Beta-tubulin Expression Research Articles

Exosome delivery of tumor suppressive microRNA-7 to cervical cancer cells: A novel approach for regulating cervical cancer cell growth (230)

<b>Objectives:</b> Exosomes are highly stable nanosized extracellular vehicles released from cells for cell-cell communication that protect their cargo from degradation in an array of environments. Adipocytederived stem cell exosomes (ACS-exos) have unique properties that allow them to be engineered as delivery vehicles in various biological settings. The ability to manipulate the exosomes and their contents allows exosomes to be a focus of cancer therapeutics. MicroRNAs (miRNAs) are small non-coding RNAs that control gene translation and expression. Abnormal expression of tissue miRNAs has been associated with significant alterations in cell growth. MicroRNA-7 (miR-7) has an inhibitory effect on X-Linked Inhibitor of Apoptosis Protein (XIAP), which is over-expressed in cervical cancer cells, inhibiting apoptosis and promoting cell proliferation. miR-7 down-regulates XIAP in cervical carcinoma cells, increasing apoptosis and suppressing colony formation. Our objective was to develop a new therapeutic approach for cervical cancer by employing ACS-exos to deliver miR-7 to cervical cancer cells so as to elicit regulatory effects on XIAP. <b>Methods:</b> The cervical cancer cell line C-33 A was used to demonstrate the effect of miR-7 on XIAP expression. An miRNA mimic hsa-miR-7 was used for transfection. The presence of XIAP in C-33 A cells was established using Western blot, and then the cells were transfected with miR-7 using Lipofectamine ^TM RNAiMAX. The transfected cells were collected 24 and 48 hours after treatment, and the expression of XIAP was tested using Western blot. To collect exosomes, adipocyte-derived stem cells (ACS) were cultured in a growth medium with exosome-depleted FBS for 72 hours, followed by differential centrifugation. The isolated exosomes were then transfected with either miR-7 or Texas-Red fluorescent-labeled siRNA (TX-Red) using Exo-Fect^TM Exosome Transfection Reagent. Transfected exosomes were added to the C-33 A cell culture media. The cells were analyzed under a fluorescence microscope after 24 and 48 hours of growth. Western blot was used to study the expression of XIAP. The presence of Beta-Tubulin was also tested as a loading control. <b>Results:</b> The presence of XIAP in C-33 A cells was confirmed by Western blot. In the cells transfected with miR-7, Western blot demonstrated downregulation of XIAP after both 24 and 48 hours. Successful nucleotide transfection of ACS-exos and oligonucleotide integration into the C-33 A cells was confirmed by fluorescence microscopy, which demonstrated the intracellular presence of TX-Red in only the positive control C-33 A cell line. A similar downregulation of XIAP in the cells treated with miR-7 transfected ACS-exos was seen as in the miR-7 transfected cells by Western blot. Beta-Tubulin expression was seen across the control and treatment cells. <b>Conclusions:</b> Delivery of tumor-suppressive miR-7 via ACS-exos elicited downregulation of XIAP. The isolation and successful transfection of exosomes demonstrated are key processes in utilizing exosomes for cancer therapeutics. Further, the exosome cargo nucleotides integrated into the target cells and resulted in alteration of protein expression. Additional studies examining the effects of exosome-delivered miR-7 on cervical cancer cell viability, proliferation, invasion are underway. These findings highlight the promising therapeutic potential of exosomes in cervical cancer.

Xenopus embryos show a compensatory response following perturbation of the Notch signaling pathway

As an essential feature of development, robustness ensures that embryos attain a consistent phenotype despite genetic and environmental variation. The growing number of examples demonstrating that embryos can mount a compensatory response to germline mutations in key developmental genes has heightened interest in the phenomenon of embryonic robustness. While considerable progress has been made in elucidating genetic compensation in response to germline mutations, the diversity, mechanisms, and limitations of embryonic robustness remain unclear. In this work, we have examined whether Xenopus laevis embryos are able to compensate for perturbations of the Notch signaling pathway induced by RNA injection constructs that either upregulate or inhibit this signaling pathway. Consistent with earlier studies, we found that at neurula stages, hyperactivation of the Notch pathway inhibited neural differentiation while inhibition of Notch signaling increases premature differentiation as assayed by neural beta tubulin expression. However, surprisingly, by hatching stages, embryos begin to compensate for these perturbations, and by swimming tadpole stages most embryos exhibited normal neuronal gene expression. Using cell proliferation and TUNEL assays, we show that the compensatory response is, in part, mediated by modulating levels of cell proliferation and apoptosis. This work provides an additional model for addressing the mechanisms of embryonic robustness and of genetic compensation.

