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A Novel Function for Semaphorin 6A

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A Novel Function for Semaphorin 6A Published OnlineFirst January 9, 2008; DOI: 10.1158/1535-7163.MCT-07-0390 233 From plasma membrane to cytoskeleton: a novel function for semaphorin 6A Silvia Prislei,1 Simona Mozzetti,1 Flavia Filippetti,2 function in the cytoskeleton and a role in modulating Marta De Donato,1 Giuseppina Raspaglio,1 tubulin isotype composition and microtubule dynamics. Lucia Cicchillitti,1 Giovanni Scambia,1,2 [Mol Cancer Ther 2008;7(1):233–41] and Cristiano Ferlini1,2 1Department of Oncology, Campobasso, and 2Laboratory of Introduction Antineoplastic Pharmacology, Department of Obstetrics and Microtubule interacting drugs are widely used in the Gynecology, Catholic University of the Sacred Heart, Rome, Italy treatment of solid tumors, such as advanced ovary, breast, and lung cancers, in which they represent a mainstay in Abstract the medical management of these diseases. Unfortunately, Class III B-tubulin (TUBB3) overexpression has been after an initial response in the first line, the vast majority reported in ovary, lung, breast, and gastric cancer of patients relapse and they do not further respond, with patients. Currently, no clinical drugs are available for a the consequent disease progression and poor outcome. specific targeting of TUBB3, whereas the investigational Study of resistance mechanisms has shown that there is drug IDN5390 specifically interacts with TUBB3. To gain not a unique factor of drug resistance but that several insight into the pathways leading to TUBB3 up-regulation, genes are involved in the generation of the resistant we did a human genome microarray analysis in A2780 phenotype, thereby precluding a successful response to cells made resistant to IDN5390 to identify selected chemotherapy (1). pathways specifically disrupted in resistant cells. Using Within the factors associated to drug resistance, in- h this approach, we discovered that semaphorin 6A creased expression of TUBB3 (class III -tubulin) seems to (SEMA6A) is down-regulated not only in IDN5390- play a prominent role in several tissues. In fact, TUBB3 has resistant cells but also in cells made resistant to cisplatin, been reported overexpressed in several human cancers, topotecan, and doxorubicin, whereas no changes were such as lung (2), ovary (3, 4), gastric (5), and breast (6) noticed in paclitaxel- and gemcitabine-resistant cells. cancers. A direct role in drug resistance is suggested by the Acute treatment with IDN5390 was able to down-regulate finding that TUBB3 is able to reduce sensitivity to bound SEMA6A in cells unselected for drug resistance. TUBB3 paclitaxel and decreased ability by the drug to suppress expression was assessed in A2780 clones with stable microtubule dynamics (7, 8). Therefore, TUBB3 appears an overexpression of SEMA6A and in a panel of clones in attractive target and TUBB3 targeting agents could improve which silencing of the protein was obtained. Quantitative our therapeutic arsenal against drug resistance. A first PCR was then used to check the modulation of SEMA6A example of this is represented by the seco-taxane IDN5390, as well as to assess the expression of TUBB3. TUBB3 was a compound able to selectively target TUBB3 (9). This drug increased (median value, 5.4) and reduced (median value, exhibits unique properties of cytotoxic, antiangiogenic 0.47) in cells with overexpression and silencing of agent and inhibitor of cell motility (10) and is currently SEMA6A, respectively. Thus, the findings indicate a undergoing preclinical development. correlation between the expression of SEMA6A and Study of the role of TUBB3 is complicated by the fact that TUBB3. Then, we found that a form of 83 kDa of this, as all the tubulin isotypes, cannot be easily overex- SEMA6A is expressed in the cytoskeleton in association pressed or silenced because it is tightly regulated in the with B-actin. These findings suggest for SEMA6A a novel cells. Therefore, to get insights into the TUBB3-dependent pathways, we used IDN5390 as a tool to investigate the genes downstream regulated by TUBB3. To this end, we generated two human ovarian adeno- carcinoma cell lines (A2780CG1 and A2780PTX) resistant to IDN5390 and paclitaxel, respectively. Then, through Received 6/11/07; revised 11/6/07; accepted 12/3/07. a full-genome microarray screening, genes diversely Grant support: Italian Ministry of University and Research grant 420111. regulated in the two drug-resistant cells were analyzed. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Using this approach, we discovered that semaphorin 6A advertisement in accordance with 18 U.S.C. Section 1734 solely to (SEMA6A) gene expression is dramatically down-regulated indicate this fact. in A2780CG1 cells. Requests for reprints: Cristiano Ferlini, Laboratory of Antineoplastic Semaphorins are secreted or transmembrane proteins Pharmacology, Department of Obstetrics and Gynecology, Catholic University of the Sacred Heart, L.go A. Gemelli 8, Rome, Italy 00168. initially identified through their role in axon guidance and Phone: 39-635508736. E-mail: [email protected] later involved in regulation of cell motility, immune res- Copyright C 2008 American Association for Cancer Research. ponse, angiogenesis, and tumor progression (reviewed in doi:10.1158/1535-7163.MCT-07-0390 refs. 11–13). Semaphorins contain a conserved extracellular MolCancerTher2008;7(1).January2008 Downloaded from mct.aacrjournals.org on September 24, 2021. © 2008 American Association for Cancer Research. Published OnlineFirst January 9, 2008; DOI: 10.1158/1535-7163.MCT-07-0390 234 TUBB3 and SEMA6A domain of f500 amino acids (Sema domain) that is shared the three independent experiments with the sigmoid-Emax with their receptors plexins and the two tyrosine kinase model using nonlinear regression, weighted by the recip- receptors MET and RON (11). The transmembrane protein rocal of the square of the predicted effect. SEMA6A exhibited elevated levels of mRNA expression in Real-timeQuantitativePCR several renal tumor tissue samples and in renal clear cell Total RNA was obtained from cultured cells using carcinoma cell lines, whereas its recombinant soluble RNeasy plus mini kit (Qiagen) according to the manufac- extracellular domain inhibited in vivo the angiogenesis turer’s directions. cDNA was prepared using iScript cDNA induced by growth factor and tumor cell line (14). Here, Synthesis Kit (Bio-Rad). Real-time quantitative PCR was we describe that SEMA6A is involved in drug resistance and done using the iCycler iQ System (Bio-Rad) and the is an inducer of TUBB3 expression. The detection of a novel iQ SYBR Green Supermix (Bio-Rad) in a final volume of cytoskeletal form of the protein supports its role in 25 AL, starting with a 3-min template denaturation step at intracellular signaling. 95jC followed by 40 cycles of 15 s at 95jC and 1 min at 60jC. The following primers were used: TUBB3 forward 5¶-GCGAGATGTACGAAGACGAC-3¶ and reverse 5¶-TTT- Materials and Methods AGACACTGCTGGCTTCG-3¶, primers 1 forward 5¶-CCCTT- Drugs CTCCGCTCGTCATTGG -3¶ and reverse 5¶-CCTTCGCAA- IDN5390 and paclitaxel were kindly provided by Indena GCCTTTGTCATTCC-3¶, primers 2 forward 5¶-AGGGAGT- and were diluted in absolute DMSO. These solutions were GATTCGGGAAAGTTACC-3¶ and reverse 5¶-ACAGACGC- further diluted at each experimental day to achieve a 0.1% AGTAGACGGTGATG-3¶, and primers 3 forward 5¶-CACA- final DMSO concentration. All the other chemicals were CATGCACACAACACATACAC-3¶ and reverse 5¶-GTATTT- purchased from Sigma unless otherwise specified. GTGTTTTGCAGGTTGGAAC-3¶. To normalize the possible Cell Cultures variation in sample concentration, in each experiment, A2780 human ovarian cancer cells were purchased from quantitative PCR was done on glyceraldehyde-3-phosphate the European Collection of Cell Cultures. Culture media dehydrogenase mRNA with primers glyceraldehyde- were selected according to the suggestions of European 3-phosphate dehydrogenase forward 5¶-CTGACCTGCCG- Collection of Cell Cultures. Cultures were incubated at TCTAGAAA-3¶ and reverse 5¶-CCACCATGGAGAAGG- j ¶ 37 C in a fully humidified atmosphere of 5% CO2/95% air. GTGG-3 . The experiments were done two or more times A2780 cells resistant to paclitaxel (A2780PTX) and IDN5390 and each time the samples were run in triplicate. The results (A2780CG1) were obtained through stepwise increase drug were analyzed as described previously (9) using the Excel concentration. To prevent the expression of P-glycoprotein spreadsheet RelQuant (Bio-Rad) and the SD among the as a factor of drug resistance in the continuous presence different experiments was calculated. of the P-glycoprotein inhibitor SB-RA (10 Amol/L; kindly Northern Blot given by Iwao Ojima), A2780PTX were then grown in Total RNA was obtained from cultured cells using TRI- the presence of 20 nmol/L paclitaxel and SB-RA and Reagent solution (Molecular Research Center) according to A2780CG1 in the presence of 50 nmol/L IDN5390 and SB- the manufacturer’s directions. mRNAs (2 Ag) purified with RA to maintain the drug-resistant phenotype. However, Oligotex mRNA Kit (Qiagen) were run on 1% agarose these cell lines do not depend on drug presence for growth. formaldehyde gel and blotted on Hybond+ nylon mem- A2780CIS and A2780ADR resistant to cisplatin and brane (Amersham Pharmacia). Digoxin-labeled probes doxorubicin were also purchased from European Collec- were obtained by PCR using 0.7 mmol/L alkali-labile tion of Cell Cultures. A2780TOP and A2780GEM, resistant DIG-11-dUTP
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