Levels Of TUBB3 Research Articles

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.

TUBB3 immunostaining improves the diagnostic accuracy of oral liquid-based cytology in squamous cell carcinoma.

Although class III beta-tubulin (TUBB3) is not expressed in normal epithelium, its expression in cancers of some organs has been reported. Herein, we investigated the expression pattern and expression levels of TUBB3 in oral squamous cell carcinoma (SCC), and assessed whether TUBB3 immunostaining could improve the diagnostic accuracy of oral scraping liquid-based cytology (LBC). Paraffin sections of biopsies from 107 patients with primary SCC and 30 patients with squamous papilloma of the tongue or gingiva were immunostained for TUBB3. In addition, 15 LBC samples obtained from the study participants with SCC were immunostained for TUBB3. Seven LBC samples were false-negative. The TUBB3 expression level in each sample was evaluated and classified as 3+, 2+, 1+, or 0. TUBB3 expression was confirmed in 91.6% of paraffin-embedded SCC specimens. Clear and diffuse positivity (2+ or above) was observed in 77.6% of the total cases. In the well-differentiated type, tumour cells in the middle layer of the parenchyma specifically expressed TUBB3. In almost LBC samples, cancerous intermediate cells showed immunopositivity similar to that of paraffin samples, even if cellular atypia was not clear in Papanicolaou staining. TUBB3 immunostaining is useful for diagnosing oral SCC in scraping LBC, especially when samples consist of intermediate cells with little morphological change. Moreover, TUBB3 immunostaining could improve the diagnostic accuracy of oral scraping LBC by reducing false-negatives.

Extensive analysis of the molecular biomarkers excision repair cross complementing 1, ribonucleotide reductase M1, β-tubulin III, thymidylate synthetase, and topoisomerase IIα in breast cancer: Association with clinicopathological characteristics.

Excision repair cross complementing 1 (ERCC1), ribonucleotide reductase M1 (RRM1), β-tubulin III (TUBB3), thymidylate synthetase (TYMS), and topoisomerase IIα (TOP2A) genes have been shown to be associated with the pathogenesis and prognosis of various types of carcinomas; however, their roles in breast cancer have not been fully validated. In this study, we evaluated the correlations among these biomarkers and the associations between their expression intensity and the clinicopathological characteristics to investigate whether the above genes are underlying biomarkers for patients with breast cancer.Ninety-seven tissue specimens collected from breast cancer patients. The expression levels of these biomarkers were measured by the multiplex branched DNA liquidchip (MBL) technology and clinicopathological characteristics were collected simultaneously.The expression levels of ERCC1, TUBB3, TYMS, and TOP2A were significantly associated with the characteristics of menopausal status, tumor size, lymph node metastasis, hormone receptor status, triple-negative status, Ki-67 index, and epidermal growth factor receptor. The expression intensity of ERCC1 negatively associated with that of TUBB3 and TYMS, and positively associated with that of RRM1. The expression intensity of TOP2A positively associated with that of TYMS. Hierarchical clustering analysis and difference test indicated that breast cancer with higher levels of TUBB3, TYMS, and TOP2A, as well as lower levels of ERCC1 and RRM1 tended to have higher histological grade and Ki-67 index.Our studies showed that ERCC1, TYMS, TUBB3, and TOP2A may be potential biomarkers for prognosis and individualized chemotherapy guidance, while there may be interactions between ERCC1 and RRM1, or TUBB3, or TYMS, as well as between TOP2A and TYMS in pathogenesis and development of breast cancer.

TUBB3 Promotes Growth and Invasion of Gallbladder Cancer Cells by Akt/mTOR Signal Pathway.

