TUBB3 Expression Research Articles

Randomized phase II trial of weekly ixabepilone with or without biweekly bevacizumab for platinum-resistant or refractory ovarian/fallopian tube/primary peritoneal cancer

<h3>Objectives:</h3> Ixabepilone is a microtubule-stabilizing agent that may retain activity in paclitaxel-treated patients. The goal of this multicenter randomized phase II study was to assess the activity and safety of ixabepilone with bevacizumab compared to ixabepilone alone in patients with platinum-resistant/refractory ovarian, fallopian tube, or primary peritoneal cancer. An exploratory objective was to examine the role of prior treatment with bevacizumab and tumor expression of class III ß-tubulin (TUBB3) by immunohistochemistry as a predictive biomarker. <h3>Methods:</h3> Participants were randomly assigned to receive ixabepilone 20 mg/m² days 1, 8, 15 with (IXA+BEV) or without (IXA) bevacizumab 10 mg/kg days 1, 15 every 28 days. Patients were stratified by receipt of prior BEV. The primary endpoint was progression-free survival (PFS). Overall survival (OS), safety, and response rates served as secondary endpoints. <h3>Results:</h3> A total of 78 patients were randomized from March 2017-July 2020. Among 76 evaluable patients who received IXA+BEV (n=39) compared to IXA (n=37), the objective response rate was 33% (n=13) versus 8% (n=3) (<i>P</i>=0.004), with clinical benefit durable at 6 months in 37% (n=14) and 3% (n=1) (<i>P</i><0.001). The addition of BEV significantly improved both PFS (median 5.5 vs 2.2 months, HR=0.33, 95% CI 0.19-0.55, <i>P</i><0.001) [Fig. 1a] and OS (median 10.0 vs 6.0 months, HR=0.52, 95% CI 0.31-0.87, <i>P</i>=0.006) [Fig. 1b]. Both regimens were well-tolerated. TUBB3 expression did not predict response in either arm. Subgroup analyses revealed minimal effect of prior BEV on response to IXA+BEV [Fig 1c/d]. <h3>Conclusions:</h3> IXA+BEV is a well-tolerated, effective combination for treatment of platinum/taxane-resistant/refractory ovarian cancer that extends both PFS/OS relative to IXA monotherapy. Prior receipt of BEV should not preclude use of IXA+BEV. TUBB3 is not a predictive biomarker for response to IXA+BEV.

Exosomal TUBB3 mRNA expression of metastatic castration-resistant prostate cancer patients: Association with patient outcome under abiraterone.

BackgroundTo use ddPCR to quantify plasma exosomal class III β‐tubulin (βIII‐tubulin, TUBB3, encoded by the TUBB3 gene) mRNA expression in metastatic castration‐resistant prostate cancer (mCRPC) patients, and study the association of this expression with abiraterone efficacy.MethodsBlood samples were prospectively collected from 52 mCRPC patients using abiraterone as first‐line therapy to measure plasma exosomal TUBB3 mRNA expression value before the initiation of abiraterone. Study endpoints were PSA response rate, PSA‐progression‐free survival (PSA‐PFS), and overall survival (OS, from CRPC to death).ResultsPatients with positive exosomal TUBB3 expression showed shorter PSA‐PFS (negative TUBB3 vs. positive TUBB3: 11.0 vs. 7.9 months; p = 0.014). Further analysis demonstrated that patients with strongly positive exosomal TUBB3 (>20 copies/20 µl) was associated with even shorter PSA‐PFS (negative TUBB3 vs. positive TUBB3 [<20 copies/20 µl] vs. strongly positive TUBB3 [>20 copies/20 µl]: 11.0 vs. 8.3 vs. 3.6 months, p = 0.005). In multivariate analyzes, TUBB3 (+) (HR: 2.114, p = 0.033) and ECOG score >2 (HR: 3.039, p = 0.006) were independent prognosticators of poor PSA‐PFS. PSA response and OS did not present significant differences.ConclusionThe exosomal TUBB3 mRNA expression level is associated with poor PSA‐PFS of abiraterone in mCRPC patients. The detection of exosomal TUBB3 can be valuable in their management.

