The Experts below are selected from a list of 3141 Experts worldwide ranked by ideXlab platform
Cristiano Ferlini - One of the best experts on this subject based on the ideXlab platform.
-
Class III β -Tubulin (TUBB3): More than a Biomarker in Solid Tumors?
Current molecular medicine, 2011Co-Authors: Marisa Mariani, Giovanni Scambia, Shohreh Shahabi, Steven Sieber, Cristiano FerliniAbstract:Class III β-tubulin (TUBB3) is a prominent mechanism of drug resistance expressed in a variety of solid tumors and particularly in lung and ovarian cancer. In the classical view, TUBB3 expression and drug resistance have been linked, and together they have been associated with a perturbation in microtubule dynamics. In keeping with this observation, TUBB3 was associated with drug resistance only when chemotherapy included a taxane in its chemical composition. In this review, we demonstrate that the classical supposition about TUBB3 is not correct, and that instead TUBB3 expression is linked to drug resistance as a complex survival mechanism activated by microenvironmental conditions such as poor nutrient supply and hypoxia.
-
Proteomic characterization of cytoskeletal and mitochondrial class III β-tubulin
Molecular cancer therapeutics, 2008Co-Authors: Lucia Cicchillitti, Flavia Filippetti, Roberta Penci, Giovanni Scambia, Michela Di Michele, Domenico Rotilio, Maria Benedetta Donati, Cristiano FerliniAbstract:Class III β-tubulin (TUBB3) has been discovered as a marker of drug resistance in human cancer. To get insights into the mechanisms by which this protein is involved in drug resistance, we analyzed TUBB3 in a panel of drug-sensitive and drug-resistant cell lines. We identified two main different isoforms of TUBB3 having a specific electrophoretic profile. We showed that the apparently higher molecular weight isoform is glycosylated and phosphorylated and it is localized in the cytoskeleton. The apparently lower molecular weight isoform is instead found exclusively in mitochondria. We observed that levels of phosphorylation and glycosylation of TUBB3 are associated with the resistant phenotype and compartmentalization into cytoskeleton. By two-dimensional nonreduced/reduced SDS-PAGE analysis, we also found that TUBB3 protein in vivo forms protein complexes through intermolecular disulfide bridges. Through TUBB3 immunoprecipitation, we isolated protein species able to interact with TUBB3. Following trypsin digestion, these proteins were characterized by mass spectrometry analysis. Functional analysis revealed that these proteins are involved in adaptation to oxidative stress and glucose deprivation, thereby suggesting that TUBB3 is a survival factor able to directly contribute to drug resistance. Moreover, glycosylation of TUBB3 could represent an attractive pathway whose inhibition could hamper cytoskeletal compartmentalization and TUBB3 function. [Mol Cancer Ther 2008;7(7):2070–9]
-
hypoxia induces class iii beta tubulin gene expression by hif 1α binding to its 3 flanking region
Gene, 2008Co-Authors: Giuseppina Raspaglio, Flavia Filippetti, Roberta Penci, Ilaria De Maria, Simona Mozzetti, Lucia Cicchillitti, Silvia Prislei, Giovanni Scambia, Cristiano FerliniAbstract:Abstract Class III β-tubulin (TUBB3) overexpression represents a major mechanism of drug resistance to microtubule interacting agents such as taxanes and Vinca alkaloids. Here, we tested hypoxia as a possible inducer of TUBB3. The effects of hypoxia on TUBB3 expression were monitored at mRNA and protein level in A2780, in its paclitaxel-resistant counterpart (TC1) and in HeLa cells. Hypoxia was a strong inducer of TUBB3 in A2780, but not in TC1 and HeLa cells. In A2780 HIF-1α was knocked down using RNA interference and TUBB3 expression was assessed in normoxia and hypoxia. The silencing abolished the hypoxia-dependent increase of TUBB3, thereby demonstrating that HIF-1α mediates TUBB3 induction in hypoxia. To investigate this phenomenon, the 5' flanking region of human TUBB3 was cloned upstream GFP as a reporter. This region contained the promoter gene, but activity of the reporter was unaffected by hypoxia. Thus, we looked at the 3' flanking region and, at + 168 nucleotides from the stop codon, an HIF-1α binding site was proven to be active in hypoxia, using a construct in which the site was cloned downstream GFP as reporter gene. Deletion of the site in the construct abolished GFP enhancement upon hypoxia. Chromatin immunoprecipitation revealed the engagement by HIF-1α of this site in hypoxia. Methylation analysis of this 3' enhancer showed that it was free of methylation in 70% of cells in A2780, while in less than 16% in both TC1 and HeLa cells, thereby suggesting that TUBB3 increase upon hypoxia is abolished through methylation of the 3' enhancer.
