High TUBB3 Expression Research Articles

Human Metastatic Melanoma Cell Lines Panel for In Vitro and In Vivo Investigations

The melanoma origin of cell lines obtained from the axillary lymph node (mel Kas, mel Pet, and mel Lap from patients with a verified diagnosis) was confirmed by the detection of the Melan A melanocyte marker expression. A hyperdiploid (2n+) for the mel Kas line; near-diploid (2n), and in some cells near-tertaploid (4n), and even hypo-octaploid (8n) set (172–179 chromosomes) in the mel Pet cell line; and a hypotetraploid (4n−) for the mel Lap line were detected by karyotypic analysis. All three cell lines are tumorigenic; however, mel Pet demonstrates tumor growth in Balb/c nude mice only in the presence of matrigel. All three lines showed a high expression of TUBB3 and PD-L1 markers, while ERa was low (minimum for mel Pet). Significant differences in the expression level were shown for the Cyt molecular marker. In the transplantation of cells to Balb/c nude mice, a stable expression level is observed only for TUBB3. For the rest of the markers, a decrease in their expression level of varying degrees was noted when the cells were growing in solid tumors in vivo. Mutations were detected in oncogenes (BRAF, EZH2, KIT, KRAS, NRAS, ROS1) and tumor suppressor genes (CDKN2A, FAT4, KMT2C, LRP1B, PTEN, PTPRB, TP53). The detailed characterization of the cell lines makes them valuable for various scientific and regulatory experiments, particularly those involving preclinical data on antiproliferative drugs for malignant melanoma or investigations into melanoma cell properties and progression.

Predictive and Prognostic Value of TUBB3, RRM1, APE1, and Survivin Expression in Chemotherapy-Receiving Patients with Advanced Non‑Small Cell Lung Cancer.

This study aimed to evaluate the expression of class III β-tubulin (TUBB3), ribonucleoside-diphosphate reductase 1 (RRM1), apurinic/apyrimidinic endonuclease 1 (APE1), and survivin in patients with advanced non-small cell lung cancer (NSCLC) to predict response to chemotherapy. TUBB3, RRM1, APE1, and survivin expression levels were determined using immunohistochemistry. Protein expression was validated in Car/Pac-resistant human H1792 and A549 cells. This study included 86 patients, among whom 34 received cisplatin (Cis)/gemcitabine (Gem) and 52 received carboplatin (Car)/paclitaxel (Pac). Patients with low TUBB3 expression and high RRM1 and survivin expression had higher response rates than those with low RRM1 and survivin expression and high TUBB3 expression in the Car/Pac regimen. The multivariate analysis indicated that TUBB3 and RRM1 were significant independent predictive biomarkers for the Car/Pac regimen; however, there was no association between any protein and overall response in patients treated with this regimen. In the Cis/Gem regimen, only high TUBB3 expression was associated with poor overall survival; however, it did not exhibit a prognostic ability. The expression levels of TUBB3 and RRM1 in NSCLC cells are potential predictive biomarkers, but not prognostic factors, of response to chemotherapy in patients with NSCLC receiving the Car/Pac regimen.

Targeting hormone-resistant breast cancer cells with docetaxel: a look inside the resistance.

Aim: The study aims to analyze the effect of long-term incubation of ERα-positive MCF7 breast cancer cells with 4-hydroxytamoxifen (HT) on their sensitivity to tubulin polymerization inhibitor docetaxel. Methods: The analysis of cell viability was performed by the MTT method. The expression of signaling proteins was analyzed by immunoblotting and flow cytometry. ERα activity was evaluated by gene reporter assay. To establish hormone-resistant subline MCF7, breast cancer cells were treated with 4-hydroxytamoxifen for 12 months. Results: The developed MCF7/HT subline has lost sensitivity to 4-hydroxytamoxifen, and the resistance index was 2. Increased Akt activity (2.2-fold) and decreased ERα expression (1.5-fold) were revealed in MCF7/HT cells. The activity of the estrogen receptor α was reduced (1.5-fold) in MCF7/HT. Evaluation of class III β-tubulin expression (TUBB3), a marker associated with metastasis, revealed the following trends: higher expression of TUBB3 was detected in triple-negative breast cancer MDA-MB-231 cells compared to hormone-responsive MCF7 cells (P < 0.05). The lowest expression of TUBB3 was found in hormone-resistant MCF7/HT cells (MCF7/HT < MCF7 < MDA-MB-231, approximately 1:2:4). High TUBB3 expression strongly correlated with docetaxel resistance: IC50 value of docetaxel for MDA-MB-231 cells was greater than that for MCF7 cells, whereas resistant MCF7/HT cells were the most sensitive to the drug. The accumulation of cleaved PARP (a 1.6-fold increase) and Bcl-2 downregulation (1.8-fold) were more pronounced in docetaxel-treated resistant cells (P < 0.05). The expression of cyclin D1 decreased (2.8-fold) only in resistant cells after 4 nM docetaxel treatment, while this marker was unchanged in parental MCF7 breast cancer cells. Conclusion: Further development of taxane-based chemotherapy for hormone-resistant cancer looks highly promising, especially for cancers with low TUBB3 expression.

