TUBB3 Expression Research Articles

High-level βIII-tubulin overexpression occurs in most head and neck cancers but is unrelated to clinical outcome.

βIII-tubulin (TUBB3) is an isotype of microtubules, which are involved in crucial cellular roles including maintenance of cell shape, intracellular transport, and mitosis. Overexpression of TUBB3 was found to be associated with poor prognosis and resistance to tubulin-binding drugs and in several solid tumors including head and neck squamous cell carcinomas (HNSCC). Considering the potential high importance of a prognostic biomarker in these cancers, this study aimed to investigate the clinical relevance of immunohistochemical TUBB3 expression in HNSCC. Tissue microarray (TMA) sections containing samples from 667 cancers of oral cavity, oro- and hypopharynx, and larynx for which follow-up data were available were analyzed for TUBB3 expression by immunohistochemistry. Over 90% of our analyzed cancers showed unequivocal cytoplasmic TUBB3 expression. Staining was considered weak in 69 (15.5%), moderate in 149 (33.5%), and strong in 188 (42.2%) of cancers. The frequent TUBB3 overexpression showed no significant correlation with pathological grading, tumor stage, nodal status, or surgical margin and had no impact on patient outcomes. Despite lacking prognostic utility in HNSCC, the remarkable high prevalence of TUBB3 expression in HNSCC emphasizes its putative relevance as a target for future drugs targeting TUBB3.

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.

Potential therapeutic genomic alterations in desmoplastic small round blue cell tumor.

11066 Background: Desmoplastic Small Round Blue Cell Tumor (DSRCT) originates from a cell with multilineage potential. A molecular hallmark of DSCRT is the EWS-WT1 reciprocal translocation. Ewing’s and DSRCT are treated similarly due to similar oncogene activation pathways and DSRCT has been represented in very limited numbers in sarcoma studies. Methods: Thirty-five DSRCT tumors were tested with a multiplatform profiling service (Caris Life Sciences, Phoenix, AZ). Specific tests performed included sequencing (NextGen), protein expression (IHC) and gene amplification (CISH or FISH). Tumor mutational load (TML) was calculated as somatic nonsynonymous missense mutations sequenced with a 592-gene panel. Molecular alterations were compared to 88 Ewing sarcomas (ES). Chi-square tests were used for comparison and statistical significance was determined as p &lt; 0.05. Results: In the 35 DSRCT tumors, high expression of TOP2A were seen in 63%, TOPO1 in 63%, PTEN in 62%, androgen receptor (AR) in 59%, EGFR in 42% of tumors; low expression of TUBB3 was seen in 44%, MGMT in 45%, TS in 48%, RRM1 in 57% and ERCC1 in 76% of tumors. When compared to ES, no significant difference was seen in protein expressions with the exception of a significantly higher overexpressson of AR in DSRCT (59% vs. 3%, p = 1.7E-10) and TUBB3 (56% vs. 29%, p = 0.03). Tumor expression of PD-L1 (Ab: SP142) was not seen in the 4 DSRCT and 10 ES tested. NextGen revealed a TP53 mutation (7%) and a FOXO3 mutation (L382fs) in DSCRT, while 6 TP53 mutations (13%), 2 APC mutations (L1129S and I1307K), 1 BRCA1(c.301+1G &gt; A) and 1 CTNNB1 (T41A) mutation were identified in ES. Tumor mutational load evaluated in the 3 DSRCT and 11 ES tumors averaged 6 and 5 mutations per megabase, respectively. Conclusions: Molecular profiling on 35 DSRCT tumors and comparison with Ewing’s sarcoma revealed low immunogenicity ( &lt; 10 Mutations/MB) and low frequency of actionable mutations including PD-L1 in both tumor types. High AR expression could present as a potential therapeutic target for DSRCT while taxanes may be more effective in Ewing’s sarcoma compared to DSCRT based on TUBB3 expression. Genomic and Molecular assessment may help determine the ideal regimen that will help achieve maximal tumor debulking.