-
Hypoxia induces class III beta-tubulin gene expression by HIF-1alpha binding to its 3' flanking region.
Gene, 2007Co-Authors: Giuseppina Raspaglio, Flavia Filippetti, Roberta Penci, Ilaria De Maria, Simona Mozzetti, Lucia Cicchillitti, Silvia Prislei, Giovanni Scambia, Cristiano FerliniAbstract:Class III beta-tubulin (TUBB3) overexpression represents a major mechanism of drug resistance to microtubule interacting agents such as taxanes and Vinca alkaloids. Here, we tested hypoxia as a possible inducer of TUBB3. The effects of hypoxia on TUBB3 expression were monitored at mRNA and protein level in A2780, in its paclitaxel-resistant counterpart (TC1) and in HeLa cells. Hypoxia was a strong inducer of TUBB3 in A2780, but not in TC1 and HeLa cells. In A2780 HIF-1alpha was knocked down using RNA interference and TUBB3 expression was assessed in normoxia and hypoxia. The silencing abolished the hypoxia-dependent increase of TUBB3, thereby demonstrating that HIF-1alpha mediates TUBB3 induction in hypoxia. To investigate this phenomenon, the 5' flanking region of human TUBB3 was cloned upstream GFP as a reporter. This region contained the promoter gene, but activity of the reporter was unaffected by hypoxia. Thus, we looked at the 3' flanking region and, at +168 nucleotides from the stop codon, an HIF-1alpha binding site was proven to be active in hypoxia, using a construct in which the site was cloned downstream GFP as reporter gene. Deletion of the site in the construct abolished GFP enhancement upon hypoxia. Chromatin immunoprecipitation revealed the engagement by HIF-1alpha of this site in hypoxia. Methylation analysis of this 3' enhancer showed that it was free of methylation in 70% of cells in A2780, while in less than 16% in both TC1 and HeLa cells, thereby suggesting that TUBB3 increase upon hypoxia is abolished through methylation of the 3' enhancer.
Giovanni Scambia - One of the best experts on this subject based on the ideXlab platform.
-
Preoperative Anti-Class III β-Tubulin Antibodies As Relevant Clinical Biomarkers in Ovarian Cancer.
Translational oncology, 2018Co-Authors: Enrica Martinelli, Giuseppina Raspaglio, Andrea Fattorossi, Alessandra Battaglia, Marco Petrillo, Gian Franco Zannoni, Mara Fanelli, Daniela Gallo, Giovanni ScambiaAbstract:Class III β-tubulin (TUBB3) overexpression in ovarian cancer (OC) associates with poor prognosis. We investigated whether TUBB3 overexpression elicited anti-TUBB3 antibody production in OC patients and whether these antibodies may have diagnostic and prognostic impact. The presence of serum anti-TUBB3 antibodies was investigated in 49 untreated OC patients and 44 healthy individuals by an in-house developed ELISA that used recombinant TUBB3 as the antigen. Receiver operating characteristic (ROC) curves were generated to assess the diagnostic accuracy of the assay. Anti-TUBB3 antibodies discriminated OC patients and healthy individuals with excellent sensitivity and specificity (91.8% and 90.9%, respectively). In multivariate analysis, anti-TUBB3 antibody level emerged as an independent prognostic factor for progression free and overall survival. The ELISA was then optimized using a biotin-labeled TUBB3 C-terminal peptide424-450 instead of recombinant TUBB3 as the antigen and streptavidin-coated plates. The diagnostic role of the anti-TUBB3 antibodies was studied in an independent series of 99 OC patients and 80 gynecological benign disease patients. ROC-curve analysis showed a valuable diagnostic potential for serum anti-TUBB3 antibodies to identify OC patients with higher sensitivity and specificity (95.3% and 97.6%, respectively). Overall, our results provide evidence that preoperative anti-TUBB3 antibody level is a promising diagnostic and prognostic biomarker for the management of OC patients.
-
Class III β -Tubulin (TUBB3): More than a Biomarker in Solid Tumors?