The combination therapy of targeting both paclitaxel and Dendrophthoe pentandra leaves extract nanoparticles for improvement breast cancer treatment efficacy by reducing TUBB3 and MAP4 expressions.

The aim of this study is to investigate the combination treatments of paclitaxel and chitosan-Dendrophthoe pentandra leaves extract nanoparticles (NPDP) on MCF-7 breast cancer cells. Chitosan-NPDP nanoparticles were characterized by Fourier-transform infrared (FTIR), scanning electron microscopy (SEM), and assessed by using immunofluorescence microscopy. MCF-7 cells are cultured and divided into six groups: group 1 was a negative control (without paclitaxel or NPDP); group 2 was treated with paclitaxel alone; groups 3-5 were treated with NPDP (2, 4, and 8 mg/mL, respectively) and group 6 was treated only by 8 mg/mL of chitosan-NPDP nanoparticles. The proliferation and cell cycle were analyzed by flow cytometry and the expression of TUBB3 and MAP4 were assessed by immunofluorescence microscopy. The combinations of paclitaxel-NPDP significantly inhibit proliferation of cells (P<0.001) and it is able to induce G2/M cell cycle arrest (P<0.001). The combination of paclitaxel-NPDP significantly decreases the expressions of TUBB3 (P<0.001) and MAP4 (P<0.001) in MCF-7 cells. These results indicate that the combination of NPDP nanoparticles could reduce the expressions of TUBB3 and MAP4. This research may provide possible sources of new therapy for NPDP.

Umbilical Cord Blood Cells Express mRNA for Neuronal Genes MAP2 and NEFL.

Hematopoietic tissue-derived cells including stem cells have been shown to differentiate into or fuse with solid organ tissue-specific cells. Among hematopoietic tissue cells, umbilical cord blood (UCB)-derived stem cells have the advantage of a higher engraftment potential. It was the purpose of this study to gather evidence on a molecular level of UCB-derived cells and stem cells generating or fusing with solid-organ tissue specific cells such as CNS-specific cells as a model for potential treatment of injured tissue.Fresh UCB was obtained by standard UCB banking procedures. Apheresis-derived mononuclear cells (adult MNC) were obtained from normal stem cells donors who underwent a 4-day G-CSF mobilization treatment. UCB cells (4 individual UCB harvests) and adult MNC (4 individuals) were selected for CD133/1+ or CD14+ or CD3+ cells by immunomagnetobead separation (autoMACS; Miltenyi Biotec). Each specimen contained between 8 and 20 × 106 selected cells at a purity of >90%.Real-time PCR was performed on the ABI Prism 7900 using the Assays-on-Demand for various neuronal genes such as Neurofilament (NEFL), Microtubule-associated protein 2 (MAP2), βTubulin 3 (TUBB3) and Nestin (NES), and the cylophilin Pre-Developed Assay Reagent (Applied Biosystems) without multiplexing. The ddCt method was used to calculate fold-difference in neuronal gene expression in the cell specimens relative to Stratagene's normal lymph node RNA.Neuronal cell-specific NEFL and MAP2 expression was found in both, UCB and adult MNC, but not TUBB3 or NES expression. The MAP2 expression was in favor of CD133−, CD14− and CD3− selected cells, whereas NEFL expression was significantly higher in CD3+ selected cells as compared with CD3− cells in both, UCB cells and adult MNC. [Display omitted] These preliminary results indicate that UCB and mobilized adult blood contain cell subsets that express mRNA for neuronal cell specific genes. NEFL expression seems to be higher in CD3-selected cells, whereas MAP2 and NEFL gene expression in progenitor cell subsets (CD133+) was found to be less.As shown by us in a clinical sex-mismatched UCB transplant setting and by others hematopoietic tissue-derived neuronal-like cells are identified in CNS tissue. Circulating cells expressing neuronal-specific genes could contribute to homeostasis of CNS tissue as known for the hematopoietic tissue.

