRF-CEM/VLB100 cells and CCRF-CEM/dEpoB140 cells. Mouse NIH/3T3 cells that expressed the mutant gamma-actin proteins were more resistant to antimicrotubule agents than cells transfected with empty plasmid. Human neuroblastoma SH-EP cells transfected with gamma-actin siRNA displayed higher relative resistance to paclitaxel (P<.001), vinblastine (P = .04), and epothilone B (P = .045) than mock-transfected cells. No gamma-actin gene mutations were identified in 37 samples of primary leukemia cells (eight from patients at diagnosis, 29 from patients at relapse). Gamma-actin gene expression was lower in acute lymphoblastic leukemia samples collected at clinical relapse (n = 25; mean gamma-actin/beta2M = 0.53) than in samples collected at diagnosis (n = 44; mean gamma-actin/beta2M = 0.68; difference = 0.15, 95% confidence interval [CI] = 0.04 to 0.27, P = .01). CONCLUSIONS: These data provide functional and associative clinical evidence of a novel form of drug resistance that involves interactions between gamma-actin and microtubules.