βIII-tubulin Expression Research Articles

Chronic Binge Alcohol Administration Dysregulates Hippocampal Genes Involved in Immunity and Neurogenesis in Simian Immunodeficiency Virus-Infected Macaques.

Alcohol use disorders (AUD) exacerbate neurocognitive dysfunction in Human Immunodeficiency Virus (HIV+) patients. We have shown that chronic binge alcohol (CBA) administration (13–14 g EtOH/kg/wk) prior to and during simian immunodeficiency virus (SIV) infection in rhesus macaques unmasks learning deficits in operant learning and memory tasks. The underlying mechanisms of neurocognitive alterations due to alcohol and SIV are not known. This exploratory study examined the CBA-induced differential expression of hippocampal genes in SIV-infected (CBA/SIV+; n = 2) macaques in contrast to those of sucrose administered, SIV-infected (SUC/SIV+; n = 2) macaques. Transcriptomes of hippocampal samples dissected from brains obtained at necropsy (16 months post-SIV inoculation) were analyzed to determine differentially expressed genes. MetaCore from Thomson Reuters revealed enrichment of genes involved in inflammation, immune responses, and neurodevelopment. Functional relevance of these alterations was examined in vitro by exposing murine neural progenitor cells (NPCs) to ethanol (EtOH) and HIV trans-activator of transcription (Tat) protein. EtOH impaired NPC differentiation as indicated by decreased βIII tubulin expression. These findings suggest a role for neuroinflammation and neurogenesis in CBA/SIV neuropathogenesis and warrant further investigation of their potential contribution to CBA-mediated neurobehavioral deficits.

Class III β-Tubulin in Colorectal Cancer: Tissue Distribution and Clinical Analysis of Chinese Patients.

BackgroundClass III β-tubulin (βIII-tubulin) has been reported to express at the invasive margin of colorectal cancer. The present study aimed to investigate the clinical implication of βIII-tubulin expression at the invasive margin of colorectal cancer.Material/MethodsWe recruited 111 patients with surgically resected colorectal carcinoma for βIII-tubulin expression analysis. The cases with βIII-tubulin-positive tumor cells found only in the invasive front tumor area were assigned to the invasive front group, while the remaining cases were all assigned to the non-invasive front group. Clinical analysis of βIII-tubulin and other clinical data was performed.ResultsThe positive staining rates and staining intensity of βIII-tubulin were significantly different between the invasive and non-invasive front groups (p=0.001 and p=0.006), and there was a significant difference in tumor differentiation between the 2 groups (p=0.032). In the non-invasive front group, staining intensity of βIII-tubulin was significantly associated with positive staining rates and lymphatic metastasis (p<0.001 and p=0.048).ConclusionsOur data showed the tissue distribution of βIII-tubulin expression at invasive margin or diffuse distribution. Expression of βIII-tubulin was correlated with tumor differentiation and lymphatic metastasis, suggesting a potential role of βIII-tubulin in tumor differentiation and metastasis. This study may shed light on βIII-tubulin as a novel potential molecular target for a new anti-cancer drug.

Changes in Cell Cycle and Up-Regulation of Neuronal Markers During SH-SY5Y Neurodifferentiation by Retinoic Acid are Mediated by Reactive Species Production and Oxidative Stress.

Human neuroblastoma SH-SY5Y cells have been used as an in vitro model for neurodegenerative disorders such as Parkinson's disease and can be induced to a mature neuronal phenotype through retinoic acid (RA) differentiation. However, mechanisms of RA-induced differentiation remain unclear. Here, we investigate the role of reactive species (RS) on SH-SY5Y neuroblastoma cells under RA differentiation, using the antioxidant Trolox® as co-treatment. We found that RA treatment for 7days reduced the cell number and proliferative capacity and induced the expression of adult catecholaminergic/neuronal markers such as tyrosine hydroxylase (TH), β-III tubulin, and enolase-2. Evaluation of intracellular RS production by DCFH oxidation assay and quantification of cell non-enzymatic antioxidant activity by TRAP demonstrated that RA increases RS production. Furthermore, mitochondrial NADH oxidation showed to be inhibited under differentiation with RA. Cells subjected to co-treatment with antioxidant Trolox® demonstrated a remaining proliferative capacity and a decrease in the pro-oxidant state and RS production. Besides, antioxidant treatment restores the mitochondrial NADH oxidation. Importantly, Trolox® co-treatment inhibited the appearance of morphological characteristics such as neurite extension and branching, and decreased the expression of TH, β-III tubulin, and enolase-2 after a seven-day differentiation with RA, indicating that RS production is a necessary step in this process. Trolox® also inhibited the phosphorylation of Akt and ERK1/2, which are involved in differentiation and survival, respectively, of these cells. Altogether, these data indicate the presence of a redox-dependent mechanism in SH-SY5Y RA-differentiation process and can be a useful insight to improve understanding of neuronal differentiation signaling.

