Overexpression Of βIII-tubulin Research Articles

Identification of novel microtubule inhibitors effective in fission yeast and human cells and their effects on breast cancer cell lines.

Microtubules are critical for a variety of cellular processes such as chromosome segregation, intracellular transport and cell shape. Drugs against microtubules have been widely used in cancer chemotherapies, though the acquisition of drug resistance has been a significant issue for their use. To identify novel small molecules that inhibit microtubule organization, we conducted sequential phenotypic screening of fission yeast and human cells. From a library of diverse 10 371 chemicals, we identified 11 compounds that inhibit proper mitotic progression both in fission yeast and in HeLa cells. An in vitro assay revealed that five of these compounds are strong inhibitors of tubulin polymerization. These compounds directly bind tubulin and destabilize the structures of tubulin dimers. We showed that one of the compounds, L1, binds to the colchicine-binding site of microtubules and exhibits a preferential potency against a panel of human breast cancer cell lines compared with a control non-cancer cell line. In addition, L1 overcomes cellular drug resistance mediated by βIII tubulin overexpression and has a strong synergistic effect when combined with the Plk1 inhibitor BI2536. Thus, we have established an economically effective drug screening strategy to target mitosis and microtubules, and have identified a candidate compound for cancer chemotherapy.

Phase II trial of eribulin in advanced/recurrent cervical cancer

<h3>Objectives:</h3> Eribulin (E), a Halichondrin B analog from the marine sponge H. okadai has clinical efficacy in pretreated metastatic breast cancer and liposarcoma as well as preclinical antitumor activity in squamous cell carcinoma (SCC). We conducted a 2-stage Phase 2 study of E in patients (pts) with advanced/recurrent cervical cancer (CC) to examine its clinical activity and evaluate potential biomarkers for predictors of response. <h3>Methods:</h3> Pts with advanced/recurrent CC after ≤1 prior chemotherapy (CT) regimen, measurable disease and ECOG performance status ≤2 were treated with E (1.4mg/m² IV day 1 and 8, every 21 days) with tumor assessments every 2 cycles. Primary endpoint was 6-month progression-free survival PFS6; secondary were best overall response (RECISTv1.1), toxicity (CTCAEv4.03) and overall survival (OS); and exploratory were associations of tumor biomarkers involved in apoptosis/proliferation, the unfolded protein response and tubulin subtypes with clinical activity. A total of 30 evaluable pts would ensure 80% power when the true PFS6=26% with a 1-sided α ≤0.1 (Ho: PFS6=10%). A prespecified futility analysis gating stage 2 was set if 0/15pts showed at least SD at 6 months (mos). Immunohistochemistry was performed on archival tumor samples. Overexpression was defined when both intensity and distribution scores are ≥2. <h3>Results:</h3> A total of 32 pts were enrolled, median age 51 years (range 29-76), 22 had SCC, the median number of cycles was 4 (range 1-77). A total of 29 pts had received prior CT with cisplatin/gemcitabine (12) and cisplatin/paclitaxel/bevacizumab (12) as the most common regimens. A total of 14 pts had received prior paclitaxel (PTX). A total of 6/32 pts (19%) achieved > PFS6; median PFS is 2.5 mos (95% CI: 1.2, 4.2). Median OS is 6.5 mos (95% CI: 4.5, 12.7). A total of 2 pts were inevaluable for response having received less than 2 cycles. Among the 30 evaluable pts, 1 (3%) had a complete response, 5 (17%) had a partial response and 13 (43%) had stable disease (clinical benefit rate 63%). Grade 3/4 adverse events occurring in > 10 % of pts are anemia (12pts), neutropenia (7pts) and leukopenia (6pts). A total of 2 pts were removed from study due to paresthesia. Pts who received prior PTX responded less favorably than those who did not (p=.002) and had a significantly shorter PFS at 1.2 mos (95% CI: 1.1, 2.0) compared to 3.5 mos (95% CI: 2.6, 5.4) for those who did not (p=.008). Analysis of correlative predictors of response revealed significant associations between βII and βIII tubulin subtypes as well as BAX. Patients who did not overexpress βII and BAX were more likely to respond to E (p=.040, p=.002, respectively). On univariate analysis, PFS was shorter in patients with BAX overexpression 1.2 mos (95% CI: 1.1, 2.6) vs 4.6 mos (95% CI: 2.7, 7.8, p=.006) in those without. This was also observed in OS with 4.3 mos (95% CI: 2.7, 6.5) in those who overexpressed BAX vs 1.3 mos (7.7, 28.3, p=.006) in those who did not overexpress. In multivariate analyses, prior PTX (HR 4.80, 95% CI: 1.12, 20.7), βIII tubulin overexpression (HR 5.22, 95% CI: 1.52, 17.93), and BAX overexpression (HR 3.61, 95% CI 1.11, 11.73) remained significant factors in PFS. Similar results were observed for OS. <h3>Conclusions:</h3> Eribulin shows some limited activity in patients with recurrent/advanced CC with a favorable toxicity profile. Prior paclitaxel exposure is associated with decreased E response. βII, βIII tubulin subtypes and BAX are predictors of response and survival. While the primary end point was not met, Eribulin may be an option for women with paclitaxel-naïve recurrent/advanced CC.

