TUBB3 Expression Research Articles

Integrating mechanisms of response and resistance against the tubulin binding agent Eribulin in preclinical models of osteosarcoma.

Osteosarcoma is the most frequently occurring bone cancer in children and adolescents. Unfortunately, treatment failures are common. Eribulin is a synthetic microtubule inhibitor that has demonstrated activity in preclinical osteosarcoma models. The effects of eribulin were evaluated in two human osteosarcoma cell lines as well as in eribulin-sensitive and -resistant osteosarcoma xenograft tumors of the Pediatric Preclinical Testing Program (PPTP) by characterizing cell viability, microtubule destabilization, mitotic arrest and mechanism of cell death. Eribulin demonstrated cytotoxic activity in vitro, through promotion of microtubule dynamic instability, arrest of cells in the G2/M phase, mitotic catastrophe and cell death. The microtubule-destabilizing protein stathmin-1 (STMN1) was coimmunoprecipitated with the cyclin-dependent kinase inhibitor p27 indicating that these cytoplasmic complexes can protect cells from the microtubule destabilizing effect of eribulin. Increased tumoral expression of P-glycoprotein (P-gp) and TUBB3 were also associated with lower drug sensitivity. In summary, eribulin successfully blocked cells in G2/M phase but interfered with mitochondria activity to inhibit proteins involved in apoptosis. Understanding the complex and inter-related mechanisms involved in the overall drug response to eribulin may help in the design of therapeutic strategies that enhance drug activity and improve benefits of eribulin in pediatric patients with osteosarcoma.

QUANTITATIVE ASSESSMENT OF BETA-III TUBULIN EXPRESSION IN LUNG ADENOCARCINOMA AND SQUAMOUS CELL CARCINOMA

Background. Beta-III tubulin (TUBB3) is a tumor-specific isoform of the microtubule protein beta-tubulin. TUBB3 is considered to be a marker of adverse prognosis and tumor resistance to therapy with taxanes and Vinka alkaloids. Association between TUBB3 expression and histological type of the tumor has not been studied properly yet. Objective. The expression level of TUBB3 in non-small cell lung cancer biopsy specimens has been measured on 2 groups of patients with adenocarcinoma and squamous cell carcinoma. Materials and methods. The samples with adenocarcinoma (n = 43) and squamous cell carcinoma (n = 39) were converted to suspension, filtered, fixed with 4 % formaldehyde, stained with monoclonal antibodies for TUBB3 and DyLight 650-conjugated secondary antibodies to mouse IgG and analyzed by flow-cytometry. The method was developed in N.N. Blokhin Russian Cancer Research Center. Results. The average level of TUBB3 expression in the adenocarcinoma group is higher than in the squamous cell carcinoma group (33.1 ± 12.4 % of cells expressing the marker in adenocarcinoma vs. 26.0 ± 13.6 % in squamous cell carcinoma; differences were statistically significant). The average level of TUBB3 expression in adenocarcinoma is 29.7 ± 8.1 % in female and is 34.9 ± 13.9 % in male (differences statistically insignificant). Since the group of patients with adenocarcinoma was presented by both men and women, while the group of patients with squamous cell carcinoma was only men, from the analysis was excluded all female patients. Differences between the groups remained statistically significant. Conclusion. TUBB3 expression in tumor tissue does not depend on the gender, at least among patients with adenocarcinoma of the lung, and at the same time, the level of TUBB3 in adenocarcinoma tissue is higher in comparison with squamous cell carcinoma tissue.

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<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<0.0001) and 23% TLE3 expression (517/2225, p<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<0.0001) were more frequent in TUBB3-positive tumors, while GNAS (2% vs. 5%, p<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.