P.2.b.055 - Changes in plasma concentrations of neurotrophin-4/5 and chemokine SDF-1 in depression

Salivary gland cells are surrounded by a complex stromal environment, in which fibroblasts are the main cells in proximity to the gland cells. In this study, the interaction between parotid gland acinar cells (PGACs), fibroblasts, and biomaterials was investigated. We prepared different biomaterials, including chitosan, polyvinyl alcohol (PVA), poly (ethylene-co-vinyl alcohol) (EVAL), polyvinylidene fluoride (PVDF), and tissue culture polystyrene (TCPS) to culture fibroblasts and then collect their conditioned media to culture PGACs. We observed no difference in AQP3, AQP5, and E-cadherin expression among different fibroblast conditioned medium treatments. Interestingly, α-amylase expression was obviously enhanced in PGACs cultured in the presence of conditioned medium from fibroblasts cultured on PVDF. Higher neurotrophin-4 (NT-4) expression was observed in PVDF-derived fibroblast conditioned medium using a growth factor protein array assay. In addition, directly adding NT-4 into the culture medium significantly promoted α-amylase expression by PGACs. Finally, nestin and βIII-tubulin expression by fibroblasts cultured on PVDF was also enhanced. Together, these results suggest that PVDF could promote α-amylase expression by PGACs via the NT-4 produced by fibroblasts.To date, there is no effective therapy for patients with dry mouth with persistent salivary hypofunction. The study made use of different biomaterials to culture fibroblasts and then collect their conditioned media to culture PGACs. It was found that the effect of fibroblast conditioned medium from PVDF on the α-amylase expression of PGACs was obviously enhanced and higher neurotrophin-4 (NT-4) expression was found in PVDF-derived fibroblast conditioned medium. In addition, directly adding NT-4 into the culture medium significantly promoted the expression of α-amylase by PGACs and the expression of nestin and βIII-tubulin of fibroblasts after being cultured on PVDF was enhanced. Therefore, the present study represents the first description of the role of NT-4 in the expression of α-amylase of PGACs and the role of PVDF in the reprogramming fibroblasts into neural progenitor-like cells, indicating that PVDF could promote the expression of α-amylase by PGACs via the NT-4 produced by fibroblasts.

Mechanism of paclitaxel resistance in a human prostate cancer cell line, PC3-PR, and its sensitization by cabazitaxel

Paclitaxel (PTX) is a microtubule-targeting drug widely used for the treatment of a variety of cancers. However, drug resistance can emerge after a series of treatments, and this can seriously affect the patient's prognosis. Here, we analyzed the mechanism of PTX resistance using a human prostate cancer cell line, PC3, and its PTX-resistant subline, PC3-PR. Compared with PC3, PC3-PR exhibited some unique phenotypes that might be associated with PTX resistance, including decreased expression of acetylated α-tubulin and the cell cycle regulator p21, and increased expression of βIII tubulin, histone deacetylase 6 (HDAC6), and the anti-apoptotic protein Bcl2. The drug exporters MDR1 and MRP1 were not involved in PTX resistance. Although cabazitaxel (CTX), a novel taxoid, has been reported to overcome PTX resistance, its mechanism of action is unknown. We found that treatment of PC3-PR cells with CTX induced expression of acetylated α-tubulin and p21, but not the related regulators p27, p15, and p16 or the Bcl2 family proteins. The pan-HDAC inhibitors trichostatin A and suberanilohydroxamic acid and the HDAC6-specific inhibitor tubacin inhibited PC3-PR proliferation and increased expression of p21 and acetylated α-tubulin in a manner similar to CTX. Our data shed light on the cellular response to PTX and CTX.

Influence of Matrices on 3D-Cultured Prostate Cancer Cells' Drug Response and Expression of Drug-Action Associated Proteins.

