Β-tubulin Mutations Research Articles

Worldwide absence of canonical benzimidazole resistance-associated mutations within β-tubulin genes from Ascaris.

The giant roundworm Ascaris is an intestinal nematode, causing ascariasis by infecting humans and pigs worldwide. Recent estimates suggest that Ascaris infects over half a billion people, with chronic infections leading to reduced growth and cognitive ability. Ascariasis affects innumerable pigs worldwide and is known to reduce production yields via decreased growth and condemnation of livers. The predominant anthelminthic drugs used to treat ascariasis are the benzimidazoles. Benzimidazoles interact with β-tubulins and block their function, and several benzimidazole resistance-associated mutations have been described in the β-tubulins of ruminant nematodes. Recent research on ascarids has shown that these canonical benzimidazole resistance-associated mutations are likely not present in the β-tubulins of Ascaris, Ascaridia or Parascaris, even in phenotypically resistant populations. To further determine the putative absence of key β-tubulin polymorphisms, we screened two β-tubulin isotypes of Ascaris, highly expressed in adult worms. Using adult and egg samples of Ascaris obtained from pigs and humans worldwide, we performed deep amplicon sequencing to look for canonical resistance-associated mutations in Ascaris β-tubulins. Subsequently, we examined these data in closer detail to study the population dynamics of Ascaris and genetic diversity within the two isotypes and tested whether genotypes appeared to partition across human and pig hosts. In the 187 isolates, 69 genotypes were found, made up of eight haplotypes of β-tubulin isotype A and 20 haplotypes of isotype B. Single nucleotide polymorphisms were seen at 14 and 37 positions for β-tubulin isotype A and isotype B, respectively. No evidence of any canonical benzimidazole resistance-associated mutations was found in either human- or pig-derived Ascaris isolates. There was, however, a difference in the genetic diversity of each isotype and distribution of β-tubulin genotypes between human- and pig-derived Ascaris. Statistical tests of population differentiation show significant differences (p < 0.001) between pig- and human-derived worms; however, more diversity was seen between worms from different populations than worms from different hosts. Our work suggests an absence of canonical β-tubulin mutations within Ascaris, but alternative modes of anthelminthic resistance may emerge necessitating continued genetic scrutiny alongside monitoring of drug efficacy.

Transcriptomic analysis of albendazole resistance in human diarrheal parasite Giardia duodenalis

Benzimidazole-2-carbamates (BZ, e.g., albendazole; ALB), which bind β-tubulin to disrupt microtubule polymerization, are one of two primary compound classes used to treat giardiasis. In most parasitic nematodes and fungi, BZ-resistance is caused by β-tubulin mutations and its molecular mode of action (MOA) is well studied. In contrast, in Giardia duodenalis BZ MOA or resistance is less well understood, may involve target-specific and broader impacts including cellular damage and oxidative stress, and its underlying cause is not clearly determined. Previously, we identified acquisition of a single nucleotide polymorphism, E198K, in β-tubulin in ALB-resistant (ALB-R) G. duodenalis WB-1B relative to ALB-sensitive (ALB-S) parental controls. E198K is linked to BZ-resistance in fungi and its allelic frequency correlated with the magnitude of BZ-resistance in G. duodenalis WB-1B. Here, we undertook detailed transcriptomic comparisons of these ALB-S and ALB-R G. duodenalis WB-1B cultures. The primary transcriptional changes with ALB-R in G. duodenalis WB-1B indicated increased protein degradation and turnover, and up-regulation of tubulin, and related genes, associated with the adhesive disc and basal bodies. These findings are consistent with previous observations noting focused disintegration of the disc and associated structures in Giardia duodenalis upon ALB exposure. We also saw transcriptional changes with ALB-R in G. duodenalis WB-1B consistent with prior observations of a shift from glycolysis to arginine metabolism for ATP production and possible changes to aspects of the vesicular trafficking system that require further investigation. Finally, we saw mixed transcriptional changes associated with DNA repair and oxidative stress responses in the G. duodenalis WB-1B line. These changes may be indicative of a role for H2O2 degradation in ALB-R, as has been observed in other G. duodenalis cell cultures. However, they were below the transcriptional fold-change threshold (log2FC > 1) typically employed in transcriptomic analyses and appear to be contradicted in ALB-R G. duodenalis WB-1B by down-regulation of the NAD scavenging and conversion pathways required to support these stress pathways and up-regulation of many highly oxidation sensitive iron-sulphur (FeS) cluster based metabolic enzymes.