Class III β-Tubulin Expression Is of Value in Selecting nab -Paclitaxel and Gemcitabine as First-Line Therapy in Unresectable Pancreatic Cancer.

Nab -paclitaxel and gemcitabine (GnP) or FOLFIRINOX (a combination of leucovorin, fluorouracil, irinotecan, and oxaliplatin [FFX]) is currently recognized as the standard first-line regimen for unresectable pancreatic ductal adenocarcinoma (PDAC). Class III β-tubulin (TUBB3) has the potential to predict resistance to taxane in various tumors; therefore, this study aimed to clarify whether TUBB3 is a predictive marker for GnP response. We retrospectively reviewed 113 patients with PDAC who received GnP or FFX as first-line chemotherapy and examined immunohistochemically the TUBB3 expression in specimens obtained by endoscopic ultrasound-guided fine-needle aspiration. High TUBB3 expression was associated with a significantly lower disease control rate ( P = 0.017) and shorter progression-free survival (PFS) ( P = 0.019), and multivariate analysis revealed that TUBB3 expression was an independent variable for PFS in the GnP first-line group ( P = 0.045). In addition, in the FFX first-line group, TUBB3 expression was not correlated with PFS or overall survival (OS). In all 113 patients, TUBB3 expression was not also associated with OS. Class III β-tubulin might be a predictive factor for the response of GnP, but not a prognostic factor for OS, helping the selection of an optimized first-line chemotherapy regimen for unresectable PDAC.

Expression of Class III Beta-Tubulin Is Associated with Invasive Potential and Poor Prognosis in Thyroid Carcinoma.

Although American Thyroid Association guidelines offer a risk stratification scheme for thyroid cancer patients, there is a continuous need for more sophisticated biomarkers that can predict disease progression. In this study, we aim to evaluate the prognostic value of class III beta-tubulin (TUBB3) and uncover the relationship between TUBB3 and invasive potential in thyroid carcinoma. Immunohistochemistry (IHC) for TUBB3 and E-cadherin was performed on a total of 254 cases of thyroid cancer specimens. Tumor budding at the invasive margin was evaluated. In vitro functional studies were also performed; the protein and mRNA levels of TUBB3 were compared among the five cell types at baseline, with transwell invasion and after blocking of TUBB3 by shRNA. IHC revealed that the levels of TUBB3 were higher in conventional papillary carcinomas (cPTCs) and anaplastic thyroid carcinomas (ATCs). In univariate analysis, high tumor budding and TUBB3 expression were associated with inferior progression-free survival in cPTC. The results of a Western blot and RT-PCR agreed with the IHC finding. The results were further validated through data from The Cancer Genome Atlas database. Our results suggest that high expression of TUBB3 in thyroid carcinoma could predict invasive potential and possibly be linked with epithelial–mesenchymal transition.

TUBB3 Is Associated with High-Grade Histology, Poor Prognosis, p53 Expression, and Cancer Stem Cell Markers in Clear Cell Renal Cell Carcinoma

Background: βIII-Tubulin, encoded by the TUBB3 gene, is a microtubule protein. Several studies have shown that overexpression of TUBB3 is linked to poor prognosis and is involved in taxane resistance in some cancers. Objective: The aim of this study was to analyze the expression and function of TUBB3 in clear cell renal cell carcinoma (ccRCC). Methods: The expression of TUBB3 was determined using immuno­histochemistry in ccRCC specimens. The effects of TUBB3 knockdown on cell growth and invasion were evaluated in RCC cell lines. We analyzed the interaction between TUBB3, p53, cancer stem cell markers, and PD-L1. Results: In 137 cases of ccRCC, immunohistochemistry showed that 28 (20%) of the ccRCC cases were positive for TUBB3. High TUBB3 expression was significantly correlated with high nuclear grade, high T stage, and N stage. A Kaplan-Meier analysis showed that high expression of TUBB3 was associated with poor overall survival after nephrectomy. In silico analysis also showed that high TUBB3 expression was correlated with overall survival. Knockdown of TUBB3 suppressed cell growth and invasion in 786-O and Caki-1 cells. High TUBB3 expression was associated with CD44, CD133, PD-L1, and p53 in ccRCC. We generated p53 knockout cells using the CRISPR-Cas9 system. Western blotting revealed that p53 knockout upregulated the expression of TUBB3. Conclusion: These results suggest that TUBB3 may play an oncogenic role and could be a potential therapeutic target in ccRCC.