Immunofluorescent analyses of tumor marker beta-III tubuilin expression in tumor and ajacent normal lung tissue derived from patients with non-small cell lung cancer

Background. Class III beta-tubulin (TUBB3) is one of the eight beta tubulin isotypes identified in human. It is constitutively expressed in brain and testis but not in lung tissue. TUBB3 is also known to appear in solid tumors, in particular in non-small cell lung cancer (NSCLC), and it is often associated with poor prognosis and resistance to taxanes and Vinka alkaloids. Objective: We have suggested that TUBB3 expression may cover not only the primary tumor but also a wide adjacent area of morphologically normal lung tissue. To check the hypothesis we have measured TUBB3 expression both in the non-small cell lung carcinoma (NSCLC) and remote lung tissue derived from the same lung. 60surgical biopsy specimens of NSCLC and morphologically normal lung tissue of 30patients were investigated. Materials and methods. Biopsy specimens were converted to suspension, fixed in 4 % formaldehyde and analyzed by flow cytometry using monoclonal antibodies to TUBB3. The method was developed in the laboratory of medicinal chemistry research Institute of experimental diagnostics and therapy of tumors (N.N. Blokhin Russian Cancer Research Center) and patented. Results. Fraction of TUBB3-positive cells in the group of adjacent lung tissue specimens was lower compared to NSCLC group but still positive in the majority of adjacent lung tissue specimens (25 of 30) and achieved up to 39 % of cells. Conclusion. We suggest that TUBB3 expression in adjacent morphologically normal lung tissue indicates presence of transformed and potentially malignant cells far from the primary site.

High levels of class III β-tubulin expression are associated with aggressive tumor features in breast cancer

Overexpression of class III β-tubulin (TUBB3), a factor that confers dynamic properties to microtubules, is a candidate biomarker for resistance to microtubule-targeting chemotherapeutics in breast and other types of solid cancer. Discrepant results from previous studies, with respect to the association of TUBB3 expression levels with breast cancer phenotype and patient prognosis, prompted the present study to investigate TUBB3 expression in a large cohort of breast cancer cases, with available clinical follow-up data. A preexisting breast cancer prognosis tissue microarray, containing a single 0.6 mm tissue core from each of 2,197 individual patients with breast cancer, was analyzed for TUBB3 expression by immunohistochemistry. The results of the present study revealed that TUBB3 expression was less frequent in lobular breast cancer cases (34%), compared with that of cancer cases of alternative histologies, including breast cancer of no special type (60%; P<0.0001). High TUBB3 positivity was associated with high tumor grade (P<0.0001), negativity for estrogen (P<0.0001) and progesterone receptors (P<0.004), as well as the presence of human epidermal growth factor 2 amplification (P<0.0001) and a triple-negative phenotype (P<0.0001). TUBB3 overexpression was additionally associated with reduced patient survival if all breast cancer cases of any histology were jointly analyzed (P=0.0088); however this link was not evident in the subset of breast cancer cases of no special type, or in a multivariate analysis including the established prognostic factors of tumor stage, grade and nodal stage. In conclusion, the present study demonstrated that TUBB3 overexpression was associated with adverse features of breast cancer, and that TUBB3 may possess a distinct role in lobular breast cancer cases, compared with alternative histological subtypes. The results of the present study do not support a clinically relevant role for TUBB3 as a prognostic marker in breast cancer.

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.

Expression of ERCC1, TUBB3, BRCA1, and TS as predictive markers of neoadjuvant chemotherapy for squamous cell carcinoma of the esophagus.