Chlamydomonas reinhardtii tubulin-gene disruptants for efficient isolation of strains bearing tubulin mutations.

The single-cell green alga Chlamydomonas reinhardtii possesses two α-tubulin genes (tua1 and tua2) and two β-tubulin genes (tub1 and tub2), with the two genes in each pair encoding identical amino acid sequences. Here, we screened an insertional library to establish eight disruptants with defective tua2, tub1, or tub2 expression. Most of the disruptants did not exhibit major defects in cell growth, flagellar length, or flagellar regeneration after amputation. Because few tubulin mutants of C. reinhardtii have been reported to date, we then used our disruptants, together with a tua1 disruptant obtained from the Chlamydomonas Library Project (CLiP), to isolate tubulin-mutants resistant to the anti-tubulin agents propyzamide (pronamide) or oryzalin. As a result of several trials, we obtained 8 strains bearing 7 different α-tubulin mutations and 12 strains bearing 7 different β-tubulin mutations. One of the mutations is at a residue similar to that of a mutation site known to confer drug resistance in human cancer cells. Some strains had the same amino acid substitutions as those reported previously in C. reinhardtii; however, the mutants with single tubulin genes showed slightly stronger drug-resistance than the previous mutants that express the mutated tubulin in addition to the wild-type tubulin. Such increased drug-resistance may have facilitated sensitive detection of tubulin mutation. Single-tubulin-gene disruptants are thus an efficient background of generating tubulin mutants for the study of the structure–function relationship of tubulin.

Benzimidazole- and QoI-resistance in Corynespora cassiicola populations from greenhouse-cultivated cucumber: An emerging problem in China

Target leaf spot caused by Corynespora cassiicola is an economically important foliar disease on cucumber. In recent years, this disease has caused a serious problem on greenhouse-cultivated cucumber in China. In this study, to explore the characteristics and possible causes of heavy occurrence of the disease, we monitored the resistance of C. cassiicola strains from different provinces of China to benzimidazole and quinone outside inhibitor (QoI) fungicides. The results from sequence comparison of target genes β-tubulin and Cytb of 619C. cassiicola strains indicate that resistance frequency to benzimidazoles and QoIs is up to 100%. Furtherly, molecular resistance mechanism of C. cassiicola to benzimidazoles and QoIs was analysed. One single mutation E198A and three double mutations E198A&M163I, E198A&F167Y and E198A&F200S were observed in target gene β-tubulin, which confers resistance to benzimidazoles. To our knowledge, this is the first report that double mutations of β-tubulin confer resistance to benzimidazoles in filamentous fungi. Compared with single mutation E198A, three double mutations significantly decreased sensitivity to benzimidazoles. Moreover, significant difference of sensitivity to benzimidazoles was observed among three double mutations. These mutation genotypes of β-tubulin have different geographical distribution and the mutation E198A&M163I is prevalent, occupying for 63.94%. In addition, strong cross resistance patterns between carbendazim, benomyl and thiabendazole were observed in C. cassiicola strains conferring different β-tubulin mutations. For QoI resistance, the only mutation G143A of Cytb was detected in tested 619C. cassiicola strains. Strong positive cross resistance was observed when comparing the EC50 values of sensitive and resistant strains of C. cassiicola for six intrinsically different QoIs such as azoxystrobin, fluoxastrobin, pyraclostrobin, fenaminstrobin, picoxystrobin and coumoxystrobin. Taken together, all the results not only provide novel insights into understanding resistance mechanism to benzimidazoles and QoIs in filamentous fungi, but also provide some important references for resistance management of target leaf spot on cucumber.