Binding modes of cabazitaxel with the different human β-tubulin isotypes: DFT and MD studies.

Taxanes (paclitaxel, docetaxel, cabazitaxel) are anticancer drugs as microtubule inhibitors. Following our previous studies on paclitaxel and docetaxel, in this work, we examine cabazitaxel and compare these three taxenes. The binding interaction of three taxanes with various β-tubulin isotypes is studied by homology modeling, molecular docking, and molecular dynamics simulations. The results show that the effects of docetaxel on βI-tubulin (- 29.5kcal/mol) and of paclitaxel on βIIa-tubulin (- 25.5kcal/mol) are much stronger than their effects on βIII-tubulin (- 17.8kcal/mol and - 8.6kcal/mol, respectively). However, the effect of cabazitaxel on βIII-tubulin (- 23.0kcal/mol) is comparable with that on βI-tubulin (- 24.0kcal/mol) and βIIa-tubulin (- 25.9kcal/mol), consistent with the fact that overexpression of βIII-tubulin increases the drug resistance to paclitaxel and docetaxel, but has little influence for cabazitaxel. This theoretical research supports the use of cabazitaxel for patients who are resistant to the action of paclitaxel and docetaxel.

Identification of a lathyrane-type diterpenoid EM-E-11-4 as a novel paclitaxel resistance reversing agent with multiple mechanisms of action.

P-glycoprotein (P-gp) and βIII-tubulin overexpression-mediated drug resistance leads to clinical therapy failure for paclitaxel. However, the development of paclitaxel-resistance reversal agents has not had much success. In this study, EM-E-11-4, a lathyrane-type diterpenoid extracted from Euphorbia micractina, demonstrated good anti-MDR (multidrug resistance) activity in paclitaxel-resistant tumor cells overexpressing either P-gp or βIII-tubulin. EM-E-11-4 was able to recover the effects of paclitaxel in inducing arrest at G2/M phase and apoptosis in both A549/Tax (P-gp overexpression) and Hela/βIII (βIII-tubulin overexpression) cells, respectively, at a non-cytotoxic dose. EM-E-11-4 could enable Flutax-1 and Rhodamine 123 be accumulated intracellularly at an accelerating rate in A549/Tax cells by inhibiting the activity of P-gp ATPase, rather than affecting the expression of P-gp. In addition, it also strengthened the effects of paclitaxel in promoting tubulin polymerization and the binding of paclitaxel to microtubules in vitro. It inhibited the expression of βIII-tubulin in Hela/βIII cells in a dose-dependent manner while not exerting influence on the other β-tubulin subtypes. As far as we know, this is the first study to report that a small molecule natural product could specifically inhibit the expression of βIII-tubulin. These results suggest EM-E-11-4 may serve as a promising MDR reversal agent, particularly for patients bearing tumors with high expression of P-gp and βIII-tubulin.

Pharmacological treatment with inhibitors of nuclear export enhances the antitumor activity of docetaxel in human prostate cancer.