Aberrant expression of β-tubulin-III (TUBB3) is known that related to aggressive tumor features and poor clinical outcomes. However, there is limited research about TUBB3 expression and its role in the outcomes and the progression of gallbladder cancer. We have measured TUBB3 level in gallbladder cancer samples and cell lines from 2012 to 2016, and tested the effects of TUBB3 in cancer cell growth, apoptosis and cell cycle arrest by using appropriate methods. The results revealed that TUBB3 was significantly over-expressed in gallbladder cancer samples and cell lines, and high TUBB3 level contributed to shorter overall survival in patients. The knockdown of TUBB3 with sh-TUBB3 inhibited the proliferation, migration and invasion of cancer cells. Meanwhile, it promotes apoptosis and changes the cell cycle distribution. Suppression of TUBB3 expression could increase p21 and cyclin B1 expression, and decrease cyclin D1. Xenograft mouse model also showed that low expression of TUBB3 reduced the growth of established gallbladder cancer xenograft in vivo. Furthermore, TUBB3 knockdown significantly decreased phosphorylation of Akt and mTOR in vitro and in vivo. TUBB3 can induce the development of gallbladder cancer by Akt/mTOR signal pathway and we point out a potential therapeutic target for gallbladder cancer treatment.

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.

THE INTRA-ASSAY PRECISION AND TIME STABILITY OF QUANTITATIVE βIII-TUBULIN EXPRESSION ASSESSMENT IN SOLID TUMOR TISSUE

Introduction. The introducing of tumor molecular profiling into clinical practice has revealed the need for development of new analytical methods for estimating marker expression in solid tumors, as routinely used method of immunohistochemistry has a number of significant drawbacks.Objective. Analytical validation of immunofluorescence staining and flow cytometry method developed by the authors for the examination of tumor protein markers in the solid tumors tissue.Materials and methods. Method validation was carried out by quantitative estimation of βIII-tubulin (TUBB3) expression in single-cell suspensions of non-small-cell lung cancer obtained from surgical tumor samples. Primary antibodies to TUBB3 (ab7751) and secondary DyLight 650-conjugated antibodies (ab98729) were used for immunofluorescent staining. The «Navios» flow cytometer (Beckman Coulter) was used to measure the fluorescence. The validation parameters were assessed by the coefficient of variation calculated as the ratio of standard deviation of TUBB3 level to its mean value.Results. Two parameters were analyzed: intra-assay precision and time stability of the results of the TUBB3 expression assessment. It was demonstrated that the mean coefficients of variation of the marker expression level in the tumor tissue did not exceed 20 % for both parameters. According to recommendations on the analytical validation of methods based on flow cytometry, it proves the validity of the method for these parameters.Conclusions. The intra-assay precision and time stability were demonstrated for the results of a quantitative estimation of TUBB3 expression in solid tumor tissue using immunofluorescence staining and flow cytometry method developed by the authors. The practical value of the time stability of immunofluorescence stain during 24 h storage of a stained cells suspension in the dark at 4 °C was highlighted. It shows the possibility of adjusting the time interval between completion of the analytical study part and flow cytometer measurement.

Hippocampal proteomic analysis reveals the disturbance of synaptogenesis and neurotransmission induced by developmental exposure to organophosphate flame retardant triphenyl phosphate

With the spread of organophosphorus flame retardants (OPFRs), the environmental and health risks they induce are attracting attention. Triphenyl phosphate (TPHP) is a popular alternative to brominated flame retardant and halogenated OPFRs. Neurodevelopmental toxicity is TPHP’s primary adverse effect, whereas the biomarkers and the modes of action have yet to be elucidated. In the present study, 0.5, 5, and 50 mg/kg of TPHP were orally administered to mice from postnatal day 10 (P10) to P70. The behavioral tests showed a compromised learning and memory capability. Proteomic analysis of the hippocampus exposed to 0.5 or 50 mg/kg of TPHP identified 531 differentially expressed proteins that were mainly involved in axon guidance, synaptic function, neurotransmitter transport, exocytosis, and energy metabolism. Immunoblot and immunofluorescence analysis showed that exposure to TPHP reduced the protein levels of TUBB3 and SYP in the synapses of hippocampal neurons. TPHP exposure also downregulated the gene expression of neurotransmitter receptors including Grins, Htr1α, and Adra1α in a dose-dependent fashion. Moreover, the calcium-dependent synaptic exocytosis governed by synaptic vesicle proteins STX1A and SYT1 was inhibited in the TPHP-treated hippocampus. Our results reveal that TPHP exposure causes abnormal learning and memory behaviors by disturbing synaptogenesis and neurotransmission.

High protein and mRNA expression levels of TUBB3 (class III ß-tubulin) are associated with aggressive tumor features in esophageal adenocarcinomas.

