Expression Of Β-tubulin Isotypes Research Articles

β-II tubulin isotype directs stiffness and differentiation of neuroblastoma SH-SY5Y cells.

β-tubulin isotypes regulate the structure and bundling of microtubule (MT) lattice, its dynamics, and resulting functions. They exhibit differential tissue expression, varying due to physical and biochemical cues. In this work, we investigated the effect of transient heat shock at 42°C on the nuclear and cytoplasmic stiffness of SH-SY5Y neuroblastoma cells through atomic force microscopy. Moreover, the variations in the expression of β-tubulin isotypes as a heat shock response were also monitored. The heat-exposed cells endured a recovery at 37°C for 24h and they manifested an increase of cytoplasmic stiffness by 130 ± 25% with respect to untreated controls. The expression of β-II tubulin isotype in heat-recovered cells is augmented by 51 ± 5% whereas the levels of total tubulin and β-III tubulin isotype remain unaltered. Upon depletion of β-II tubulin isotype using shRNA, the increase in cytoplasmic stiffness was dampened. However, it remained unaffected upon depletion with β-III tubulin isotype shRNA. This features the role of the β-II tubulin isotype in regulating cellular stiffness. In addition, neuroblastoma SH-SY5Y cells undergo differentiation by initiating neuritogenesis and prior evidence suggests the indispensable role of β-II tubulin isotype in this process. The heat-recovered cells which expressed higher levels of β-II tubulin isotype expedited the differentiation process in 3-day which was around 5-day for control cells, however, upon depletion of β-II tubulin isotype, the cells almost lost their differentiation potential. Altogether, this work highlights the role of β-II tubulin isotype as a biomarker for cellular stiffness.

Delineating the interaction of combretastatin A-4 with αβ tubulin isotypes present in drug resistant human lung carcinoma using a molecular modeling approach

Tubulin isotypes are known to regulate microtubule dynamic instability and contribute to the development of drug resistance in certain types of cancers. Combretastatin-A4 (CA-4) has a potent anti-mitotic, vascular disrupting and anti-angiogenic activity. It binds at the interface of αβ tubulin heterodimers and inhibits microtubules assembly. Interestingly, the CA-4 resistant human lung carcinoma shows alteration of βI and βIII isotype levels, a higher expression of βI tubulin isotype and a decreased expression of βIII tubulin isotypes has been reported in drug resistant cell lines. However, the origin of CA-4 resistance in lung carcinoma is not well understood. Here, we investigate the interaction and binding affinities of αβI, αβIIb, αβIII and αβIVa tubulin isotypes with CA-4, employing molecular modeling approaches. Sequence analysis shows that variations in residue composition at the CA-4 binding pocket of βI, βIII and βIVa tubulin isotypes when compared to template βIIb isotype. Molecular docking result shows that the CA-4 prefers ‘cis’ conformation in all αβ-tubulin isotypes. Molecular dynamics simulation reveal role of H7 helix, T7 loop and H8 helix of β-tubulin in lower binding affinity of αβI and αβIII isotypes for CA-4. The order of binding energy for CA-4 is αβIIb > αβIVa > αβI > αβIII. This suggest that drug resistance is induced in human lung carcinoma cells by altering the expression of β-tubulin isotypes namely βI and βIII which show lowest binding affinities. Our present study can help in designing potential CA-4 analogs against drug-resistant cancer cells showing altered expression of tubulin isotypes.Abbreviations:CA-4combretastatin-A4MDmolecular dynamicsRMSDroot mean square deviationDSSPdictionary of secondary structure of proteinsVMDvisual molecular dynamicsCommunicated by Ramaswamy H. Sarma

2-(m-Azidobenzoyl)taxol binds differentially to distinct β-tubulin isotypes

There are seven β-tubulin isotypes present in distinct quantities in mammalian cells of different origin. Altered expression of β-tubulin isotypes has been reported in cancer cell lines resistant to microtubule stabilizing agents (MSAs) and in human tumors resistant to Taxol. To study the relative binding affinities of MSAs, tubulin from different sources, with distinct β-tubulin isotype content, were specifically photolabeled with a tritium-labeled Taxol analog, 2-(m-azidobenzoyl)taxol, alone or in the presence of MSAs. The inhibitory effects elicited by these MSAs on photolabeling were distinct for β-tubulin from different sources. To determine the exact amount of drug that binds to different β-tubulin isotypes, bovine brain tubulin was photolabeled and the isotypes resolved by high-resolution isoelectrofocusing. All bands were analyzed by mass spectrometry following cyanogen bromide digestion, and the identity and relative quantity of each β-tubulin isotype determined. It was found that compared with other β-tubulin isotypes, βIII-tubulin bound the least amount of 2-(m-azidobenzoyl)taxol. Analysis of the sequences of β-tubulin near the Taxol binding site indicated that, in addition to the M-loop that is known to be involved in drug binding, the leucine cluster region of βIII-tubulin contains a unique residue, alanine, at 218, compared with other isotypes that contain threonine. Molecular dynamic simulations indicated that the frequency of Taxol-accommodating conformations decreased dramatically in the T218A variant, compared with other β-tubulins. Our results indicate that the difference in residue 218 in βIII-tubulin may be responsible for inhibition of drug binding to this isotype, which could influence downstream cellular events.