Mutations in Human Tubulin Proximal to the Kinesin-Binding Site Alter Dynamic Instability at Microtubule Plus- and Minus-Ends

The assembly of microtubule-based cellular structures depends on regulated tubulin polymerization and directional transport. Here, we purify and characterize tubulin heterodimers that have human β-tubulin isotype III (TUBB3), as well as heterodimers with one of two β-tubulin mutations (D417H or R262H). Both point mutations are proximal to the kinesin-binding site and have been linked to an ocular motility disorder in humans. Compared to wild-type, microtubules with these mutations have decreased catastrophe frequencies and increased average lifetimes of plus- and minus-end-stabilizing caps. Importantly, the D417H mutation does not alter microtubule lattice structure or Mal3 binding to growing filaments. Instead, this mutation reduces the affinity of tubulin for TOG domains and colchicine, suggesting that the distribution of tubulin heterodimer conformations is changed. Together, our findings reveal how residues on the surface of microtubules, distal from the GTP-hydrolysis site and inter-subunit contacts, can alter polymerization dynamics at the plus- and minus-ends of microtubules.

Structural basis for drug resistance conferred by β-tubulin mutations: a molecular modeling study on native and mutated tubulin complexes with epothilone B

Using molecular modeling, we have investigated the structure and dynamic properties of epothilone B–tubulin complexes with wild-type and mutated tubulin, aimed at identifying the molecular factors involved in the emergence of drug resistance induced by four protein mutations at Phe270Val, Thr274Ile, Arg282Gln, and Gln292Glu. Our results revealed that tubulin mutations render significant changes in the protein conformation in regions involved either in the binding of the ligand or in interdimer contacts that are relevant to the assembly of stable microtubules. In addition, point mutations induce drastic changes in the binding pose of the ligand and in the interaction networks responsible for the epothilone–tubulin association. Large ligand displacements inside the binding pocket and an overall decrease in the strength of drug-receptor polar contacts suggest a looser binding of the ligand in tubulin mutants. These results explain the loss of activity for epothilone B against cancer cells that contain tubulin mutants and provide valuable information to enhance the understanding of the atomic source of epothilones’ activity, which can be helpful to conduct further research on the rational design of more potent therapeutic tubulin-binding agents.

Investigation of Paclitaxel Resistant R306C Mutation in β-Tubulin—A Computational Approach.

Paclitaxel is the most effective chemotherapeutic agent used for the treatment of a broad spectrum of solid tumors. However, observed paclitaxel resistance in clinical trials presents one of the major obstacles for cancer chemotherapy. Most importantly, resistance due to β-tubulin mutations (R306C) has been intensely debated in recent years. Despite all efforts, mechanism of resistance is still not well understood. In this study, computational techniques were employed to uncover the effect of R306C mutation in the β-tubulin structure and its function. The tools such as I-Mutant, CUPSAT and Fold-X were employed to address the consequence of R306C mutation in the structural stability of β-tubulin. Further, molecular docking and molecular dynamics study was employed to understand the functional impact of β-tubulin mutation. Our results suggest that the R306C mutation causes a significant reduction in the binding affinity between β-tubulin and paclitaxel. Further, docked complex analysis indicates that destruction of conservative hydrogen bond maintained by the residues Arg282 and Gly360 should be responsible for the large conformation changes of the binding pocket in R306C mutant. Finally, molecular dynamics simulations study confirms the stable binding of paclitaxel with native type β-tubulin structure rather than mutant (R306C) type. We certainly believe that this study will provide useful guidance for the development of novel inhibitors that are less susceptible to drug resistance.

Exploring the impact of F270V mutation in the β-tubulin (Bos Taurus) structure and its function: a computational perspective

Paclitaxel is the most effective chemotherapeutic agent used for the treatment of a broad spectrum of solid tumors. However, observed paclitaxel resistance in clinical trials presents one of the major obstacles for cancer chemotherapy. Most importantly, resistance due to β-tubulin mutations (F270V) has been intensely debated in recent years. Despite all efforts, mechanism of resistance is still not well understood. In this study, computational techniques were employed to uncover the effect of F270V mutation in the β-tubulin structure and its function. The tools such as MuStab, CUPSAT and I-Mutant were employed to address the consequence of F270V mutation in the structural stability of β-tubulin. Further, molecular simulation study was employed to understand the functional impact of β-tubulin mutation. We believe that this study will provide useful guidance for the development of novel inhibitors that are less susceptible to drug resistance.