Expression of Class III Beta-tubulin Predicts Prognosis in Patients with Cisplatin-resistant Bladder Cancer Receiving Paclitaxel-based Second-line Chemotherapy.

Class III beta-tubulin (TUBB3) expression is recognized as a predictive marker for chemosensitivity to cisplatin- and taxane-based chemotherapies in various malignancies. The aim of this study was to clarify the predictive value of TUBB3 expression for the anticancer effects of first-line cisplatin-based chemotherapy and second-line paclitaxel-based chemotherapy in patients with urothelial cancer (UC). We reviewed 116 patients with UC (90 with bladder cancer and 27 with upper urinary tract cancer) treated with first-line cisplatin-based chemotherapy. Among them, 42 patients received a paclitaxel-based regimen as second-line chemotherapy for advanced cisplatin-resistant UC. TUBB3 expression was evaluated using immunohistochemistry, and survival analyses were performed using Kaplan-Meier survival curves and multivariate Cox proportional hazard analysis. TUBB3 was mainly detected in the cytoplasm of cancer cells, and 64 patients (55.2%) were judged as having positive TUBB3 expression. TUBB3 expression was significantly associated with tumour grade (p<0.001). TUBB3 expression was not associated with time to progression after first-line cisplatin-based chemotherapy. However, positive expression of TUBB3 was significantly associated with unfavourable overall survival in patients receiving second-line paclitaxel-based chemotherapy (p=0.021). In addition, a multivariate analysis model including T-stage and metastasis at the beginning of second-line therapy and regimen showed that TUBB3 expression was an independent predictor of poorer survival (hazard ratio(HR)=3.44, 95% confidential interval(CI)=1.15-10.33, p=0.027). TUBB3 expression was identified as a useful predictive factor for survival after second-line paclitaxel-based therapy in patients with cisplatin-resistant UC. Our results are useful for determining treatment strategies for such patients.

Prognostic and predictive factors in patients with metastatic or recurrent cervical cancer treated with platinum-based chemotherapy.

BackgroundRecognizing resistance or susceptibility to the current standard cisplatin and paclitaxel treatment could improve therapeutic outcomes of metastatic or recurrent cervical cancer.MethodsForty-five tissue samples from patients participating in a phase II trial of cisplatin and ifosfamide, with or without paclitaxel were collected for retrograde analysis. Immunohistochemistry and genotyping was performed to test ERCC1, III β-tubulin, COX-2, CD4, CD8 and ERCC1 (C8092A and N118 N) and MDR1 (C3435T and G2677 T) gene polymorphisms, as possible predictive and prognostic markers. Results were statistically analyzed and correlated with patient characteristics and outcomes.ResultsPatients with higher levels of ERCC1 expression had shorter PFS and OS than patients with low ERCC1 expression (mPFS:5.1 vs 10.2 months, p = 0.027; mOS:10.5 vs. 21.4 months, p = 0.006). Patients with TT in the site of ERCC1 N118 N and GT in the site of MDR1 G2677 T polymorphisms had significantly longer PFS (p = 0.006 and p = 0.027 respectively). ERCC1 expression and the ERCC1 N118 N polymorphism remained independent predictors of PFS. Interestingly, high III beta tubulin expression was associated with chemotherapy resistance and fewer responses [5/20 (25%)] compared to lower III β-tubulin expression [15/23 (65.2%)] (p = 0.008). Finally, ΙΙΙ β-tubulin levels and chemotherapy regimen were independent predictors of response to treatment.ConclusionsERCC1 expression proved to be a significant prognostic factor for survival in our metastatic or recurrent cervical cancer population treated with cisplatin based chemotherapy. ERCC1 N118 N and MDR1 G2677 T polymorphism also proved of prognostic significance for disease progression, while overexpression of III β-tubulin was positively correlated with chemotherapy resistance.

Expression of the Class II and III Beta-Tubulin in Neoplastic and Non-Neoplastic Lymphoid Tissues