Abstract 2932: Impaired taxane binding to MT pore sites mediates intrinsic drug resistance in diffuse gastric cancer

Abstract Successful clinical application of taxanes (microtubule-stabilizers) is limited due to intrinsic or acquired drug resistance. Thus, it is critical to unveil the molecular mechanisms of taxane resistance to significantly improve clinical outcomes. Retrospective analysis of the TAX-325 gastric cancer (GC) trial revealed that the addition of docetaxel (DTX) to cisplatin/fluorouracil increased progression-free plus overall survival, in intestinal (INT) but not in diffuse (DIF) GC subtypes. Our preclinical data confirmed that DIF GC cell lines are intrinsically resistant to taxanes. The incidence of DTX resistance in DIF GC cell lines was 2.5 times higher than INT GC cell lines. Drug efflux, tubulin posttranslational modification and differential β-tubulin isotype expression were ruled out as potential mechanisms of intrinsic taxane resistance. Thus, a new molecular mechanism must underlie the intrinsic taxane resistance in DIF GC. To quantify the kinetics of taxol binding to cellular MTs, we treated both DIF and INT groups of GC lines with the fluorescein-conjugated paclitaxel analog, Flutax-2. The Flutax-2 staining intensity of cellular MTs was assessed by live-cell confocal microscopy at different time points. Following a 3h incubation, there was less than 20% decrease in Flutax-2 intensity in the sensitive cell lines, compared to 59∼89% decrease in the resistant cell lines. These data suggested different binding kinetics between sensitive and resistant cells. We added 1μM Flutax-2 to sensitive/resistant cells’ native cytoskeletons for 0∼60 seconds to determine the association rate (kon) of Flutax-2 binding to MTs. The kon of Flutax-2 in the sensitive cell lines was significantly higher in sensitive (5.8×104M−1s−1) versus resistant cells (0.3∼2.6×104M−1s−1). Next, we measured the dissociation rates (koff) by competing the pre-bound Flutax-2 with DTX (0∼600 seconds). Although 20∼40% faster koff was observed in resistant cells, it appeared that the association rate of Flutax-2 was the dominant factor of differential taxane binding to MTs in GC cells. The binding mode of taxanes to MTs involves 1) binding to MT pores and 2) internalization to the high-affinity binding site at the MT lumen. To differentiate between the two, we used hexaflutax, which binds exclusively to the MT pores. We observed hexaflutax decorating radial MT arrays in sensitive but not in the resistant cells. As tubulin mutations around the high-affinity taxane binding site or tubulin posttranslational modifications/differential expression of β-tubulin isotype is not the case in DIF GC cell lines, our data suggest that modifications of the MT pore conformation or occlusion of the pore site is responsible for the intrinsic taxane resistance in DIF GC. Determining the origin of the defect at the pores will help design better MT-stabilizing drugs to overcome chemo-resistance, the major obstacle hindering overall survival of patients. Citation Format: Katsuhiro Kita, Giuseppe Galletti, Kyle Cleveland, Chao Zhang, Isabel Barasoain, J. Fernando Díaz, Doron Betel, Manish A. Shah, Paraskevi Giannakakou. Impaired taxane binding to MT pore sites mediates intrinsic drug resistance in diffuse gastric cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2932.