This study investigated the effects of matrix on the behaviors of 3D-cultured cells of two prostate cancer cell lines, LNCaP and DU145. Two biologically-derived matrices, Matrigel and Cultrex BME, and one synthetic matrix, the Alvetex scaffold, were used to culture the cells. The cell proliferation rate, cellular response to anti-cancer drugs, and expression levels of proteins associated with drug sensitivity/resistance were examined and compared amongst the 3D-cultured cells on the three matrices and 2D-cultured cells. The cellular responses upon treatment with two common anti-cancer drugs, Docetaxel and Rapamycin, were examined. The expressions of epidermal growth factor receptor (EGFR) and β-III tubulin in DU145 cells and p53 in LNCaP cells were examined. The results showed that the proliferation rates of cells cultured on the three matrices varied, especially between the synthetic matrix and the biologically-derived matrices. The drug responses and the expressions of drug sensitivity-associated proteins differed between cells on various matrices as well. Among the 3D cultures on the three matrices, increased expression of β-III tubulin in DU145 cells was correlated with increased resistance to Docetaxel, and decreased expression of EGFR in DU145 cells was correlated with increased sensitivity to Rapamycin. Increased expression of a p53 dimer in 3D-cultured LNCaP cells was correlated with increased resistance to Docetaxel. Collectively, the results showed that the matrix of 3D cell culture models strongly influences cellular behaviors, which highlights the imperative need to achieve standardization of 3D cell culture technology in order to be used in drug screening and cell biology studies.

A Rationally Optimized Nanoparticle System for the Delivery of RNA Interference Therapeutics into Pancreatic Tumors in Vivo.

Pancreatic cancer is a devastating disease with a dismal prognosis. Short-interfering RNA (siRNA)-based therapeutics hold promise for the treatment of cancer. However, development of efficient and safe delivery vehicles for siRNA remains a challenge. Here, we describe the synthesis and physicochemical characterization of star polymers (star 1, star 2, star 3) using reversible addition-fragmentation chain transfer polymerization (RAFT) for the delivery of siRNA to pancreatic cancer cells. These star polymers were designed to contain different lengths of cationic poly(dimethylaminoethyl methacrylate) (PDMAEMA) side-arms and varied amounts of poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA). We showed that star-POEGMA polymers could readily self-assemble with siRNA to form nanoparticles. The star-POEGMA polymers were nontoxic to normal cells and delivered siRNA with high efficiency to pancreatic cancer cells to silence a gene (TUBB3/βIII-tubulin) which is currently undruggable using chemical agents, and is involved in regulating tumor growth and metastases. Notably, systemic administration of star-POEGMA-siRNA resulted in high accumulation of siRNA to orthotopic pancreatic tumors in mice and silenced βIII-tubulin expression by 80% at the gene and protein levels in pancreatic tumors. Together, these novel findings provide strong rationale for the use of star-POEGMA polymers as delivery vehicles for siRNA to pancreatic tumors.

βIII-Tubulin alters glucose metabolism and stress response signaling to promote cell survival and proliferation in glucose-starved non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) survival rates are dismal and high βIII-tubulin expression is associated with chemotherapy drug resistance and tumor aggressiveness in this disease. Mounting evidence supports a role for βIII-tubulin in promoting cell survival in the harsh tumor microenvironment, which is characterized by poor nutrient supply. This study aimed to investigate the role of βIII-tubulin in glucose stress response signaling and the survival and proliferation of NSCLC cells. This study revealed that βIII-tubulin regulates cellular metabolism and glucose stress response signaling in NSCLC cells to promote cell survival and proliferation in glucose starvation. βIII-Tubulin decreases the reliance of cells on glycolytic metabolism, priming them to cope with variable nutrient supply present within the tumor microenvironment. βIII-Tubulin protects cells from endoplasmic reticulum (ER) stress and reduces both basal and glucose starvation-induced autophagy to maintain cell survival and proliferation. βIII-Tubulin enables rapid Akt activation in response to glucose starvation and co-immunoprecipitates with the master regulator of the ER stress response GRP78. Furthermore, suppression of βIII-tubulin delays the association of GRP78 with Akt in response to glucose starvation with the potential to influence Akt activation and ER homeostasis under these conditions. Together these results identify that βIII-tubulin regulates glucose metabolism and alters glucose starvation stress signaling to promote cell proliferation and survival in NSCLC cells. This elucidates a hitherto unknown role for this microtubule protein and provides insight into correlations between high βIII-tubulin expression and poor patient outcome in this disease.

Pioglitazone Enhances Survival and Regeneration of Pelvic Ganglion Neurons After Cavernosal Nerve Injury

To investigate the effects of pioglitazone on pelvic ganglion neurons in a rat model of bilateral cavernosal nerve crush injury (BCNI), thereby elucidating the actions of pioglitazone in preventing post-prostatectomy neurogenic erectile dysfunction. Sprague-Dawley rats aged 12 weeks were divided into four groups: (a) sham procedure, (b) BCNI, (c) BCNI + postsurgical pioglitazone, and (d) BCNI + pre and postsurgical pioglitazone (preventive therapy). Preoperative injection of Fluoro-Gold (FG) fluorescent tracer into the cavernosal tissue was performed for retrograde labeling of pelvic ganglion cells. Pelvic ganglia were resected at 2 weeks in all rats and processed for real-time polymerase chain reaction, immunohistochemistry, and Western blot to examine the expression of FG, neuronal nitric oxide synthase, β-III tubulin, neurturin, and glial cell line-derived neurotrophic factor family receptor alpha-2 (GFRα2). Animals treated with pre- and postsurgical pioglitazone demonstrated increased staining for FG similar to sham levels. Gene expression of neuronal nitric oxide synthase, neurturin, GFRα2, and β-III tubulin was also upregulated in the group receiving preventive therapy. Pioglitazone provides a protective effect on pelvic ganglion neurons after BCNI.