Aim:Abnormal expression patterns of beta-tubulin isotypes may provide a molecular rationale for the behaviour of lymphoma subtypes. In the present study class II and III beta-tubulin expression was assessed in non-neoplastic and neoplastic lymphoid tissues with reference to potential utility as new tumour biomarkers.Methods and results:In this cross-sectional study class II and III beta-tubulin expression was assessed in 304 neoplastic and 20 normal lymphoid tissues using qualitative and semi-quantitative immunohistochemistry. Class II beta-tubulin was found to be positive in the germinal centres, mantle zone and interfollicular regions of normal lymphoid tissues. It was also expressed in 15/15 (100%) lymphoblastic lymphomas, 229/231 (99%) mature B cell lymphomas, 22/22 (100%) T/NK-cell lymphomas and 36/36(100%) classical Hodgkin lymphomas. Class III beta-tubulin in contrast was germinal centre restricted and more selective, being found mainly in classical Hodgkin lymphomas (34/36 (94%)). It was also expressed in 58/171(34%) DLBCL, 11/12 (92%) mantle cell lymphomas and 6/6 (100%) Burkitt lymphomas. Other mature B cell, T/NK cell lymphomas and precursor lymphoblastic lymphomas were usually negative.Conclusions:Class II beta-tubulin shows ubiquitous expression in neoplastic and non-neoplastic lymphoisd tissues. In contrast, Class III beta-tubulin is germinal centre-restricted. Its consistent expression in classical Hodgkin lymphomas may point to use in the identification of Reed-Sternberg and Hodgkin cells. Its expression in a proportion of DLBCL, Burkitt and mantle cell lymphomas is of interest as this may be related to their aggressiveness.

A phase II evaluation of ixabepilone in the treatment of recurrent/persistent carcinosarcoma of the uterus, an NRG Oncology/Gynecologic Oncology Group study

BackgroundThe primary objectives were to determine the objective response rate (ORR) and safety profile of ixabepilone in women with recurrent or persistent uterine carcinosarcoma (UCS). Secondary objectives included progression-free survival (PFS) and overall survival (OS). Exploratory translational objectives included characterization of class III beta tubulin expression and its association with response, PFS, and OS. MethodsPatients had measurable disease; up to two prior chemotherapeutic regimens were allowed, but must have included a taxane. Women received ixabepilone 40mg/m2 as a 3hour IV infusion on day 1 of a 21daycycle. Treatment was continued until disease progression or unacceptable toxicity occurred. ResultsForty-two women were enrolled, with 34 eligible and evaluable. Median age was 68years. ECOG performance status was 0 in 56% of women, 38% had received radiation, and 15% had received 2 lines of chemotherapy. Overall ORR was 11.8% (4/34, 90% CI 4.2–25.1%); all were partial responses. Stable disease for at least 8weeks was achieved in 8 patients (23.5%). Median PFS and OS were 1.7mo and 7.7mo, respectively, with a median follow-up of 37mo. Six month PFS was 20.6%. Major grade≥3 toxicities were neutropenia (47%), fatigue (15%), dehydration (15%), hypertension (15%), and hyponatremia (15%); grade 2 peripheral neuropathy was reported in 18%. In this small sample size, class III beta tubulin expression in the primary tumor was not associated with the response to ixabepilone, PFS, or OS. ConclusionIn this cohort of women, single agent ixabepilone showed modest but insufficient clinical activity.

41P - The gelatinases-stimuli nanoparticles reverse docetaxel resistance and epithelial to mesenchymal transition in lung cancer cell line

Drug resistance is a main obstacle for the successful cancer therapy. Emerging evidence suggests that miR-200c functions as an effective cancer stem cells (CSCs) inhibitor and restores sensitivity to microtubule-targeting drugs. In the present work, we engineered the intelligent gelatinases-stimuli nanoparticles (NPs) to co-delivery miR-200c and antitumor drug docetaxel (DOC) to verify their synergetic effects on inhibition of CSCs and cancer cells. After tumor cells were treated with miR-200c NPs, miR-200c and its targeted gene class III beta-tubulin (TUBB3) expression were evaluated. The effects of (miR-200c + DOC) NPs on DOC-resistant lung cancer cells viability as well as the expression of E-cadherin and CD44 were studied. We found that the (miR-200c + DOC) NPs significantly overcome DOC resistance, possibly by elevated miR-200c expression,low TUBB3 level, and reversed the EMT through upregulation E-cadherin and inhibition lung CSCs. The (miR-200c + DOC) NPs may provide a new modality for co-delivery of nucleic acid and drugs to inhibit CSCs and reverse drug resistance.