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.

Tubulin β‐III modulates seizure activity in epilepsy

Tubulin β-III (TUBB3) is the most dynamic β-tubulin isoform expressed in neurons, and is highly expressed in the central nervous system. However, the relationship between TUBB3 and epileptic seizures has not been thoroughly investigated. The aims of this study were to investigate the expression of TUBB3 in patients with temporal lobe epilepsy and two different rat models of chronic epilepsy, and to determine the specific roles of TUBB3 in epilepsy. TUBB3 expression was upregulated in human and rat epileptic tissue. Moreover, TUBB3 expression was associated with inhibitory GABAergic neurons and the inhibitory postsynaptic scaffold protein gephyrin. TUBB3 downregulation attenuated the behavioural phenotypes of epileptic seizures during the pilocarpine-induced chronic phase of epileptic seizures and the pentylenetetrazole kindling process, whereas TUBB3 overexpression had the opposite effect. Whole-cell clamp recordings and western blotting revealed that the amplitude of GABA-A receptor-mediated miniature inhibitory postsynaptic currents and the surface expression of the GABA-A receptor were increased in rats in which TUBB3 expression was downregulated. Importantly, TUBB3 interacted with GABA-A receptor-associated protein, which is known to be involved in GABA-A receptor trafficking. These results indicate that TUBB3 plays a critical role in the regulation of epileptic seizures via GABA-A receptor trafficking, suggesting a molecular mechanism for new therapeutic strategies. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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.

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 ...

The influence of TSA and VPA on the in vitro differentiation of bone marrow mesenchymal stem cells into neuronal lineage cells: Gene expression studies.

Epigenetic mechanisms regulate the transcription of genes, which can affect the differentiation of MSCs. The aim of the current work is to determine how the histone deacetylase inhibitors TSA and VPA affect the expression of neuronal lineage genes in a culture of rat MSCs (rMSCs). We analyzed the expression of early neuron marker gene (Tubb3), mature neuron markers genes (Vacht, Th, Htr2a) and the oligodendrocyte progenitor marker gene (GalC). Moreover, changes in the gene expression after three different periods of exposure to TSA and VPA were investigated for the first time. After six days of exposition to TSA and VPA, the expression of Tubb3 and GalC decreased, while the expression of Th increased. The highest increase of VAChT expression was observed after three days of TSA and VPA treatment. A decrease in Htr2a gene expression was observed after TSA treatment and an increase was observed after VPA treatment. We also observed that TSA and VPA inhibited cell proliferation and the formation of neurospheres in the rMSCs culture. The central findings of our study are that TSA and VPA affect the expression of neuronal lineage genes in an rMSCs culture. After exposure to TSA or VPA, the expression of early neuronal gene decreases but equally the expression of mature neuron genes increases. After TSA and VPA treatment ER of the oligodendrocyte progenitor marker decreased. TSA and VPA inhibit cell proliferation and the formation of neurospheres in rMSCs culture.

Partial Reprogramming of Pluripotent Stem Cell-Derived Cardiomyocytes into Neurons

Direct reprogramming of somatic cells has been demonstrated, however, it is unknown whether electrophysiologically-active somatic cells derived from separate germ layers can be interconverted. We demonstrate that partial direct reprogramming of mesoderm-derived cardiomyocytes into neurons is feasible, generating cells exhibiting structural and electrophysiological properties of both cardiomyocytes and neurons. Human and mouse pluripotent stem cell-derived CMs (PSC-CMs) were transduced with the neurogenic transcription factors Brn2, Ascl1, Myt1l and NeuroD. We found that CMs adopted neuronal morphologies as early as day 3 post-transduction while still retaining a CM gene expression profile. At week 1 post-transduction, we found that reprogrammed CMs expressed neuronal markers such as Tuj1, Map2, and NCAM. At week 3 post-transduction, mature neuronal markers such as vGlut and synapsin were observed. With single-cell qPCR, we temporally examined CM gene expression and observed increased expression of neuronal markers Dcx, Map2, and Tubb3. Patch-clamp analysis confirmed the neuron-like electrophysiological profile of reprogrammed CMs. This study demonstrates that PSC-CMs are amenable to partial neuronal conversion, yielding a population of cells exhibiting features of both neurons and CMs.