Bioengineered Human Pyloric Sphincters Using Autologous Smooth Muscle and Neural Progenitor Cells.

Gastroparesis leads to inadequate emptying of the stomach resulting in severe negative health impacts. Appropriate long-term treatments for these diseases may require pyloric sphincter tissue replacements that possess functional smooth muscle cell (SMC) and neural components. This study aims to bioengineer, for the first time, innervated human pylorus constructs utilizing autologous human pyloric sphincter SMCs and human neural progenitor cells (NPCs). Autologous SMCs and NPCs were cocultured in dual-layered hydrogels and formed concentrically aligned pylorus constructs. Innervated autologous human pylorus constructs were characterized through biochemical and physiologic assays to assess the phenotype and functionality of SMCs and neurons. SMCs within bioengineered human pylorus constructs displayed a tonic contractile phenotype and maintained circumferential alignment. Neural differentiation within bioengineered constructs was verified by positive expression of βIII-tubulin, neuronal nitric oxide synthase (nNOS), and choline acetyltransferase (ChAT). Autologous bioengineered innervated human pylorus constructs generated a robust spontaneous basal tone and contracted in response to potassium chloride (KCl). Contraction in response to exogenous neurotransmitter acetylcholine (ACh), relaxation in response to vasoactive intestinal peptide (VIP), and electrical field stimulation (EFS) were also observed. Neural network integrity was demonstrated by inhibition of EFS-induced relaxation in the presence of a neurotoxin or nNOS inhibitors. Partial inhibition of ACh-induced contraction and VIP-induced relaxation following neurotoxin treatment was observed. These studies provide a proof of concept for bioengineering functional innervated autologous human pyloric sphincter constructs that generate a robust basal tone and contain circumferentially aligned SMCs, which display a tonic contractile phenotype and functional differentiated neurons. These autologous constructs have the potential to be used as (1) functional replacement organs and (2) physiologically relevant models to investigate human pyloric sphincter disorders.

Combination Therapy Using Human Adipose-derived Stem Cells on the Cavernous Nerve and Low-energy Shockwaves on the Corpus Cavernosum in a Rat Model of Post-prostatectomy Erectile Dysfunction

To investigate combined therapeutic efficacy of human adipose-derived stem cells (h-ADSCs) application on injured cavernous nerve and low-energy shockwave therapy (SWT) on the corpus cavernosum in a rat model of post-prostatectomy erectile dysfunction. Rats were randomly divided into 5 groups: control, bilateral cavernous nerve injury (BCNI), adipose-derived stem cell (ADSC) (BCNI group with h-ADSCs on the cavernous nerve), SWT (BCNI group with low-energy SWT on the corpus cavernosum), and ADSC/SWT (BCNI group with a combination of h-ADSCs and low-energy SWT). After 4 weeks, erectile function was assessed using intracavernosal pressure. The cavernous nerves and penile tissue were evaluated through immunostaining, Western blotting, and a cyclic guanosine monophosphate assay. ADSC/SWT significantly improved intracavernosal pressure compared to the other experimental group. ADSC had significantly increased β-III tubulin expression of the cavernous nerve, and SWT had a markedly enhanced vascular endothelial growth factor expression in corpus cavernosum. The ADSC/SWT group had a significantly increased in alpha smooth muscle actin content (P < .05), neural nitric oxide synthase (nNOS) of the dorsal penile nerve (P < .05), endothelial nitric oxide synthase (eNOS) protein expression (P < .05), and cyclic guanosine monophosphate level (P < .05) compared to the ADSC or SWT alone group. In addition, ADSC/SWT reduces the apoptotic index in the corpus cavernosum. In this study, h-ADSCs showed an effect on the recovery of injured cavernous nerve and low-energy SWT improved angiogenesis in the corpus cavernosum. The h-ADSCs combined with low-energy SWT showed beneficial effect on the recovery of erectile function in a rat model of postprostatectomy erectile dysfunction.

Mesenchymal Stem Cell-Like Properties of Orbital Fibroblasts in Graves' Orbitopathy.