Bcl-2/Bcl-xL inhibitor ABT-737 sensitizes pancreatic ductal adenocarcinoma to paclitaxel-induced cell death.

Pancreatic ductal adenocarcinoma (PDA) is an aggressive malignant disease that is resistant to various chemotherapeutic agents and commonly relapses. Efficient elimination of metastasized PDA is critical for a positive post-surgical treatment outcome. The present study analyzed the effect of the B-cell lymphoma-2 (Bcl-2)/B-cell lymphoma extra-large (Bcl-xL) inhibitor, ABT-737, on paclitaxel-induced PDA cell death. A total of 8 PDA cell lines were subjected to immunoblotting to compare the expression of Bcl-2/Bcl-xL and other factors associated with taxane resistance, including myeloid cell leukemia 1 and βIII-tubulin (TUBB3). The viability of PDA cells was analyzed following treatment with paclitaxel alone or a combination treatment with ABT-737 and paclitaxel. Treatment with the ABT-737/paclitaxel combination induced PDA cell death at a lower concentration of paclitaxel compared with paclitaxel alone. In addition, the viable cell population at the saturation point of paclitaxel was also decreased by co-treatment with ABT-737. ABT-737 lowered the half maximal inhibitory concentration (IC50) by >2-fold in PDA cells with high Bcl-2/Bcl-xL expression, but not in PDA cells with low Bcl-2/Bcl-xL expression and high TUBB3 expression. Knockdown of Bcl-xL lowered the IC50 of paclitaxel, but knockdown of TUBB3 did not. ABT-737 sensitized PDA to paclitaxel-induced cell death, and Bcl-xL expression was a key determinant of its sensitivity. ABT-737 is potential candidate for combination chemotherapy of PDA with high Bcl-xL expression levels.

MP88-02 EXPRESSION OF CLASS III BETA-TUBULIN PREDICTS PROGNOSIS IN CISPLATIN-RESISTANT BLADDER CANCER PATIENTS RECEIVING PACLITAXEL-BASED SECOND-LINE CHEMOTHERAPY

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology IV1 Apr 2017MP88-02 EXPRESSION OF CLASS III BETA-TUBULIN PREDICTS PROGNOSIS IN CISPLATIN-RESISTANT BLADDER CANCER PATIENTS RECEIVING PACLITAXEL-BASED SECOND-LINE CHEMOTHERAPY Tomohiro Matsuo, Yasuyoshi Miyata, Yuichiro Nakamura, Takuji Yasuda, Kojiro Ohba, and Hideki Sakai Tomohiro MatsuoTomohiro Matsuo More articles by this author , Yasuyoshi MiyataYasuyoshi Miyata More articles by this author , Yuichiro NakamuraYuichiro Nakamura More articles by this author , Takuji YasudaTakuji Yasuda More articles by this author , Kojiro OhbaKojiro Ohba More articles by this author , and Hideki SakaiHideki Sakai More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2727AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Class III beta-tubulin (TUBB3) is associated with malignant aggressiveness and prognosis, in addition, its expression is recognized as a predictive marker for taxane-based chemotherapy (CTx) in several cancers. In urothelial cancer (UC), standard first-line therapy for advanced disease is cisplatin (CDDP)-based CTx. On the other hand, although second-line regimen for CDDP-resistant tumor is not still established, several studies showed paclitaxel (PTX)-based regimens were effective to improve the prognosis. The main aim is to clarify the predictive value of TUBB3 expression for anti-cancer effect by first-line CDDP-based CTx and second-line PTX-based one in patients with UC. METHODS We reviewed 116 UC (bladder cancer = 90 and upper urinary tract cancer = 26) patients treated with CDDP-based regimen as first-line CTx. Among these patients, 53 patients were received PTX-based second-line CTx. As PTX-based CTx, the combination of gemcitabine and PTX were performed in 42 patients. TUBB3 expression was evaluated by immunohistochemical technique, and survival analyses were performed by using Kaplan-Meier survival curves and multivariate COX proportional hazard analysis. RESULTS Positively stained ratio of TUBB3 in grade 3 tumors (50 / 74 = 67.6%) was significantly higher (P < 0.001) than that in grade 1 (0 / 5 = 0.0%) and grade 2 (14 / 37 = 37.8%). A similar trend was found in T stage; however, it did not reach the significant level (P = 0.062). No significant relationship was found in age, sex, and metastasis. When the predictive value of TUBB3 expression for CDDP-based first-line CTx was investigated, it is not recognized as a significant predictive factor for progression-free survival (P = 0.796). On the other hand, high expression of TUBB3 is significantly associated with unfavorable overall survival from starting of second-line CTx (P = 0.013). Multivariate analysis model including all pathological features, part of a tumor, and regimen of second-line therapy showed that TUBB3 expression was identified as an independent predictor (hazard ratio = 5.18, 95% confidential intervals = 1.85 - 14.53, P = 0.002). CONCLUSIONS The TUBB3 expression is associated with malignant potential in UC. TUBB3 expression was identified as a useful predictive factor for anti-cancer effects of second-line PTX-based CTx in advanced UC patients with CDDP-resistant tumors. Out information is useful to discuss treatment strategies for patients with CDDP-resistant UC. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1174-e1175 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Tomohiro Matsuo More articles by this author Yasuyoshi Miyata More articles by this author Yuichiro Nakamura More articles by this author Takuji Yasuda More articles by this author Kojiro Ohba More articles by this author Hideki Sakai More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Quantitative proteomic analysis of TUBB3 to identify gastric cancer patients who may benefit from docetaxel: A reevaluation of the ITACA-S trial.