Paclitaxel resistance by random mutagenesis of α‐tubulin

Many mammalian β-tubulin mutations that confer paclitaxel resistance have been characterized, but little is currently known about the role of α-tubulin mutations in drug resistance. Previous studies using two-dimensional gel electrophoresis showed that α-tubulin mutations occur with a frequency equal to β-tubulin mutations among CHO cells selected for resistance to paclitaxel but the identities of those mutations are largely unknown. We have now sequenced the major α-tubulin gene in several paclitaxel resistant CHO cell lines with lesions in genomic DNA and identified five mutations that predominately affect the amino terminal part of the protein. We also used random mutagenesis and transfection of α-tubulin cDNA to select further paclitaxel resistant mutants in an effort to remove genomic constraints that may limit the diversity of mutations. This approach led to the identification of 16 additional mutations that were distributed throughout the α-tubulin sequence. The mutations were confirmed as sufficient to confer resistance by site-directed mutagenesis, and they acted by a mechanism that involved reductions in microtubule assembly. One mutation prevented the acetylation of α-tubulin but otherwise produced a phenotype similar to the other mutations. A scan of the literature revealed that a significant number of drug resistance mutations overlap or lie close to lesions that have been reported in patients with brain disorders suggesting that alterations in microtubule assembly underlie both cellular resistance and developmental defects.

β-Tubulin mutations that cause severe neuropathies disrupt axonal transport

Microtubules are fundamental to neuronal morphogenesis and function. Mutations in tubulin, the major constituent of microtubules, result in neuronal diseases. Here, we have analysed β-tubulin mutations that cause neuronal diseases and we have identified mutations that strongly inhibit axonal transport of vesicles and mitochondria. These mutations are in the H12 helix of β-tubulin and change the negative charge on the surface of the microtubule. This surface is the interface between microtubules and kinesin superfamily motor proteins (KIF). The binding of axonal transport KIFs to microtubules is dominant negatively disrupted by these mutations, which alters the localization of KIFs in neurons and inhibits axon elongation in vivo. In humans, these mutations induce broad neurological symptoms, such as loss of axons in the central nervous system and peripheral neuropathy. Thus, our data identified the critical region of β-tubulin required for axonal transport and suggest a molecular mechanism for human neuronal diseases caused by tubulin mutations.

An inherited TUBB2B mutation alters a kinesin-binding site and causes polymicrogyria, CFEOM and axon dysinnervation

Microtubules are essential components of axon guidance machinery. Among β-tubulin mutations, only those in TUBB3 have been shown to cause primary errors in axon guidance. All identified mutations in TUBB2B result in polymicrogyria, but it remains unclear whether TUBB2B mutations can cause axon dysinnervation as a primary phenotype. We have identified a novel inherited heterozygous missense mutation in TUBB2B that results in an E421K amino acid substitution in a family who segregates congenital fibrosis of the extraocular muscles (CFEOM) with polymicrogyria. Diffusion tensor imaging of brains of affected family members reveals aberrations in the trajectories of commissural projection neurons, implying a paucity of homotopic connections. These observations led us to ask whether axon dysinnervation is a primary phenotype, and why the E421K, but not other, TUBB2B substitutions cause CFEOM. Expression of exogenous Tubb2b-E421K in developing callosal projection neurons is sufficient to perturb homotopic connectivity, without affecting neuronal production or migration. Using in vitro biochemical assays and yeast genetics, we find that TUBB2B-E421K αβ-heterodimers are incorporated into the microtubule network where they alter microtubule dynamics and can reduce kinesin localization. These data provide evidence that TUBB2B mutations can cause primary axon dysinnervation. Interestingly, by incorporating into microtubules and altering their dynamic properties, the E421K substitution behaves differently than previously identified TUBB2B substitutions, providing mechanistic insight into the divergence between resulting phenotypes. Together with previous studies, these findings highlight that β-tubulin isotypes function in both conserved and divergent ways to support proper human nervous system development.