Abstract 4743: Preclinical studies characterize tumor type sensitivity to FASN inhibition and the mechanism and efficacy of novel drug combinations with TVB-2640

Abstract Tumor cells have an increased dependence on FASN-synthesized palmitate compared to non-tumor cells, which obtain many of their required lipids from the extracellular milieu. Palmitate and palmitate-derived lipids comprise diverse cellular components and function in processes required for tumor cell proliferation and survival. Previously we showed that FASN inhibition results in tumor cell apoptosis in vitro and xenograft tumor growth inhibition in vivo. Our studies demonstrated that diverse tumor types exhibit sensitivity to FASN inhibition and characterized mechanisms of action that associate with the antitumor activity of highly selective small molecule FASN inhibitors. In vitro studies with diverse tumor cell types elucidated a mechanism of action that includes plasma membrane remodeling, signal transduction pathway inhibition, and gene expression reprogramming. TVB-2640, TVB-3166, and TVB-3664 belong to a series of orally available, reversible, potent, and selective FASN inhibitors discovered and developed by 3-V Biosciences. Analysis of gene expression data from tumor cell lines and human tumors, both primary and patient-derived xenografts, has allowed for the classification of FASN sensitivity by tumor type, histology, and molecular genetic markers. Discoveries from these analyses are being characterized further using in vitro and in vivo studies. Combined inhibition of FASN and microtubule function with taxane treatment, e.g. paclitaxel, results in synergistic inhibition of tumor growth. Indeed, in Phase I clinical investigation, TVB-2640 combined with paclitaxel has shown promising early signs of clinical activity. Previous in vitro studies revealed that FASN inhibition causes changes in beta-tubulin expression and disrupts the organization of cellular microtubule structures in varied tumor cell types such as CALU-6 non-small-cell lung and 22Rv1 prostate tumor cell lines. Extending our investigation of the mechanism of FASN/taxane synergy, we now show that FASN inhibition prevents beta-tubulin palmitoylation. This likely plays a significant role in the observed effects on beta-tubulin expression and microtubule architecture. As disruption of protein palmitoylation is believed to contribute significantly to the anti-tumor activity of FASN inhibition in general, we expanded the analysis of protein palmitoylation following inhibition of FASN with TVB-3166 and TVB-3664 to include key oncogenic drivers of cell growth, proliferation, and survival such as K-Ras and EGFR. Additionally, the efficacy of FASN inhibition in combination with additional, non-taxane approved cancer therapies, including immunomodulatory agents and bevacizumab, is being investigated. Citation Format: Timothy S. Heuer, Richard Ventura, Julie Lai, Joanna Waszczuk, Claudia Rubio, Glenn Hammonds, Marie O’ Farrell, Douglas Buckley, George Kemble. Preclinical studies characterize tumor type sensitivity to FASN inhibition and the mechanism and efficacy of novel drug combinations with TVB-2640. [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 4743.

Characterization of Beta Tubulin Expression During Human Foam Cell Formation and in Atherosclerotic Lesions of the Aorta

Foam cells arise when monocytes accumulate lipids and as a result get larger. It is assumed that the cytoskeleton compensates for this morphological change. Beta tubulin is essential to the formation of microtubules, which are an important component of the cytoskeleton. The purpose of this research was to elucidate the expression patterns of beta tubulin isotypes 1–4 in human monocytes during foam cell formation and in vivo in mouse atherosclerotic lesions to determine what relationship exists between beta tubulin expression and 1) a monocyte's ability to undergo foam cell formation or 2) lesion development in a mouse model of atherosclerosis. We hypothesized that the distribution of and amount of at least one beta tubulin isotype changes throughout the three stages of foam cell induction and within an atherosclerotic lesion, relative to a normal aorta. Further, we expect that monocytes with manipulated levels of a certain isotype(s) of beta tubulin will exhibit differences in the uptake of low‐density lipoprotein when compared to wild‐type human monocytes. Levels of beta tubulin 1–4 were measured by western blot and immunofluorescence throughout the stages of foam cell differentiation, and several beta tubulin isotypes were manipulated by siRNA to determine the effects of diminished beta tubulin expression on foam cell formation. Additionally, we performed immunohistochemistry, using the superior portion of the heart, and qPCR, using the abdominal aortic arch; both from ApoE−/− mice fed a high cholesterol diet to induce atherosclerotic lesions. In cultured monocytes, it was found that the distribution of beta tubulin 1, 3, and 4 changes throughout the stages of foam cell induction, and manipulation of beta tubulins altered foam cell formation. Furthermore, the silencing or decreasing of beta tubulin enhanced lipid aggregation. Based on our immunohistochemical analyses, the distribution of the beta‐tubulins appears to be unique and differs between wildtype mice without lesions and ApoE knockout mice with lesions. In particular, beta‐tubulin II was less abundant and diffuse, while beta‐tubulin III appeared more abundant but diffuse. Based on initial qPCR analyses, beta‐tubulin I and III expression was highest compared to wildtype mice. Based on the qPCR data, beta‐tubulin III has the highest rate of expression thus far; we intend to analyze expression of additional genes. This study could provide further knowledge on the development of foam cell formation and atherosclerotic lesions that may be influenced by beta‐tubulin, which may provide a possible target for treatment or prevention of atherosclerosis.Support or Funding InformationSupported by a grant to Veronica Contreras‐Shannon from the Semp Russ Foundation and Lila G. and Vesey F. Taylor Fund, San Antonio Area Foundation; a summer 2015 SURF to Andrea De Los Santos from the Undergraduate Research Office, St. Mary's University; and a summer 2014 SURF to Adriana Torres from the Undergraduate Research Office, St. Mary's University

Re-expression of LKB1 in LKB1-mutant EKVX cells leads to resistance to paclitaxel through the up-regulation of MDR1 expression.