BackgroundEsophageal adenocarcinomas show an increasing incidence in the Western world and their overall survival remains low. Microtubules are multifunctional cytoskeletal proteins involved in crucial cellular roles, including maintenance of cell shape, intracellular transport, meiosis, and mitosis. Microtubulus-TUBB3 was found overexpressed in several carcinomas suggesting a significant role in cancer development. High levels of TUBB3 expression were also described to be associated with poor clinical outcome in various cancers. It was shown that overexpression of TUBB3 could be related to reduced efficiency of taxane-based targeting anticancer drugs in several cancer types.ResultsThere is a statistically significant association between high TUBB3 protein and TUBB3 mRNA expression and shortened survival (p<0,0001). Prognostic impact of TUBB3 expression is seen in patients with and without multimodal treatment. Multivariate analysis revealed a strong TUBB3 expression to be an independent prognosis factor. Validation of protein expression by mRNA in situ hybridization underlines the credibility of the immunohistochemical results.DiscussionOur study emphasized the significant importance of TUBB3 in esophageal adenocarcinoma. TUBB3 serves as an independent prognostic marker and may be a valuable biomarker for routine application in esophageal adenocarcinoma especially to address the need for adjuvant treatment in individuals following neoadjuvant therapy and surgery. Future prospective studies are needed which include the results of TUBB3 in preoperative biopsy material to proof the prognostic impact of TUBB3.Materials and Methods280 esophageal adenocarcinomas that underwent primary surgical resection or resection after neoadjuvant therapy were analyzed by mRNA-in-situ-hybridization (RNAscope®) and by immunohistochemistry (TUBB3 rabbit monoclonal antibody; Epitomics).

146P - Predicting response to chemotherapy in gastric cancer patients randomized to docetaxel: A reevaluation of the ITACA-S trial

Background: No predictive biomarker for chemotherapy has been validated for clinical use. Clinical studies suggest tumor expression of the proteins class III β-tubulin (TUBB3) and thymidine phosphorylase (TYMP) predict resistance to taxane and response to 5-FU, respectively. Immunohistochemical definitions of protein status vary widely. We evaluated relationships between survival and expression of TUBB3 and TYMP as quantitated by mass spectrometry in the archived tumor samples of 247 patients from the Intergroup Trial of Adenocarcinoma of the Stomach (ITACA-S). Patients had been randomized to monotherapy with 5-FU/LV or to FOLFIRI plus docetaxel and cisplatin. Methods: Gastric tumor tissues were microdissected and solubilized for proteomic quantitation of 45 protein biomarkers. The cutoff for TUBB3 (750 amol/µg of total protein) was predetermined based on the assay’s limit of detection. An experimental cutoff for TYMP (1335 amol/ug) was derived in the 5-FU/LV-treated patients using Monte Carlo 2-fold cross-validation. The Mantel-Cox log-rank test was used for survival comparisons. Results: Among gastric cancer (GC) patients treated with docetaxel-containing chemotherapy (n = 125), those with TUBB3 protein levels below the cutoff had a longer median overall survival (mOS) than patients with TUBB3 levels above the cutoff (1563 vs 886 days, p < 0.04). TUBB3 level made no difference in survival among patients who received 5-FU/LV (p = 0.64). Of note, among patients with high TUBB3 levels, those treated with 5-FU/LV survived 3 years longer than patients treated with docetaxel (mOS = 1991 vs 886 days, p = 0.048). 5-FU/LV-treated patients (n = 122) with TYMP protein expression above the cutoff survived longer than those with lower TYMP levels (mOS: 2362 vs 1062 days, p < 0.02). A combination of TUBB3 and TYMP expression predicted the longest survival in patients treated with FOLFIRI plus docetaxel (p < 0.001). Conclusions: Quantitative proteomic analysis identified subsets of trial patients who benefitted from specific adjuvant chemotherapy regimens. Personalized chemotherapy based on quantitated TYMP and TUBB3 is promising and warrants broader evaluation. Legal entity responsible for the study: NantOmics, LLC Funding: IRCCS and NantOmics, LLC Disclosure: F. Cecchi, S. Schwartz, S. Sellappan, T. Hembrough, Y. Tian: Employee at NantOmics. All other authors have declared no conflicts of interest.