Comparative Binding Energy (COMBINE) Analysis Supports a Proposal for the Binding Mode of Epothilones to β‐Tubulin

The conformational preferences of epothilone A (EPA) and a 12,13-cyclopropyl C12-epimerized analogue were explored in aqueous solution using molecular dynamics simulations. The simulated conformers that provided an optimal fit in the paclitaxel binding site of mammalian β-tubulin were then selected. The resulting modeled complexes were simulated before and after refinement of the M-loop to improve the fitting and assess ligand stability within the binding pocket. The tubulin-bound conformation of EPA was found to be unlike a previously reported solution obtained through mixed crystallographic/NMR/modeling studies. However, our conformation was in agreement with an NMR-based proposal although the exact binding pose within the site was different. Molecular models were built for the complexes of 14 epothilone derivatives with β-tubulin. A projection to latent structures regression method succeeded in providing a good prediction of the experimentally measured binding enthalpies for the whole set of ligands by assigning weights to a selection of interaction energy terms. These receptor-based, quantitative structure-activity relationships support the proposed binding mode, help confirm and interpret previously acquired experimental data, shed additional light on the effect of several β-tubulin mutations on ligand binding, and can potentially direct further experimental studies.

Taxane-Induced Blockade to Nuclear Accumulation of the Androgen Receptor Predicts Clinical Responses in Metastatic Prostate Cancer

Prostate cancer progression requires active androgen receptor (AR) signaling which occurs following translocation of AR from the cytoplasm to the nucleus. Chemotherapy with taxanes improves survival in patients with castrate resistant prostate cancer (CRPC). Taxanes induce microtubule stabilization, mitotic arrest, and apoptotic cell death, but recent data suggest that taxanes can also affect AR signaling. Here, we report that taxanes inhibit ligand-induced AR nuclear translocation and downstream transcriptional activation of AR target genes such as prostate-specific antigen. AR nuclear translocation was not inhibited in cells with acquired β-tubulin mutations that prevent taxane-induced microtubule stabilization, confirming a role for microtubules in AR trafficking. Upon ligand activation, AR associated with the minus-end-microtubule motor dynein, thereby trafficking on microtubules to translocate to the nucleus. Analysis of circulating tumor cells (CTC) isolated from the peripheral blood of CRPC patients receiving taxane chemotherapy revealed a significant correlation between AR cytoplasmic sequestration and clinical response to therapy. These results indicate that taxanes act in CRPC patients at least in part by inhibiting AR nuclear transport and signaling. Further, they suggest that monitoring AR subcellular localization in the CTCs of CRPC patients might predict clinical responses to taxane chemotherapy.

Abstract 2555: Class I β-tubulin mutations induce resistance to microtubule destabilizer through stabilization of the microtubule networks

Abstract Anti-mitotic compounds such as vincristine, colchicine and paclitaxel have been developed to target microtubules in cancers. Although these compounds have been shown successful in treating various cancers, development of drug-resistance cells limits their efficacies in clinical situations. To determine possible mechanisms that are responsible for drug-resistance to microtubule de-stabilizers, KB-derived drug-resistant KB-L30 cells were developed. KB-L30 cells showed resistance to various microtubule de-stabilizers through multiple-drug resistant (MDR)-independent mechanisms. In contrast, this specific clone showed hyper-sensitized to the microtubule-stabilizer, paclitaxel. Single-strand conformation polymorphism (SSCP) analysis, RT-PCR and DNA sequencing revealed no mutation on exon 1, 2 and 3 of βI-tubulin. Surprisingly, six novel mutation sites were found present in the exon 4 of βI-tubulin gene. Transfection of the βI-tubulin-mutated construct into KB cells demonstrated that these mutations could alter its sensitivity to the microtubule de-stabilizer, BPR0L075, in vitro. On the other hand, western blot analysis revealed increased microtubule assembly and reduced microtubule dynamic instability in KB-L30 cells when compared to its parental KB cells. In addition, computational modeling indicated that a direct relationship exists between tubulin mutations and alteration in the microtubule assembly and dynamic instability in KB-L30 cells. In conclusion, we showed for the first time that mutations in exon 4 of βI-tubulin induced resistance to microtubule de-stabilizers and hyper-sensitivity to microtubule stabilizer through an alteration in the microtubule assembly and dynamics in cancer cells. The results of this study have important implications for the development of future anti-mitotic compounds that are able to target microtubule mutation-induced drug-resistant cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2555.