The tumor suppressor LKB1 has recently been shown to be involved in the regulation of microtubule dynamics, thus cancer cells with inactivated LKB1 may have developed a means to overcome dysregulated microtubule functions, making them intrinsically resistant to microtubule targeting agents. Here, we generated isogenic LKB1-wild type and mutant non-small cell lung cancer (NSCLC) cell lines to evaluate the role of LKB1 in paclitaxel resistance. SRB, flow cytometry and immunoblotting were used to assess cell proliferation and apoptosis in NSCLC cell lines after paclitaxel treatment. Expression of LKB1 was restored in LKB1-null cells by retrovirus infection and was reduced in LKB1-wild type cells by shRNA knock down. The restoration of LKB1 in LKB1-null cells failed to promote paclitaxel-induced apoptosis in both p53-wild type and p53-mutant backgrounds, indicating that LKB1 was not required for paclitaxel-induced apoptosis. Interestingly, the re-establishment of LKB1 expression led to the up-regulation of class III beta-tubulin and MDR1 in EKVX cells. The up-regulation of MDR1 protein and transcripts in EKVX cells was specifically associated with the expression of wild-type LKB1 and mainly responsible for the increased cellular resistance to paclitaxel. However, the presence of LKB1 protein was not required to maintain this increased MDR1 expression even though there was no genetic amplification or promoter de-methylation of the ABCB1 locus in EKVX-LKB1-WT cells. These data suggest that LKB1 does not promote paclitaxel-induced apoptosis in most NSCLC cell lines. In contrast, in some NSCLC, the presence of LKB1 may facilitate increases in either MDR1 or class III beta-tubulin expression which can lead to paclitaxel resistance.

Class III beta-tubulin expression in uterine cancer correlates with worse outcomes

Objectives: Increased tumor expression of the class III beta-tubulin isotype (b3T), correlates with poor clinical outcomes in patients with various malignancies. Our objective was to determine the association between b3T expression and clinical outcomes in uterine cancer patients treated with chemotherapy.

Different protein expression associated with chemotherapy response in oropharyngeal cancer according to HPV status.

BackgoundOropharyngeal cancer (OPC) associated with human papilloma virus (HPV OPC) shows better treatment outcomes than non-HPV OPC. We investigated the expression of p53, β-tubulin, bcl-2 and ERCC 1, which are well-known biomarkers to predict the chemotherapy response, according to HPV status in OPC patients.MethodsPatients who treated with at least 2 cycles of induction chemotherapy followed by concurrent chemoradiotherapy for locally advanced oropharyngeal cancer were reviewed. HPV PCR and immunohistochemical stain was done in paraffin embedded tumor tissue and evaluated the relation with the chemotherapy response and survival outcomes according to HPV status.ResultsSeventy-four patients were enrolled for this study and all patients received induction chemotherapy with docetaxel, 5-FU and cisplatin. After induction chemotherapy, complete response (CR) was shown in 22 patients (30%) and partial response (PR) in 46 patients (62%). HPV + was detected in 21 patients (28%), while 35 patients (47%) showed p16+ expression by IHC analysis. p16 positive patients showed better overall response, PFS and OS than p16 negative patients. p53 and class III beta-tubulin expression were significantly higher in HPV- and p16- than HPV + and p16+ patients. Conversely, bcl-2 expression was greater in HPV + or p16+ than HPV- or p16- patients. ERCC1 expression did not differ significantly according to HPV status. In multivariate analyses, early T stage (p = 0.036) and good PS (PS 0) (p = 0.029) showed a better 3Y-PFS rate, and low p53 expression (p = 0.012) and complete response after induction chemotherapy (p = 0.026) were highly associated with 3Y-OS rate. Low expression of p53 and p16 positive patients showed significantly prolonged OS than others (p = 0.010).ConclusionP53, class III beta-tubulin and bcl-2 were differently expressed in OPC according to HPV status and present study suggested the underlying mechanism of better response to chemotherapy in case of HPV OPC than non-HPV OPC. Among these biomarkers, p53 is the strongest prognostic marker in OPC and p53 in addition to p16 support the rationale to study of de-escalation strategy for OPC.