47 Background: No predictive biomarker of the response to neoadjuvant chemotherapy with docetaxel and cisplatin plus 5-fluorouracil (NAC-DCF) is available for patients with squamous cell carcinoma of the esophagus (SCCE) in a clinical setting. The aim of this study was to identify the biomarkers associated with chemotherapeutic efficacy and long-term survival for patients with advanced SCCE who had received NAC-DCF followed by surgery. Methods: This study included 45 patients with advanced SCCE who received NAC-DCF between January 2008 and December 2012. The NAC-DCF was conducted as a phase II study (UMIN000007408). The expressions of excision repair cross-complementing-1 (ERCC1), class III beta-tubulin, breast cancer susceptibility gene I (BRCA1), and thymidylate synthase were investigated simultaneously in the pre-treatment endoscopic tumor biopsy samples. Results: The multivariate logistic regression analysis indicated that pathological responses were significantly associated with tumors with low ERCC1 expression (P = 0.016) and with tumors with high BRCA1 expression (P = 0.014). The incidence of pathological responses to DCF in patients with low ERCC1 and high BRCA1 expressions was 100%. The multivariate Cox proportional hazard model analysis for relapse-free survival (RFS) revealed tumors with high BRCA1 expression (P = 0.031, hazards ratio, 4.39) as independent prognostic factors. Conclusions: Low ERCC1 expression and high BRCA1 expression in patients with SCCE were predictive biomarkers for chemotherapeutic efficacy. High BRCA1 expression was considered as prognostic factors for long-term RFS. These results may be a helpful tool for tailoring chemotherapy of patients with SCCE. Prospective validation of this biomarker panel is warranted. Clinical trial information: UMIN000007408.

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.

Clinicopathological significance of β -tubulin isotype III gene expression in breast cancer patients.

The molecular classification of breast cancer mainly focuses on estrogen receptor (ER), Progesterone receptor (PgR), and Human Epidermal Growth Factor Receptor 2(HER2/Neu) status detected by immunohistochemistry (IHC) analysis. The β -tubulin isotype III (TUBB3) gene was thought to be a marker of taxane resistance or cancer aggressiveness. To evaluate the clinicopathological significance of TUBB3 expression in breast cancer patients, we measured TUBB3 mRNA levels in 92 breast cancer patients by Quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and examined their correlation with ER, PgR, and HER2 status detected by IHC. We observed a significant positive correlation between the TUBB3 mRNA expression and the immunohistochemical positivity of both PgR (p= 0.000) and HER2 (p= 0.001). In addition, TUBB3 mRNA expression was associated with lymph nodes status (P= 0.008) and tumor stages (0.029), but no correlation was found with other clinicopathological features, such as age, pathohistological grades and tumor size. In conclusion, TUBB3 expression correlated significantly with molecular markers of breast cancer, such as PgR and HER2, suggesting that TUBB3 mRNA level facilitate the identification of a subset of patients who respond to Taxane treatment in addition to hormonal therapy and trastuzumab.

Analysis of ERCC1, BRCA1, RRM1 and TUBB3 as predictors of prognosis in patients with non-small cell lung cancer who received cisplatin-based adjuvant chemotherapy: A prospective study.

Although adjuvant platinum-based chemotherapy has been demonstrated to improve survival in patients with completely resected non-small cell lung cancer (NSCLC), individualized approaches to therapy are urgently required to improve the treatment efficacy and reduce unnecessary toxicity. It was hypothesized in the present study that the protein levels of excision repair cross-complementation group 1 (ERCC1), breast cancer 1 (BRCA1), ribonucleotide reductase M1 (RRM1) and class III β-tubulin (TUBB3) may influence the therapeutic effect of adjuvant cisplatin-based chemotherapy. The expression of ERCC1, BRCA1, RRM1 and TUBB3 in tissues obtained from 84 patients with NSCLC was analyzed in the present non-interventional study by immunohistochemistry prior to adjuvant chemotherapy. All patients received adjuvant cisplatin-based chemotherapy. The primary endpoint in the present study was disease free survival (DFS). Out of the 84 tumors, the expression of ERCC1, BRCA1, RRM1 and TUBB3 was identified in 46 (55%), 11 (13%), 73 (87%) and 76 (90%) tissues, respectively. A beneficial response to adjuvant cisplatin-based chemotherapy in DFS was associated with the absence of the expression of ERCC1 [hazard ratio (HR), 2.166; 95% confidence interval (CI), 1.049-4.474; P=0.037] and BRCA1 (HR, 2.419; 95% CI, 1.127-5.193; P=0.023), but not with the expression status of RRM1 (HR, 0.568; 95% CI, 0.234-1.379; P=0.212) or TUBB3 (HR, 1.874; 95% CI, 0.448-7.842; P=0.39). In addition, patients lacking the expression of ERCC1 and BRCA1 benefited more from adjuvant cisplatin-based chemotherapy compared with patients that expressed either ERCC1 or BRCA1 (HR, 3.102; 95% CI, 1.343-7.163; P=0.008). The expression of ERCC1 and BRCA1 was significantly associated with the DFS time in patients with NSCLC treated with adjuvant cisplatin-based chemotherapy, respectively. The combination of the ERCC1 and BRCA1 expression levels may be a promising prognostic prediction for adjuvant cisplatin-based chemotherapy.