Graves' orbitopathy (GO) is a sight-threatening autoimmune disorder causing extraocular muscle fibrosis, upper lid retraction and eye bulging due to orbital fat expansion. These clinical features are mediated by aspects of orbital fibroblasts differentiation, including adipogenesis and fibrosis. Our previous work suggested that this dual phenotype might be a manifestation of mixed cell populations, partially linked to the expression of mesenchymal stem cell (MSC) marker CD90. Thus, we set out to determine whether GO orbital fibroblasts displayed MSC properties. Control and GO orbital fibroblasts previously characterized for CD90 and CD45 expression were analyzed by flow cytometry for classical MSC positive (CD73, CD105) and negative (CD14, CD19, HLA-DR, and CD34) markers. Graves' orbitopathy fibroblasts were tested further for their ability to undergo lineage specific differentiation following standard protocols. Control and GO fibroblasts strongly expressed CD73 and CD105, with a higher percentage of positive cells and stronger expression levels in GO. Neither cell type expresses CD14, CD19, and HLA-DR. Protein CD34 was expressed at low levels by 45% to 70% of the cells, with its expression significantly lower in GO cells. Graves' orbitopathy fibroblasts displayed features of osteogenesis (calcium deposits, and osteocalcin [BGLAP] and osteonectin [SPARC] expression), chondrogenesis (glycosaminoglycan production; SOX9 and aggrecan [ACAN] expression), myogenesis (α-smooth muscle actin expression), and neurogenesis (β-III tubulin expression) upon differentiation. Our findings suggest that orbital fibroblasts contain a population of cells that fulfil the criteria defining MSC. This subpopulation may be increased in GO, possibly underlying the complex differentiation phenotype of the disease.

Effects of biomaterial-derived fibroblast conditioned medium on the α-amylase expression of parotid gland acinar cells

Salivary gland cells are surrounded by a complex stromal environment, in which fibroblasts are the main cells in proximity to the gland cells. In this study, the interaction between parotid gland acinar cells (PGACs), fibroblasts, and biomaterials was investigated. We prepared different biomaterials, including chitosan, polyvinyl alcohol (PVA), poly (ethylene-co-vinyl alcohol) (EVAL), polyvinylidene fluoride (PVDF), and tissue culture polystyrene (TCPS) to culture fibroblasts and then collect their conditioned media to culture PGACs. We observed no difference in AQP3, AQP5, and E-cadherin expression among different fibroblast conditioned medium treatments. Interestingly, α-amylase expression was obviously enhanced in PGACs cultured in the presence of conditioned medium from fibroblasts cultured on PVDF. Higher neurotrophin-4 (NT-4) expression was observed in PVDF-derived fibroblast conditioned medium using a growth factor protein array assay. In addition, directly adding NT-4 into the culture medium significantly promoted α-amylase expression by PGACs. Finally, nestin and βIII-tubulin expression by fibroblasts cultured on PVDF was also enhanced. Together, these results suggest that PVDF could promote α-amylase expression by PGACs via the NT-4 produced by fibroblasts. Statement of SignificanceTo date, there is no effective therapy for patients with dry mouth with persistent salivary hypofunction. The study made use of different biomaterials to culture fibroblasts and then collect their conditioned media to culture PGACs. It was found that the effect of fibroblast conditioned medium from PVDF on the α-amylase expression of PGACs was obviously enhanced and higher neurotrophin-4 (NT-4) expression was found in PVDF-derived fibroblast conditioned medium. In addition, directly adding NT-4 into the culture medium significantly promoted the expression of α-amylase by PGACs and the expression of nestin and βIII-tubulin of fibroblasts after being cultured on PVDF was enhanced. Therefore, the present study represents the first description of the role of NT-4 in the expression of α-amylase of PGACs and the role of PVDF in the reprogramming fibroblasts into neural progenitor-like cells, indicating that PVDF could promote the expression of α-amylase by PGACs via the NT-4 produced by fibroblasts.

Abstract 3600: Impaired taxane binding to MTs in intrinsically taxane resistant gastric cancer cells without β-tubulin mutation