PAX3 inhibits β-Tubulin-III expression and neuronal differentiation of neural stem cell

PAX3 functions at the nodal point in neural stem cell maintenance and differentiation. Using bioinformatics methods, we identified PAX3 as a potential regulator of β-Tubulin-III (TUBB3) gene transcription, and the results indicated that PAX3 might be involved in neural stem cell (NSC) differentiation by orchestrating the expression of cytoskeletal proteins. In the present study, we reported that PAX3 could inhibit the differentiation of NSCs and the expression of TUBB3. Further, using luciferase and electrophoretic mobility shift assays, we demonstrated that PAX3 could bind to the promoter region of TUBB3 and inhibit TUBB3 transcription. Finally, we confirmed that PAX3 could bind to the promoter region of endogenous TUBB3 in the native chromatin of NSCs. These findings indicated that PAX3 is a pivotal factor targeting various molecules during differentiation of NSCs in vitro.

Abstract PD8-04: Racial differences in the molecular landscape of breast cancer

Abstract Background:African Americans (AA) have a higher mortality associated with breast cancer (BC) when compared to Caucasians (CC).This has been attributed to diverse factors that include access to care, reproductive factors and different somatic genomic profiles. We aimed to compare the racial mutational landscape of 565 BC samples. Methods:DNA from formalin-fixed paraffin-embedded samples was sequenced using the Illumina NextSeq (Agilent SureSelect XT, 592 gene selected based on COSMIC database) and MiSeq (TruSeq, 47 gene) for mutation and gene amplification analyses. Protein expression was evaluated by Immunohistochemistry (IHC). The exome aggregation consortium database was assessed for known ethnicity associations. Results:Tumor samples from 118 AA and 447 CC female patients were included in this analysis. AA were younger (median age 56 vs58y, p0.005) and had higher proportion of triple negative BC (TNBC) (32% vs17%, p0.001). 50.3% of the samples were obtained from primary tumors and the remainder from metastatic sites. This was similar in AA and CC (48.8% vs51.8% primary tumors, pNS). The two genes with highest mutation prevalence were TP53 and PIK3CA. AA had fewer PIK3CA mutations (14.7% vs28.2%, p0.03). Within HR+/HER2+ and HR+/HER2- subtypes there was a similar trend in the number of PIK3CA mutations but it was no longer significant. The remainder mutation analysis did not differ between races. In terms of protein expression there were significant differences in the androgen receptor(AR), RRM1, EGFR and TS expression (table). AR positivity defined as ≥10% was less frequent in AA (40.0% vs60.4%, p0.0001 and when adjusted for age, p0.005) and associated with PIK3CA mutations in both AA and CC (p0.01 and p0.007). AR expression in TNBC was positive in 17.8% of CC and 5.4% of AA (pNS). Copy number variation (CNV) data assessed by NextGen revealed significantly higher gene copy number in AA compared to CC in CCND1 (16% vs4%, p0.04), FGF19 (16% vs1.3%, p0.01) and FGF4 (16% vs3%, p0.02). When only TNBC was considered, RRM1, TOPO1 and TUBB3 expression was significantly higher in AA than CC (table) and there were no differences in the mutational analyses. Evaluation of other BC subtypes (HER2, HR positive) is currently underway. Conclusions:In this large cohort of AA who underwent genomic profiling there were relatively few differences in the mutation analysis compared to CC. The only significant difference seen was the lower number of PIK3CA mutations in AA, which had been previously reported in a cohort of 105 AA from the TCGA data (Keenan et al. JCO 2015). Protein expression by IHC revealed lower expression of AR in AA, even after adjustment for age, which could have therapeutic implications. Some of the racial differences found in the molecular landscape of BC including PIK3CA mutations, AR, EGFR expression and CNV may contribute to a more aggressive tumor biology in AA. Protein expression by IHCBCTNBCAA %CC %pAA %CC %pHER2/neu912ns00nsAR40600.005180.07ER44620.0000nsPR35460.0900nsEGFR30160.036050nsERCC133460.063626nsMGMT6163ns6571nsPD-150340.066548nsPD-L106ns011nsPGP76ns1015nsPTEN5658ns4247nsRRM139270.0459270.01TLE350590.104357nsTOP2A7164ns86470.08TOPO16358ns69460.03TS44300.016349nsTUBB355410.0677510.03 Citation Format: Lynce F, Xiu J, Nunes MR, Swain SM, Gatalica Z, Isaacs C, Pohlmann P. Racial differences in the molecular landscape of breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD8-04.