Airway basal stem cells generate distinct subpopulations of PNECs.

SUMMARYPulmonary neuroendocrine cells (PNECs) have crucial roles in airway physiology and immunity by producing bioactive amines and neuropeptides (NPs). A variety of human diseases exhibit PNEC hyperplasia. Given accumulated evidence that PNECs represent a heterogenous population of cells, we investigate how PNECs differ, whether the heterogeneity is similarly present in mouse and human cells, and whether specific disease involves discrete PNECs. Herein, we identify three distinct types of PNECs in human and mouse airways based on single and double positivity for TUBB3 and the established NP markers. We show that the three PNEC types exhibit significant differences in NP expression, homeostatic turnover, and response to injury and disease. We provide evidence that these differences parallel their distinct cell of origin from basal stem cells (BSCs) or other airway epithelial progenitors.

TUBB3 is associated with PTEN, neuroendocrine differentiation, and castration resistance in prostate cancer

BackgroundTubulin-β3 encoded by the Tubulin-β3 (TUBB3) gene is a microtubule protein. Previous studies have shown that TUBB3 expression is upregulated in castration-resistant prostate cancer (CaP) and is involved in taxane resistance. However, the biological mechanism of TUBB3 involvement in the progression to castration-resistant CaP is not fully elucidated. This study aimed to analyze the expression and function of TUBB3 in localized and metastatic CaP. MethodsTUBB3 expression was determined using immunohistochemistry in localized and metastatic CaP. We also investigated the association between TUBB3, phosphatase and tensin homolog (PTEN), and neuroendocrine differentiation and examined the involvement of TUBB3 in new antiandrogen drugs (enzalutamide and apalutamide) resistance in metastatic CaP. ResultsIn 155 cases of localized CaP, immunohistochemistry showed that 5 (3.2%) of the CaP cases were positive for tubulin-β3. Kaplan–Meier analysis showed that high expression of tubulin-β3 was associated with poor prostate-specific antigen recurrence-free survival after radical prostatectomy. In 57 cases of metastatic CaP, immunohistochemistry showed that 14 (25%) cases were positive for tubulin-β3. Tubulin-β3 expression was higher in metastatic CaP than in localized CaP. High tubulin-β3 expression was correlated with negative PTEN expression. TUBB3 expression was increased in neuroendocrine CaP based on several public databases. PTEN knockout decreased the sensitivity to enzalutamide and apalutamide in 22Rv-1 cells. TUBB3 knockdown reversed the sensitivity to enzalutamide and apalutamide in PTEN-CRISPR 22Rv-1 cells. High expression of tubulin-β3 and negative expression of PTEN were significantly associated with poor overall survival in metastatic CaP treated with androgen deprivation therapy. ConclusionsThese results suggest that TUBB3 may be a useful predictive biomarker for survival and play an essential role in antiandrogen resistance in CaP.

Optic Nerve Head and Retinal Abnormalities Associated with Congenital Fibrosis of the Extraocular Muscles.

Congenital fibrosis of the extraocular muscles (CFEOM) is a congenital cranial dysinnervation disorder caused by developmental abnormalities affecting cranial nerves/nuclei innervating the extraocular muscles. Autosomal dominant CFEOM arises from heterozygous missense mutations of KIF21A or TUBB3. Although spatiotemporal expression studies have shown KIF21A and TUBB3 expression in developing retinal ganglion cells, it is unclear whether dysinnervation extends beyond the oculomotor system. We aimed to investigate whether dysinnervation extends to the visual system by performing high-resolution optical coherence tomography (OCT) scans characterizing retinal ganglion cells within the optic nerve head and retina. Sixteen patients with CFEOM were screened for mutations in KIF21A, TUBB3, and TUBB2B. Six patients had apparent optic nerve hypoplasia. OCT showed neuro-retinal rim loss. Disc diameter, rim width, rim area, and peripapillary nerve fiber layer thickness were significantly reduced in CFEOM patients compared to controls (p < 0.005). Situs inversus of retinal vessels was seen in five patients. Our study provides evidence of structural optic nerve and retinal changes in CFEOM. We show for the first time that there are widespread retinal changes beyond the retinal ganglion cells in patients with CFEOM. This study shows that the phenotype in CFEOM extends beyond the motor nerves.