59 Background: Chemotherapy (CTX) becomes targeted therapy when biomarkers can predict a patient’s response. A relationship between resistance to taxanes and overexpression of class III b-tubulin (TUBB3) has been suggested by small clinical studies, but not confirmed in randomized trials. The Intergroup Trial of Adjuvant CTX in Adenocarcinoma of the Stomach (ITACA-S) evaluated the survival advantage of postoperative sequential CTX with FOLFIRI followed by docetaxel plus cisplatin in comparison to monotherapy with 5-FU/LV in patients with radically resected gastric cancer (N = 1106). The results showed no difference in survival between the two CTX arms. In a random subset of patients (N = 247) from the ITACA-S trial, we applied mass spectrometry-based proteomics to assess the role of TUBB3 as a predictive biomarker for response to taxane-containing therapy. Methods: Archived tumor tissues were microdissected and solubilized for proteomic analysis. TUBB3 and 44 other protein biomarkers were quantified using a mass spectrometry-based selected reaction monitoring assay. A predetermined TUBB3 cutoff of 700 amol/µg was based on the assay’s limit of detection. The Mantel-Cox log-rank test was used for survival comparisons. Results: Among patients treated with taxane-containing CTX (n = 125), those with TUBB3 levels below the cutoff had nearly twice the median overall survival (OS) as patients with TUBB3 levels above the cutoff (1566 vs. 801 days, p = 0.0282). TUBB3 levels made no statistical difference in survival among patients who did not receive taxane. Of note, among patients with high TUBB3 levels ( &gt; 700 amol), those treated without taxane-containing CTX survived far longer than patients in the taxane arm (OS = 1991 vs 801 days, p = 0.048). Conclusions: Quantitative proteomic analysis of TUBB3 expression identified a subset of gastric cancer patients who benefitted from the addition of docetaxel to adjuvant CTX. Patients with high TUBB3 expression levels had worse outcomes on a taxane-containing regimen than on CTX without taxane. Personalized CTX based on the TUBB3 biomarker is promising and warrants further validation. Clinical trial information: NCT01989858.

Abstract 4923: Expression of class III beta-tubulin (TUBB3) in 3580 colorectal cancers (CRCs) and correlation with clinico-pathological and molecular features