Differential expression of β-tubulin isotypes in different life stages of Parascaris spp after exposure to thiabendazole

Anthelmintic resistance (AR) to macrocyclic lactones (ML) has been described in Parascaris of horses world-wide. In contrast, benzimidazoles (BZ) are still effective, although reduced efficacy to this drug class was recently reported. The mode of action of BZ is binding to β-tubulin, which prevents polymerisation of microtubules. In this study, β-tubulin gene expression of isotypes 1 and 2 was investigated at seven time points (0, 6, 24, 72, 96 and 120h) during embryogenesis and in adult worms. In addition, an in ovo larval developmental test was developed to study β-tubulin gene expression of both isotypes in parasacaris eggs after exposure to different concentrations of thiabendazole (TBZ) for five days at 25°C. A strong pattern of differential expression of β-tubulin and isotype 1 was observed in all stages, while isotype 2 expression was mainly found at an early phase of the embryogenesis. For isotype 1, a 5-fold increase was observed during the first 48h, but gene expression gradually decreased after 72, 96 and 120h. Isotype 2 was only expressed during the first 24h, followed by a 130-fold decrease at (time points) 72, 96 and 120h. The in ovo larval developmental test, in which we exposed initially unembryonated eggs to increased concentrations of TBZ, did affect isotype 1 gene expression but not isotype 2. This assumes that each isotype has specific functions in different life stages. This is in agreement with the ‘multi-tubulin’ hypothesis, which states that different tubulin isotypes are required for specialised microtubule functions. Isotype 1 is the most likely drug target for BZs, as isotype 2 was only expressed at very low levels later in development. Increasing concentrations of TBZ altered β-tubulin isotype 1 gene expression after exposure of the eggs for five days, but this was not seen for isotype 2.

Abstract 833: Photolabeling of β-tubulin isotypes by radiolabeled 2-(m-azidobenzoyl)taxol

Abstract There are seven β-tubulin isotypes present in distinct quantities in cells of different origin. Altered expression of β-tubulin isotypes were found in cells resistant to microtubule stabilizing agents (MSAs), such as Taxol, epothilone B, ixabepilone and discodermolide, all of which bind to β-tubulin. For example, the level of βIII-tubulin was very low in the human ovarian cancer cell line, Hey, but increased 8- to 15-fold in two drug resistant daughter cell lines, EpoB8 and Ixab80, respectively. The βV-tubulin level was increased by 2-fold in EpoB8 cells. In contrast, levels of βI- and βIV-tubulin were unchanged and decreased, respectively, in EpoB8. Therefore, it is important to measure the binding affinity of MSAs to different β-tubulin isotypes and study their influence on the drug resistant phenotype. To study the relative binding affinities of MSAs, tubulins from HeLa cells, bovine brain, porcine brain and chicken erythrocytes were specifically photolabeled with the tritium labeled 2-(m-azidobenzoyl)taxol, in the presence and absence of Taxol, Taxotere, epothilone B, ixabepilone and discodermolide. β-tubulin isotype content from these sources was different, particularly for chicken erythrocytes that contain only one β-tubulin isotype, βVI. The inhibitory effects elicited by these MSAs on photolabeling were distinct for β-tubulins from different sources. Taxol had a minimal inhibitory effect on HeLa, bovine and porcine brain β-tubulin, but a strong inhibitory effect on chicken erythrocyte β-tubulin. Discodermolide exhibited opposite effects on photolabeling, compared to those of Taxol. However, Taxotere appeared to have a stronger inhibitory effect than Taxol on photolabeling of β-tubulins from all sources tested. Ixabepilone had the strongest inhibitory effect on HeLa β-tubulin. The inhibition pattern for β-tubulin from porcine brain correlated very well with that from bovine brain. To determine drug binding affinities for the different β-tubulin isotypes, tubulins from different sources were photolabeled and the isotypes resolved by high resolution isoelectrofocusing (IEF). Approximately 20 bands were seen with bovine and porcine brain tubulin, but only 3-5 bands were resolved with chicken erythrocyte tubulin. Many bands represent posttranslationally modified tubulins. IEF patterns for bovine and porcine brain tubulin were very similar. All bands were analyzed by mass spectrometry following CNBr digestion, and the identity and relative quantity of each β-tubulin isotype determined. After measuring the radioactivity associated with each band, it was found that the binding affinity of 2-(m-azidobenzoyl)taxol to βIII-tubulin was the lowest, compared to other β-tubulin isotypes. Citation Format: Chia-Ping H. Yang, Hui Xiao, Susan Band Horwitz. Photolabeling of β-tubulin isotypes by radiolabeled 2-(m-azidobenzoyl)taxol. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 833. doi:10.1158/1538-7445.AM2014-833

βIII-Tubulin Overexpression Is an Independent Predictor of Prostate Cancer Progression Tightly Linked to ERG Fusion Status and PTEN Deletion