Background and aimsDocetaxel (DTX) modestly increases patient survival of metastatic castration-resistant prostate cancer (mCRPC) due to insurgence of pharmacological resistance. Deregulation of Chromosome Region Maintenance (CRM-1)/ exportin-1 (XPO-1)-mediated nuclear export may play a crucial role in this phenomenon.Material and methodsHere, we evaluated the effects of two Selective Inhibitor of Nuclear Export (SINE) compounds, selinexor (KPT-330) and KPT-251, in association with DTX by using 22rv1, PC3 and DU145 cell lines with their. DTX resistant derivatives.Results and conclusionsWe show that DTX resistance may involve overexpression of β-III tubulin (TUBB3) and P-glycoprotein as well as increased cytoplasmic accumulation of Foxo3a. Increased levels of XPO-1 were also observed in DTX resistant cells suggesting that SINE compounds may modulate DTX effectiveness in sensitive cells as well as restore the sensitivity to DTX in resistant ones. Pretreatment with SINE compounds, indeed, sensitized to DTX through increased tumor shrinkage and apoptosis by preventing DTX-induced cell cycle arrest. Basally SINE compounds induce FOXO3a activation and nuclear accumulation increasing the expression of FOXO-responsive genes including p21, p27 and Bim causing cell cycle arrest. SINE compounds-catenin and survivin supporting apoptosis. βdown-regulated Cyclin D1, c-myc, Nuclear sequestration of p-Foxo3a was able to reduce ABCB1 and TUBB3 H2AX levels, prolonged γ expression. Selinexor treatment increased DTX-mediated double strand breaks (DSB), and reduced the levels of DNA repairing proteins including DNA PKc and Topo2A. Our results provide supportive evidence for the therapeutic use of SINE compounds in combination with DTX suggesting their clinical use in mCRPC patients.

Modification of C-seco taxoids through ring tethering and substituent replacement leading to effective agents against tumor drug resistance mediated by βIII-Tubulin and P-glycoprotein (P-gp) overexpressions

In our efforts to improve the efficacy of taxane-based microtubule (MT) stabilizing agents against tumor drug resistance mediated by multiple mechanisms, two clinically relevant factors were focused: i.e., P-glycoprotein and βIII-tubulin overexpression. Based on the structure of C-seco taxoid 1 m (IDN5390) which was believed to more selectively interact with βIII-tubulin than paclitaxel, we prepared a series of C-seco taxoids bearing various 7,9-O-linkages and/or different substituents at C2 and C3′ positions. Some of them exhibited much more potent binding affinity to MTs and cytotoxicity than their C-seco parent compounds in drug resistant cells with both mechanisms. SAR analysis indicated that C2 modifications significantly enhanced MT binding but brought ambiguous influence to cytotoxicity whereas 7,9-linkage and C3′ modifications enhance cytotoxicity more efficiently than improve MT binding. These observations illustrate a better translation of molecular binding effect to cellular activity by C ring closure and C3′ modification than C2 modification in C-seco taxoids.

βIII-tubulin overexpression is linked to aggressive tumor features and genetic instability in urinary bladder cancer

Development of genetic instability is a hallmark of tumor progression. Type III β-tubulin (TUBB3) is a component of microtubules involved in chromosome segregation. Its overexpression has been linked to adverse features of urinary bladder cancer. To investigate the role of TUBB3 for development of genetic instability, we compared TUBB3 expression with histopathological features and surrogate markers of genetic instability and tumor aggressiveness; copy number changes of HER2, TOP2A, CCND1, RAF1, and FGFR1; nuclear accumulation of p53, and cell proliferation in a tissue microarray (TMA) with more than 700 bladder cancers. TUBB3 expression was linked to high-grade and advanced-stage cancers (P<.0001), rapid cell proliferation (P<.0001), presence of multiple gene copy number alterations (P=.0008), and nuclear accumulation of p53 (P=.0008). Strong TUBB3 staining was found in 43% of urothelial cancers harboring copy number alterations as compared with 28% of genetically stable cancers, and in 50% of p53-positive cancers as compared with 30% of p53-negative tumors. The fraction of tumors with concomitant TUBB3 and p53 positivity increased with tumor stage and grade: 2% in pTaG1-2, 11% in pTaG3, 17% in pT1G2, 23% in pT1G3, and 32% in pT2-4 cancers (P<.0001). Importantly, strong TUBB3 overexpression was detectable in about 20% of low-grade, noninvasive cancers. In summary, our study demonstrates that TUBB3 overexpression is linked to an aggressive subtype of urinary bladder cancers, which is characterized by increased genetic instability, p53 alterations, and rapid cell proliferation. Detection of TUBB3 overexpression in genetically stable, low-grade, and noninvasive bladder cancers may be clinically useful to identify patients requiring particular close monitoring.