Abstract Class III beta-tubulin plays a crucial role in intracellular transport, meiosis and mitosis. High expression of TUBB3 has been shown to associate with poor prognosis and taxane resistance in various cancer types. CRC is known to be generally resistant to taxane therapy. We investigated expression of TUBB3 in 3580 CRCs and made correlations with clinicopathological and molecular parameters. 3580 CRC samples were evaluated by tumor profiling (Caris Life Sciences, Phoenix, AZ). Tests included Sanger or next generation sequencing (NGS), protein expression by immunohistochemistry (IHC) and gene amplification by in situ hybridization (ISH). TUBB3 expression was evaluated by immunohistochemistry (Ab: POLY, Covance) and expression higher than 2+, 30% was scored as positive. TUBB3 positive expression was observed in 37% (1320/3580) of the complete CRC cohort, specifically 27% in mucinous histology (164/617) and 17% in signet ring histology (29/171). While the expression was not associated with average patient age (59 years old) or gender (53% vs. 50% male), TUBB3 was more frequently expressed in tumors that originated from the left colon (370/1016 or 36%) than from the right colon (235/790 or 30%, p = 0.003). In the 1847 tumors taken from metastatic sites, 40% (746) overexpressed TUBB3 while in 1629 CRCs taken from primary tumors, only 34% (547) overexpressed TUBB3 (p&amp;lt;0.0001). Interestingly, in tumors that overexpressed TUBB3, 65% also overexpressed cMET (828/1275) and 33% also overexpressed TLE3 (431/1299). This compares to 50% cMET expression (1096/2207, p&amp;lt;0.0001) and 23% TLE3 expression (517/2225, p&amp;lt;0.0001) in tumors that were negative for TUBB3. Microsatellite instability detected by fragment analysis was more prevalent in the TUBB3-negative cohort than the TUBB3-positive cohort (7.4% or 77/1046 vs. 2.9% or 16/558, p = 0.0002). Mutations in APC (62% vs. 56%, p = 0.0003) and KRAS (55% vs. 46%, p&amp;lt;0.0001) were more frequent in TUBB3-positive tumors, while GNAS (2% vs. 5%, p&amp;lt;0.0001) and SMAD4 (10.1% vs. 14.6%, p = 0.0005) mutations were significantly more frequent in tumors that were negative for TUBB3 expression. Thus, high expression of TUBB3 was found in 37% of CRCs, and was significantly associated with tumors that originated from the left colon and with tumors taken from metastatic sites. Distinct biomarker features detected by IHC and sequencing suggest that TUBB3 expression may carry theranostic importance which warrants further investigation in clinical trials. Citation Format: Joanne Xiu, Sandeep Reddy, Wafik S. El-Deiry. Expression of class III beta-tubulin (TUBB3) in 3580 colorectal cancers (CRCs) and correlation with clinico-pathological and molecular features. [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 4923.

Decreased expression of class III β-tubulin is associated with unfavourable prognosis in patients with malignant melanoma

Class III β-tubulin (TUBB3) has been recognized as being associated with resistance to taxane-based regimens in several cancers. However, little is known about the clinicopathological significance of TUBB3 expression in patients with cutaneous malignant melanoma. The aim of this study was to examine the prognostic significance of TUBB3 expression in cutaneous malignant melanoma. A total of 106 patients with surgically resected cutaneous malignant melanoma were assessed. Tumour sections were immunohistochemically stained for TUBB3, Ki-67 and microvessel density with CD34. TUBB3 was highly expressed in 80% (85/106) of patients. No statistically significant relationship was observed between the high expression of TUBB3 and any variables. On univariate analysis, ulceration, disease stage, TUBB3 and CD34 revealed a significant relationship with overall survival and progression-free survival. Multivariate analysis confirmed that a low TUBB3 expression was an independent prognostic factor for poor prognosis of cutaneous malignant melanoma. The decreased expression of TUBB3 could be a significant marker for predicting unfavourable prognosis in patients with cutaneous malignant melanoma.

High expression of class III β-tubulin in small cell lung carcinoma

Class III β-tubulin (TUBB3) is emerging as a biomarker in a number of cancers. TUBB3 has been shown to be a prognostic indicator of more aggressive disease and a predictor of resistance to taxanes and vinca alkaloids. To date, there is little data on TUBB3 expression in small cell lung carcinoma (SCLC). The primary objective of this study was to determine the expression of TUBB3 in SCLC. Immunohistochemical staining of SCLC tumor specimens was performed using standard procedures. Expression of TUBB3 was determined as a composite of the percentage of malignant cells staining positive and the intensity of staining. Clinical and tumor data for each patient was compared with the degree of TUBB3 expression. A total of 66 SCLCs were evaluable for TUBB3 expression. The majority of specimens (n=56, 85%) had high expression of TUBB3. Only 4.5% (n=3) had low expression of TUBB3. The mean distribution of positive staining for the specimens was 87.3±1.8% (mean ± SE). Specimens from core biopsies were significantly more likely to have high TUBB3 expression when compared with fine needle aspirates (P=0.004). There were no other significant findings when comparing clinical or tumor characteristics. Overall, we found that expression of TUBB3 in SCLC is higher than expected. Innate resistance to microtubule inhibitors, such as the taxanes and vinca alkaloids, may be associated with this finding. Attempts at microtubule inhibition with novel agents may be able to overcome this resistance mechanism. Further evaluation of TUBB3 as a biomarker in SCLC is warranted.