Prognostic effect of class III β-tubulin and Topoisomerase-II in patients with advanced thymic carcinoma who received combination chemotherapy, including taxanes or topoisomerase-II inhibitors.

Class III β-tubulin (TUBB3) and Topoisomerase-II (topo-II) are considered to be the predictors of therapeutic efficacy and outcome in several types of human neoplasm. However, whether TUBB3 or topo-II may predict the response to combination chemotherapy and prognosis in patients with advanced thymic carcinoma (ATC) remains unclear. The aim of the present study was to investigate the prognostic significance of TUBB3 and topo-II expression levels in ATC. A total of 34 patients with ATC who received combination chemotherapy were enrolled in the present study. Immunohistochemical analysis was used to examine the expression of TUBB3, topo-II and Ki-67 in tumor specimens obtained by surgical resection or biopsy. TUBB3 and topo-II were highly expressed in 38 and 53% of the tumors, respectively. Progression-free survival (PFS) was significantly shorter in patients with high levels of TUBB3 compared with those with low levels of TUBB3 (P<0.01), whereas no significant difference in PFS between patients with high and low topo-II expression levels was observed (P=0.31). Patients with overexpression of TUBB3 or topo-II exhibited significantly shorter overall survival rates (OS) compared with those patients with low levels of expression of these proteins (TUBB3; P=0.01, topo-II; P=0.01). Multivariate analysis demonstrated that a high level of TUBB3 expression was an independent unfavorable prognostic factor for OS, and a high level of topo-II expression tended to correlate with poor prognosis without statistical significance. Additionally, a subset analysis demonstrated that the treatment with taxanes, but not topo-II inhibitors, tended to prolong OS in patients with TUBB3 overexpression and there was significant survival advantage of chemoradiotherapy over chemotherapy in patients with topo-II overexpression. It was revealed that an enhanced expression of TUBB3 or topo-II was clearly associated with clinical outcomes in patients with ATC who received combination chemotherapy, including taxanes or topo-II inhibitors, suggesting the prognostic significance of these markers.

Predicting survival in gastric cancer patients randomized to docetaxel with mass spectrometric quantitation of TUBB3.

4068 Background: No predictive biomarker for chemotherapy has been validated for clinical use. A relationship between resistance to taxanes and expression of class III β-tubulin (TUBB3) protein has been suggested by small clinical studies, but not confirmed in randomized trials. Immunohistochemical definitions of “TUBB3 positive” vary widely between studies and depend on subjective measures of strong vs weak staining. We retrospectively evaluated the relationship between survival and TUBB3 protein quantitated by mass spectrometry in the tumor samples of 247 patients from the Intergroup Trial of Adenocarcinoma of the Stomach (ITACA-S). Patients had been randomized to a docetaxel-containing adjuvant regimen or to monotherapy with fluorouracil and leucovorin (5-FU/LV). Methods: Archived tumor tissues were microdissected and solubilized for proteomic analysis. TUBB3 and 44 other protein biomarkers were quantified with a mass spectrometry-based assay. A TUBB3 protein cutoff of 750 amol/ug was predetermined based on the assay’s limit of detection. The Mantel-Cox log-rank test was used for survival comparisons. Results: Among gastric cancer (GC) patients treated with docetaxel-containing chemotherapy (n = 125), those with TUBB3 levels below the cutoff (750 amol/µg of total protein) had a longer median overall survival (mOS) than patients with TUBB3 levels above the cutoff (1563 vs 886 days, p = 0.04). TUBB3 level made no difference in survival among patients who received 5-FU/LV. Of note, among patients with high TUBB3 levels ( &gt; 750 amol), those treated with 5-FU/LV survived 3 years longer than patients in the docetaxel arm (mOS = 1991 vs 886 days, p = 0.048). Conclusions: Quantitative proteomic analysis of TUBB3 identified a subset of GC patients who benefitted from the addition of docetaxel to adjuvant chemotherapy. GC patients with TUBB3-expressing tumors had worse outcomes on a docetaxel-containing regimen than on 5-FU/LV. An ongoing study of triple-negative breast cancer patients treated with docetaxel suggests that proteomic TUBB3 may be predictive in other cancer indications. Personalized chemotherapy based on quantitated TUBB3 is promising and warrants broader evaluation.