Current molecular medicine, 2011Co-Authors: Marisa Mariani, Giovanni Scambia, Shohreh Shahabi, Steven Sieber, Cristiano FerliniAbstract:Class III β-tubulin (TUBB3) is a prominent mechanism of drug resistance expressed in a variety of solid tumors and particularly in lung and ovarian cancer. In the classical view, TUBB3 expression and drug resistance have been linked, and together they have been associated with a perturbation in microtubule dynamics. In keeping with this observation, TUBB3 was associated with drug resistance only when chemotherapy included a taxane in its chemical composition. In this review, we demonstrate that the classical supposition about TUBB3 is not correct, and that instead TUBB3 expression is linked to drug resistance as a complex survival mechanism activated by microenvironmental conditions such as poor nutrient supply and hypoxia.
-
Proteomic characterization of cytoskeletal and mitochondrial class III β-tubulin
Molecular cancer therapeutics, 2008Co-Authors: Lucia Cicchillitti, Flavia Filippetti, Roberta Penci, Giovanni Scambia, Michela Di Michele, Domenico Rotilio, Maria Benedetta Donati, Cristiano FerliniAbstract:Class III β-tubulin (TUBB3) has been discovered as a marker of drug resistance in human cancer. To get insights into the mechanisms by which this protein is involved in drug resistance, we analyzed TUBB3 in a panel of drug-sensitive and drug-resistant cell lines. We identified two main different isoforms of TUBB3 having a specific electrophoretic profile. We showed that the apparently higher molecular weight isoform is glycosylated and phosphorylated and it is localized in the cytoskeleton. The apparently lower molecular weight isoform is instead found exclusively in mitochondria. We observed that levels of phosphorylation and glycosylation of TUBB3 are associated with the resistant phenotype and compartmentalization into cytoskeleton. By two-dimensional nonreduced/reduced SDS-PAGE analysis, we also found that TUBB3 protein in vivo forms protein complexes through intermolecular disulfide bridges. Through TUBB3 immunoprecipitation, we isolated protein species able to interact with TUBB3. Following trypsin digestion, these proteins were characterized by mass spectrometry analysis. Functional analysis revealed that these proteins are involved in adaptation to oxidative stress and glucose deprivation, thereby suggesting that TUBB3 is a survival factor able to directly contribute to drug resistance. Moreover, glycosylation of TUBB3 could represent an attractive pathway whose inhibition could hamper cytoskeletal compartmentalization and TUBB3 function. [Mol Cancer Ther 2008;7(7):2070–9]
-
hypoxia induces class iii beta tubulin gene expression by hif 1α binding to its 3 flanking region
Gene, 2008Co-Authors: Giuseppina Raspaglio, Flavia Filippetti, Roberta Penci, Ilaria De Maria, Simona Mozzetti, Lucia Cicchillitti, Silvia Prislei, Giovanni Scambia, Cristiano FerliniAbstract:Abstract Class III β-tubulin (TUBB3) overexpression represents a major mechanism of drug resistance to microtubule interacting agents such as taxanes and Vinca alkaloids. Here, we tested hypoxia as a possible inducer of TUBB3. The effects of hypoxia on TUBB3 expression were monitored at mRNA and protein level in A2780, in its paclitaxel-resistant counterpart (TC1) and in HeLa cells. Hypoxia was a strong inducer of TUBB3 in A2780, but not in TC1 and HeLa cells. In A2780 HIF-1α was knocked down using RNA interference and TUBB3 expression was assessed in normoxia and hypoxia. The silencing abolished the hypoxia-dependent increase of TUBB3, thereby demonstrating that HIF-1α mediates TUBB3 induction in hypoxia. To investigate this phenomenon, the 5' flanking region of human TUBB3 was cloned upstream GFP as a reporter. This region contained the promoter gene, but activity of the reporter was unaffected by hypoxia. Thus, we looked at the 3' flanking region and, at + 168 nucleotides from the stop codon, an HIF-1α binding site was proven to be active in hypoxia, using a construct in which the site was cloned downstream GFP as reporter gene. Deletion of the site in the construct abolished GFP enhancement upon hypoxia. Chromatin immunoprecipitation revealed the engagement by HIF-1α of this site in hypoxia. Methylation analysis of this 3' enhancer showed that it was free of methylation in 70% of cells in A2780, while in less than 16% in both TC1 and HeLa cells, thereby suggesting that TUBB3 increase upon hypoxia is abolished through methylation of the 3' enhancer.