Sigma-1 receptor stimulation protects retinal ganglion cells from ischemia-like insult through the activation of extracellular-signal-regulated kinases 1/2

Sigma-1 receptor (σ-1) activation and mitogen-activated protein kinases (MAPKs) have been shown to protect retinal ganglion cells (RGCs) from cell death. The purpose of this study was to determine if σ-1 receptor stimulation with pentazocine could promote neuroprotection under conditions of an ischemia-like insult (oxygen glucose deprivation (OGD)) through the phosphorylation of extracellular signal regulated kinase (pERK)1/2. Primary RGCs were isolated from P3–P7 Sprague–Dawley rats and purified by sequential immunopanning using Thy1.1 antibodies. RGCs were cultured for 7 days before subjecting the cells to an OGD insult (0.5% oxygen in glucose-free medium) for 6 h. During the OGD, RGCs were treated with pentazocine (σ-1 receptor agonist) with or without BD 1047 (σ-1 receptor antagonist). In other experiments, primary RGCs were treated with pentazocine in the presence or absence of an MEK1/2 inhibitor, PD098059. Cell survival/death was assessed by staining with the calcein-AM/ethidium homodimer reagent. Levels of pERK1/2, total ERK1/2, and beta tubulin expression were determined by immunoblotting and immunofluorescence staining. RGCs subjected to OGD for 6 h induced 50% cell death in primary RGCs (p < 0.001) and inhibited pERK1/2 expression by 65% (p < 0.001). Cell death was attenuated when RGCs were treated with pentazocine under OGD (p < 0.001) and pERK1/2 expression was increased by 1.6 fold (p < 0.05) compared to OGD treated RGCs without pentazocine treatment. The co-treatment of PD098059 (MEK1/2 inhibitor) with pentazocine significantly abolished the protective effects of pentazocine on the RGCs during this OGD insult. Activation of the σ-1 receptor is a neuroprotective target that can protect RGCs from an ischemia-like insult. These results also established a direct relationship between σ-1 receptor stimulation and the neuroprotective effects of the ERK1/2 pathway in purified RGCs subjected to OGD. These findings suggest that activation of the σ-1 receptor may be a therapeutic target for neuroprotection particularly relevant to ocular neurodegenerative diseases that effect RGCs.

Class III beta-tubulin, drug resistance and therapeutic approaches in cancers

Class III beta-tubulin is one of the critical proteins associated with microtubule assembly, important to many cellular functions including mitochondrial respiration and intracellular trafficking. Widely regarded as a specific neuronal marker in developmental neurobiology and stem cell research, it is also highly expressed in a wide range of tumors of both neuronal and non-neuronal origin. The expression of class III beta-tubulin is tightly controlled at multiple levels with tissue-dependent mechanisms of regulation. For instance, class III beta-tubulin expression is under the control of estrogens in breast cancer cells but is influenced by exposure to hypoxia and poor-nutrient supply in ovarian cancer. In some but not all cancers, class III beta-tubulin expression is purely a prognostic biomarker, predicting poor outcome of patients regardless of chemotherapy treatment. Moreover, the expression of class III beta-tubulin does not confer an aggressive phenotype by itself. Instead, class III beta-tubulin functions like a cytoskeletal gateway, which enhances the incorporation of pro-survival kinases into the cytoskeleton and protects them from degradation. The associations of class III beta-tubulin with survival kinase PIM-1, RNA-binding protein HuR, microRNAs are examples highlighting the functional complexity of this protein. The utility of class III beta-tubulin as a prognostic biomarker can also greatly improve if combined with these pro-survival partners. Subsequently, pharmacogenetic approaches, designed to counteract and target these pathways and associated-factors concurrently, might lead to better therapies and prognostic tools for class III beta-tubulin expressing cancers.