βIII-Tubulin Regulates Breast Cancer Metastases to the Brain.

Brain metastases occur in about 10% to 30% of breast cancer patients, which culminates in a poor prognosis. It is, therefore, critical to understand the molecular mechanisms underlying brain metastatic processes to identify relevant targets. We hypothesized that breast cancer cells must express brain-associated markers that would enable their invasion and survival in the brain microenvironment. We assessed a panel of brain-predominant markers and found an elevation of several neuronal markers (βIII-tubulin, Nestin, and AchE) in brain metastatic breast cancer cells. Among these neuronal predominant markers, in silico analysis revealed overexpression of βIII-tubulin (TUBB3) in breast cancer brain metastases (BCBM) and its expression was significantly associated with distant metastases. TUBB3 knockdown studies were conducted in breast cancer models (MDA-Br, GLIM2, and MDA-MB-468), which revealed significant reduction in their invasive capabilities. MDA-Br cells with suppressed TUBB3 also demonstrated loss of key signaling molecules such as β3 integrin, pFAK, and pSrc in vitro. Furthermore, TUBB3 knockdown in a brain metastatic breast cancer cell line compromised its metastatic ability in vivo, and significantly improved survival in a brain metastasis model. These results implicate a critical role of TUBB3 in conferring brain metastatic potential to breast cancer cells.

Emerging Microtubule Targets in Glioma Therapy

Major advances in the genomics and epigenomics of diffuse gliomas and glioblastoma to date have not been translated into effective therapy, necessitating pursuit of alternative treatment approaches for these therapeutically challenging tumors. Current knowledge of microtubules in cancer and the development of new microtubule-based treatment strategies for high-grade gliomas are the topic in this review article. Discussed are cellular, molecular, and pharmacologic aspects of the microtubule cytoskeleton underlying mitosis and interactions with other cellular partners involved in cell cycle progression, directional cell migration, and tumor invasion. Special focus is placed on (1) the aberrant overexpression of βIII-tubulin, a survival factor associated with hypoxic tumor microenvironment and dynamic instability of microtubules; (2) the ectopic overexpression of γ-tubulin, which in addition to its conventional role as a microtubule-nucleating protein has recently emerged as a transcription factor interacting with oncogenes and kinases; (3) the microtubule-severing ATPase spastin and its emerging role in cell motility of glioblastoma cells; and (4) the modulating role of posttranslational modifications of tubulin in the context of interaction of microtubules with motor proteins. Specific antineoplastic strategies discussed include downregulation of targeted molecules aimed at achieving a sensitization effect on currently used mainstay therapies. The potential role of new classes of tubulin-binding agents and ATPase inhibitors is also examined. Understanding the cellular and molecular mechanisms underpinning the distinct behaviors of microtubules in glioma tumorigenesis and drug resistance is key to the discovery of novel molecular targets that will fundamentally change the prognostic outlook of patients with diffuse high-grade gliomas.

βIII-tubulin overexpression is linked to left-sided tumor localization and nuclear β-catenin expression in colorectal cancer

Abstract Introduction/Background βIII-tubulin expression correlates with poor outcome in various malignancies. Materials and methods βIII-tubulin expression was analyzed by immunohistochemistry on a tissue microarray containing 1800 colorectal cancers. Results were compared to clinic-pathological and molecular parameters. Results βIII-tubulin expression was detectable in 79.2% of 1619 interpretable colorectal cancers. Whole tumor slide analysis showed that βIII-tubulin is homogenously expressed in CRC. High βIII-tubulin expression was associated with left-sided tumor localization ( p =0.0303) and nuclear β-catenin expression ( p =0.003). High βIII-tubulin expression was not linked to the gender of the patient ( p =0.5842). When all tumors were analyzed the prognostic role of βIII-tubulin expression was not independent of pT stage, pN stage, tumor grade or tumor localization ( p =0.0517). Conclusion βIII-tubulin expression is not an independent prognostic parameter in colorectal cancer. The significant association with left-sided tumor localization and a key genomic alteration of colorectal cancer such as β-catenin suggest interaction with important pathways involved in colorectal cancer.