Response of Human Retinal Pigment Epithelial Cells to the Effect of the Conditioned Media of Newt Retinal Regenerates

The development of the retina and the signaling pathways involved in this process are conserved among vertebrates. Therefore, it was hypothesized that active factors stimulating the retinal regeneration in newts can cause transdifferentiation of human retinal pigment epithelial (RPE) cells. To verify this, we studied the effect of conditioned media (CM) obtained from in vitro regenerating retinas of newts on the phenotype, expression of a number of genes, and localization of cell proteins of human RPE cells of the ARPE-19 line. Within 120 h of cultivation, a change in the morphology of human RPE cells was observed, which manifested in a change in shape and an increase in cell size, which was accompanied by a decrease in proliferative activity. 24 hours after exposure to CM, ARPE-19 showed a short-term increase in the expression of TUBB3 (a panneuronal marker) and a decrease in the expression of BMP2 and BMP4 (neural diffe-rentiation inhibitors). Using immunocytochemical (ICC) methods, we observed an increase in the intensity of staining of cells with antibodies to βIII-tubulin protein and a decrease in the intensity of staining with antibodies to the Cx43 protein of gap junctions (compared to the control) during the period between 24 and 72 h after exposure. At the same time, we observed a decrease in the levels of mRNA expression of the RPE markers OTX2 and KRT18, as well as the KLF4 gene, which is responsible for proliferation and differentiation. Additionally, the shift in the distribution of β-catenin to cytoplasmic localization was recorded using the ICC method. To exclude mesenchymal (and osteogenic in particular) differentiation under the effect of CM, we studied expression of the SPP1 and RUNX2 genes by qPCR. No SPP1 transcription was found in ARPE-19 cells in the control and under the effect of CM, and a decrease in the expression level of RUNX2 mRNA under the effect of CM was observed. Therefore, an attempt to shift to neuronal differentiation was observed in ARPE-19 cells during the period between 24 and 72 h after exposure to the CM of newt retinal regenerates, as indicated by an increase in transcription and translation of βIII-tubulin, a weakening of intercellular adhesion, and a decrease in the expression of inhibitors of neural differentiation BMP2 and BMP4, as well as RPE markers OTX2 and KRT18. However, after that, due to the short-term effect of CM, during the period between 72 and 120 h after exposure, the cells returned to the initial differentiation, as evidenced by a decrease in the expression of neuronal differentiation markers (NES, TUBB3, PAX6) and an increase in the expression of RPE markers (OTX2, MITF).

In vitro investigation of growth factors including MGF and IGF-1 in neural stem cell activation, proliferation, and migration

Neurogenesis is mainly activated after damage in adult tissues. This destruction activates the neural stem cells (NSCs) by exiting from a quiescent state and initiating proliferation, differentiation, and migration towards the damaged area. Although studies have investigated to clarify the process of NSC biology and neurogenesis, there are still significant artifacts in understanding the primary mechanism. It is known that only a small percentage of NSC become neurons and integrate into the brain tissue after this process. The significant proportion differentiates to become either astrocytes or oligodendrocytes. Furthermore, the quiescent stem cells in the niche are mainly activated by the stimuli affect. In recent years, many studies have been conducted with varying hormones, some of which might provide neuro-stimulation effect and/or involved in the regeneration of the brain tissue and/or neuroprotection from traumatic or ischemic pathologies, including Insulin-like growth factor 1 (IGF-1), Mechano Growth Factor (MGF), Basic Fibroblast Growth Factor (FGF-2), Erythropoietin (EPO), Epidermal Growth Factor (EGF), Nerve Growth Factor (NGF) and Brain-Derived Neurotrophic Factor (BDNF).In this study, we examined the effects of FGF-2, MGF, IGF-1, EPO, EGF, NGF, and BDNF alone or with various combinations on rat hippocampal NSC by tracking the changes in the expression of Nestin, GFAP, TUBB3, and DCX genes during 24 h (h), 72 h and 168 h time frame. The apoptosis analysis revealed that FGF-2 and FGF-2 coupled growth factors effectively protect NSCs against apoptosis, whereas MGF coupled growth factors failed in this protection. The cell cycle analysis demonstrated that these growth factors had accumulated the NSCs exit from the quiescent phase to the Mitosis phase, mostly without being long in the Synthesis Phase. Neurosphere sizes were increased with MGF, signifying MGF being effective in neural progenitor cells. The combined use of MGF with FGF-2 was more effective in postmitotic neurons than MGF alone.We have comparatively demonstrated the effect of cytokines alone and combined administration on activation, proliferation, and migration of NSCs. Although many issues are still waiting to be investigated in adult neurogenesis, neural regeneration, and adult neural stem cell biology, the results provide vital resources to the researchers that are interested in the varying effect of growth factor on NSC.