Abstract The taxanes (i.e., paclitaxel, docetaxel (DTX) and cabazitaxel) are microtubule (MT)-stabilizing drugs widely used to treat solid tumor malignancies. Their success is limited by the presence of intrinsic or acquired drug resistance. Understanding the molecular mechanisms of taxane resistance is a key to significantly improve clinical outcomes of taxane-based chemotherapy. In gastric cancer (GC), retrospective analysis of the TAX-325 trial revealed that the addition of docetaxel (DTX) to standard cisplatin/fluorouracil increased progression-free and overall survival, primarily only in intestinal subtype (INT) GC, suggesting that diffuse (DIF) GC may be intrinsically taxane resistant. In fact, our previous data supported this hypothesis; DIF GC cell lines had higher incidence (63%) of DTX resistance than INT GC cell lines (25%). Flow cytometric analyses showed negligible P-glycoprotein expression on the cell surface of all cell types, and accumulation of C14-labeled DTX in cells was observed in both sensitive and resistant GC cell lines. These results ruled out drug efflux as a possible mechanism that confers taxane resistance in DIF GC cells. Next, we used fluorescein-conjugated paclitaxel (Flutax) as a probe to investigate the affinity of taxanes to MTs. Live cell imaging showed strong binding of Flutax to MTs in a sensitive cell line at least for 60 minutes. In contrast, Flutax failed to stay bound to MTs in resistant GC cell lines,. In addition, discontinuous decoration of MTs with Flutax was seen in resistant but not in sensitive cell lines. Our observation suggests that MTs of resistant GC cell lines have lower affinity for taxanes, and there might be subtle structural changes of MTs. We are currently conducting the competition of Flutax/DTX to quantify the affinity of DTX in both types of GC cell lines. Because tubulin mutations have been reported in many taxane-resistant cell lines, we sequenced the different tubulin isotypes in GC cell lines using both Sanger and next generation sequencing. No mutations were identified. In addition, we did not see any significant changes in the expression of βIII-tubulin or tubulin post-translational modifications between the sensitive and resistant cell lines. Pathway analyses on the RNA-Seq datasets derived from the panels of sensitive and resistant cell lines before and after taxane treatment is ongoing to identify the key molecular events underlying taxane resistance.Taken together, our data indicate that new molecular pathway(s) or change of overall MT structure and/or function may contribute to the impaired taxane binding to MTs in resistant GC cells. Our study will lead us to novel mechanistic insights in intrinsic drug resistance, and will ultimately allow us to target key molecule(s) to overcome chemo-resistance, the key determinant to improve overall survival of patients. Citation Format: Katsuhiro Kita, Giuseppe Galletti, Kyle Cleveland, Prashant V. Thakkar, Ada Gjyrezi, Chao Zhang, Isabel Barasoain, J. Fernando Díaz, Doron Betel, Manish A. Shah, Paraskevi Giannakakou. Impaired taxane binding to MTs in intrinsically taxane resistant gastric cancer cells without β-tubulin mutation. [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 3600. doi:10.1158/1538-7445.AM2015-3600

Abstract 730: The role of ZEB1/ZEB2 and βIII-tubulin in mediating docetaxel-resistant prostate cancer

Abstract Docetaxel therapy is the gold standard treatment for advanced castrate-resistant prostate cancer (CRPC). However, patients either do not respond or develop resistance over time. Transcriptomic and proteomic analysis of docetaxel-resistant prostate cancer sub-lines developed by our group revealed multiple mechanisms of resistance in line with advanced disease, including over-expression of anti-apoptotic proteins and alterations of NF-KB activation. The sub-lines also demonstrated a coordinated loss and gain of epithelial and mesenchymal markers respectively; a process characteristic of Epithelial-Mesenchymal Transition (EMT). Studies have highlighted a role of EMT in prostate cancer progression, metastasis and docetaxel resistance. However, the role of EMT drivers in mediating resistance is not defined. We hypothesise EMT to be a central mechanism of apoptotic resistance in advanced docetaxel-resistant prostate cancer, representing a target for therapeutic manipulation. EMT was characterised in the PC-3 D12 and DU145 R docetaxel-resistant sub-lines through an increased invasive capacity, MMP-1 secretion and protein expression of E-cadherin transcriptional repressors ZEB1 and ZEB2, in comparison to parental cell lines. This was associated with an increased expression of βIII-tubulin; a tubulin isotype linked to taxane resistance and tumour aggressiveness. Upon treatment with the apoptotic trigger docetaxel (20nM), the PC-3 D12 and DU145 R sub-lines demonstrated significant resistance compared to parental controls. Simultaneous siRNA knockdown of ZEB1 and ZEB2 resulted in both a partial reversal in apoptotic resistance and a decrease in cell viability; this was also associated with a down-regulation of βIII-tubulin and a re-expression of E-cadherin. Our results provide evidence of the process of EMT in in vitro models of docetaxel-resistant prostate cancer, which is associated with differential susceptibility to docetaxel and is partially reversed through siRNA knockdown of the EMT drivers, ZEB1 and ZEB2. In addition, we have identified a link between EMT and βIII-tubulin in our models of docetaxel resistance. Current experiments are investigating the tissue expression of ZEB1 and βIII-tubulin in prostate cancer metastases following docetaxel therapy, which will determine the clinical relevance of EMT and βIII-tubulin as mediators of docetaxel resistance in CRPC. Citation Format: Karen Hanrahan, Maria Prencipe, Jane Bugler, Lisa Murphy, Amanda O'Neill, R. William Watson. The role of ZEB1/ZEB2 and βIII-tubulin in mediating docetaxel-resistant prostate 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 730. doi:10.1158/1538-7445.AM2015-730