-
Hypoxia induces class III beta-tubulin gene expression by HIF-1alpha binding to its 3' flanking region.
Gene, 2007Co-Authors: Giuseppina Raspaglio, Flavia Filippetti, Roberta Penci, Ilaria De Maria, Simona Mozzetti, Lucia Cicchillitti, Silvia Prislei, Giovanni Scambia, Cristiano FerliniAbstract:Class III beta-tubulin (TUBB3) overexpression represents a major mechanism of drug resistance to microtubule interacting agents such as taxanes and Vinca alkaloids. Here, we tested hypoxia as a possible inducer of TUBB3. The effects of hypoxia on TUBB3 expression were monitored at mRNA and protein level in A2780, in its paclitaxel-resistant counterpart (TC1) and in HeLa cells. Hypoxia was a strong inducer of TUBB3 in A2780, but not in TC1 and HeLa cells. In A2780 HIF-1alpha was knocked down using RNA interference and TUBB3 expression was assessed in normoxia and hypoxia. The silencing abolished the hypoxia-dependent increase of TUBB3, thereby demonstrating that HIF-1alpha mediates TUBB3 induction in hypoxia. To investigate this phenomenon, the 5' flanking region of human TUBB3 was cloned upstream GFP as a reporter. This region contained the promoter gene, but activity of the reporter was unaffected by hypoxia. Thus, we looked at the 3' flanking region and, at +168 nucleotides from the stop codon, an HIF-1alpha binding site was proven to be active in hypoxia, using a construct in which the site was cloned downstream GFP as reporter gene. Deletion of the site in the construct abolished GFP enhancement upon hypoxia. Chromatin immunoprecipitation revealed the engagement by HIF-1alpha of this site in hypoxia. Methylation analysis of this 3' enhancer showed that it was free of methylation in 70% of cells in A2780, while in less than 16% in both TC1 and HeLa cells, thereby suggesting that TUBB3 increase upon hypoxia is abolished through methylation of the 3' enhancer.
Sarah Mackinnon - One of the best experts on this subject based on the ideXlab platform.
-
Two unique TUBB3 mutations cause both CFEOM3 and malformations of cortical development.
American Journal of Medical Genetics Part A, 2015Co-Authors: Mary C. Whitman, Wai-man Chan, Caroline Andrews, Max A. Tischfield, Steven F. Stasheff, Francesco Brancati, Xilma R. Ortiz-gonzalez, Sara Nuovo, Francesco Garaci, Sarah MackinnonAbstract:One set of missense mutations in the neuron specific beta tubulin isotype 3 (TUBB3) has been reported to cause malformations of cortical development (MCD), while a second set has been reported to cause isolated or syndromic Congenital Fibrosis of the Extraocular Muscles type 3 (CFEOM3). Because TUBB3 mutations reported to cause CFEOM had not been associated with cortical malformations, while mutations reported to cause MCD had not been associated with CFEOM or other forms of paralytic strabismus, it was hypothesized that each set of mutations might alter microtubule function differently. Here, however, we report two novel de novo heterozygous TUBB3 amino acid substitutions, G71R and G98S, in four patients with both MCD and syndromic CFEOM3. These patients present with moderately severe CFEOM3, nystagmus, torticollis, and developmental delay, and have intellectual and social disabilities. Neuroimaging reveals defective cortical gyration, as well as hypoplasia or agenesis of the corpus callosum and anterior commissure, malformations of hippocampi, thalami, basal ganglia and cerebella, and brainstem and cranial nerve hypoplasia. These new TUBB3 substitutions meld the two previously distinct TUBB3-associated phenotypes, and implicate similar microtubule dysfunction underlying both.