Evidence suggests that class III β-tubulin (βIII-tubulin) may represent a prognostic and predictive molecular marker in prostate cancer. βIII-Tubulin expression was determined by IHC in 8179 prostate cancer specimens in a TMA format. Results were compared with tumor phenotype, biochemical recurrence, v-ets avian erythroblastosis virus E26 oncogene homolog (ERG) status, and deletions on PTEN, 3p13, 5q21, and 6q15. βIII-Tubulin expression was detectable in 25.6% of 8179 interpretable cancers. High βIII-tubulin expression was strongly associated with both TMPRSS2:ERG rearrangement and ERG expression (P<0.0001 each). High βIII-tubulin expression was tightly linked to high Gleason grade, advanced pT stage, and early prostate-specific antigen (PSA) recurrence in all cancers (P<0.0001 each), but also in the subgroups of ERG-negative and ERG-positive cancers. When all tumors were analyzed, the prognostic role of βIII-tubulin expression was independent of Gleason grade, pT stage, pN stage, surgical margin status, and preoperative PSA. Independent prognostic value became even more evident if the analysis was limited to preoperatively available features, such as biopsy specimen Gleason grade, preoperative PSA, cT stage, and βIII-tubulin expression (P<0.0001 each). βIII-Tubulin expression was associated with PTEN (P<0.0001) when all tumors were analyzed, but also in the subgroups of ERG-negative and ERG-positive cancers. βIII-Tubulin expression is an independent prognostic parameter. The significant associations with key genomic alterations of prostate cancer, such as TMPRSS2:ERG fusions and PTEN deletions, suggest interactions with several pivotal pathways involved in prostate cancer.

Abstract A268: TAS-119, a selective Aurora A inhibitor, enhanced the antitumor efficacy of taxanes in multiple human tumor cell lines including paclitaxel-resistant cells.

Abstract Background: Aurora A is a serine/ threonine kinase that plays a critical role in regulating multiple mitosis processes. Aberrant expression of Aurora A has been associated with spindle checkpoint dysfunction and increased resistance to the microtubule-polymerizing agents, paclitaxel and docetaxel (1). Aurora A has been identified as one of the genes associated with enhancement of paclitaxel cytotoxicity in a siRNA library screen (2). Moreover, Aurora A overexpression/ gene amplification has been reported in various types of human cancers. In this report, we demonstrate that a selective Aurora kinase A inhibitor, TAS-119, synergistically enhances the activity of paclitaxel in multiple cell lines in vitro. Materials and Methods: Evaluation of synergistic effects in combination was conducted by curve-shift analysis and Bliss additivity model, based on cell viability. Cell viability was assayed by using CellTiter-Glo™ which measures cellular ATP. Selectivity of TAS-119 for three isotypes of Aurora kinases was determined via monitoring the auto-phosphorylation of kinases by Western blot. Apoptosis induction was assessed by cleaved-PARP detection. As paclitaxel-resistant cell lines, both A549.T12 (3) and PTX10 (4) were used in combination experiments. A549.T12 and PTX10 were reported to have altered expression of β-tubulin isotypes and β-tubulin mutation, respectively. Results: TAS-119 selectively inhibited cellular auto-phosphorylation of Aurora A without affecting the activity of both Aurora B and Aurora C. TAS-119 showed a synergistic effect with paclitaxel or docetaxel in multiple tumor cell lines from different origins, even in cells insensitive to TAS-119 treatment alone. Furthermore, TAS-119 also showed synergistic effects in paclitaxel-resistant cell lines. Cleaved-PARP analysis revealed that the cytotoxic synergism in combination with paclitaxel was induced by promoting the caspase-dependent apoptotic cell death. Conclusion: The unique characteristic of TAS-119 as a highly selective Aurora A inhibitor contributed to the synergistic enhancement of antiproliferative effects of taxanes in multiple cell lines, possibly mediated by apoptotic pathways. These findings provide a rationale for the clinical development of TAS-119 as a combination therapy with taxanes. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A268. Citation Format: Akihiro Miura, Hiroshi Sootome, Keiko Ishihara, Norio Masuko, Hiroshi Hirai, Teruhiro Utsugi. TAS-119, a selective Aurora A inhibitor, enhanced the antitumor efficacy of taxanes in multiple human tumor cell lines including paclitaxel-resistant cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A268.