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.

Differentiation of rat adipose tissue-derived stem cells into neuron-like cells by valproic acid, a histone deacetylase inhibitor.

Valproic acid (VPA) is a widely used antiepileptic drug, which has recently been reported to modulate the neuronal differentiation of adipose tissue-derived stem cells (ASCs) in humans and dogs. However, controversy exists as to whether VPA really acts as an inducer of neuronal differentiation of ASCs. The present study aimed to elucidate the effect of VPA in neuronal differentiation of rat ASCs. One or three days of pretreatment with VPA (2 mM) followed by neuronal induction enhanced the ratio of immature neuron marker βIII-tubulin-positive cells in a time-dependent manner, where the majority of cells also had a positive signal for neurofilament medium polypeptide (NEFM), a mature neuron marker. RT-PCR analysis revealed increases in the mRNA expression of microtubule-associated protein 2 (MAP2) and NEFM mature neuron markers, even without neuronal induction. Three-days pretreatment of VPA increased acetylation of histone H3 of ASCs as revealed by immunofluorescence staining. Chromatin immunoprecipitation assay also showed that the status of histone acetylation at H3K9 correlated with the gene expression of TUBB3 in ASCs by VPA. These results indicate that VPA significantly promotes the differentiation of rat ASCs into neuron-like cells through acetylation of histone H3, which suggests that VPA may serve as a useful tool for producing transplantable cells for future applications in clinical treatments.

Tubb3 regulation by the Erk and Akt signaling pathways: a mechanism involved in the effect of arginine ADP-ribosyltransferase 1 (Art1) on apoptosis of colon carcinoma CT26 cells.

The influence of the most important classical mono-ADP-ribosyltransferase, arginine ADP-ribosyltransferase 1 (Art1), on survival and apoptosis of colon carcinoma cells and the potential mechanisms have been partly discussed in our previous study but still need to be further studied. In this present study, Art1 of colon carcinoma CT26 cells was silenced with lentiviral vector-mediated short hairpin RNA (shRNA) or overexpressed with lentiviral vector-mediated complementary DNA (cDNA) and allograft transplant tumors are established in Balb/c mice. We verified Art1 knockdown increases apoptosis of CT26 cells transplant tumor; Art1 overexpression acts oppositely. Accordingly, growth of transplant tumors is inhibited in Art1 knockdown transplant tumors and increases in Art1 overexpression transplant tumors. Furthermore, activity of Akt and Erk cell signal pathways and expression of an apoptosis biomarker, βIII-tubulin (Tubb3), decrease when Art1 was silenced and increase when Art1 was overexpressed. Inhibiting Akt pathway or Erk pathway both downregulates expression of Tubb3 on protein and messenger RNA (mRNA) level, indicating that Tubb3 could be regulated by both Akt and Erk pathways, and plays a role in the influence of Art1 on apoptosis of Balb/c mice allograft transplant tumor. We also demonstrated that Bcl-2 family is not the responsible downstream factor of the Erk pathway in colon carcinoma cells which is undergoing apoptosis. These findings enrich the molecular mechanism for the function of Art1 in colon carcinoma and provide a complementary support for Art1 to be a potential therapeutic target of the treatment of this kind of malignant tumor.