The Expression of P53, &amp;Bgr;-Tubulin, Bcl-2 According to P16 Status in Advanced Oropharyngeal Cancer After Induction Chemotherapy Followed By Concurrent Chemoradiotherapy

ABSTRACT Aim: Oropharyngeal cancer (OPC) associated with human papilloma virus (HPV) shows better treatment outcomes than non-HPV OPC. We investigated the expression of p53, b-tubulin, bcl-2 and ERCC 1 according to p16 status in OPC patients receiving induction chemotherapy followed by concurrent chemoradiotherapy. Methods: Patients who were treated with at least 2 cycles of induction chemotherapy for locally advanced oropharyngeal cancer from 2004 to 2011 were reviewed. Immunohistochemical staining of p53, b-tubulin, bcl-2, ERCC, which are well known to be associated with chemotherapy response, and p16, as a surrogate marker for HPV, was done in paraffin-embedded tumor tissue. We analyzed the response for chemotherapy and survival outcomes according to p16 expression. Results: Seventy-four patients were enrolled for this study. All patients received induction chemotherapy with docetaxel, 5-FU, cisplatin (n = 72) or 5-FU and cisplatin (n = 2). One patient received a salvage operation and eight patients were not given radiotherapy due to poor general condition after induction chemotherapy. Therefore, 65 patients received chemoradiotherapy subsequently (total radiation dose; ≥60Gy n = 54, Conclusions: OPC associated with HPV (HPV OPC) showed lower expression of p53, beta tubulin and higher expression of bcl-2 than non-HPV OPC. In addition, the expressions of p53 and beta tubulin were prognostic factors for OPC. This result suggested the underlying mechanism of better response to chemotherapy in case of HPV OPC than non-HPV OPC and de-escalation of treatment in patients with HPV OPC could be considered based on clinical trial results. Disclosure: All authors have declared no conflicts of interest.

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.

Epithelial-to-Mesenchymal Transition Leads to Docetaxel Resistance in Prostate Cancer and Is Mediated by Reduced Expression of miR-200c and miR-205

Docetaxel is a standard chemotherapy for patients with metastatic prostate cancer. However, the response is rather limited and not all of the patients benefit from this treatment. To uncover key mechanisms of docetaxel insensitivity in prostate cancer, we have established docetaxel-resistant sublines. In this study, we report that docetaxel-resistant cells underwent an epithelial-to-mesenchymal transition during the selection process, leading to diminished E-cadherin levels and up-regulation of mesenchymal markers. Screening for key regulators of an epithelial phenotype revealed a significantly reduced expression of microRNA (miR)-200c and miR-205 in docetaxel-resistant cells. Transfection of either microRNA (miRNA) resulted in re-expression of E-cadherin. Functional assays confirmed reduced adhesive and increased invasive and migratory abilities. Furthermore, we detected an increased subpopulation with stem cell-like properties in resistant cells. Tissue microarray analysis revealed a reduced E-cadherin expression in tumors after neoadjuvant chemotherapy. Low E-cadherin levels could be linked to tumor relapse. The present study uncovers epithelial-to-mesenchymal transition as a hallmark of docetaxel resistance. Therefore, we suggest that this mechanism is at least in part responsible for chemotherapy failure, with implications for the development of novel therapeutics.

Cross-validation study of class III beta-tubulin as a predictive marker for benefit from adjuvant chemotherapy in resected non-small-cell lung cancer: analysis of four randomized trials

The IALT, JBR.10, ANITA and Cancer and Leukemia Group B 9633 trials compared adjuvant chemotherapy with observation for patients with resected non-small-cell lung cancer (R-NSCLC). Data from the metastatic setting suggest high tumor class III beta-tubulin (TUBB3) expression is a determinant of insensitivity to tubulin-targeting agents (e.g. vinorelbine, paclitaxel). In 265 patients from JBR.10 (vinorelbine-cisplatin versus observation), high TUBB3 was an adverse prognostic factor and was associated (nonsignificantly) with 'greater' survival benefit from chemotherapy. We explored this further in additional patients from JBR.10 and the other three trials. TUBB3 immunohistochemical staining was scored for 1149 patients on the four trials. The original JBR.10 cut-off scores were used to classify tumors as TUBB3 high or low. The prognostic and predictive value of TUBB3 on disease-free survival (DFS) and overall survival (OS) was assessed by Cox models stratified by trial and adjusted for clinical factors. High TUBB3 expression was prognostic for OS [hazard ratio (HR)=1.27 (1.07-1.51), P=0.008) and DFS [HR=1.30 (1.11-1.53), P=0.001). TUBB3 was not predictive of a differential treatment effect [interaction P=0.20 (OS), P=0.23 (DFS)]. Subset analysis (n=420) on vinorelbine-cisplatin gave similar results. The prognostic effect of high TUBB3 expression in patients with R-NSCLC has been validated. We were unable to confirm a predictive effect for TUBB3.

Abstract B100: Preclinical evaluation of a novel HSP90 inhibitor HSP990 in glioblastoma multiforme.