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.

Molecular variances between rectal and left-sided colon cancers.

522 Background: Recent analysis of CALGB 80405 showed that left sided colon tumors (LT) respond differently to biologics compared with right-sided tumors, likely due to molecular differences. Molecular variations between LT and rectal tumors remain undefined. Herein, we report our exploration of these variations. Methods: Tumors with origins clearly defined as splenic flexure to descending colon (SFT), sigmoid colon (SgT), or rectum (RT) were included. Protein expression, gene amplification and NextGen sequencing was tested. Microsatellite instability (MSI) was measured by PCR. Tumor mutational load (TML) was calculated using only somatic nonsynonymous missense mutations. Chi-square tests were used for comparative analyses. Results: In total, 1,457 primary tumors (SFT 125; SgT 460, and RT 872) were examined. When compared with SFT, RT had a higher frequency of TP53 (71% vs. 57%, p = 0.03) and APC (66% vs. 49%, p = 0.01); a lower frequency of PIK3CA (11% vs. 22%, p = 0.02), BRAF (3% vs. 15% p = 0.0001), GNAS (0.9% vs. 4%, p = 0.04), HNF1A (0.7% vs. 5%, p = 0.01), and CTNNB1(0.3% vs. 4%, p = 0.003); and a higher expression of TOPO1 (52% vs. 31%, p = 0.0001), ERCC1 (29% vs. 15%, p = 0.03), and MGMT (64% vs. 53%, p = 0.048). When compared with SgT, RT had higher expression of TLE3 (33% vs. 23%, p = 0.007), TOPO1 (52% vs. 35%, p &lt; 0.001), TUBB3 (41% vs. 28%, p = 0.003), and MGMT (64% vs. 54%, p = 0.003). There were no differences between SFT, SgT, and RT in the frequency of PD-L1 expression (5%, 2%, and 2%) on tumor cells, PD-1 expression on tumor-infiltrating lymphocytes (54%, 42%, and 42%), or Her-2 expression (1%, 2%, and 3%) and amplification (3%, 3%, and 5%). MSI was seen in 7% of SFT, 4% of SgT, and 0.7% of RT (total LT vs. RT, p = 0.01). Mean TML was 23, 6.5, and 7 mutations (mut)/MB (332 tumors), and the portion of tumors carrying a TML of &gt; 17mut/MB was 9%, 1.6%, and 4% for SFT, SgT, and RT, respectively. In all 3 cohorts, a TML &gt; 17 mut/MB was highly concordant with MSI. There was a correlation between PD-1 and TML in RT (p = 0.04) but not in SFT or SgT. There were no correlations between PD-L1 and TML. Conclusions: Tumors arising in the rectum may carry genetic alterations that are distinct from LT. A better understanding of disease biology may help to identify therapeutic targets and advance precision medicine

Molecular characterization of squamous cell carcinoma of the anal canal (SCCA).