βIII-tubulin overexpression is linked to aggressive tumor features and shortened survival in clear cell renal cell carcinoma.

βIII-tubulin (TUBB3) is a microtubule component overexpression of which is found in many solid cancer types, often linked to poor patient prognosis, and has been suggested to predict failure of microtubule-targeting chemotherapeutics. This study was designed to determine prevalence and prognostic impact of TUBB3 expression in kidney cancers. A tissue microarray (TMA) containing more than 1,200 renal tumors was analyzed by immunohistochemistry. TUBB3 expression varied markedly between the different histological subtypes and was more frequent in 105 papillary cancers (75.2 %, p < 0.0001), 38 oncocytomas (52.6 %, p < 0.0001), and 22 chromophobic carcinomas (36.4 %, p = 0.1221) than in 555 clear cell RCC (16.4 %). In clear cell cancers, strong TUBB3 positivity was linked to high Fuhrman grade (p < 0.0001), advanced stage (0.002), nodal metastases (p = 0.0433), hematogenous metastases (p = 0.0016), and shortened overall survival (p < 0.0001). Associations with outcome and tumor phenotype were inversely for papillary RCC, where TUBB3 immunostaining was linked to low tumor stage (p = 0.0012) and prolonged survival (p = 0.0043). TUBB3 expression levels and their effects are strikingly different between ccRCC and papillary RCC. These differences may be caused by differences in VHL function between these RCC subtypes, because VHL (like TUBB3) is another strong regulator of microtubule function.

Abstract 3214: C-seco taxanes with 7, 9-linkages exhibit better activity in drug-resistant tumor cells

Abstract Taxanes are a group of very successful MT stabilizing agents in cancer chemotherapy, although they suffer from tumor drug resistance, which compromises clinical efficacy. Many efforts were made in our labs to the structural modification of paclitaxel to improve efficacy against tumor drug resistance mediated by multiple mechanisms, with the emphasis on two clinically relevant ones: P-glycoprotein (Pgp) and β-III tubulin overexpression. While Pgp mediated multidrug resistance has been extensively explored and applied to the drug discovery projects, β-III tubulin overexpression related drug resistance was much less explored. The β-III tubulin isotype was found to play an important role in conferring resistance to MT stabilizing agents only in recent decade. It has also been identified as a survival factor in several types of cancers, thus conferring resistance to various anticancer agents, such as MT destabilizing agents and DNA interacting agents. We have found some high affinity taxanes (e.g. LX2-32C, Yg-3-46a) exhibited their activity toward Pgp efflux pump and also β-III tubulin mediated drug resistance. However, they did not show greater specificity for the β-III tubulin isotype. Based on a C-seco taxane IDN5390 which was reported to be more selectively interacted with β-III tubulin than paclitaxel, we have succeeded in the design and synthesis of 7,9-O-linked C-seco taxanes with different linkages. Some of them exhibited better activity than IDN5390, and the activities were found sensitive to the structural changes of the linkers. Citation Format: Yong Tang, Pei Cai, Javier Rodríguez-Salarisch, J. Fernando Díaz, Weishuo Fang. C-seco taxanes with 7, 9-linkages exhibit better activity in drug-resistant tumor cells. [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 3214. doi:10.1158/1538-7445.AM2014-3214

In vivo and in vitro properties of STX2484: a novel non-steroidal anti-cancer compound active in taxane-resistant breast cancer.