Longitudinal assessment of TUBB3 expression in non-small cell lung cancer patients

Class-III-beta-tubulin (TUBB3) expression may be a potential predictive factor for treatment with microtubule interfering cytotoxic drugs in non-small cell lung cancer (NSCLC). Potential changes in TUBB3 expression during chemotherapy may be of interest if future choice of chemotherapy is to be based on TUBB3 expression. If the biomarker expression changes during chemotherapy, biopsies before initiation of chemotherapy beyond first line may be needed if treatment decision is to be based on TUBB3 expression. Thus, the aim was to explore TUBB3 expression heterogeneity and changes during chemotherapy. TUBB3 expression was investigated by immunohistochemistry performed on diagnostic biopsies and on available subsequent resection specimens in 65 NSCLC patients stage T1-3N0-2 who received neoadjuvant carboplatin and paclitaxel (NAC-group). Another group of 53 NSCLC patients stage T1-4N0-1 was treated with surgery alone without preceding chemotherapy (OP-group). Paired repeated samples were compared in order to evaluate for changes in TUBB3 expression. No statistically significant change in TUBB3 expression was observed between initial diagnostic biopsies and subsequent surgical resections of primary tumors in either the OP-group (p = 0.124) or the NAC-group (p = 0.414). When dichotomized into high and low TUBB3 expression, discordance between diagnostic biopsies and resection specimens of the primary tumors occurred in 22 % and 40 % in the OP-group and NAC-group, respectively (p = 0.169). Significantly more patients having low TUBB3 expression experienced down-staging during neoadjuvant chemotherapy compared to patients having high TUBB3 expression (p = 0.022). A high degree of discordance of TUBB3 expression between paired repeated tumor samples was observed, which likely reflects intratumoral heterogeneity. This emphasizes a need for optimal tumor tissue samples in order to stratify patients based on TUBB3 expression. No significant changes in TUBB3 expression after neoadjuvant carboplatin and paclitaxel chemotherapy occurred, suggesting no need for rebiopsy in case second-line chemotherapy with microtubule interfering cytotoxic treatments is necessary.

Correlation between expression of TUBB3/STMN1 and EGFR signaling pathway in non-small cell lung cancer

背景与目的已有研究表明分子标志物在指导肺癌个体化治疗中起着重要作用。本研究旨在探讨非小细胞肺癌（non-small cell lung cancer, NSCLC）抗微管类药物靶点TUBB3（tubulin, beta 3 class Ⅲ)/STMN1（stathmin 1）基因表达水平与EGFR（epidermal growth factor receptor）、KRAS（Kirsten rat sarcoma viral oncogene homolog）、BRAF（v-raf murine sarcoma viral oncogene homolog B）、PI3K（phosphatidylinositol-4, 5-bisphosphate 3-kinase）基因突变的相关性，为NSCLC预后及药物疗效判断提供参考依据。方法回顾性分析我院经病理确诊的46例NSCLC患者手术切除的肿瘤标本，通过分支DNA-液相芯片法检测TUBB3、STMN1基因mRNA表达水平，通过xTAG液相芯片法检测EGFR、KRAS、BRAF、PI3K基因突变，对检测结果应用SPSS 19.0软件采用Spearman相关进行分析基因表达与基因突变的关系。结果抗微管靶点TUBB3和STMN1存在很强的共表达性，TUBB3基因高表达时，STMN1趋向于高表达（P=0.006）。EGFR E21突变与TUBB3存在负相关关系：EGFR E21无突变时TUBB3趋向于高表达（P=0.004, 6）。KRAS E2突变与STMN1存在正相关关系：KRAS E2突变时STMN1趋向于高表达（P=0.038, 6）。结论抗微管耐药相关因子TUBB3和STMN1与EGFR通路的关键因子突变相关，提示了EGFR突变和KRAS突变是调控TUBB3/STMN1表达的重要因素，为研究化疗药物耐药性提供了重要的参考因素。

Effect of TUBB3, TS and ERCC1 mRNA expression on chemoresponse and clinical outcome of advanced gastric cancer by multiplex branched-DNA liquid chip technology

To analyze the impact of β-tubulin-III (TUBB3), thymidylate synthase (TS) and excision repair cross complementation group 1 (ERCC1) mRNA expression on chemoresponse and clinical outcome of patients with advanced gastric cancer treated with TXT/CDDP/FU (DCF) regimen chemotherapy. The study population consisted of 48 patients with advanced gastric cancer. All patients were treated with DCF regimen palliative chemotherapy. The mRNA expressions of TUBB3, TS and ERCC1 of primary tumors were examined by multiplex branched-DNA liquid chip technology. The patients with low TUBB3 mRNA expression had higher response rate to chemotherapy than patients with high TUBB3 expression (P=0.011). There were no significant differences between response rate and TS or ERCC1 expression pattern. Median overall survival (OS) and median time to progression (TTP) were significantly longer in patients with low TUBB3 mRNA expression (P=0.002, P<0.001). TS or ERCC1 expression was not correlated with TTP and OS. In the combined analysis including TUBB3, TS and ERCC1, the patients with 0 or 1 high expression gene had better response rate, TTP and OS than the remaining patients (all P<0.001). Multivariate analysis revealed that ECOG (Eastern Cooperative Oncology Group)≥2 (HR=2.42, P=0.009) and TUBB3 (HR=2.34, P=0.036) mRNA expression significantly impacted on OS. High TUBB3 mRNA expression is correlated with resistance to DCF regimen chemotherapy. TUBB3 might be a predictive and prognostic factor in patients with advanced gastric cancer treated with TXT-based chemotherapy. The combined evaluation of TUBB3, TS and ERCC1 expression can promote the individual treatment in advanced gastric cancer.