TUBB3 overexpression has a negligible effect on the sensitivity to taxol in cultured cell lines.

Microtubules are cellular targets for a variety of anticancer therapies because of their critical function in mitosis. Taxol belongs to a class of microtubule targeting agents that suppresses microtubule dynamics and interferes with the functioning of the mitotic spindle, thereby effectively blocking cell cycle progression of rapidly proliferating tumor cells. Despite its antitumor activity, drug resistance remains a common obstacle in improving its overall clinical efficacy. Previous studies have shown that the expression of a specific β-tubulin isotype, βIII-tubulin/TUBB3, is dysregulated in drug-refractory tumors. However, whether enhanced TUBB3 expression is directly involved in promoting taxol resistance remains a subject of debate. Here, we have used several approaches to assess the functional relation of TUBB3 overexpression and taxol resistance. First, we generated a number of taxol-resistant cell lines, to find that TUBB3 expression was elevated in a resistant cell line (RPE-20) derived from untransformed retinal pigment epithelial (RPE) cells, but the abundance of TUBB3 remained unchanged in four other cell lines after taxol treatment. However, although RPE-20 cells displayed enhanced TUBB3 levels, we find that simultaneous up-regulation of the P-glycoprotein (P-gP) drug-efflux pump is responsible for the resistance to taxol. Indeed, we could show that TUBB3 levels were dynamically regulated upon taxol exposure and withdrawal, unrelated to the resistance phenotype. Next, we generated cell lines in which we could induce robust overexpression of TUBB3 from its endogenous locus employing the CRISPRa system. We demonstrate that solely enhancing TUBB3 expression results in a very minor decrease in the sensitivity to taxol. This was further substantiated by selective depletion of TUBB3 in a series of breast cancer cell lines expressing high levels of TUBB3. We find that TUBB3 depletion had a minimal effect on the sensitivity to taxol in one of these cell lines, but had no effect in all of the others. Based on these findings we propose that TUBB3 overexpression can only marginally affect the sensitivity to taxol in cultured cell lines.

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.

β-III tubulin modulates the behavior of Snail overexpressed during the epithelial-to-mesenchymal transition in colon cancer cells

Class III β-tubulin (TUBB3) is a marker of drug resistance expressed in a variety of solid tumors. Originally, it was described as an important element of chemoresistance to taxanes. Recent studies have revealed that TUBB3 is also involved in an adaptive response to a microenvironmental stressor, e.g. low oxygen levels and poor nutrient supply in some solid tumors, independently of the microtubule targeting agent. Furthermore, it has been demonstrated that TUBB3 is a marker of biological aggressiveness associated with modulation of metastatic abilities in colon cancer. The epithelial-to-mesenchymal transition (EMT) is a basic cellular process by which epithelial cells lose their epithelial behavior and become invasive cells involved in cancer metastasis. Snail is a zinc-finger transcription factor which is able to induce EMT through the repression of E-cadherin expression. In the presented studies we focused on the analysis of the TUBB3 role in EMT-induced colon adenocarcinoma cell lines HT-29 and LS180. We observed a positive correlation between Snail presence and TUBB3 upregulation in tested adenocarcinoma cell lines. The cellular and behavioral analysis revealed for the first time that elevated TUBB3 level is functionally linked to increased cell migration and invasive capability of EMT induced cells. Additionally, the post-transcriptional modifications (phosphorylation, glycosylation) appear to regulate the cellular localization of TUBB3 and its phosphorylation, observed in cytoskeleton, is probably involved in cell motility modulation.

Abstract B41: The gelatinases-stimuli nanoparticles reverse docetaxel resistance and epithelial to mesenchymal transition in lung cancer cell line

Abstract 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 TUBB3 expression were evaluated. The effects of (miR-200c+DOC) NPs on docetaxel-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. Citation Format: Qin Liu, Rutian Li, Baorui Liu. The gelatinases-stimuli nanoparticles reverse docetaxel resistance and epithelial to mesenchymal transition in lung cancer cell line. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B41.

Tissue-specific control of midbody microtubule stability by Citron kinase through modulation of TUBB3 phosphorylation.

