Reduced LIMK2 expression in colorectal cancer reflects its role in limiting stem cell proliferation

Objective: Colorectal cancer (CRC) is a major contributor to cancer mortality and morbidity. LIM kinase 2 (LIMK2) promotes tumour cell invasion and metastasis. The objectives of this study were to determine how LIMK2 expression is associated with CRC progression and patient outcome, and to use genetically modified Drosophila and mice to determine how LIMK2 deletion affects gastrointestinal stem cell regulation and tumour development.<p></p> Design: LIMK2 expression and activity were measured by immunostaining tumours from CRC-prone mice, human CRC cell lines and 650 human tumours. LIMK knockdown in Drosophila or Limk2 deletion in mice allowed for assessment of their contributions to gastrointestinal stem cell homeostasis and tumour development.<p></p> Results: LIMK2 expression was reduced in intestinal tumours of cancer-prone mice, as well as in human CRC cell lines and tumours. Reduced LIMK2 expression and substrate phosphorylation were associated with shorter patient survival. Genetic analysis in Drosophila midgut and intestinal epithelial cells isolated from genetically modified mice revealed a conserved role for LIMK2 in constraining gastrointestinal stem cell proliferation. Limk2 deletion increased colon tumour size in a colitis-associated colorectal mouse cancer model.<p></p> Conclusions: This study revealed that LIMK2 expression and activity progressively decrease with advancing stage, and supports the hypothesis that there is selective pressure for reduced LIMK2 expression in CRC to relieve negative constraints imposed upon gastrointestinal stem cells.<p></p&gt

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

Role of Actin and its regulating proteins in drug response

Antimicrotubule drugs are used in the treatment of childhood neuroblastoma and acute lymphoblastic leukaemia (ALL). Resistance to these agents can be a major clinical problem and mechanisms mediating resistance are not fully understood. Previous studies have reported an association between the actin cytoskeleton and resistance to antimicrotubule drugs. Thus, the aim of this study was to investigate the role of the actin regulating proteins, LIM kinases (LIMK1 and LIMK2) in drug resistance. In addition, the role of γ actin, a major actin isoform, in drug resistance was also examined.Chapter 1 reviewed the known mechanisms of antimicrotubule drug resistance and the interaction between the microtubules and actin cytoskeleton. The methodologies used in this study are described in chapter 2.LIMKs are known to regulate the actin cytoskeleton via phosphorylation of cofilin. Real Time RT PCR and western blotting was used in chapter 3 and showed that expression of LIMKs and their downstream target cofilin was altered in antimicrotubule resistant neuroblastoma and leukaemia cells. Moreover, altered LIMK expression was detected in in vivo derived vincristine resistant ALL xenografts and ALL clinical samples, further demonstrating that alterations in LIMKs and cofilin are associated with antimicrotubule drug resistance.Importantly, in chapter 4, gene silencing and drug treated clonogenic assays were performed to elucidate the functional role of LIMK1 and LIMK2 in drug response. Silencing of LIMK1 and/or LIMK2 increased sensitivity of neuroblastoma cells to microtubule targeting drugs and DNA damaging agents, suggesting that LIMKs may be useful targets to improve the efficacy of anticancer drugs.γ-Actin has been associated with drug resistance and chapter 5 used gene silencing and drug treated clonogenic assays to show that decreased γ actin expression conferred resistance to anitmicrotubule drugs but not to DNA damaging agents. Microscopy and tubulin polymerisation assays showed that reduced γ-actin protects microtubules from paclitaxel induced polymerisation. This data supports a functional role for γ actin in antimicrotubule drug action. In conclusion, this study showed that LIMKs and γ actin mediate the action of antimicrotubule drugs and other anticancer agents, demonstrating that the actin cytoskeleton may serve as a useful drug target to improve the efficacy of anticancer drugs

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 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

Relative resistance of CEM/AKB4 cells to cytotoxic agents compared to parental CCRF-CEM cells.

a<p>Determined by dividing the IC₅₀ for the resistant (CEM/AKB4) cell line by the IC₅₀ of the parent (CEM) cell line.</p

Resistance levels of CEM/AKB4.

<p>(A) Plot of cell viability against concentration of ZM447439 for both CEM/AKB4 and parental CEM cells as determined by cytotoxicity assay. (B) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030734#s3" target="_blank">Results</a> of the same experiment performed in the presence of the P-glycoprotein inhibitor verapamil. Points are the means, and bars are the SEM of at least three independent experiments.</p

Proliferation timecourse of CEM and CEM/AKB cells in the presence and absence of ZM447439.

<p>Cells were grown either in vehicle alone or in 4 µM ZM447439 and proliferation was determined at indicated timepoints as the corrected absorbance using the Alamar blue assay measured spectrophotometrically. Error bars represent the SEM of three independent experiments.</p

Levels of phosphorylated Histone H3 in CEM and CEM/AKB cells in the presence or absence of 16 µM ZM447439.

<p>Levels of phosphorylated Histone H3 in CEM and CEM/AKB cells in the presence or absence of 16 µM ZM447439.</p