Background:STX2484 is a novel non-steroidal compound with potent anti-proliferative activity. These studies aimed to identify STX2484's mechanism of action, in vivo efficacy and activity in taxane-resistant breast cancer models.Methods:Effects of STX2484 and paclitaxel on proliferation, cell cycle and apoptosis were assessed in vitro in drug-resistant (MCF-7DOX) and non-resistant cells (MCF-7WT). STX2484 efficacy in βIII tubulin overexpression in MCF-7 cells was also determined. Anti-angiogenic activity was quantified in vitro by a co-culture model and in vivo using a Matrigel plug assay. An MDA-MB-231 xenograft model was used to determine STX2484 efficacy in vivo.Results:STX2484 is a tubulin disruptor, which induces p53 expression, Bcl2 phosphorylation, caspase-3 cleavage, cell cycle arrest and apoptosis. In addition, STX2484 is a potent anti-angiogenic agent in vitro and in vivo. In breast cancer xenografts, STX2484 (20 mg kg−1 p.o.) suppressed tumour growth by 84% after 35 days of daily dosing, with limited toxicity. In contrast to paclitaxel, STX2484 efficacy was unchanged in two clinically relevant drug-resistant models.Conclusions:STX2484 is an orally bioavailable microtubule-disrupting agent with in vivo anti-angiogenic activity and excellent in vivo efficacy with no apparent toxicity. Crucially, STX2484 has superior efficacy to paclitaxel in models of clinical drug resistance.

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.

A semisynthetic taxane Yg-3-46a effectively evades P-glycoprotein and β-III tubulin mediated tumor drug resistance in vitro

Tumor resistance, especially that mediated by P-glycoprotein (P-gp) and β-III tubulin, is a major obstacle to the efficacy of most microtubule-targeting anticancer drugs in clinics. A novel semisynthetic taxane, 2-debenzoyl-2-(3-azidobenzyl)-10-propionyldocetaxel (Yg-3-46a) was shown to be highly cytotoxic to breast cancer cell lines MCF-7 and MCF/ADR which overexpressed P-gp via long term culture with doxorubicin, and cervical cancer cell lines Hela and Hela/βIII which overexpressed βIII-tubulin via stable transfection with TUBB3 gene. siRNA transfection experiments also confirmed that Yg-3-46a can circumvent P-gp and β-III tubulin mediated drug resistance. In addition, its cytotoxicity was lower than that of paclitaxel in the human mammary cell line HBL-100 and the human telomerase-immortalized retinal pigment epithelium cell line (hTERT-RPE1), suggesting a better safety margin for this compound in vivo. It exhibited more potent microtubule polymerization ability than paclitaxel in vitro, and also induced G2/M phase arrest in MCF-7/ADR cells. Moreover, it was found to induce apoptosis in MCF-7/ADR cells through the caspase-dependent death-receptor pathway by enhancing levels of Fas and FasL, and activating caspase-8 and 3. Yg-3-46a was found to be a poorer substrate of P-gp compared to paclitaxel, in both binding and ATPase experiments, which is likely responsible for its ability to circumvent P-gp mediated multidrug resistance (MDR). All of these results indicate that Yg-3-46a is a novel microtubule-stabilizing agent that has the potential to evade drug resistance mediated by P-gp and β-III tubulin overexpression.

BCL2 and BCLxL are key determinants of resistance to antitubulin chemotherapeutics in melanoma cells

Malignant melanoma is refractory to various chemotherapeutics including antitubulin agents such as paclitaxel. Previous studies have suggested a link between βIII-tubulin overexpression and paclitaxel resistance through alterations in the properties of the mitotic spindle. We found that paclitaxel treatment induced temporary mitotic arrest in 7 melanoma cell lines irrespective of the βIII-tubulin level, suggesting that βIII-tubulin had no significant influence on spindle properties. On the other hand, the amount of BCL2, an anti-apoptotic protein, was well correlated with paclitaxel resistance. Treatment of the paclitaxel-resistant cell lines with ABT-737, an inhibitor of BCL2 and BCLxL, or simultaneous knock-down of BCL2 and BCLxL dramatically increased the cells' sensitivity, while knock-down of MCL1, another member of the BCL2 family, had only a minimal effect. Our results suggest that the paclitaxel sensitivity of melanoma cells is attributable to apoptosis susceptibility rather than a change in spindle properties and that BCL2 and BCLxL play a pivotal role in the former.