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.

Individualized chemotherapy guided by the expression of ERCC1, RRM1, TUBB3, TYMS and TOP2A genes versus classic chemotherapy in the treatment of breast cancer: A comparative effectiveness study.

ERCC1, RRM1, TUBB3, TYMS and TOP2A genes have been shown to be associated with drug resistance in various types of tumors; however, their roles in breast cancer chemotherapy have not been fully validated. In the present study, 140 well-matched patients with breast cancer, comprising 70 patients receiving individualized chemotherapy and 70 receiving classic chemotherapy, were analyzed. In the individualized chemotherapy group, the mRNA expression levels of ERCC1, RRM1, TUBB3, TYMS and TOP2A in breast cancer tissues were measured using multiplex branched DNA liquidchip technology prior to chemotherapy; an individualized chemotherapy regimen was developed for each patient according to the results. As a control, patients in the classic chemotherapy group received a docetaxel + epirubicin + cyclophosphamide regimen. Survival analyses were performed using the Kaplan-Meier method. The prognostic factors for disease-free survival (DFS) and overall survival (OS) in the patients were identified via Cox's proportional hazards regression model. Adverse reactions were evaluated according to the National Cancer Institute Common Toxicity Criteria 4. Compared with the classic chemotherapy group, the DFS and OS of the individualized chemotherapy group were significantly longer (DFS, 77.4 vs. 67.1 months, P=0.039; OS, 81.4 vs. 75.4 months, P=0.031), and the incidence of grade 2 or 3 palpitations and chest tightness was lower (12.9 vs. 27.1%, P=0.035). The chemotherapy strategy guided by genetic detection was an independent protection factor for DFS [hazard ratio (HR)=0.389, 95% confidence interval (CI): 0.153, 0.989, P=0.047], but not an independent protection factor for OS (HR=0.340, 95% CI: 0.107, 1.078, P=0.067). The results indicate that the combined detection of ERCC1, RRM1, TUBB3, TYMS and TOP2A gene expression and use of the results to guide individualized chemotherapy can improve treatment efficacy and reduce unnecessary toxicity.

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.

Radiation-Related Deregulation of TUBB3 and BRCA1/2 and Risk of Secondary Lung Cancer in Women With Breast Cancer