Cytokinesis, the physical separation of daughter cells at the end of cell cycle, is commonly considered a highly stereotyped phenomenon. However, in some specialized cells this process may involve specific molecular events that are still largely unknown. In mammals, loss of Citron-kinase (CIT-K) leads to massive cytokinesis failure and apoptosis only in neuronal progenitors and in male germ cells, resulting in severe microcephaly and testicular hypoplasia, but the reasons for this specificity are unknown. In this report we show that CIT-K modulates the stability of midbody microtubules and that the expression of tubulin β-III (TUBB3) is crucial for this phenotype. We observed that TUBB3 is expressed in proliferating CNS progenitors, with a pattern correlating with the susceptibility to CIT-K loss. More importantly, depletion of TUBB3 in CIT-K-dependent cells makes them resistant to CIT-K loss, whereas TUBB3 overexpression increases their sensitivity to CIT-K knockdown. The loss of CIT-K leads to a strong decrease in the phosphorylation of S444 on TUBB3, a post-translational modification associated with microtubule stabilization. CIT-K may promote this event by interacting with TUBB3 and by recruiting at the midbody casein kinase-2α (CK2α) that has previously been reported to phosphorylate the S444 residue. Indeed, CK2α is lost from the midbody in CIT-K-depleted cells. Moreover, expression of the nonphosphorylatable TUBB3 mutant S444A induces cytokinesis failure, whereas expression of the phospho-mimetic mutant S444D rescues the cytokinesis failure induced by both CIT-K and CK2α loss. Altogether, our findings reveal that expression of relatively low levels of TUBB3 in mitotic cells can be detrimental for their cytokinesis and underscore the importance of CIT-K in counteracting this event.

6-Methoxy Podophyllotoxin Induces Apoptosis via Inhibition of TUBB3 and TOPIIA Gene Expressions in 5637 and K562 Cancer Cell Lines.

Podophyllotoxin (PTOX), a natural compound in numerous plants, contains remarkable biological properties that include anti-tumor, anti-viral such as anti-human im- munodeficiency virus (HIV) activities. In order to avoid its adverse effects, various com- pounds have been derived from PTOX. 6-methoxy PTOX (MPTOX) is one of the natural PTOX derivatives with an extra methoxy group. MPTOX is mostly isolated from the Linum species. This study has sought to determine the biological effects of MPTOX on cancer cell lines, 5637 and K562. In this experimental study, we treated the 5637 and K562 cancer cell lines with MPTOX in a doseand time-dependent manner. Apoptosis was examined by flow cytometry and viability rate was analyzed by the MTT assay. Expressions of the tubulin (TUBB3) and topoisomerase II (TOPIIA) genes were determined by real-time poly- merase chain reaction (PCR). Treatment with MPTOX led to significant induction of apoptosis in cancer cells compared to control cells. Gene expression analysis showed reduced levels of TUBB3 and TOPIIA mRNA following MPTOX treatment. MPTOX inhibited TUBB3 and TOPIIA gene expression and subsequently induced cell death through apoptosis. These results suggested that MPTOX could be considered a potential anti-tumor agent.

Expression of ERCC1, TYMS, TUBB3, RRM1 and TOP2A in patients with esophageal squamous cell carcinoma: A hierarchical clustering analysis

The aim of the present study was to investigate the correlation between the expression levels of excision repair cross complementing 1 (ERCC1), thymidylate synthase (TYMS), class III β-tubulin (TUBB3), ribonucleoside-diphosphate reductase (RRM1) and topoisomerase IIα (TOP2A) with the clinical characteristics of patients with esophageal squamous cell carcinoma (ESCC). A total of 29 ESCC tissue samples were collected from patients that had not previously received systematic treatment. The expression levels of ERCC1, TYMS, TUBB3, RRM1 and TOP2A were determined using a microarray technique, while Spearman’s rank correlation analysis was used to determine the strength of the correlations between the expression levels of the biomarkers and the pathogenesis of esophageal cancer. High expression levels of TYMS and TOP2A were observed in 24% of the samples and high expression levels of TUBB3 and RRM1 were identified in 7% of the samples. Hierarchical clustering analysis of these biomarkers enabled the samples to be grouped. Group 1 patients exhibited low expression levels of TYMS, RRM1 and TOP2A and high expression of ERCC1 and TUBB3, while group 2 samples had low expression levels of ERCC1 and TUBB3 and high expression levels of TYMS, RRM1 and TOP2A. Analysis using Fisher’s exact test demonstrated a statistically significant difference in the severity of carcinoma invasion between the two groups (P<0.05), however, no significant differences were identified with regard to the clinical stage or lymphatic metastasis (P>0.05). Therefore, hierarchical clustering analysis indicated that the expression levels of ERCC1, TYMS, TUBB3, RRM1 and TOP2A were closely associated with the clinical characteristics of patients with ESCC.