RRM1, TUBB3, TOP2A, CYP19A1, CYP2D6: Difference between mRNA and protein expression in predicting prognosis of breast cancer patients.

The study investigated the clinical significance of RRM1 (ribonucleoside reductase subunit M1), TUBB3 (tubulin-β-III), TOP2A (DNA topoisomerase II), CYP19A1 (cytochrome P450, family 19, subfamily A, polypeptide 1) and CYP2D6 (cytochrome P450, family 2, subfamily D, polypeptide 6) for the diagnosis and possible predictive roles in breast cancer. Tissue microarray detected the expression of RRM1, tubulin-β-III, Topo IIα, CYP19A1 and CYP2D6 protein in breast cancer tissue and tissue adjacent to tumors (TATs). In addition, a publically available tool, was used to assess the prognostic value of their gene expression in breast cancer (http://kmplot.com). Analysis for relapse-free survival (RFS), disease-free survival (DFS) and overall survival (OS) was performed. Cytoplasmic RRM1, tubulin-β-III, CYP19A1 and Topo IIα staining were significantly higher in breast cancer tissues compared with TATs (P<0.050). Significant correlation occurred between RRM1 expression with pathological classification (P=0.018), lymph node involvement (P=0.035) and ER status (P=0.003). Tubulin-β-III and CYP2D6 expression correlated significantly with tumor grade (P=0.021 for tubulin-β-III and P=0.029 for CYP2D6, respectively). Cox analysis showed that the protein expression of CYP2D6, CYP19A1, RRM1, Topo IIα or tubulin-β-III was not an independent prognostic factor. A significant association occurred between RFS and TUBB3, TOP2A, CYP19A1, and CYP2D6 mRNA expression. With CYP19A1 (P<0.001) and CYP2D6 (P<0.001), a high expression was associated with good clinical outcome. Conversely, a low expression of TUBB3 (P<0.001) and TOP2A (P<0.001) was associated with good clinical outcome. TUBB3 (P=0.0004) and TOP2A (P<0.001) were significant prognostic factors in predicting the patient OS. The expression of RRM1, tubulin-β-III, Topo IIα and CYP19A1 in tumor tissues was significantly higher than that in TATs. TUBB3, TOP2A, CYP19A1 and CYP2D6 gene expression, but not protein expression, was associated with patient survival.

Abstract 2083: TFAP4 inhibits differentiation of MYCN-amplified neuroblastoma

Abstract Background. Despite the identification of MYCN-amplification as an adverse prognostic marker in neuroblastoma, no drugs that target MYCN have yet been developed. An alternative approach is to identify genes synthetically lethal to MYCN-amplified neuroblastoma. Through a whole genome shRNA screen, we have previously identified transcription factor AP4 (TFAP4) as a synthetic lethal gene of MYCN-amplified neuroblastoma. Silencing of TFAP4, both in vitro and in vivo inhibited the growth of MYCN-amplified neuroblastoma. TFAP4 has previously reported to repress neuronal specific genes in non-neuronal cells (Kim et al, 2006). Here, we investigate the function of TFAP4 in inhibiting differentiation of MYCN-amplified neuroblastoma. Methods: Master regulators specifically associated to MYCN amplified patients were identified by MARINA (Master Regulator Inference algorithm). TFAP4 expression was measured by real-time PCR, Western blot and immunohistochemistry. GAP43 expression was detected by Western blot. Gene expression changes after silencing of TFAP4 or retinoic acid treatment was determined by RNAseq. Results: TFAP4 was identified as a master regulator specifically in MYCN amplified neuroblastoma patients in both NCI-TARGET dataset and a published GEO dataset (Kocak et al, 2013). We determined that TFAP4 is a direct target of MYCN and TFAP4 expression was highly correlated with Stage 4, MYCN-amplified neuroblastoma in both patient datasets. Neuronal markers TUBB3 and GAP43 expression were inversely correlated with TFAP4 expression in TARGET (P = 9.77e-03 and P = 7.78e-08). Silencing TFAP4 in MYCN amplified cells induced a differentiated phenotype, as shown by markedly increased the number of neurites, and an increase in the neuronal differentiation marker GAP43. Forty hours after silencing TFAP4, we observed 415 genes that were significantly up-regulated and 457 genes down-regulated (P&amp;lt;0.01). Genes with increased expression were enriched for gene ontology terms associated with neuronal activity. Gene signature of TFAP4 also showed a strong correlation with the treatment signature of the differentiation agent retinoic acid in SKNDZ. Conclusions. We demonstrated that TFAP4 is an important downstream effector of MYCN, that inhibits the neuronal differentiatin of MYCN-amplified neuroblastoma. This may provide novel targets for the treatment of neuroblastoma. Citation Format: Shuobo Zhang, Gonzalo Lopez, Jiyang Yu, Jose Silva, Angela Kadenhe-Chiweshe, Andrea Califano, Darrell Yamashiro. TFAP4 inhibits differentiation of MYCN-amplified neuroblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2083. doi:10.1158/1538-7445.AM2015-2083