538 Background: Nivolumab has shown promising results in SCCA patients. The majority of SCCA cases have been linked to prior human papillomavirus (HPV) infection. However, HPV negative tumors are frequently TP53 mutated and often resistant to therapy. Molecular characteristics of SCCA are largely undefined. Here we explored the underlying biology of SCCA and the differences between TP53-wild type ( TP53-WT) and TP53-mutated ( TP53-MT) tumors. Methods: SCCA specimens underwent multiplatform testing with protein expression (IHC), gene amplification (ISH), and sequencing (NGS). Tumor mutational burden (TMB) was calculated using only somatic nonsynonymous missense mutations. Chi-square tests were used for comparative analyses. Results: In total, 253 tumors were studied. The most frequently mutated genes included PIK3CA (24%), BRCA2 (14%), FBXW7 (12.4%), TP53 (9.7%), and PTEN (8.9%). In a subset of 23 tumors subjected to Illumina NextSeq (592 gene) testing, the most common mutations were NOTCH2 (30%), NOTCH1 (27.3%), POLE (21.7%) , TSC2 (17.4%), PTEN (14.3%), BRAF (13.6%), BRCA2 (13.0%), PIK3CA (13.0%), and FBXW7 (9.5%). Tumors frequently expressed MRP1 (97.6%), EGFR (92.7%), TOP2A (88.5%), TOPO1 (69.5%), MGMT (67.8%), and RRM1 (59.9%). Expression of PD-1 was seen in 55.8% (24/43) of tumors, and PD-L1 in 15.4% (9/34). HER2 was amplified in 2% (3/147) of samples, which has not been previously described in SCCA. When compared with TP53-WT (n = 93) tumors, T P53-MT (n = 10) had higher rates of BRAF (22% vs. 1%, p &lt; 0.001) and RB1 mutations (44% vs. 0%, p &lt; 0.001), whereas TP53-WT had higher expression of TOPO1 (76% vs. 40%, p = 0.01) and TUBB3 (19% vs. 50%, p = 0.02). There were no differences between the two groups in the frequency of PD-1 or PD-L1 expression. Mean TMB was 8.6 mutations/megabase and, using a TMB cut-off &gt; 17 mutations/megabase to define high vs. low TMB, 6.7% of tumors were TMB-High. High TMB did not correlate with PD-1 (p = 0.50) or PD-L1 status (p = 0.52). Conclusions: Molecular profiling differences between TP53-MT and TP53-WT SCCA indicate different carcinogenic pathways and biology, which may influence response to therapy. Low frequency mutations in several druggable genes may provide therapeutic opportunities for patients with SCCA.

Clinical significance of UGT1A1 polymorphism and expression of ERCC1, BRCA1, TYMS, RRM1, TUBB3, STMN1 and TOP2A in gastric cancer

BackgroundIndividualized therapeutic regimen is a recently intensively pursued approach for targeting diseases, in which the search for biomarkers was considered the first and most important. Thus, the goal of this study was to investigate whether the UGT1A1, ERCC1, BRCA1, TYMS, RRM1, TUBB3, STMN1 and TOP2A genes are underlying biomarkers for gastric cancer, which, to our knowledge, has not been performed.MethodsNinety-eight tissue specimens were collected from gastric cancer patients between May 2012 and March 2015. A multiplex branched DNA liquidchip technology was used for measuring the mRNA expressions of ERCC1, BRCA1, TYMS, RRM1, TUBB3, STMN1 and TOP2A. Direct sequencing was performed for determination of UGT1A1 polymorphisms. Furthermore, correlations between gene expressions, polymorphisms and clinicopathological characteristics were investigated.ResultsThe expressions of TYMS, TUBB3 and STMN1 were significantly associated with the clinicopathological characteristics of age, gender and family history of gastric cancer, but not with differentiation, growth patterns, metastasis and TNM staging in patients with gastric cancer. No clinical characteristics were correlated with the expressions of ERCC1, BRCA1, RRM1 and TOP2A. Additionally, patients carrying G allele at −211 of UGT1A1 were predisposed to developing tubular adenocarcinoma, while individuals carrying 6TAA or G allele respectively at *28 or −3156 of UGT1A1 tended to have a local invasion.ConclusionsThe UGT1A1 polymorphism may be useful to screen the risk population of gastric cancer, while TYMS, TUBB3 and STMN1 may be potential biomarkers for prognosis and chemotherapy guidance.