IntroductionBreast cancer survivors are at increased risk of developing unrelated primary cancers, particularly lung cancer. Evidence indicates that sex hormones as well as a deregulation of DNA-repair pathways may contribute to lung cancer onset. We investigated whether the hormone status and expression of markers involved in DNA repair (BRCA1/2, ERCC1, and P53R2), synthesis (TS and RRM1), and cell division (TUBB3) might be linked to lung cancer risk. Patients and MethodsThirty-seven breast cancer survivors with unrelated lung cancer and 84 control subjects comprising women with breast cancer (42/84) or lung cancer (42/84) were enrolled. Immunohistochemistry on tumor tissue was performed. Geometric mean ratio was used to assess the association of marker levels with patient groups. ResultsEstrogen receptor was expressed in approximately 90% of the breast cancer group but was negative in the majority of the lung cancer group, a result similar to the lung cancer control group. Likewise, ER isoform β was weakly expressed in the lung cancer group. Protein analysis of breast cancer versus control had a significantly lower expression of BRCA1, P53R2, and TUBB3. Likewise, a BRCA1 reduction was observed in the lung cancer group concomitant with a BRCA2 increase. Furthermore, BRCA2 and TUBB3 increased in ipsilateral lung cancer in women who had previously received radiotherapy for breast cancer. ConclusionThe decrease of DNA-repair proteins in breast cancer could make these women more susceptible to therapy-related cancer. The increase of BRCA2 and TUBB3 in lung cancer from patients who previously received radiotherapy for breast cancer might reflect a tissue response to exposure to ionizing radiation.

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

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

Ex Vivo Radiation Leads to Opposing Neurite Growth in Whole Ganglia vs Dissociated Cultured Pelvic Neurons

BackgroundProstatic radiation therapy (RT) often causes erectile dysfunction (ED) and the mechanisms governing RT-induced ED are unclear with a lack of therapeutic strategies. AimTo determine the effects of ex vivo RT on major pelvic ganglion (MPG) neuron survival, and neurite growth in whole vs dissociated culture. MethodsMPGs were removed and irradiated (0 or 8 Gy) from male Sprague Dawley rats. For dissociated culture, MPG neurons were digested in collagenase/dispase and cultured on coverslips. Immunofluorescent staining for beta-tubulin III (TUBB3; neuron marker), neuronal nitric oxide synthase (nNOS; nitrergic marker), tyrosine hydroxylase (TH; sympathetic marker), and terminal deoxynucleotidyl transferase dUTP nick end labeling assessed neurite length, branching, autonomic neuron density, and apoptosis. For whole organ culture, MPGs were grown in Matrigel. Gene expression of apoptotic markers (caspase 1, 3), TUBB3, nNOS, TH, and Schwann cells (Sox10, Krox20, glial fibrillary acid protein) was measured in whole organ cultured MPGs by quantitative polymerase chain reaction. OutcomesAfter 72 hours, neurite length, branching, autonomic neuron density, and apoptosis were assessed, and gene expression was measured. ResultsRT increased apoptosis in dissociated neurons measured by terminal deoxynucleotidyl transferase dUTP nick end labeling (P < .001) and whole MPG culture via upregulation of caspase 3 gene expression (P < .05). Nitrergic neurons were markedly decreased in irradiated dissociated culture (P < .05), while nNOS gene expression was upregulated in irradiated whole organ culture (P < .05). The proportion of dissociated sympathetic neurons and whole organ TH gene expression remained unchanged after RT. Interestingly, RT dissociated neurites were 22% shorter than controls, while RT whole organ neurites were 15% longer than controls (P < .01). MPG Schwann cells markers (Sox10, Krox20) were elevated after RT in whole organ culture. Clinical TranslationProstatic RT leads to increased neuronal cell death and less erectogenic nitrergic neurons contributing to ED. Strengths & LimitationsThe advantages of dissociated neuron culture include distinct neurites which are easily measured for apoptosis, length/branching, and specific neuron types. In contrast, whole MPG culture is advantageous as it contains all the supporting cells present in vivo. ConclusionThe 2 different culture methods demonstrated opposing neurite growth after RT indicating the importance of supporting cell network to promote pelvic neuron neuritogenesis and survival following RT.Randolph JT, Pak ES, Koontz BF, et al. Ex Vivo Radiation Leads to Opposing Neurite Growth in Whole Ganglia vs Dissociated Cultured Pelvic Neurons. J Sex Med 2020;17:1423–1433.

Outcomes of GDPT (gemcitabine, cisplatin, prednisone,thalidomide) versus CHOP in newly diagnosed peripheral T-cell lymphoma patients.