Abstract 3076: Post-transcriptional regulatory mechanisms contributing to the evolution of paclitaxel resistance in breast cancer model cell line, SKBR3

Abstract Introduction: Breast Cancer (BCa) treatments often include taxanes in the treatment regimens and resistance to these drugs is common in a clinical setting. Several mechanisms were put forth to explain the resistance. These include induction of multidrug (MDR) resistance family of transporters, differential regulation of tubulin isotypes, microRNA (miR) mediated post-transcriptional regulation and autophagy. Aims: (i) to create a panel of breast cancer cell lines from SKBR3 (Her2+ and ER-ve) with varying levels of drug resistance to paclitaxel; (ii) to profile differentially expressed miRs in these panels of cell lines to understand the evolution of drug resistance with common and unique signatures associated with each of the cell lines in the panel and (iii) to assess β-tubulin (TUBB3) expression and its regulation by miRs. Methods: Drug resistant SKBR-3 cells were generated by a stepwise increase in drug concentration to the parental cells until a maximum tolerable dose was reached. Control cells were those without any treatment (wild type, WT) and serial passage of SKBR3 cells for the same numbers of generations as in each of the drug resistant lines. Total RNA extracted from the cell lines was subjected to small RNAome profiling (with focus on miRs) using Next Generation Sequencing (Illumina Genome Analyzer IIx). Partek Genomics Suite 6.6 was used for analyzing sequencing files. Data normalized (RPKM) from triplicate experiments (three independent cell growth experiments) were adjusted for potential batch effects. We report miRs showing statistically significant differences in expression levels (&amp;gt;2-fold and FDR adjusted p&amp;lt;0.05) between WT SKBR3 and drug resistant cell line(s) with concordant profiles from triplicate sequencing experiments. Protein expression levels of β-tubulin (TUBB3) were assessed using Western blots. Results and conclusions: The resistant cell lines expressed TUBB3 (graded up-regulation with the level of resistance) as expected as well as down-regulation of hsa-miR-200c, a known miR directly regulating the expression of TUBB3. The resistant cell lines also showed up-regulation of miRs 221-3p and 99a known to modulate ER and its mediated gene regulatory pathways, consistent with the ER-ve phenotype of SKBR3 cells. Overall, 27 miRs are differentially expressed when all resistant cells are considered (irrespective of the level of resistance conferred). As expected, some of the identified miRs were reported to have a role in Epithelial-Mesenchymal Transition vis-à-vis acquisition of cancer stem cell properties. We also observed unique miRs in cells expressing graded levels of resistance and the data is subject to further analysis for potential hierarchy of programmed expression of miRs facilitating transitioning of cells from sensitive to highly drug resistant phenotype. Citation Format: Sambasivarao Damaraju, Preethi Krishnan, Marc St George, Jack Tuszynski, Carol Cass, IngSwie Goping, Olga Kovalchuk. Post-transcriptional regulatory mechanisms contributing to the evolution of paclitaxel resistance in breast cancer model cell line, SKBR3. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3076. doi:10.1158/1538-7445.AM2015-3076