Expression of β-tubulin isotypes in urothelial carcinoma of the bladder

Our study aims to investigate the expressions of β-tubulin isotypes and their significances in urothelial carcinoma of the bladder (UCB) as altered expression of a specific β-tubulin isotype is associated with chemoresistance and poor prognosis in other malignancies. Expression of β-tubulin isotypes was retrospectively examined in 342 UCB samples obtained from 1995 to 2010 by immunohistochemistry. TUBB1 (307/342, 89.8 %) was most frequently overexpressed in the cytoplasm of UCB cases, followed by TUBB4 (101/342, 29.5 %), TUBB2 (85/342, 24.9 %), and TUBB3 (60/342, 17.5 %). TUBB1 overexpression was associated with older age (p = 0.032), high WHO grade (p = 0.001), and advanced TNM stage (p = 0.006). High levels of TUBB2 expression were associated with high WHO grade (p < 0.001), advanced TNM stage (p < 0.001), and non-papillary growth pattern (p = 0.007). TUBB3 overexpression was related to high WHO grade (p = 0.029). In univariate and multivariate survival analyses, TUBB1 overexpression was associated with poor recurrence-free survival (RFS) rates of all cases (hazard ratio 1.98, p = 0.031) and of the patients with transurethral and/or partial resection (hazard ratio 2.12, p = 0.031). TUBB2 overexpression was correlated with a short RFS of the patients with T2-T4 stages (hazard ratio 3.48, p = 0.007). TUBB3 overexpression was related to a poor RFS of the patients undergoing radical cystectomy (hazard ratio 5.90, p = 0.002). High TUBB1, TUBB2, and TUBB3 expressions are associated with unfavorable clinicopathologic factors and are independent prognostic factors for recurrence-free survival of UCB.

Expression of β‐tubulin isotypes in classical Hodgkin's lymphoma

Microtubules consist of heterodimers of α- and β-tubulin. Aberrant expression of specific β-tubulin isotype is associated with resistance to chemotherapy in malignant tumors. In this study, we examined the expressions of β-tubulin isotypes in classical Hodgkin's lymphoma (cHL) by immunohistochemistry. Among the β-tubulin isotypes, class II β-tubulin (31/34, 91%) was most frequently overexpressed in the cytoplasm of almost all Hodgkin's and Reed-Sternberg (HRS) cells, followed by class I β-tubulin (18/34, 53%) and class III β-tubulin (12/34, 35%). Class IV β-tubulin was not expressed in any cHL case. Class I β-tubulin was expressed in the background lymphoid cells as well as the HRS cells. Thus, our results indicate that class II β-tubulin may be very useful for immunohistochemical diagnosis of cHL, and provide valuable information for the potential application of β-tubulin isotype-specific targeting.

Modelling the microtubule: towards a better understanding of short-chain fatty acid molecular pharmacology

Systems biology combines experimental data with computational modelling to describe complex biological mechanisms and pathways. Short-chain fatty acids (SCFAs-chemopreventive compounds produced in the colon lumen) impair microtubule (MT) function in colon cancer cells by altering the relative expression of β-tubulin isotypes. The β-tubulin isotype composition along MT fibres is believed to contribute to a "tubulin code" defining which microtubule-associated proteins (MAPs) and kinesins are recruited and the arrangement of tubulin post-transcriptional modifications (PTMs) along the fibre, which in turn dictate many critical cellular functions. SCFAs drive acetylation of many proteins by virtue of being histone deacetylase inhibitors (HDACi's). Known acetyl-proteins include transcription factors and cytoplasmic cytoskeletal keratins as well as histones. Disruption of the MT cytoskeleton is a prime target of many cancer therapies including anti-microtubule drugs (AMD). This review focuses on SCFAs as HDACi's and how they might affect tubulin dynamics, modifications and isotypes. It discusses the evolution of mechanistic models that have helped improve understanding of tubulin-MT structure and dynamics and how to develop these models, combined with those describing transcription and the cell cycle, could provide hypotheses for how SCFAs disrupt cytoskeletal function. The review demonstrates how systems biology could offer potentially novel ideas for therapies in the prevention and treatment of cancers through the continued development and elaboration of such models.

Abstract IA9-1: Is taxol resistance related to tubulin isotypes?