Suppression of PTEN/AKT signaling decreases the expression of TUBB3 and TOP2A with subsequent inhibition of cell growth and induction of apoptosis in human breast cancer MCF-7 cells via ATP and caspase-3 signaling pathways.

The aim of the present study was to evaluate the effects of PTEN/AKT signaling on TUBB3 and TOP2A expression and on the subsequent cell growth of human breast cancer MCF-7 cells. We found that the disease-free survival (DFS) and overall survival (OS) of breast cancer patients with TUBB3‑positive tumors were lower than these rates in the patients with TUBB3-negative tumors. Meanwhile, DFS and OS of breast cancer patients with TOP2A-positive tumors were also lower than these rates in patients with TOP2A-negative tumors. Suppression of PTEN reduced the protein expression of TUBB3 and TOP2A in MCF-7 cells. Suppression of PTEN also reduced cell proliferation and induced apoptosis and caspase-3 activity in MCF-7 cells. Moreover, an increase in ATP also reduced TUBB3 and TOP2A protein expression, reduced cell proliferation and induced apoptosis and caspase-3 activity in the MCF-7 cells following suppression of PTEN. Suppression of phosphorylation-AKT (p-AKT) reduced the protein expression of TUBB3 and TOP2A in the MCF-7 cells. Suppression of p-AKT also reduced cell proliferation and induced apoptosis and caspase-3 activity in the MCF-7 cells. Then, ATP also reduced TUBB3 and TOP2A protein expression, reduced cell proliferation and induced apoptosis and caspase-3 activity in MCF-7 cells following suppression of p-AKT. These results suggest that PTEN/AKT signaling affects the expression of TUBB3 and TOP2A reducing cell growth and inducing apoptosis of human breast cancer MCF-7 cells through ATP and caspase-3 signaling pathways. TUBB3 and TOP2A may be promising prognostic markers for the efficacy of adjuvant cisplatin-based chemotherapy.

PROGNOSTIC SIGNIFICANCE OF MONORESISTANCE GENE EXPRESSION IN TUMORS OF PATIENTS WITH NON-SMALL CELL LUNG CANCER AFTER PREOPERATIVE CHEMOTHERAPY

A significant role in the development of lung tumor resistance to drug therapy play so called mono resistance genes that determine resistance/sensitivity of tumor cells to distinct chemotherapeutic agents, they include BRCA1, RRM1, ERCC1, TOP1, TOP2a, TUBB3, TYMS, ABCC5 genes. It is shown that the use of these biomarkers as a criteria to appoint the adequate postoperative chemotherapy is associated with the forecast. However in the course of neoadjuvant chemotherapy change the expression profile of these genes can occur, which can lead to misinterpretation of results. In this regard perspective is the development of new relevant prognostic factors, which take into account occurred molecular genetic alterations in lung tumors after preoperative therapy. We presented the results of the combined treatment of 52 patients with NSCLC IIA - IIIB stage, based on the analysis of chemotherapy resistance/sensitivity gene expression for appointment the suitable drugs. It was found that the lack of expression of ERCC1 gene in lung tumor derived after neoadjuvant chemotherapy is associated with high rates of disease-free survival (P. = 0,00913). Low level expression of RRM1 gene (less than 0.5) is also associated with high rates of patient's survival (log-rank test P. = 0,01808), while a high level is a marker of poor prognosis. It is found that the combination of enhanced expression (&gt;0.5) of any two or three genes: RRM1, ABCC5, TYMS, are the indicators of poor disease-free survival. We have shown that expression of ERCC1 gene is the independent prognostic factor. At zero ERCC1 expression in lung tumors, regardless of high or low level of expression of RRM1, ABCC5, TYMS genes, a high recurrence-free survival is observed. Thus, the data suggest the prognostic significance of the expression of ERCC1, TUBB3, RRM1, ABCC5 and TYMS genes in the lung tumor after neoadjuvant chemotherapy.