Abstract 3660: Discovery of a small molecule TUBB3/βIII-tubulin modulator in lung cancer

Abstract Background: Non-small Cell Lung Cancer (NSCLC) survival rates are dismal and chemotherapy resistance is a significant clinical problem. βIII-tubulin (encoded by TUBB3 gene) is aberrantly expressed and is associated with chemoresistance and tumor aggressiveness in NSCLC (1), where it has been identified as a bona fide target for chemosensitisation (2,3). In order to understand how TUBB3/βIII-tubulin is regulated in NSCLC cells we sought to identify chemical small molecules that can modulate its expression. Methods: H460 cells expressing a TUBB3 or GAPDH promoter-luciferase reporter construct were generated and used in drug screening and promoter activity testing. To isolate modulators of TUBB3 promoter activity, a cell-based screen of libraries of diverse chemical small molecules (30K) and FDA-approved bioactives (3680) was performed. Cell viability, growth and proliferation were measured using standard methods. Cell cycle was assessed using flow cytometry, and gene and protein expression by RT-PCR and Western blotting, respectively. Microtubule morphology was assessed using immunostaining and confocal microscopy. Drug-treated clonogenic assays were used to quantitate changes in drug sensitivity. Results: Based upon their ability to modulate TUBB3 promoter activity, we identified two hit compounds, CCI01 and CCI02, as well as the bioactive compound, RITA. For all three leads we observed: 1) repression in TUBB3 promoter activity which was not a result of cell cytotoxicity; 2) no effects on cell cycle or viability, but instead cytostatic effects; 3) significantly enhanced TUBB3 expression in a time and dose-dependent manner. TUBB3 gene enhancement was translated at the protein level in CCI01 treated H460, but not in CCI02 or RITA treated cells. Additionally, CCI01 did not alter microtubule morphology but enhanced βIII-tubulin immunostaining in two independent NSCLC cell lines, H460 and H1299, compared to control. Importantly, CCI01 enhanced βIII-tubulin expression was functional and led to a significant decrease in in vitro sensitivity to DNA-damaging and tubulin-binding agents in H460 cells. Conclusion: A novel lead small molecule TUBB3/βIII-tubulin enhancer has been identified that is able to increase expression of βIII-tubulin in NSCLC and significantly reduce sensitivity to tubulin binding and DNA-damaging agents. Implications: Identification of a modulator of TUBB3/βIII-tubulin expression will provide a valuable research tool to probe βIII-tubulin regulation. (1) Kavallaris. Nature Rev Cancer, 10:194-204, 2010 (2) McCarroll et al., Cancer Res 70:4995-5003, 2010 (3) Gan et al., Cancer Res. 67:9356-9363, 2007 Citation Format: Felicity Chao Lin Kao, Tim Failes, Greg M. Arndt, Murray Norris, Maria Kavallaris. Discovery of a small molecule TUBB3/βIII-tubulin modulator in lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3660. doi:10.1158/1538-7445.AM2015-3660

EGFR and K-RAS mutations and ERCC1, TUBB3, TYMS, RRM1 and EGFR mRNA expression in non-small cell lung cancer: Correlation with clinical response to gefitinib or chemotherapy.