βIII-tubulin expression as a predictor of docetaxel resistance in metastatic castrate-resistant prostate cancer.

e15174 Background: Docetaxel is a tubulin-targeting cytotoxic that remains first-line therapy in metastatic castrate-resistant prostate cancer (mCRPC) patients (pts) even though half of pts are reported to be non-responders. A predictive marker to identify those who will benefit from docetaxel-therapy will assist clinical decision making. High βIII-tubulin (TUBB3) expression has previously been reported to correlate with lack of response to taxanes in other cancers. We evaluated TUBB3 expression as a predictor of docetaxel-resistance in mCRPC. Methods: mCRPC pts treated with at least 3 cycles of docetaxel between 1990 and 2011 were identified retrospectively. TUBB3 immunostaining was performed on archival formalin-fixed, paraffin-embedded tissue. Stain intensity was scored from 0 to 3; 2 and 3 were interpreted as positive. Rates of PSA response were compared between pts with positive (+) and negative (-) TUBB3 expression. Two definitions of PSA response were evaluated (any PSA decline and at least 50% decline). Overall survival (OS) distribution between TUBB3+ and TUBB3- pts was estimated by the Kaplan-Meier method. Results: Of 73 pts, 26 (35%) expressed TUBB3. At diagnosis, the mean age was 65.7 years and the median Gleason score was 8. At the time of docetaxel therapy, the mean age was 71.2 years, the median PSA level was 70.9 (range, 0.2-5253) and 76% had ECOG performance status ≤1. The median number of docetaxel cycles was 7 (range, 3-18). The total dose of docetaxel was not different between groups (p=0.705). The median OS was 19.2 mo. TUBB3 expression was not correlated with any clinical or pathological characteristic (age, Gleason score, stage, ECOG, PSA, LDH, alkaline phosphatase, hemoglobin, visceral disease or chemotherapy before docetaxel). 65% of TUBB3+ pts had any PSA decline compared to 89% of pts with TUBB3- (p=0.0267). 52% of TUBB3+ pts had a PSA decline of ≥ 50% compared to 70% of TUBB3- pts (p=0.0144). Median OS for TUBB3+ pts was 16.8 mo compared to 20.4 mo in TUBB3- pts (p=0.039). Conclusions: High TUBB3 expression was associated with shorter OS and lower PSA response rates in mCRPC pts treated with docetaxel. These findings need to be validated prospectively.

High TUBB3 Expression, an Independent Prognostic Marker in Patients with Early Non–Small Cell Lung Cancer Treated by Preoperative Chemotherapy, Is Regulated by K-Ras Signaling Pathway

We assessed the prognostic and predictive value of β-tubulin III (TUBB3) expression, as determined by immunohistochemistry, in 412 non-small cell lung cancer (NSCLC) specimens from early-stage patients who received neoadjuvant chemotherapy (paclitaxel- or gemcitabine-based) in a phase III trial (IFCT-0002). We also correlated TUBB3 expression with K-Ras and EGF receptor (EGFR) mutations in a subset of 208 cryopreserved specimens. High TUBB3 protein expression was associated with nonsquamous cell carcinomas (P < 0.001) and K-Ras mutation (P < 0.001). The 127 (30.8%) TUBB3-negative patients derived more than 1 year of overall survival advantage, with more than 84 months median overall survival versus 71.7 months for TUBB3-positive patients [HR, 1.58; 95% confidence interval (CI), 1.11-2.25)]. This prognostic value was confirmed in multivariate analysis (adjusted HR for death, 1.51; 95% CI, 1.04-2.21; P = 0.031) with a bootstrapping validation procedure. TUBB3 expression was associated with nonresponse to chemotherapy (adjusted HR, 1.31; 95% CI, 1.01-1.70; P = 0.044) but had no predictive value (taxane vs. gemcitabine). Taking account of these clinical findings, we further investigated TUBB3 expression in isogenic human bronchial cell lines only differing by K-Ras gene status and assessed the effect of K-Ras short interfering RNA (siRNA) mediated depletion, cell hypoxia, or pharmacologic inhibitors of K-Ras downstream effectors, on TUBB3 protein cell content. siRNA K-Ras knockdown, inhibition of RAF/MEK (MAP-ERK kinase) and phosphoinositide 3-kinase (PI3K)/AKT signaling, and hypoxia were shown to downregulate TUBB3 expression in bronchial cells. This study is the first one to identify K-Ras mutations as determinant of TUBB3 expression, a chemoresistance marker. Our in vitro data deserve studies combining standard chemotherapy with anti-MEK or anti-PI3K drugs in patients with TUBB3-overexpressing tumors.