Abstract 4416: Pursuing semi-synthetic taxanes as antitumor agents effective against drug-resistant tumors

Abstract Despite the renowned success of Paclitaxel (Taxol) in cancer chemotherapy, it suffers from some disadvantages, especially ineffectiveness against drug-resistant tumors. About ten paclitaxel analogues have been evaluated in clinical trials worldwides, however, none of these drug candidates have shown promising results against drug-resistant tumors in clinical trials. Drug resistance, or multidrug resistance (MDR), is a complex phenomenon, in which various physiological or pathological factors are involved. It has been reasoned that targeting a single factor related to MDR may be disappointing. For patients resistant to taxane chemotherapy, overexpression of ABC transporters, especially P-glycoprotein (P-gp), as well as mutations in β-tubulin and/or overexpression of β-III subtype tubulin may account for the drug resistance. So we decided to pursue highly active paclitaxel analogues and simultaneously effective against drug-resistant tumors through targeting multiple factors related to drug resistance. We designed and synthesized some C-2, 7, 10, 13 modified paclitaxel analogues, based on the rationale at the very beginning to improve interactions with its receptor β-tubulin, as well as decrease interactions with P-gp. Those analogues were subjected to evaluations for the ligand-protein interactions with β-tubulin and P-gp, as well as antitumor activities in drug-sensitive and MDR tumor cells and tumor-bearing animal models. On the basis of a comprehensive analysis of the interactions of paclitaxel analogues with tubulin, it was found that ΔG (free energy change) for drug-tubulin binding at different positions is additive to that of the whole molecule, and the optimal modification for one molecule can be predicted from the free energy contributions of different positions. The more interesting observation for the SARs of these paclitaxel analogues is that we discovered the high affinity taxanes, although exhibiting similar dissociation constants to P-gp as paclitaxel, are still active in P-gp overexpressed MDR cells, in contrast to paclitaxel. On the basis of this observation, we reasoned that enhancement of the binding affinity of taxanes to microtubules may counteract the exportation of those molecules by P-gp from MDR tumor cells, thus make those molecules more effective against MDR. In addition, these high affinity taxanes are also effective against drug resistant tumor cells bearing β-tubulin mutations, as well as β-III overexpression tumor cells. One of the high affinity taxanes also showed effectiveness in nude mice exhibiting drug resistance phenotypes. All above results support that seeking for high affinity taxanes may be an promising approach to pursuing novel antitumor agents effective against drug resistance. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4416.

Class I β-tubulin mutations in 2-methoxyestradiol-resistant acute lymphoblastic leukemia cells: implications for drug-target interactions

2-Methoxyestradiol (2ME2) is a naturally occurring derivative of estradiol that has been shown to be an active small molecule that has antitumor and antiangiogenic properties. 2ME2 binds to beta-tubulin near the colchicine-binding site, inhibits microtubule polymerization, and induces mitotic arrest. To improve understanding of the mechanisms of action and resistance to 2ME2, we selected leukemia cells, CCRF-CEM, that display increasing resistance to 2ME2, and three of the highly resistant sublines were chosen for detailed analysis. The 2ME2 cells selected in 7.2 to 28.8 micromol/L were found to be 47- to 107-fold resistant to 2ME2 and exhibited low levels of cross-resistance to vinblastine. Two of the lowest 2ME2-resistant sublines were significantly hypersensitive to colchicine and epothilone B, but the hypersensitive effects were lost in the highest 2ME2-resistant subline. Moreover, 2ME2-resistant cells require 10-fold higher concentrations of 2ME2 to induce G(2)-M cell cycle arrest and have higher amounts of tubulin polymer compared with parental cells. Gene and protein sequencing revealed four class I beta-tubulin mutations, S25N, D197N, A248T, and K350N, in the 2ME2-resistant cells. The S25N mutation is within the paclitaxel-binding site, whereas A248T and K350N are within the colchicine-binding site on beta-tubulin, yet the resistant cells were not cross-resistant to paclitaxel or colchicine. This strongly suggests that the mutations have induced conformational changes to the binding site that resulted in 2ME2 resistance. The 2ME2-resistant leukemia cells provide novel insights into microtubule stability and drug-target interactions.