Abstract Microtubules are an important component of the euraryotic cytoskeleton and are essential for normal cell division. They are also a validated target for antitumor drugs, such as taxol, that bind to the – tubulin subunit in the microtubule polymer. Endogenous resistance or the development of drug resistance during treatment with taxol is a serious clinical problem. Understanding the emergence of taxol resistance and being able to predict patient response would enhance the efficacy of the drug. A number of mechanisms, such as overexpression of P-glycoprotein that acts as an ATP-dependent drug-efflux pump, or mutations in the drug-binding site in tubulin, have been described. Of particular interest to our research, is the aberrant expression of β-tubulin isotypes in cancer. Tubulin is composed of α- and β-tubulin subunits that exist as heterodimers. In humans, there are seven β-tubulins and eight β-tubulins, each the product of a distinct gene located on different chromosomes. Each isotype can undergo extensive posttranslational modifications such as acetylation, phosphorylation, polyglutamylation, polyglycylation and reversible tyrosination, all of which add to the complexity of the tubulin/microtubule system. Most of the divergence between the isotypes is found in the last 15-20 amino acids at the carboxyl end of tubulin and in addition, most posttranslational modifications of tubulin occur in this region. For the most part, tubulin isotypes have been detected at the mRNA level or at the protein level with isotype-specific antibodies. Our laboratory has developed proteomic methods, including highresolution isoelectrofocusing, CNBr cleavage and mass spectrometry that allow us to determine the isotype content and posttranslational modifications of tubulin in cells and tissues in a single experiment. We have utilized taxol, which enhances tubulin polymerization, to isolate sufficient tubulin from cells and are developing methods to quantify tubulin isotype expression by mass spectrometry. The overexpression of βIII-tubulin has been associated with resistance to taxanes in a range of tumor types and in taxol-resistant cells generated in our laboratory. βIII-tubulin is normally expressed only in neuronal cells. However, it is aberrantly expressed in numerous malignant tissues and could be a meaningful prognostic factor for treatment outcome. As has been shown by others in different tumors, in our laboratory an immunohistochemical analysis of 47 tumor specimens from head and neck patients determined that overexpression of β-III tubulin was highly prognostic of survival. Citation Information: Clin Cancer Res 2010;16(14 Suppl):IA9-1.

Abstract LB-64: Synuclein gamma inhibits BubR1 function and renders resistance to antimicrotubule drug

Abstract The microtubule (MT) cytoskeleton is an effective and validated target for cancer chemotherapeutic drugs. Currently, anti-MT drug taxane, e.g. docetaxel, is the first-line chemotherapeutic agent to treat patients with locally advanced or metastatic breast cancer and is also one of the most effective chemotherapeutic agents in clinical use in six different cancers (breast, ovarian, non-small cell lung, prostate, gastric, and head and neck). Nearly half of the treated breast and ovarian cancer patients, however, do not respond to chemotherapy. Although general mechanisms of drug resistance may apply to docetaxel resistance, more specifically, the current research paradigm on docetaxel resistance focus on acquired -tubulin mutations and altered expression of β-tubulin isotypes that interfere with drug target binding. Synuclein gamma (SNCG) is a new unfavorable prognostic marker for breast cancer and a potential target for cancer treatment. At the cellular level, SNCG increases metastasis and promotes genetic instability. At the molecular level, SNCG functions like a tumor specific chaperone and regulates many pathways in growth and progression of cancer, including an interaction with mitotic checkpoint kinase BubR1. Here, we show that expression of SNCG is sufficient to induce specific resistance to docetaxel-caused mitotic arrest and apoptosis in breast cancer cells and in breast cancer xenografts. SNCG binds to spindle checkpoint kinase BubR1, interferes with its interaction with other checkpoint proteins of p55cdc20-APC and Mda2, reduces BubR1's kinase activity, and thus attenuates docetaxel-caused mitotic cell death. The working mechanism of anti-MT drugs heavily relies on the normal function of the checkpoint machinery in which BubR1 is a critical component, this inhibitory effect of SNCG on BubR1 function may lead to the inactivation of checkpoint and thus renders docetaxel resistance. To test the clinical relevance, in a pilot neoadjuvant trial, 20 women with histological proven locally advanced breast cancer were treated with 3 cycles of neoadjuvant TE regimen (docetaxel + epirubicin). SNCG-positive tumors are resistant to anticancer effect of neoadjuvant therapy as assayed by reduction in primary tumor size, an increase in apoptotic index, and a decrease in Ki67 proliferation index. These data show that SNCG renders docetaxel resistance by inhibiting BubR1 activity and is new biomarker for predicting anti-MT 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 LB-64.

Class III .BETA.-Tubulin Expression in Tumor Cells is Correlated with Resistance to Docetaxel in Patients with Completely Resected Non-Small-Cell Lung Cancer

To assess the relationship between in vitro chemosensitivity evaluated by the histoculture drug response assay (HDRA) and the expression of beta-tubulin isotypes in tumors of patients with completely resected NSCLC in order to determine the predictive value of beta-tubulin in chemotherapy for NSCLC. Expression of beta-tubulin isotypes was immunohistochemically analyzed in a series of 58 tumor samples from patients with completely resected NSCLC. The sensitivity of individual tumors to anticancer agents was evaluated by HDRA. Class III beta-tubulin expression by tumor cells was significantly correlated with resistance to docetaxel (p=0.0250), but not related with resistance to gemcitabine. Patient characteristics (age, gender, histology, and stage) were not associated with class III beta-tubulin expression. An abundance of class III beta-tubulin in tumor cells could be a biomarker for resistance to docetaxel in patients with completely resected NSCLC.