-
Expanding the Phenotypic Spectrum and Variability of Endocrine Abnormalities Associated With TUBB3 E410K Syndrome
The Journal of clinical endocrinology and metabolism, 2015Co-Authors: Ravikumar Balasubramanian, Sheena Chew, Wai-man Chan, Peter B. Kang, Caroline Andrews, Sarah Mackinnon, Elizabeth C. EngleAbstract:Context: A heterozygous de novo c.1228G>A mutation (E410K) in the TUBB3 gene encoding the neuronal-specific β-tubulin isotype 3 (TUBB3) causes the TUBB3 E410K syndrome characterized by congenital fibrosis of the extraocular muscles (CFEOM), facial weakness, intellectual and social disabilities, and Kallmann syndrome (anosmia with hypogonadotropic hypogonadism). All TUBB3 E410K subjects reported to date are sporadic cases. Objective: This study aimed to report the clinical, genetic, and molecular features of a familial presentation of the TUBB3 E410K syndrome. Design: Case report of a mother and three affected children with clinical features of the TUBB3 E410K syndrome. Setting: Academic Medical Center. Main Outcome Measures: Genetic analysis of the TUBB3 gene and clinical evaluation of endocrine and nonendocrine phenotypes. Results: A de novo TUBB3 c.1228G>A mutation arose in a female proband who displayed CFEOM, facial weakness, intellectual and social disabilities, and anosmia. However, she underwent no...
-
A novel syndrome caused by the E410K amino acid substitution in the neuronal β-tubulin isotype 3.
Brain, 2013Co-Authors: Sheena Chew, Ravikumar Balasubramanian, Wai-man Chan, Peter B. Kang, Caroline Andrews, Bryn D. Webb, Sarah Mackinnon, Darren T. Oystreck, Jessica K. Rankin, Thomas O. CrawfordAbstract:Missense mutations in TUBB3, the gene that encodes the neuronal-specific protein β-tubulin isotype 3, can cause isolated or syndromic congenital fibrosis of the extraocular muscles, a form of complex congenital strabismus characterized by cranial nerve misguidance. One of the eight TUBB3 mutations reported to cause congenital fibrosis of the extraocular muscles, c.1228G>A results in a TUBB3 E410K amino acid substitution that directly alters a kinesin motor protein binding site. We report the detailed phenotypes of eight unrelated individuals who harbour this de novo mutation, and thus define the 'TUBB3 E410K syndrome'. Individuals harbouring this mutation were previously reported to have congenital fibrosis of the extraocular muscles, facial weakness, developmental delay and possible peripheral neuropathy. We now confirm by electrophysiology that a progressive sensorimotor polyneuropathy does indeed segregate with the mutation, and expand the TUBB3 E410K phenotype to include Kallmann syndrome (hypogonadotropic hypogonadism and anosmia), stereotyped midface hypoplasia, intellectual disabilities and, in some cases, vocal cord paralysis, tracheomalacia and cyclic vomiting. Neuroimaging reveals a thin corpus callosum and anterior commissure, and hypoplastic to absent olfactory sulci, olfactory bulbs and oculomotor and facial nerves, which support underlying abnormalities in axon guidance and maintenance. Thus, the E410K substitution defines a new genetic aetiology for Moebius syndrome, Kallmann syndrome and cyclic vomiting. Moreover, the c.1228G>A mutation was absent in DNA from ∼600 individuals who had either Kallmann syndrome or isolated or syndromic ocular and/or facial dysmotility disorders, but who did not have the combined features of the TUBB3 E410K syndrome, highlighting the specificity of this phenotype-genotype correlation. The definition of the TUBB3 E410K syndrome will allow clinicians to identify affected individuals and predict the mutation based on clinical features alone.
Sheena Chew - One of the best experts on this subject based on the ideXlab platform.
-
Neuronal-Specific TUBB3 Is Not Required for Normal Neuronal Function but Is Essential for Timely Axon Regeneration
Cell reports, 2018Co-Authors: Alban Latremoliere, Sheena Chew, Long Cheng, Michelle M. Delisle, Elizabeth B. Hutchinson, Andrew Sheridan, Chloe Alexandre, Frederic Latremoliere, Shu Hsien SheuAbstract:Summary We generated a knockout mouse for the neuronal-specific β-tubulin isoform TUBB3 to investigate its role in nervous system formation and maintenance. TUBB3−/− mice have no detectable neurobehavioral or neuropathological deficits, and upregulation of mRNA and protein of the remaining β-tubulin isotypes results in equivalent total β-tubulin levels in TUBB3−/− and wild-type mice. Despite similar levels of total β-tubulin, adult dorsal root ganglia lacking TUBB3 have decreased growth cone microtubule dynamics and a decreased neurite outgrowth rate of 22% in vitro and in vivo. The effect of the 22% slower growth rate is exacerbated for sensory recovery, where fibers must reinnervate the full volume of the skin to recover touch function. Overall, these data reveal that, while TUBB3 is not required for formation of the nervous system, it has a specific role in the rate of peripheral axon regeneration that cannot be replaced by other β-tubulins.