Abstract PD07-01: Phase 2 Study of Ixabepilone Versus Paclitaxel as Neoadjuvant Therapy for Early Stage Breast Cancer with Comparative Biomarker Analysis

Abstract Background: Anthracyclines (A) and taxanes (T) are standard neoadjuvant treatments for breast cancer (BC), achieving pathologic complete response (pCR) rates of 20-30% in unselected patient (pt) populations. Ixabepilone (ixa) is approved for treatment of metastatic BC: plus capecitabine (Cap) in pts progressing after A and T or as monotherapy after progression on A, T and Cap. Prior data suggest that overexpression of βIII tubulin is associated with resistance to paclitaxel (P) while activity of ixa was unaffected. We present the first randomized comparison of neoadjuvant ixa and P in early stage BC. Primary objectives were to estimate pathologic complete response rate (pCR) in the overall population and in biomarker-defined populations. Methods Pts with early stage BC were biopsied for immunohistochemical (IHC) and mRNA biomarker analyses prior to chemotherapy. Following 4 cycles of doxorubicin/cyclophosphamide (AC), pts were randomized to either every 3 week ixa (40mg/m2:4 cycles) or weekly P (80mg/m2:12 doses). Post-therapy surgery and pathological reports were used to assess pCR. Baseline βIII expression was assessed via a standardized IHC assay (Dako, CA) and predefined single gene mRNA markers (including TUBB3, CAPG, TACC3) were assessed via Affymetrix gene expression profilling. The pCR rate and cutoff for biomarker positivity were estimated using a cross-validation method. Secondary endpoints in the study included clinical objective response rate and safety. Results Pts (N=384) were enrolled in 15 countries: 313 pts were treated with AC. 295 pts were randomized; 289 were treated with either ixa or P. Of these, 247 (123, ixa; 124, P) had βIII IHC data and 231 (114, ixa; 117, P) had both pathologic and βIII IHC data. Baseline characteristics were balanced between arms, including triple negative pts (TN, 49%). The pCR rate in all randomized pts was 24.3% (90% CI: 18.6-30.8) with ixa and 25.2% (90% CI: 19.4-31.7) with P. pCR rates were similar regardless of sub-group. Table 1. pCR rates No significant interaction was observed between βIII expression and treatment arms (logistic regression analysis). Secondary efficacy measures were consistent with the pCR results. No clinically meaningful differences in efficacy endpoints were noted between ixa and P with mRNA markers (TUBB3, CAPG and TACC3; analyses are ongoing with others). The safety profiles of ixa and P were similar, including incidence of peripheral neuropathy (Grade 3/4: ixa 4.1% vs P 3.5%). An exception was greater neutropenia with ixa (Grade 3/4; 41.3% versus 8.4% with P) although there was no difference in the rate of febrile neutropenia (0.7%). Summary Overall, the results indicated that ixa had similar efficacy to P when measured by pCR in the neoadjuvant BC setting. No clinically meaningful differences were noted in the efficacy profile of ixa compared to P across the subsets analyzed. Ixa or P following AC was well tolerated with similar safety profiles. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD07-01.

Abstract PD07-02: βIII Tubulin Expression by Immunohistochemistry (IHC) and mRNA in a Randomized Phase 2 Neoadjuvant Breast Cancer (BC) Study of Ixabepilone (ixa) Compared to Paclitaxel (pac)