Tubulin-Targeted Drug Action: Functional Significance of Class II and Class IVb β-Tubulin in Vinca Alkaloid Sensitivity

Aberrant expression of beta-tubulin isotypes is frequently described in tumor tissues and tubulin-binding agent (TBA)-resistant cell lines. There is limited understanding of the role of specific beta-tubulin isotypes in cellular sensitivity to TBAs, and to gain insights into the functional role of betaII- and betaIVb-tubulin, we examined these isotypes in lung cancer cell lines NCI-H460 (H460) and Calu-6. Drug-treated clonogenic assays revealed that small interfering RNA-mediated knockdown of either betaII- or betaIVb-tubulin hypersensitized the lung cancer cell lines to Vinca alkaloids, with the effects more pronounced following betaIVb-tubulin knockdown. In contrast, there was no change in paclitaxel sensitivity following knockdown of either isotype. Cell cycle analysis revealed a greater propensity for the betaII- and betaIVb-tubulin knockdown cells to undergo G2-M cell cycle block following 5 nmol/L vincristine treatment, with the betaIVb knockdown cells being more sensitive than the betaII-tubulin knockdown cells compared with control. In contrast to betaII-tubulin knockdown, betaIVb-tubulin knockdown cells showed a significant increase in the sub-G1 population (cell death) following treatment with both 5 and 40 nmol/L of vincristine compared with controls. Importantly, betaIVb-tubulin knockdown in H460 cells caused a significant dose-dependent increase in Annexin V staining in response to vincristine but not paclitaxel. Therefore, increased sensitivity to induction of apoptosis is one mechanism underlying the Vinca alkaloid hypersensitivity. This study provides direct evidence that betaII- or betaIVb-tubulins have functionally distinct roles and expression of these isotypes may serve as strong predictors of Vinca alkaloid response and resistance.

Src Tyrosine Kinase and Multidrug Resistance Protein-1 Inhibitions Act Independently but Cooperatively to Restore Paclitaxel Sensitivity to Paclitaxel-Resistant Ovarian Cancer Cells

Src tyrosine kinase has been found to be overexpressed in both mouse and human ovarian cancer cells as well as in human primary ovarian cancers. Furthermore, Src inhibition sensitizes ovarian cancer cells to chemotherapeutic agents such as paclitaxel and cisplatin. Interestingly, Src inhibition has also been shown to resensitize paclitaxel-resistant cells to the cytotoxic effects of paclitaxel. The current study was undertaken in an effort to determine the mechanism by which Src resensitizes drug-resistant ovarian cancer cells. The paclitaxel-resistant human (CaOV3TaxR) and mouse (ID8TaxR) ovarian cancer cell lines express large amounts of the multidrug resistance-1 (MDR-1) protein compared with the paclitaxel-sensitive parent cell lines. Src inhibition had no effect on MDR-1 protein expression. Furthermore, Src inhibition did not affect MDR-1 function as determined by rhodamine 123 and paclitaxel uptake or retention. Coinhibition of both Src and MDR-1 synergistically enhanced paclitaxel-induced cytotoxicity in paclitaxel-resistant ovarian cancer cell lines. Inhibition of Src enhanced microtubule stabilization in paclitaxel-resistant ovarian cancer cells treated with paclitaxel without affecting expression of beta-tubulin isotypes and resulted in multipolar spindle formation and apoptosis. These results show that Src inhibition restores paclitaxel sensitivity to paclitaxel-resistant ovarian cancer cells by an MDR-independent mechanism, possibly by decreasing the critical intracellular concentration at which paclitaxel induces tubulin stabilization and bundling. Src tyrosine kinase may provide a viable target for therapeutic intervention in drug-resistant ovarian cancer.