Osteopontin and basic fibroblast growth factor are associated with prognosis in advanced non-small cell lung cancer (JMTO LC 0004)

19025 Background: A prospective randomized phase III trial for advanced non-small cell lung cancer (NSCLC) compared paclitaxel (P) plus carboplatin (C) vs. vinorelbine (V), gemcitabine (G) followed by docetaxel (D) in the Japan-Multinational Trial Organization (JMTO LC0003) (Kawahara ASCO 06). This trial was conducted jointly with SWOG 0003 using a common arm of PC. Pharmacogenomic studies of samples from both trials demonstrated significant associations between genotype and clinical outcome (Gandara ASCO 07). Previous analysis of SWOG 0003 samples showed that elevated osteopontin (OPN) plasma levels were highly prognostic (Mack ASCO 05). Our correlative study (JMTO LC 0004) of JMTO LC 0003 was originally designed to assess β-tubulin mutations, which was determined to be insignificant. We elected to study and validate OPN as a prognostic marker, using stock samples under additional approval of JMTO IRB. Method: A total of 20 ml of blood was collected prior to treatment from patients enrolled in JMTO LC0003. Serum concentrations of OPN and basic fibroblast growth factor (bFGF) were measured by ELISA. The relationships between OPN, bFGF levels and patients characteristics were examined using χ2 test, including treatment arm, age, smoking status, histology, clinical stage, performance status, weight loss and LDH. Effect of OPN and bFGF levels on progression-free survival (PFS) and overall survival (OS) were examined by multivariate analysis adjusted by treatment arm, smoking status, clinical stage, performance status, weight loss and LDH. Results: Seventy-one samples were obtained, including 32 specimens from the PC arm and 39 from the VGD arm. There were no significant relationships between either OPN or bFGF levels with patient characteristics. However, smokers tended to have higher OPN levels (p=0.083). In analysis of clinical outcome, low OPN levels correlated with better PFS (p<0.001), and high bFGF correlated with better OS (p=0.003). Conclusion: Consistent with findings from SWOG 0003, low OPN serum levels were significantly associated with a favorable prognosis in the JMTO LC 0004. Additionally, high bFGF levels were associated with improved survival. No significant financial relationships to disclose.

2-Methoxyestradiol Inhibits the Anaphase-Promoting Complex and Protein Translation in Human Breast Cancer Cells

2-methoxyestradiol (2ME2), an estradiol metabolite with antiproliferative and antiangiogenic activities, is in phase I/II clinical trials for breast cancer. 2ME2 inhibits microtubule polymerization and causes cells to arrest in G2-M. The purpose of this study was to further elucidate the molecular mechanism of 2ME2. MDA-MB-435 breast cancer cells were treated with 2ME2 (2 micromol/L) or vehicle alone. RNA was extracted and genomic profiling was done using 22k Agilent microarrays. Expression Analysis Systematic Explorer was used to determine enrichment of Gene Ontology categories. Protein isolates were subjected to Western blot analysis. Protein synthesis was measured with a [35S]methionine pulse assay. An MDA-MB-435 cell line with two beta-tubulin mutations (2ME2R) was used to determine whether novel mechanisms were tubulin-dependent. Gene Ontology categories enriched include genes that regulate the mitotic spindle assembly checkpoint, apoptosis, and the cytosolic ribosome. The target of the mitotic spindle assembly checkpoint is the anaphase-promoting complex (APC). APC inhibition was confirmed by measuring protein levels of its targets securin and cyclin B1, which were increased in 2ME2-treated cells. Because gene expression in the cytosolic ribosome category was decreased, we evaluated whether 2ME2 decreases protein translation. This was confirmed with a pulse assay, which showed decreased isotope incorporation in 2ME2-treated cells, which was maintained in the tubulin-resistant 2ME2R cells. APC inhibition was not maintained in 2ME2R cells. 2ME2 induces tubulin-dependent cell cycle arrest through regulation of genes involved in the mitotic spindle assembly checkpoint, which results in inhibition of the APC and tubulin-independent inhibition of protein translation.

