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1639.Full.Pdf Cancer Therapy: Preclinical Stomatin-like Protein 2 Is Overexpressed in Cancer and Involved in Regulating Cell Growth and Cell Adhesion in Human Esophageal Squamous Cell Carcinoma Liyong Zhang,1, 2 Fang Ding,1Wenfeng Cao,1Zhongmin Liu,1Wei Liu,3 ZaichengYu,3 Yu Wu,1 Wendong Li,1Yan d a L i, 2 and Zhihua Liu1 Abstract Purpose: Stomatin-like protein 2 (SLP-2) is a novel and unusual stomatin homologue of unknown functions. It has been implicated in interaction with erythrocyte cytoskeleton and presumablyotherintegralmembrane proteins, but not directly with the membrane bilayer.Weshow here the involvement of SLP-2 inhuman esophageal squamous cell carcinoma (ESCC), lung cancer, laryngeal cancer, and endometrial adenocarcinoma and the effects of SLP-2 on ESCC cells. Experimental Design: Previous work of cDNA microarray in our laboratory revealed that SLP-2 was significantly up-regulated in ESCC. The expression of SLP-2 was further evaluated in human ESCC, lung cancer, laryngeal cancer, and endometrial adenocarcinoma by semiquan- titative reverse transcription-PCR, Western blot, and immunohistochemistry. Mutation detection of SLP-2 exons was done by PCR and automated sequencing. Antisense SLP-2 eukaryotic expression plasmids were constructed and transfected into human ESCC cell line KYSE450. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, clonogenecity assay, flow cytometry assay, nude mice tumorigenetic assay, and cell attachment assay were done to investigate the roles of SLP-2 gene. Results: All tumor types we tested showed overexpression of SLP-2 compared with their normal counterparts (P V 0.05). Moreover, immunohistochemistry analysis of mild dysplasia, severe dys- plasia, and ESCC showed that overexpressionof SLP-2 occurredinpremalignant lesions. Mutation analysis indicated that no mutation was found in SLP-2 exons. KYSE450 cells transfected with antisense SLP-2 showed decreased cell growth, proliferation, tumorigenecity, and cell adhesion. Conclusions: SLP-2 was first identified as a novel cancer-related gene overexpressed in human ESCC, lung cancer, laryngeal cancer, and endometrial adenocarcinoma. Decreased cell growth, cell adhesion, and tumorigenesis in the antisense transfectants revealed that SLP-2 may be important in tumorigenesis. Esophageal squamous cell carcinoma (ESCC), the major multistage process involving multifactorial etiology and histologic form of esophageal cancer, is one of the most genetic-environment interactions (1, 2). Genetic changes are frequent fatal malignancies in the world, especially in the speculated to be one of the most important causes for the high northern part of China. Human ESCC carcinogenesis is a prevalence and familial aggregation of ESCC in China (3). It is known that some of the complicated genetic alternations occur frequently in the tumorigenesis of esophagus, such as point Authors’ Affiliations: 1National Laboratory of Molecular Oncology, Cancer mutations of p53 and p16; amplification of cyclin D, c-myc, Institute and Hospital, Chinese Academy of Medical Sciences and Peking Union hst-1, int-2, and epidermal growth factor receptor; as well as Medical College; 2MOE Key Laboratory of Bioinformatics, Department of Automation, Tsinghua University, Beijing, P.R. China; and 3Department of Thoracic allelic loss on chromosomes 3p, 4p, 4q, 5q, 9p21-22, 11p, 11q, Surgery, the First Affiliate Hospital of Anhui Medical University, Hefei, P.R. China 13q, 17p, 18q, and 21q (4–7). However, this is not sufficient Received 8/24/05; revised 12/4/05; accepted 12/27/05. to understand the common pathway of tumorigenesis and Grant support: National Natural Science Foundation of China grants 60234020 progression of ESCC. Previous study in our lab has also and 30425027, China Key Program on Basic Research grant 2004CB518604, National Hi-Tech R&D Program of China grant 2004AA231041, and China revealed that a Mendelian autosomal recessive major gene may Postdoctoral Science Foundation grant 2004035306. play a significant role in the etiology of ESCC in a moderate The costs of publication of this article were defrayed in part by the payment of page high-incidence area of northern China (1). However, the charges. This article must therefore be hereby marked advertisement in accordance molecular pathways involved in the pathogenesis of ESCC with 18 U.S.C. Section 1734 solely to indicate this fact. Note: Supplementary data for this article are available at Clinical Cancer Research remain poorly defined. To better understand the molecular online (http://clincancerres.aacrjournals.org/). mechanisms that underlie the tumor formation and progres- Requests for reprints: Zhihua Liu, National Laboratory of Molecular Oncology, sion, we previously analyzed gene expression