Abstract 1666: Increased sensitivity of βIII tubulin-containing microtubules to cabazitaxel compared with docetaxel: A possible role of βIII tubulin in specifying good efficacy of cabazitaxel for docetaxel-resistant tumors

Abstract Tumor progression and resistance to taxane-based therapies have been shown to correlate with overexpression of the class III β-tubulin isotype (βIII tubulin). Elevated expression of βIII tubulin is often associated with tumor aggressiveness in patients with metastatic docetaxel-resistant prostate cancer. Cabazitaxel is a novel taxane recently approved for the treatment of metastatic prostate cancer that failed docetaxel therapy. To understand the mechanistic distinctions between cabazitaxel and docetaxel, we analyzed the effects of both drugs on bovine brain microtubules consisting of unfractionated tubulin, which contains 25% βIII tubulin, and with microtubules depleted of βIII tubulin. BetaIII tubulin was depleted using immunoaffinity chromatography purification technique. Both cabazitaxel and docetaxel enhanced polymerization of unfractionated tubulin to similar extents. The stoichiometry of 14C-cabazitaxel and 14C-docetaxel binding to microtubules composed of unfractionated tubulin were similar at very low concentrations (≤ 10 μM). The Kds for binding were 8.4 μM and 7.5 μM for 14C-cabazitaxel and 14C-docetaxel, respectively. However, at higher concentrations (10 - 40 μM) significantly more 14C-cabazitaxel bound to the microtubules than 14C-docetaxel, with an apparent higher affinity. We also analyzed the effects of cabazitaxel and docetaxel on in vitro dynamic instability of microtubules assembled from unfractionated and βIII-depleted tubulin. We found that 100 nM cabazitaxel or docetaxel stabilized the dynamics of microtubules assembled from unfractionated tubulin similarly. However, cabazitaxel suppressed the dynamics of microtubules containing βIII tubulin more strongly than docetaxel. For example, 100 nM cabazitaxel suppressed the shortening rate of unfractionated microtubules by 49% and the shortening rate of βIII-depleted microtubules by only 31%. The overall suppression of dynamicity of microtubules assembled from unfractionated βIII-rich tubulin was 49% vs. 33% for βIII-depleted microtubules. In contrast with cabazitaxel, 100 nM docetaxel suppressed the shortening rate of microtubules assembled from unfractionated and βIII-depleted tubulin by 48% and 52%, respectively. The overall suppression of dynamicity of microtubules assembled from unfractionated βIII-rich tubulin was 56% vs. 50% for βIII-depleted microtubules. Experiments to examine the binding of cabazitaxel and docetaxel to microtubules assembled from βIII-depleted tubulin are ongoing. Our results indicate that the more potent activity of cabazitaxel for microtubules containing βIII tubulin may contribute to its superior efficacy in prostate tumors overexpressing βIII tubulin as compared with that of docetaxel. Supported by a research award from Sanofi Oncology. Citation Format: Leslie Wilson, Olga Azarenko, Gregoriy Smiyun, Herbert Miller, Mary Ann Jordan. Increased sensitivity of βIII tubulin-containing microtubules to cabazitaxel compared with docetaxel: A possible role of βIII tubulin in specifying good efficacy of cabazitaxel for docetaxel-resistant tumors. [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 1666. doi:10.1158/1538-7445.AM2015-1666

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

Bioengineered fibrin-based niche to direct outgrowth of circulating progenitors into neuron-like cells for potential use in cellular therapy

Objective. Autologous cells are considered to be the best choice for use in transplantation therapy. However, the challenges and risks associated with the harvest of transplantable autologous cells limit their successful therapeutic application. The current study explores the possibility of isolating neural progenitor cells from circulating multipotent adult progenitor cells for potential use in cell-based and patient-specific therapy for neurological diseases. Approach. To enable the selection of neural progenitor cells from human peripheral blood mononuclear cells, and to support their lineage maintenance, the composition of a fibrin-based niche was optimized. Morphological examination and specific marker analysis were carried out, employing a qualitative/quantitative polymerase chain reaction followed by immunocytochemistry to: (i) characterize neural progenitor cells in culture; (ii) monitor proliferation/survival; and (iii) track their differentiation status. Main results. The presence of neural progenitors in circulation was confirmed by the presence of nestin+ cells at the commencement of the culture. The isolation, proliferation and differentiation of circulating neural progenitors to neuron-like cells were directed by the engineered niche. Neural cell isolation to near homogeneity was confirmed by the expression of β-III tubulin in ∼95% of cells, whereas microtubule associated protein-2 expression confirmed their ability to differentiate. The concentration of potassium chloride in the niche was found to favour neuron-like cell lengthening, cell-cell contact, and expressions of synaptophysin and tyrosine hydroxylase. Significance. The purpose of this research was to find out if peripheral blood could serve as a potential source of neural progenitors for cell based therapy. The study established that neural progenitors could be selectively isolated from peripheral blood mononuclear cells using a biomimetic niche. The selected cells could multiply and slowly differentiate into neuron-like cells. These neuron-like cells expressed functional proteins—tyrosine hydroxylase and synaptophysin. Early progenitors that proliferate while expressing β-III tubulin could be harvested from the culture, suggesting their potential use in cell transplantation therapy.