The Roles of β-Tubulin Mutations and Isotype Expression in Acquired Drug Resistance

The antitumor drug paclitaxel stabilizes microtubules and reduces their dynamicity, promoting mitotic arrest and eventually apoptosis. Upon assembly of the α/β-tubulin heterodimer, GTP becomes bound to both the α and β-tubulin monomers. During microtubule assembly, the GTP bound to β-tubulin is hydrolyzed to GDP, eventually reaching steady-state equilibrium between free tubulin dimers and those polymerized into microtubules. Tubulin-binding drugs such as paclitaxel interact with β-tubulin, resulting in the disruption of this equilibrium. In spite of several crystal structures of tubulin, there is little biochemical insight into the mechanism by which anti-tubulin drugs target microtubules and alter their normal behavior. The mechanism of drug action is further complicated, as the description of altered β-tubulin isotype expression and/or mutations in tubulin genes may lead to drug resistance as has been described in the literature. Because of the relationship between β-tubulin isotype expression and mutations within β-tubulin, both leading to resistance, we examined the properties of altered residues within the taxane, colchicine and Vinca binding sites. The amount of data now available, allows us to investigate common patterns that lead to microtubule disruption and may provide a guide to the rational design of novel compounds that can inhibit microtubule dynamics for specific tubulin isotypes or, indeed resistant cell lines. Because of the vast amount of data published to date, we will only provide a broad overview of the mutational results and how these correlate with differences between tubulin isotypes. We also note that clinical studies describe a number of predictive factors for the response to anti-tubulin drugs and attempt to develop an understanding of the features within tubulin that may help explain how they may affect both microtubule assembly and stability.

Preclinical Pharmacologic Evaluation of MST-997, an Orally Active Taxane with Superior In vitro and In vivo Efficacy in Paclitaxel- and Docetaxel-Resistant Tumor Models

Because resistance to paclitaxel and docetaxel is frequently observed in the clinic, new anti-microtubule agents have been sought. The aim of this study was to evaluate the efficacy and oral activity of a novel taxane (MST-997) in paclitaxel- and docetaxel-resistant tumor models in vitro and in vivo. Tubulin polymerization assays, immunohistochemistry, and cell cycle analysis was used to evaluate mechanism of action of MST-997. The effect of MST-997 on growth inhibition in a panel of paclitaxel- and docetaxel-resistant cell lines that overexpressed P-glycoprotein (MDR1) or harbored beta-tubulin mutations were assayed in vitro and in murine xenografts. MST-997 induced microtubule polymerization (EC50 = 0.9 micromol/L) and bundling, resulting in G2-M arrest and apoptosis. In addition, MST-997 was a potent inhibitor of paclitaxel- and docetaxel-sensitive tumor cell lines that did not have detectable P-glycoprotein (IC50 = 1.8 +/- 1.5 nmol/L). Minimal resistance (1- to 8-fold) to MST-997 was found in cell lines that either overexpressed MDR1 or harbored point mutations in beta-tubulin. Most notable, MST-997 displayed superior in vivo efficacy as a single i.v. or p.o. dose either partially or completely inhibited tumor growth in paclitaxel- and docetaxel-resistant xenografts. MST-997 represents a potent and orally active microtubule-stabilizing agent that has greater pharmacologic efficacy in vitro and in vivo than the currently approved taxanes. Our findings suggest that MST-997, which has entered phase I clinical trials, may have broad therapeutic value.