8018 Background: Peripheral T-cell lymphoma(PTCL) is highly heterogeneous invasive NHL.There is no consensus standard treatment for it now. So outcomes of GDPT versus CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) in treating newly diagnosed PTCL were compared. Methods: An open-label prospective clinical trial with 153 newly diagnosed PTCL patients conducted between January 2010 and December 2018 was designed. Patients were randomly assigned to the GDPT group (77 cases) and CHOP group (76 cases). Patients in each group were further divided into four subgroups: PTCL-NOS, ALCL, AITL, and an other types, in accordance with pathological patterns. Based on expression of RRM1, TOP2A, TUBB3 and ERCC1, patients were divided into groups with high and low gene expression levels. Clinical characteristics, side effects, efficacy, PFS and OS were compared. Results: There were no significant differences in the basic clinical features or side effects between the GDPT and CHOP groups. The ORR of the GDPT group was better than that of the CHOP group (66.3%vs. 50.0%, P= 0.042), as was the CR rate (42.9% vs. 27.6%, P= 0.049). Patients in the GDPT group had a longer PFS and OS than the CHOP group. The 4-year PFS and OS rates in the GDPT group were both superior to those in the CHOP group (63.6% vs. 53.0% for PFS, P= 0.035; 66.8% vs. 53.6% for OS, P= 0.039).In the GDPT group, the difference in CR between the four subgroups was statistically significant (P = 0.046).In the CHOP group, differences in both CR and ORR among the four subgroups were statistically significant ( P= &lt; 0.001 and P= 0.005, respectively).There were also statistically significant differences in CR between patients treated with CHOP and GDPT in the PTCL-NOS subgroup, AITL subgroup, and the other types subgroup( P= 0.015; P= 0.003; P= 0.005, respectively).The data also showed a significant difference in OS among the four subgroups within the GDPT group ( P= 0.001).The OS of AITL was shorter than that of the other three subgroups. Four subgroups of CHOP showed a significant difference in PFS ( P= 0.019). There was no statistical association between responses and the gene expression levels of RRM1, ERCC1, TUBB3 and TOP2A. Conclusions: The GDPT group had better response rates and prolonged the patients’ PFS and OS. As a promising new regimen, GDPT is expected to become the first-line therapy for PTCL. New agents should be applied to patients who do not achieve good responses with previous treatment, such as those diagnosed with angioimmunoblastic T cell lymphoma. Clinical trial information: NCT01664975 .

Proteomic profiling to identify therapeutics targets in glioblastoma (GBM).

2555 Background: Glioblastoma (GBM) is an aggressive primary brain tumor with poor prognosis. Treatment at diagnosis is largely confined to surgery, radiation and temozolomide (TMZ) with median progression-free survival (PFS) of 7 months and median overall survival (mOS) of 15 months. GBM tumors recur in most cases and in patients with recurrent GBM, the mOS is 6.2 months. The lack of effective therapies underscores the importance of exploring other agents. We propose that quantitating therapy-associated protein biomarkers can improve treatment personalization for GBM. Methods: 97 FFPE GBM tissues were microdissected and solubilized for mass spectrometry-based proteomic analysis of therapy-associated protein biomarkers in our CLIA certified lab. We quantified protein levels of MGMT, hENT1, RRM1, TOPO1 and EGFR/TUBB3 (antibody target and payload resistance markers, respectively, for anti-EGFR ADCs) simultaneously. The multiplexed assay also quantified additional 24 clinically relevant proteins. Results: 43/57 patients were predicted to respond to TMZ based on undetectable levels of MGMT, confirming wide utility of this agent. 42/97(43%) patients were predicted to have gemcitabine sensitivity based on high expression of the response marker (hENT1 &gt; 100 amol/ug) and low expression of the resistance marker (RRM1 &lt; 700 amol/ug). 11/97(11%) patients expressed TOPO1 &gt; 1350 amol/ug (75th percentile of all indications tested by author’s laboratory), suggesting likely response to irinotecan and topotecan. EGFR expression ranged from &lt; 100 amol/ug to &gt; 25000 amol/ug, including overexpression (&gt; 1500 amol/ug) in 22%(21/97) of cases. While expression of EGFR(81/97, 84%) suggested likely response to anti-EGFR ADC, concurrent expression of TUBB3(78/81) may indicate resistance to several known payloads, such as taxanes and MMAE. Conjugation with another payload that targets sensitivity marker TOPO1 (68% expression) is a likely option. Proteomic analysis also revealed detectable levels of multiple RTKs (FGFR(4), AXL(20), IGF1R(10), MET overexpression(1), and HER2 overexpression(2)), indicating potential response to RTK inhibitors. Exploratory investigation in tumor vs TME using proteomics and metabolomics is ongoing. Conclusions: In this population of GBM patients, proteomic analysis identified protein targets of multiple approved and investigational therapies. Gemcitabine, which crosses the blood-brain barrier, may be considered as a salvage option after TMZ failure. Proteomic quantitation of EGFR and TUBB3 may improve patient selection for EGFR-targeting ADCs.