Potential Neurogenesis of Human Adipose-Derived Stem Cells on Electrospun Catalpol-Loaded Composite Nanofibrous Scaffolds.

Catalpol, a natural active ingredient extracted from the traditional Chinese medicine, was verified exhibiting beneficial effects on neural differentiation compared with commonly used chemical inducers by our previous studies. The aim of this study was to evaluate the effects of catalpol-loaded scaffold on guiding neuronal differentiation of human adipose tissue-derived stem cells (hASCs). Fabrication technique of catalpol loading into the electrospun poly(lactic-co-glycolic acid)/multi-walled carbon nanotubes/silk fibroin nanofibrous scaffolds was successfully established. The topographical and mechanical properties of the nanofibers scaffolds were characterized by scanning electron microscopy and tensile instrument, respectively. In vitro catalpol release was studied in phosphate-buffered solution at 37°C. Immunnocytochemistry, RT-PCR, and western blot assays were performed to estimate hASCs neuronal differentiation, and it was shown that catalpol has significantly upregulated the expressions of βIII-tubulin and Nissl. Our experiments demonstrated that catalpol, as a traditional Chinese medicine extract, could be encapsulated into composite nanofibers and induce differentiation of hASCs into neural-like cells, which might offer new avenues in nerve regeneration.

Toll like receptor-2 regulates production of glial-derived neurotrophic factors in murine intestinal smooth muscle cells.

Gut microbiota-innate immunity axis is emerging as a key player to guarantee the structural and functional integrity of the enteric nervous system (ENS). Alterations in the composition of the gut microbiota, derangement in signaling of innate immune receptors such as Toll-like receptors (TLRs), and modifications in the neurochemical coding of the ENS have been associated with a variety of gastrointestinal disorders. Indeed, TLR2 activation by microbial products controls the ENS structure and regulates intestinal neuromuscular function. However, the cellular populations and the molecular mechanisms shaping the plasticity of enteric neurons in response to gut microbes are largely unexplored. In this study, smooth muscle cells (SMCs), enteric glial cells (EGCs) and macrophages/dendritic cells (MΦ/DCs) were isolated and cultured from the ileal longitudinal muscle layer of wild-type (WT) and Toll-like receptor-2 deficient (TLR2(-/-)) mice. Quantification of mRNA levels of neurotrophins at baseline and following stimulation with TLR ligands was performed by RT-PCR. To determine the role of neurotrophins in supporting the neuronal phenotype, we performed co-culture experiments of enteric neurons with the conditioned media of cells isolated from the longitudinal muscle layer of WT or TLR2(-/-) mice. The neuronal phenotype was investigated evaluating the expression of βIII-tubulin, HuC/D, and nNOS by immunocytochemistry. As detected by semi-quantitative RT-PCR, SMCs expressed mRNA coding TLR1-9. Among the tested cell populations, un-stimulated SMCs were the most prominent sources of neurotrophins. Stimulation with TLR2, TLR4, TLR5 and TLR9 ligands further increased Gdnf, Ngf, Bdnf and Lif mRNA levels in SMCs. Enteric neurons isolated from TLR2(-/-) mice exhibited smaller ganglia, fewer HuC/D(+ve) and nNOS(+ve) neurons and shorter βIII-tubulin axonal networks as compared to neurons cultured from WT mice. The co-culture with the conditioned media from WT-SMCs but not with those from WT-EGCs or WT-MΦ/DCs corrected the altered neuronal phenotype of TLR2(-/-) mice. Supplementation of TLR2(-/-) neuronal cultures with GDNF recapitulated the WT-SMC co-culture effect whereas the knockdown of GDNF expression in WT-SMCs using shRNA interference abolished the effect on TLR2(-/-) neurons. These data revealed that by exploiting the repertoire of TLRs to decode gut-microbial signals, intestinal SMCs elaborate a cocktail of neurotrophic factors that in turn supports neuronal phenotype. In this view, the SMCs represent an attractive target for novel therapeutic strategies.