profiles of Cancer Institute, Chinese Academy of Medical Sciences and Peking Union Medical normal and tumor cells systematically (8–10). In addition, College, Beijing 100021, P.R. China. Phone/Fax: 86-10-67723789; E-mail: previous work on cDNA microarry representing 34,176 clones [email protected]. F 2006 American Association for Cancer Research. revealed that SLP-2 was up-regulated over six times in ESCC doi:10.1158/1078-0432.CCR-05-1858 tissues (11, 12). www.aacrjournals.org 1639 Clin Cancer Res 2006;12(5) March 1, 2006 Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2006 American Association for Cancer Research. Cancer Therapy: Preclinical SLP-2 is a novel and unusual member of the stomatin gene TTCTCTGGAT-3V(antisense), both at annealing temperature of 57jC superfamily (13–15). The vertebrate homologues identified are for 27 cycles. The expression of the housekeeping gene GAPDH was SLP-1, SLP-2, and SLP-3 (13, 14, 16). SLP-1 is most abundant used as an internal control. in brain and shares many similarities with UNC-24 (13, 14). Antibody production and Western blot analysis. Amino acid sequences of SLP-2 were obtained from National Center for Biotech- SLP-3 is specifically expressed in olfactory sensory neurons nology Information Protein database and subjected to analysis of (13, 16). All of these proteins as well as the stomatin from other secondary structure, hydrophilicity profile, and antigenecity using the species share a characteristic NH2-terminal hydrophobic program Protean of DNAStar software (DNAStar, Inc., Madison, WI). domain as well as a consensus stomatin signature sequence The polypeptide composed of 13 amino acids (ASLDEELDRVKMS) at that defines the stomatin gene family (13). Similar to other the COOH-terminal of SLP-2 (corresponding to amino acids 344-356) family members, SLP-2 shares the cognate stomatin signature was chosen and synthesized, coupled with keyhole limpet hemocyanin sequence. However, it is the first member of this family to be at the end of the NH2-terminal, and emulsified in complete Freund’s recognized that lacks an NH2-terminal hydrophobic domain adjuvant used as antigens for immunization with New Zealand White (13). These features distinguish it from stomatin, SLP-1, and rabbit and chicken, respectively. Immune response was enhanced with SLP-3. SLP-2 may link stomatin or other integral membrane antigens emulsified in incomplete Freund’s adjuvant after 14 and 21 proteins to the peripheral cytoskeleton and play a role in days of immunization, respectively. Antibodies were recovered after being subjected to ELISA by Zhuhai Bioinforbody Inc. (Zhuhai, China). regulating ion channel conductance or the organization of The rabbit SLP-2 antibody was then purified by protein A. sphingolipid and cholesterol-rich lipid rafts (13). For Western blot analysis, cells or tissues were lysed with the buffer In this study, the expression of SLP-2 in four disparate tumor [1% SDS, 10 mmol/L Tris-Cl (pH 7.6), 20 Ag/AL aprotinin, 20 Ag/AL types, ESCC, lung cancer, laryngeal carcinoma, and endome- leupeptin, and 1 mmol/L 4-(2-aminoethyl)benzenosulfonyl fluoride]. trium adenocarcinoma, was evaluated by semiquantitative The protein concentrations were determined using the Bicinchoninic reverse transcription-PCR (RT-PCR), Western blot, and immu- Acid Protein Assay kit (Pierce, Rockford, IL). Ten micrograms of protein nohistochemistry. To investigate the role of SLP-2 in tumori- were separated on 12% of SDS-PAGE gels and transferred to genesis, antisense-oriented SLP-2 was stably transfected into polyvinylidene difluoride membranes. After blocking, the membranes KYSE450 cells. Antisense transfection of SLP-2 in KYSE450 were incubated with the appropriate primary antibody, anti-SLP-2 cells not only obviously reduced cell growth and proliferation antibody (1:1,000 dilution) or mouse monoclonal antibody against in vitro but also inhibited tumor formation and growth in vivo. fibronectin 1 (1:1,000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), at 4jC overnight. After washing, the membranes were The identification of SLP-2 as a novel cancer-related gene may incubated with secondary antibody at a dilution of 1:3,000 at room have implications for understanding tumorigenesis. temperature for 1 hour. Proteins were detected with the enhanced chemiluminescence kit (Amersham Pharmacia Biotechnology, Inc., Piscataway, NJ) and anti-h-actin antibody (Sigma, St. Louis, MO) was Materials and Methods used as loading control. Immunohistochemical staining. Tissue sections were dewaxed with Tissue specimens. Fresh tissue specimens were taken from patients xylene and rehydrated through gradient ethanol into water. After presented to Cancer Hospital of Chinese Academy of Medical Sciences endogenous
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