Personalizing medicines has refined the traditional treatments for non-small-cell lung cancer (NSCLC). In the present study, efforts towards personalizing delivery of care based on the status of EGFR and K-RAS mutations, and mRNA expression levels of ERCC1, TUBB3, TYMS, RRM1 and EGFR by choosing appropriate treatments for 52 patients with NSCLC were discussed. Among these 52 NSCLC patients, there were 14 patients treated with gefitinib. Ten patients with EGFR exon 21 point mutations or exon 19 deletions had better treatment outcomes following gefitinib treatment (71.4%). There were 38 patients treated with platinum-based chemotherapy. Docetaxel-platinum based chemotherapy was chosen as the first-line treatment when the patients had low or median ERCC1/TUBB3 expression and gemcitabine-platinum based chemotherapy was chosen when the patients had low or median ERCC1/RRM1 expression. In total, 26 cases had mRNA expression levels of ERCC1/TUBB3 or ERCC1/RRM1 that could be used to predict the treatment outcomes of chemotherapy (68.4%). The present results indicated that the mutation status of EGFR, as well as the mRNA expression levels of ERCC1, TUBB3 and RRM1, could be used as predictors of the response to gefitinib or chemotherapy.

Prognostic significance of the mRNA expression of ERCC1, RRM1, TUBB3 and TYMS genes in patients with non-small cell lung cancer.

The present study aimed to investigate the prognostic value of excision repair cross-complementing 1 (ERCC1), ribonucleotide reductase subunit M1 (RRM1), class III β-tubulin (TUBB3) and thymidylate synthase (TYMS) in patients with non-small cell lung cancer (NSCLC) receiving platinum-based adjuvant chemotherapy. The mRNA expression of these genes was assessed in 72 tumor tissue samples obtained following surgery, using multiplex branched-DNA technology. Subsequent to surgery, all 72 patients with NSCLC were treated with platinum-based chemotherapy. The expression of these five genes was analyzed and the correlation with clinical characteristics and patient survival was investigated. Among the 72 samples, the incidence rate of mRNA expression of ERCC1 was 38.9% (28/72), RRM1 was 55.6% (40/72), TUBB3 was 47.2% (34/72) and TYMS was 62.5% (45/72). The incidence rate of ERCC1 expression in adenocarcinoma (34.2%) was significantly lower than that in non-adenocarcinoma (44.1%; P<0.05). Furthermore, the incidence rates of TYMS and TUBB3 expression in the high-median differentiation tissue samples were significantly lower than those in the low differentiation tissue samples (P<0.05). When the correlation of gene expression and patient survival was analyzed, high expression of ERCC1, RRM1, TUBB3 or TYMS was found to be associated with poor prognosis (P<0.001, P=0.001, P=0.001 and P=0.001, respectively). ERCC1, RRM1, TUBB3 and TYMS are key factors involved in survival following surgical treatment in patients with NSCLC. The mRNA expression of these genes may have prognostic value for patients with NSCLC treated with platinum-based chemotherapy.

Association of expression ofβ-tubulinIIIwith neoadjuvant chemotherapy efficacy of paelitaxel in advanced esophageal carcinoma

Objective To explore the association of the expression levels ofβ-tubulinⅢ(TuBB3) in esophageal cancer with the neoadjuvant chemotherapy efficacy of paelitaxel. Methods The expression levels of TuBB3 were detected by immnohistochemistry. The association of the TuBB3 expression with the neoadjuvant chemotherapy efficacy of paelitaxel, the radical resection rate of tumor, the postoperative pathological staging and one-year survival rate was analyzed. Results The response rate of chemotherapy in the group with lower TuBB3 expression was higher than that with higher expression(68. 4% and 41. 2%).The group with lower TuBB3 also had higher radical resection rate of tumor(94. 6% and 78. 7%), lower postoperative pathological staging and higher one-year survival rate(89. 2% and 68. 8%)than that with higher expression(P< 0. 05). Conclusion TuBB3 can be a predictor for the efficacy of paelitaxel-based chemotherapy in the neoadjuvant treatment of esophageal cancer.Esophageal cancer patients with lowerl TuBB3 expression has higher radical resection rate of tumor, and more survival benefit postoperatively when treatment with paelitaxel. Key words: Esophageal carcinoma; Paelitaxel; β-tubulinⅢ; Chemotherapy