3D Printed Conductive Nanocellulose Scaffolds for the Differentiation of Human Neuroblastoma Cells

We prepared cellulose nanofibrils-based (CNF), alginate-based and single-walled carbon nanotubes (SWCNT)-based inks for freeform reversible embedding hydrogel (FRESH) 3D bioprinting of conductive scaffolds. The 3D printability of conductive inks was evaluated in terms of their rheological properties. The differentiation of human neuroblastoma cells (SH-SY5Y cell line) was visualized by the confocal microscopy and the scanning electron microscopy techniques. The expression of TUBB3 and Nestin genes was monitored by the RT-qPCR technique. We have demonstrated that the conductive guidelines promote the cell differentiation, regardless of using differentiation factors. It was also shown that the electrical conductivity of the 3D printed scaffolds could be tuned by calcium–induced crosslinking of alginate, and this plays a significant role on neural cell differentiation. Our work provides a protocol for the generation of a realistic in vitro 3D neural model and allows for a better understanding of the pathological mechanisms of neurodegenerative diseases.

Exosomal TUBB3 mRNA expression level to predict prognosis for abiraterone treatment in metastatic prostate patients.

244 Background: TUBB3 (Class III β-tubulin), a dynamic component of microtubules, is considered a prognostic biomarker of tumor aggressiveness and poor survival. Previous studies have researched into its indication of resistance to chemotherapeutic drugs. However, its association with abiraterone, a next-generation androgen-axis targeting drug in prostate cancer has not been studied yet. In this study, we aimed to find out if exosomal TUBB3 expression could predict the efficacy of abiraterone in metastatic prostate cancer patients. Methods: Blood samples of metastatic prostate cancer patients during different disease stages were collected. We isolated the exosomes and extracted the RNA for analysis of TUBB3 by ddPCR. Absolute target mRNA concentration was measured by ddPCR as copies per milliliter (copies/20ul). Clinical data were collected for all patients. Primary study endpoint was progression-free survival (PFS). Statistical analyses were performed with SPSS 25.0. Results: A total of 54 metastatic prostate cancer patients who have received abiraterone treatment were enrolled. Baseline characteristics are comparable between high (≥10 copies/20ul) and low (&lt;10 copies/20ul) TUBB3 expression groups. Univariate analyses revealed that baseline hemoglobin≤120g/L, baseline sodium concentration &gt;140 mmol/L, baseline potassium concentration &gt;4.39 mmol/L and high exosomal TUBB3 expression were associated with shorter PFS, while only baseline sodium concentration and exosomal TUBB3 expression were independent prognosticators in multivariate analyses. In subsequently subgroup analyses, high exosomal TUBB3 expression demonstrates particularly high hazard ratio in multiple subgroups (patients younger than 70 years old, without neuroendocrine differentiation and immunohistochemical androgen-receptor splice variant 7 expression, with baseline PSA &gt;100 ng/mL, and patients receiving abiraterone as first-line therapy). Conclusions: Exosomal TUBB3 expression has potential in making treatment plan and predict prognosis in metastatic prostate cancer patients. High exosomal TUBB3 expression indicates poor response to abiraterone, especially as first line therapy.