Class III β-Tubulin Overexpression Is a Prominent Mechanism of Paclitaxel Resistance in Ovarian Cancer Patients

Abstract The vast majority of women with advanced ovarian cancer will ultimately relapse and develop a drug-resistant disease with an overall 5-year survival of &amp;lt;50%. Unfortunately, the mechanisms of drug resistance actually operating in patients are still unknown. To address this issue, in 41 patients affected by advanced ovarian cancer the three main mechanisms of paclitaxel resistance were investigated: overexpression of MDR-1 gene, point mutations at prominently expressed α-tubulin and β-tubulin genes and selective alterations in the expression of β-tubulin isotypes. MDR-1 and the β-tubulin isotypes expression were evaluated by semiquantitative and real-time PCR. On the same specimens, quantitative immunohistochemistry was also done in the tumor area. No statistically significant changes of MDR-1 expression were noticed between the sensitive and resistant patients either at the mRNA or protein level. The tubulin mutations for the ubiquitous α-tubulin and β-tubulin genes were evaluated by automated DNA sequencing, and in all patients, no mutations were detected in both resistant and sensitive cases. With regard to the expression of tubulin isoforms, a statistically significant up-regulation of class III β-tubulin was found in the resistant subset. It is worth noting that this statistically significant increase of the expression of class III β-tubulin was detectable at the mRNA and protein level. By a direct comparison of the three main known mechanisms of paclitaxel resistance, this study indicates that overexpression of class III β-tubulin is the most prominent mechanism of paclitaxel resistance in ovarian cancer.

Predicting response to paclitaxel/carboplatin-based therapy in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is a systemic illness. The majority of newly diagnosed patients depend on systemic chemotherapy to improve outcome. Among the new chemotherapeutic agents recently tested, paclitaxel (Taxol; Bristol-Myers Squibb Company, Princeton, NJ) has shown convincing single-agent activity in advanced NSCLC. The two initial phase II studies using paclitaxel alone showed a 1-year survival rate of 40%, comparable to that seen with combination regimens. Paclitaxel/carboplatin is one of several standard regimens for patients with stage IIIB to IV disease. It is as effective as any other new agent/platinum combination studied to date; it is easy to administer and well tolerated. Identification of the molecular and genetic events involved in each step of tumor progression seems to be crucial both to understanding lung cancer and for the development of new pharmacologic compounds that target specific cellular processes affecting growth and proliferation. An innovative strategy is to combine established chemotherapy with these new compounds. Resistance to available chemotherapy drugs is the major obstacle to effective chemotherapy. Genetic abnormalities could play a role in outlining some patterns of chemoresistance. Acquired resistance to paclitaxel can be mediated by several mechanisms, including overexpression of p-glycoprotein, altered expression of β-tubulin isotypes, intrinsic or acquired mutations in β-tubulin, and expression of novel genes. Beta-tubulin mutations were recently identified in 33% of 49 NSCLC patients, none of whom had an objective response to paclitaxel treatment. Cisplatin resistance is associated with several molecular alterations, including overexpression of metallothionein and the mRNA level of the excision repair cross-complementing ( ERCCI) gene. Early detection of circulating cancer cells in peripheral blood would enable more accurate lung cancer staging. Furthermore, sequential measurements of DNA concentration may be used to monitor the effects of therapy. Serum DNA can be used as a surrogate for detecting genetic abnormalities and as a potential guide for customizing treatment. We analyzed the presence of β-tubulin mutations in serum DNA from NSCLC patients and from healthy individuals. Beta-tubulin mutations were detected in 42% of the 131 patients and in none of the control group. Several clinical studies are proposed to develop more customized approaches in lung cancer.

Differentiation of human colon cancer cells changes the expression of beta-tubulin isotypes and MAPs.

The human colon adenocarcinoma HT29-D4 cell line is an interesting model for studies on epithelial cell differentiation. Undifferentiated cells are malignant proliferating cells, whereas differentiated cells act like epithelial polarized cells. In the present study, we first characterized the action of taxoids on the microtubular network of HT29-D4 cells according to the state of differentiation. Microtubular bundles were found in undifferentiated cells but not in differentiated cells, even with 500-fold higher taxoid concentrations for 96 h. This finding led us to study changes in microtubules according to the polarity status of the cell. E-MAP-115 was expressed only in differentiated cells; expression of β-tubulin isotypes was altered in them relative to undifferentiated cells. Classes I, II, III, IVa and IVb isotypes were expressed in both phenotypes; however, differentiated epithelial cells displayed a specific increase in class III β-tubulin. Thus, the increase in expression of this β-tubulin isotype in differentiated cells is not restricted to neuronal cells. Moreover, these expression changes may reflect a higher stability of microtubular network in differentiated cells, which may explain the lower activity of anti-microtubule agents, independently of the mitotic process. These results indicate that the composition of microtubules should be considered as one of the criteria involved in the response of tumour cells to chemotherapy with anti-microtubule agents. © 1999 Cancer Research Campaign