Targeted delivery of miR-200c/DOC to inhibit cancer stem cells and cancer cells by the gelatinases-stimuli nanoparticles

Cancer stem cells (CSCs) are recently discovered as vital obstacles for the successful cancer therapy. Emerging evidences suggest that miR-200c functions as an effective CSCs inhibitor and can restore sensitivity to microtubule-targeting drugs. In the present work, the intelligent gelatinases-stimuli nanoparticles (NPs) was set up to co-deliver miR-200c and docetaxel (DOC) to verify their synergetic effects on inhibition of CSCs and non-CSC cancer cells. After tumor cells were treated with miR-200c NPs, miR-200c and its targeted gene class III beta-tubulin (TUBB3)TUBB3 expression were evaluated. The effects of miR-200c/DOC NPs on tumor cell viability, migration and invasion as well as the expression of E-cadherin and CD44 were studied. The antitumor effects of miR-200c/DOC NPs were compared with DOC NPs in xenograft gastric cancer mice. Moreover, the residual tumors after treatment were subcutaneously seeded into nude mice to further investigate the effective maintenance of NPs. We found that the gelatinases-stimuli NPs facilitated miR-200c into cells, achieving sustained miR-200c expression in tumor cells during 9 days. The miR-200c/DOC NPs significantly enhanced cytotoxicity of DOC, possibly by decreasing TUBB3 level, and reversing EMT. The miR-200c NPs achieved high levels of in vivo accumulation and long retention in gastric cancer xenografts after intravenous administration. The miR-200c/DOC NPs prominently suppressed in vivo tumor growth with elevated miR-200c and E-cadherin levels and down-regulated TUBB3 and CD44 expressions. When the residual tumors after miR-200c/DOC NPs treatment were re-transplanted into nude mice, the tumors demonstrated the slowest growth speed. The miR-200c/DOC NPs may provide a promising modality for co-delivery of nucleic acid and drugs to simultaneously inhibit CSCs and non-CSC cancer cells.

Serum levels of TUBB3 correlate with clinical outcome in Chinese patients with advanced gastric cancer receiving first-line paclitaxel plus capecitabine

The overexpression of β-tubulin III (TUBB3) in tumor tissues was reversely related with the efficacy of paclitaxel and clinical outcome in different cancers. In this study, we aimed to investigate the association between serum levels of TUBB3 and clinical outcome in advanced gastric cancer patients receiving first-line paclitaxel plus capecitabine. One hundred and twenty-eight advanced gastric cancer patients receiving first-line paclitaxel plus capecitabine in Peking University Cancer Hospital from December 2006 to October 2010 were enrolled in the study. Serum samples from 32 healthy individuals were used as controls. TUBB3 expression level in advanced gastric cancer was significantly higher than that in healthy control group (31.6 ± 17.8 ng/mL vs. 16.9 ± 3.8 ng/mL, p < 0.001). For all patients, the clinical benefit rate (CBR), median progression-free survival (PFS), and overall survival (OS) were 55.6 %, 179 and 306 days, respectively. The CBR, median PFS, and OS in patients with low (n = 27) and high levels (n = 101) of TUBB3 were 95.8 %/45.1 % (low vs. high, p < 0.001), 190 days/166 days (p = 0.064), and 360 days/297 days (p = 0.023), respectively. Cox multivariate regression analysis demonstrated that the serum levels of TUBB3 were an independent prognostic factor for advanced gastric cancer patients (HR = 1.950; 95 % CI, 1.242-3.062; p = 0.004). This study indicated that low levels of TUBB3 in serum could predict better response and survival for advanced gastric cancer patients receiving paclitaxel plus capecitabine, which could be used to select patients who would benefit from this regimen.