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.

Differential in vitro sensitivity to patupilone versus paclitaxel in uterine and ovarian carcinosarcoma cell lines is linked to tubulin-beta-III expression

ObjectiveTo compare the in vitro sensitivity/resistance to patupilone versus paclitaxel in uterine and ovarian carcinosarcomas (CS). MethodsFive primary carcinosarcoma cell lines, two from uterine and three of ovarian origin, were evaluated for growth rate and tested for their in vitro sensitivity/resistance to patupilone versus paclitaxel by MTS assays. To identify potential mechanisms underlying the differential sensitivity/resistance to patupilone, expression levels of β-tubulin III (TUBB3) were determined with quantitative-real-time-polymerase-chain-reaction (q-RT-PCR) in primary uterine and ovarian CS cell lines and in 26 uterine and 9 ovarian CS fresh-frozen-tissues. ResultsNo appreciable difference in sensitivity to patupilone versus paclitaxel was noted in ovarian CS cell lines, or when uterine and ovarian CS cell lines were compared in their response to paclitaxel. In contrast, uterine CS cell lines were found to be significantly more sensitive to patupilone than to paclitaxel (P<0.002) and demostrated lower IC50s to patupilone (range 0.76–0.93nM) when compared to ovarian CS (range 1.9–3.4 nM, p<0.05). Higher levels of TUBB3 were detected in uterine CS cell lines and fresh frozen tissues when compared to ovarian CS (P<0.05). ConclusionsUterine CS cell lines are significantly more sensitive than ovarian CS cell lines to patupilone versus paclitaxel. High expression of TUBB3 is associated with sensitivity to patupilone in primary CS cell lines and may act as a genetic marker to predict chemotherapy efficacy. Patupilone may represent a promising drug in the treatment of this subset of rare but highly aggressive gynecological tumors.

High β-III tubulin expression in triple-negative (TN) breast cancer (BC) subtype and correlation to ixabepilone response: A retrospective analysis

3587 Background: High βIII-tubulin (βIII) expression confers resistance to many anti-microtubule agents (Sève, Lancet 2008; 9: 168). The anti-microtubule agent ixabepilone (Ixa) demonstrates efficacy in βIII over-expressing tumor models resistant to taxanes and has clinical activity in patients with TN BC. The incidence and expression of βIII by BC subtype have not been delineated. Methods: Retrospective analyses of βIII mRNA expression were conducted in two microarray datasets of BC cohorts. Using intrinsic gene clustering, BC tumors from the Ixa neoadjuvant study CA163–080 (080) and the International Genome Consortium (IGC) were defined as basal-like (BL), luminal-like, Her2-overexpressing (Her2+) or normal-like subtypes. For study 080, IHC status subtyping defined TN, Her2+ and Her2-ER+PR+ patients. Mean expression of TUBB3, the gene encoding for βIII, was determined for each subtype. Clinical responses for 080 patients were compared to βIII expression levels. Results: For both cohorts, mean expression of TUBB3 was higher in BL and Her2+ tumors (one-way ANOVA and Tukey's HSD test; both p &lt; 0.001). TUBB3 expression was rank-ordered: low, medium and high. In 080 and IGC cohorts, high expressors represent &gt;50% of patients in BL and Her2+ subtypes and ∼20% of other BC subtypes. Subtyping 080 patients by receptor expression status demonstrated mean TUBB3 expression was higher for TN compared to Her2+ and Her2-ER+PR+ patients (t-test; p = 0.009). TN patients (n = 11/46) in study 080 had a trend to higher pCR rate than the overall population (n = 29/161), 26 vs. 18%. Receiver operating characteristics (ROC) analysis with AUC=0.66 demonstrates that high TUBB3 expression may predict for Ixa response in the overall 080 population. Conclusions: BC patients with TN, BL and Her2+ tumors have higher βIII expression. For the 080 study, responses to Ixa were higher for TN BC patients, and ROC analysis correlated response to βIII levels in the overall population. These data suggest high βIII in BL and TN BC may contribute to the aggressiveness of these subtypes and predict for Ixa clinical response. Additional studies are needed to address the activity of taxanes relative to βIII expression in TN BC. [Table: see text]