Abstract Background: Overexpression of βIII tubulin has been shown to correlate with poor prognosis and reduced response to taxanes. Ixa exhibits activity in βIII overexpressing tumor models and BC patients (pts) with high βIII mRNA levels. We report the prevalence of βIII by a validated IHC assay and the correlation of βIII protein and mRNA expression in BC tissue samples. Methods: In this neoadjuvant BC study CA163-100, 295 patients were randomized 1:1 to ixa (40 mg/m2 IV x 4 cycles) or pac (80 mg/m2 IV wkly x 12) after all pts received 4 cycles of doxorubicin (60 mg/m2) plus cyclophosphamide (600 mg/m2). Pre-chemotherapy formalin-fixed paraffin-embedded pt tissue sections were assessed for intensity and percentage of tumor cell staining of βIII by IHC. RNA was extracted from additional pre-chemotherapy core needle biopsy specimens and profiled for gene expression using Affymetrix HG-U133Av2 gene chips. Tumors were classified as basal-like, Her2-enriched, luminal-like A and B and normal-like using a 50-gene subtype predictor. In addition, Her2 and hormone receptor status was used for tumor subtyping. Results: βIII staining data were obtained for 261 pts, of which 247 were randomized to either ixa or pac arms of study CA163-100. The prevalence of βIII positivity was 39.1% (102/261) using the prespecified cut-off of ≥50% (2+, 3+) positive cells. The distribution of IHC scores was bimodal. 64% of specimens classified as βIII negative had no observable staining, while 77% of βIII positive specimens had a majority of tumor cells staining at the highest intensity (3+). IHC and gene expression profiling data were available for 235 pts and a correlation between βIII tubulin protein and mRNA expression was observed. The relative βIII mRNA expression levels were significantly higher in pts classified as βIII positive vs. βIII negative by IHC (P&amp;lt;0.0001) and 80% of tumors classified as βIII tubulin positive by IHC, had high relative TUBB3 mRNA levels, using a median cut-off. An association between βIII IHC status and breast cancer subtypes was also seen with 69% (62/90) of βIII positive specimens also classified as basal-like and conversely, 55% (62/113) of basal-likes classified as βIII positive. Nearly half (48%) the specimens in this cohort were basal-like, a classification that was quite concordant with triple negative status and as such, triple negative tumors were also enriched for βIII positivity. Conclusion: βIII is reliably measured by a validated IHC assay, providing a bimodal distribution of scores in this BC study. Correlative analyses of βIII status by BC subtype suggest that βIII positivity is significantly associated with aggressive basal-like/TN breast cancers. The clinical utility of βIII as a potential tool to select patients who may benefit from ixa in this study will be presented separately. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD07-02.

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 5472: Class III beta-tubulin in castration resistant human prostate cancer

Abstract BACKGROUND: Class III β-tubulin (βIII-tubulin) is expressed in tissues of neuronal lineage but it is also expressed in several human malignancies including non small cell lung carcinoma, breast and ovarian cancer. Overexpression of βIII-tubulin in these tumors is associated with an unfavorable outcome and resistance to taxane-based therapies. βIII-tubulin deregulation however is still poorly documented in prostate cancer. Previously, we reported that βIII-tubulin expression is increased in prostate cancer (PCa) cells by androgen ablation. Here we sought to better characterize the contribution of βIII-tubulin to castration resistant PCa, the stage of the tumor most responsible for mortality from PCa. METHODS: βIII-tubulin expression was assayed by immunohistochemistry in prostate tumors obtained from patients with castration resistant PCa. Protein expression was scored as null (0), weak (1), moderate (2) and strong (3) and considered significant when the score was 2 or more in more than 10% of cancer cells. Consecutive sections were also immunostained for neuroendocrine biomarkers, neuron specific enolase (NSE) and chromogranin A (CgA). An androgen independent (AI) variant of LNCaP cells (AI-LNCaP) was assessed for the effects of docetaxel treatment on βIII-tubulin expression. Finally, DU145 cells stably overexpressing βIII-tubulin were tested for their sensitivity to docetaxel relative to control vector-transfected cells. RESULTS: 24/40 specimens of castration resistant tumors were found to express significant levels of βIII-tubulin. In 6 out of 22 cases analyzed, regions of the tumor positively co-stained for both βIII tubulin and NSE, however none of 8 evaluated cases showed co-staining for βIII-tubulin and CgA. AI-LNCaP cells treated with 5 to 10 nM docetaxel showed a time dependent increase in βIII-tubulin expression. DU145 cells stably overexpressing βIII-tubulin were found to be more resistant to docetaxel than the parental counterpart with a 2-fold increase in the IC50. CONCLUSION: Our data indicate that βIII-tubulin is expressed in the majority of castration resistant prostate tumors. Although βIII-tubulin immunostaining of prostate tumor cells sometimes coincides with staining for neuroendocrine markers, most βIII-tubulin positive tumor cells examined did not show coincidental staining so increased βIII-tubulin expression may occur independently of NE differentiation. Regardless, our in vitro studies support a role for βIII-tubulin in the emergence of doxetaxel resistance by androgen independent human PCa 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 5472.