P2.04-034 SPARC/β-Tubulin III Expressions for Clinical Outcomes of ESCC Patients Receiving Nab-Paclitaxel plus DDP Neoadjuvant Chemotherapy: Topic: Esophageal Cancer and Other Malignancies

To assess the role of secreted protein, acidic and rich in cysteine (SPARC) and β-tubulin III (TUBB3) in predicting the clinical outcomes of Chinese ESCC patients receiving nab-paclitaxel plus cisplatin neoadjuvant chemotherapy. The clinical data and tumor biopsies prior treatment from 35 stage II-III ESCC patients receiving nab-paclitaxel plus cisplatin from July 2011 to December 2012 were retrospectively collected and analyzed for SPARC and TUBB3 expressions by immunohistochemistry. The relationships between expressions of SPARC/TUBB3 and response or survival were determined by statistical analysis. All patients received two cycles neoadjuvant CT. 30/35 patients accepted surgery (85.7%). 24/30 patients (80.0%) received adjuvant CT, and 7 patients (23.3%) among them received adjuvant radiotherapy. 30 had R0 resection (100%). Pathological complete response (pCR) was achieved in 4 patients (13.3%). Near pCR (microfoci of tumor cells on the primary tumor without lymph nodal metastases) was acquired in 2 patients (6.7%). Down-staging was observed in 19 of 30 patients (63.3%). SPARC and TUBB3 statuses were evaluated in 22 patients. There was no significant correlation between TUBB3/SPARC status and clinical response. The median PFS in TUBB3 negative staining samples was longer than those in TUBB3 positive staining samples, although there was no statistical difference of OS between two groups. The median PFS/OS in SPARC negative staining samples and SPARC positive staining samples have no statistical difference. Not applicable

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

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

BIX01294, an inhibitor of histone methyltransferase, induces autophagy-dependent differentiation of glioma stem-like cells.

Glioblastoma (GBM) contains rare glioma stem-like cells (GSCs) with capacities of self-renewal, multi-lineage differentiation, and resistance to conventional therapy. Drug-induced differentiation of GSCs is recognized as a promising approach of anti-glioma therapy. Accumulating evidence suggests that unique properties of stem cells depend on autophagy. Here we demonstrate that BIX01294, an inhibitor of a G9a histone methyltransferase (introducing H3K9me2 and H3K27me3 repressive marks) triggers autophagy in human glioma cells. Pharmacological or genetic inhibition of autophagy decreased LC3-II accumulation and GFP-LC3 punctation in BIX01294-treated cells. GSCs-enriched spheres originating from glioma cells and GBM patient-derived cultures express lower levels of autophagy related (ATG) genes than the parental glioma cell cultures. Typical differentiation inducers that upregulate neuronal and astrocytic markers in sphere cultures, increase the level of ATG mRNAs. G9a binds to the promoters of autophagy (LC3B, WIPI1) and differentiation-related (GFAP, TUBB3) genes in GSCs. Higher H3K4me3 (an activation mark) and lower H3K9me2 (the repressive mark) levels at the promoters of studied genes were detected in serum-differentiated cells than in sphere cultures. BIX01294 treatment upregulates the expression of autophagy and differentiation-related genes in GSCs. Pharmacological inhibition of autophagy decreases GFAP and TUBB3 expression in BIX01294-treated GSCs suggesting that BIX01294-induced differentiation of GSCs is autophagy-dependent.