[CANCER RESEARCH 62, 801– 808, February 1, 2002] The CLN3 Gene is a Novel Molecular Target for Cancer Drug Discovery 1 Svetlana N. Rylova, Andrea Amalfitano, Dixie-Ann Persaud-Sawin, Wei-Xing Guo, Jerry Chang, Paul J. Jansen, Alan D. Proia, and Rose-Mary Boustany 2 Departments of Pediatrics and Neurobiology [S. N. R., D-A. P-S., W-X. G., J. C., P. J. J., R-M. B.], Genetics [A. A.], and Pathology [A. D. P.], Duke University Medical Center, Durham, North Carolina, 27710 ABSTRACT Juvenile Batten disease is a neurodegenerative disease caused by accel- erated apoptotic death of photoreceptors and neurons attributable to defects in the CLN3 gene. CLN3 is antiapoptotic when overexpressed in NT2 neuronal precursor cells. CLN3 negatively modulates endogenous ceramide levels in NT2 cells and acts upstream of ceramide generation. Because defects in regulation of apoptosis are involved in the development of cancer, we evaluated the expression of CLN3 on both mRNA and protein levels in a variety of cancer cell lines and solid colon cancer tissue. We also observed the effect of the blocking of CLN3 protein expression on cancer cell growth, survival, ceramide production, and apoptosis by using an adenovirus-bearing antisense CLN3 construct. We show that CLN3 mRNA and protein are overexpressed in glioblastoma (U-373G and T98g), neuroblastoma (IMR-32 and SK-N-MC), prostate (Du145, PC-3, and LNCaP), ovarian (SK-OV-3, SW626, and PA-1), breast (BT-20, BT-549, and BT-474), and colon (SW1116, SW480, and HCT 116) cancer cell lines but not in pancreatic (CAPAN and As-PC-1) or lung (A-549 and NCI- H520) cancer cell lines. CLN3 is also up-regulated in mouse melanoma and breast carcinoma cancer cell lines. We found CLN3 expression is 22–330% higher than in corresponding normal colon control tissue in 8 of 10 solid colon tumors. An adenovirus-expressing antisense CLN3 (Ad-AS- CLN3) blocks CLN3 protein expression in DU-145, BT-20, SW1116, and T98g cancer cell lines as seen by Western blot. Blocking of CLN3 expres- sion using Ad-AS-CLN3 inhibits growth and viability of cancer cells. It also causes elevation in endogenous ceramide production through de novo ceramide synthesis and results in increased apoptosis as shown by pro- pidium iodide and JC-1 staining. This suggests that Ad-AS-CLN3 may be an option for therapy in some cancers. More importantly these results suggest that CLN3 is a novel molecular target for cancer drug discovery. INTRODUCTION Mutations in the CLN3 gene are responsible for the JNCL 3 (1). JNCL is a recessively inherited neurodegenerative disorder of childhood (1–3). The clinical hallmarks are progressive loss of vision, seizures, and mental deterioration. These symptoms are attributable to massive cortical neuronal death and gradual loss of photoreceptor cells (2, 3). Apoptosis has been shown to be the mechanism of neurodegeneration in the brain of patients with the juvenile form of Batten disease (4). Moreover, up-regulation of Bcl-2 and elevation of endogenous ceramide levels in the brain from affected individuals provides mechanistic evidence for apoptotic death of neurons in this disorder (5). We have demonstrated previously that stable CLN3 overexpression protects NT2 neuronal precursor cells from serum starvation-induced growth inhibition and also rescues these cells from death caused by treatment with vincristine, etoposide, and staurosporine (6). We have also shown that CLN3 up-regulation decreases the level of the lipid second messenger, ceramide, in these cells and also attenuates vinc- ristine-induced activation of ceramide (6). However, overexpression of CLN3 fails to protect NT2 cells from exogenous ceramide-induced killing. These facts place CLN3 upstream of ceramide in apoptosis signaling and suggest that CLN3 plays an important role in mecha- nisms of cell death and survival. The fact that CLN3 is highly conserved across species from human to yeast and also in Caenorh- abditis elegans and Drosophila underscores its importance for cell function. CLN3 has also been found to be developmentally regulated in differentiating hNT neurons and in neonatal rat brain. Peaks of expression are noted just after hNT cells exit the cell cycle, and on day Po in neonatal rat brain, which corresponds to the period of maximum neuronal growth (7). This suggests that CLN3 is an oncofetal, anti- apoptotic gene. These facts led us to investigate whether CLN3 could be differentially expressed in some cancers. Defects in proapoptotic events can contribute to cancer formation by allowing cells to survive and to proliferate beyond their normal life span (8 –12). Regulation of apoptosis is involved in the development of tumors and plays an essential role in their treatment. A variety of chemotherapeutic agents and radiation kill tumor cells by inducing apoptosis (10, 13, 14). It has been established that many tumor types are resistant to chemotherapy-induced apoptosis. This can occur either because of inactivation of tumor suppressor genes, such as p53 or retinoblastoma, or because of overexpression of antiapoptotic onco- genes, such as Bcl-2 (B-cell lymphoma), Bcr-Abl (myelogenous leu- kemia), BUG-1 (breast cancer), and survivin in a number of cancers and lymphomas (15–18). Identification of novel antiapoptotic genes expressed in cancer cells provides a basis for a better understanding of the biology of these tumors and may lead to discovery of new targets for anticancer drug development (9, 19 –21). Direct inactivation of antiapoptotic gene expression may promote cancer cell death. Sup- pression of antiapoptotic genes can be achieved by antisense strate- gies, which in some instances may improve the efficacy of conven- tional chemotherapy (9). Antisense-based therapies have already been developed to block Bcl-2 overexpression in non-Hodgkin’s lym- phoma (22). In this study we establish that CLN3 mRNA and protein are overexpressed in a number of cancer cell lines including breast, colon, malignant melanoma, prostate, ovarian, neuroblastoma, and glioblas- toma multiforme but not lung or pancreatic cancer cell lines. We also show that CLN3 is overexpressed in 8 of 10 solid human colon cancer cases. Additionally, we demonstrate that blocking CLN3 protein expression in these cancer cell lines, using adenovirus-mediated an- tisense CLN3 methodology, inhibits cancer cell growth and viability. Treatment of cancer cells with Ad-AS CLN3 virus also affects the de novo ceramide synthetic pathway and results in ceramide elevation and cancer cell death by apoptosis. MATERIALS AND METHODS Cell Culture. Cancer cell lines were obtained from American Type Culture Collection (Manassas, VA) and maintained at 37°C and 5% CO 2 except for Received 4/18/01; accepted 12/4/01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by NIH Grant RO-2NS30170 (to R-M. B.) and in part by NIH Grant RO-IDK52925 (to A. A.). The work was also supported by a grant from the Batten Disease Support and Research Association foundation and by a grant from the Battin’ for Betsy Charities Inc. 2 To whom requests for reprints should be addressed, at Duke University Medical Center, MSRB Box 2604, Durham, NC, 27710. Phone: (919) 681-6220; Fax: (919) 681- 8090; E-mail: boust001@mc.duke.edu. 3 The abbreviations used are: JNCL, juvenile form of neuronal ceroid lipofuscinosis; FBS, fetal bovine serum; BSS, balanced salt solution; Ad-AS-CLN3, adenovirus anti- sense-CLN3 vector; MOI, multiplicity of infection; RT-PCR, reverse transcription-PCR; PI, propidium iodide. 801 Research. on November 27, 2021. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from