Abstract Heat shock protein (HSP) 90 is a molecular chaperone that modulates stability of several client proteins, many of which are key regulators of signaling pathways in glioblastoma multiforme (GBM). The potential for targeting multiple effectors makes HSP90 an appealing therapeutic target for GBM. The biological activity of HSP990, a novel HSP90 inhibitor (Novartis Pharmaceuticals) as single agent was evaluated in a panel of short-term explant cultures derived from primary GBM xenografts (n=10). HSP990 treatment resulted in a dose-dependent growth inhibition in the all xenograft lines, with an IC50 ranging from 5 to 30 nM. A 72 h exposure to HSP990 (10–30 nM) resulted in a dose-dependent decrease in the HSP90 client proteins Akt, Cdc2 and Cdc25C, a decrease in associated post-translational modifications p-Akt (S473), p-CDC2 (Y15), p-CDC25 (S216), and increased expression of HSP70. Additionally, a dose-dependent increase in HSP990-induced apoptosis (Annexin-V positivity), was observed within 24–72 hours of drug treatment. GBM tumors harbor a subpopulation of glioma stem cells that grow as neurospheres in neurobasal media and are considered important for tumor maintenance and progression. The effects of HSP990 were tested in neurospheres derived from 4 of the xenografts tested above (GBM12, GBM39, GB59, GBM12TMZ). These cultures demonstrated potential for tri-lineage differentiation upon serum challenge and had robust secondary neurosphere formation, demonstrating two hallmark features of cancer stem cells. In a bulk-culture neurosphere formation assay, co-incubation with 5 to 10 nM HSP990 was highly effective at suppressing neurosphere formation in all 4 xenograft lines. Notably, in GBM12 neurosphere cultures maintained in either serum-free media or challenged with serum, 30 nM HSP990 was equally effective at promoting up-regulation of HSP70, but exhibited less effect on Akt or Cdc2 in serum-free cultures as compared to cultures supplemented with serum. Incubation of serum-free neurosphere cultures with 5 nM HSP990 (5 days) resulted in increased glioneuronal differentiation exhibiting elevated GFAP, Oct-4 and beta-tubulin expression compared to untreated controls. To test ‘self renewal’ GBM12 neurospheres were treated with HSP990 (10, 30 nM) for 7 days followed by dissociation and plating equal numbers of viable cells at low density in fresh drug-free neurobasal media. Interestingly, HSP990 treatment did not affect the number of secondary neurospheres (surviving fraction: control = 1.00, 10 nM = 1.30, 30 nM = 1.05). Treatment of GBM12TMZ flank tumor xenografts with weekly HSP990 (13 mg/kg P.O.) resulted in a modest 9 day delay in tumor re-growth (40.3 and 49.3 respectively) compared to placebo treatment. In conclusion, HSP990 (a) had anti-proliferative, pro-apoptotic effect on differentiated serum-supplemented xenograft cultures (b) a pro-differentiation effect on serum-free GBM stem cell cultures (c) markedly suppressed neurosphere formation with prolonged drug exposure but not with more limited drug exposure (d) inhibited GBM growth in vivo. Thus, HSP990 may inhibit GBM growth both through direct growth inhibition and through pro-differentiation effects on GBM stem-like cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B100.

Postmortem proteomic analysis in human amygdala of drug addicts: possible impact of tubulin on drug-abusing behavior

Besides the ventral tegmental area and the nucleus accumbens as the most investigated brain reward structures, several reports about the relation between volume and activity of the amygdala and drug-seeking behavior have emphasized the central role of the amygdala in the etiology of addiction. Considering its proposed important role and the limited number of human protein expression studies with amygdala in drug addiction, we performed a human postmortem proteomic analysis of amygdala tissue obtained from 8 opiate addicts and 7 control individuals. Results were validated by Western blot in an independent postmortem replication sample from 12 opiate addicts compared to 12 controls and 12 suicide victims, as a second "control sample". Applying 2D-electrophoresis and MALDI-TOF-MS analysis, we detected alterations of beta-tubulin expression and decreased levels of the heat-shock protein HSP60 in drug addicts. Western blot analysis in the additional sample demonstrated significantly increased alpha- and beta-tubulin concentrations in the amygdala of drug abusers versus controls (P = 0.021, 0.029) and to suicide victims (P = 0.006, 0.002). Our results suggest that cytoskeletal alterations in the amygdala determined by tubulin seem to be involved in the pathophysiology of drug addiction, probably via a relation to neurotransmission and cellular signaling. Moreover, the loss of neuroprotection against stressors by chaperons as HSP60 might also contribute to structural alteration in the brain of drug addicts. Although further studies have to confirm our results, this might be a possible pathway that may increase our understanding of drug addiction.