-
Expanding the Phenotypic Spectrum and Variability of Endocrine Abnormalities Associated With TUBB3 E410K Syndrome
The Journal of clinical endocrinology and metabolism, 2015Co-Authors: Ravikumar Balasubramanian, Sheena Chew, Wai-man Chan, Peter B. Kang, Caroline Andrews, Sarah Mackinnon, Elizabeth C. EngleAbstract:Context: A heterozygous de novo c.1228G>A mutation (E410K) in the TUBB3 gene encoding the neuronal-specific β-tubulin isotype 3 (TUBB3) causes the TUBB3 E410K syndrome characterized by congenital fibrosis of the extraocular muscles (CFEOM), facial weakness, intellectual and social disabilities, and Kallmann syndrome (anosmia with hypogonadotropic hypogonadism). All TUBB3 E410K subjects reported to date are sporadic cases. Objective: This study aimed to report the clinical, genetic, and molecular features of a familial presentation of the TUBB3 E410K syndrome. Design: Case report of a mother and three affected children with clinical features of the TUBB3 E410K syndrome. Setting: Academic Medical Center. Main Outcome Measures: Genetic analysis of the TUBB3 gene and clinical evaluation of endocrine and nonendocrine phenotypes. Results: A de novo TUBB3 c.1228G>A mutation arose in a female proband who displayed CFEOM, facial weakness, intellectual and social disabilities, and anosmia. However, she underwent no...
-
A novel syndrome caused by the E410K amino acid substitution in the neuronal β-tubulin isotype 3.
Brain, 2013Co-Authors: Sheena Chew, Ravikumar Balasubramanian, Wai-man Chan, Peter B. Kang, Caroline Andrews, Bryn D. Webb, Sarah Mackinnon, Darren T. Oystreck, Jessica K. Rankin, Thomas O. CrawfordAbstract:Missense mutations in TUBB3, the gene that encodes the neuronal-specific protein β-tubulin isotype 3, can cause isolated or syndromic congenital fibrosis of the extraocular muscles, a form of complex congenital strabismus characterized by cranial nerve misguidance. One of the eight TUBB3 mutations reported to cause congenital fibrosis of the extraocular muscles, c.1228G>A results in a TUBB3 E410K amino acid substitution that directly alters a kinesin motor protein binding site. We report the detailed phenotypes of eight unrelated individuals who harbour this de novo mutation, and thus define the 'TUBB3 E410K syndrome'. Individuals harbouring this mutation were previously reported to have congenital fibrosis of the extraocular muscles, facial weakness, developmental delay and possible peripheral neuropathy. We now confirm by electrophysiology that a progressive sensorimotor polyneuropathy does indeed segregate with the mutation, and expand the TUBB3 E410K phenotype to include Kallmann syndrome (hypogonadotropic hypogonadism and anosmia), stereotyped midface hypoplasia, intellectual disabilities and, in some cases, vocal cord paralysis, tracheomalacia and cyclic vomiting. Neuroimaging reveals a thin corpus callosum and anterior commissure, and hypoplastic to absent olfactory sulci, olfactory bulbs and oculomotor and facial nerves, which support underlying abnormalities in axon guidance and maintenance. Thus, the E410K substitution defines a new genetic aetiology for Moebius syndrome, Kallmann syndrome and cyclic vomiting. Moreover, the c.1228G>A mutation was absent in DNA from ∼600 individuals who had either Kallmann syndrome or isolated or syndromic ocular and/or facial dysmotility disorders, but who did not have the combined features of the TUBB3 E410K syndrome, highlighting the specificity of this phenotype-genotype correlation. The definition of the TUBB3 E410K syndrome will allow clinicians to identify affected individuals and predict the mutation based on clinical features alone.
Maria Kavallaris - One of the best experts on this subject based on the ideXlab platform.
-
Abstract 3660: Discovery of a small molecule TUBB3/βIII-tubulin modulator in lung cancer
Cancer Chemistry, 2015Co-Authors: Felicity Chao Lin Kao, Tim Failes, Greg M. Arndt, Murray D. Norris, Maria KavallarisAbstract: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
-
abstract 3660 discovery of a small molecule TUBB3 βiii tubulin modulator in lung cancer
Cancer Research, 2015Co-Authors: Felicity Kao, Tim Failes, Greg M. Arndt, Murray D. Norris, Maria KavallarisAbstract: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
