[CANCER RESEARCH 60, 6526 – 6530, November 15, 2000] Treatment of Prostate Cancer by Radioiodine Therapy after Tissue-specific Expression of the Sodium Iodide Symporter 1 Christine Spitzweg, 2 Michael K. O’Connor, Elizabeth R. Bergert, Donald J. Tindall, Charles Y. F. Young, and John C. Morris Departments of Endocrinology [C. S., E. R. B., J. C. M.], Nuclear Medicine [M. K. O.], and Urology [D. J. T., C. Y. F. Y.], Mayo Clinic, Rochester, Minnesota 55905 ABSTRACT Causing prostate cancer cells to express functionally active sodium iodide symporter (NIS) by targeted NIS gene transfer might offer the possibility of radioiodine therapy of prostate cancer. Therefore, we inves- tigated radioiodine accumulation and therapeutic effectiveness of 131 I in NIS-transfected prostate cancer cells in vitro and in vivo. The human prostatic adenocarcinoma cell line LNCaP was stably transfected with NIS cDNA under the control of the prostate-specific antigen promoter. The stably transfected LNCaP cell line NP-1 showed perchlorate-sensitive, androgen-dependent iodide uptake in vitro that resulted in selective killing of these cells by 131 I in an in vitro clonogenic assay. Xenografts were established in athymic nude mice and imaged using a gamma camera after i.p. injection of 500 Ci of 123 I. In contrast to the NIS-negative control tumors (P-1) which showed no in vivo uptake of 123 I, NP-1 tumors accu- mulated 25–30% of the total 123 I administered with a biological half-life of 45 h. In addition, NIS protein expression in LNCaP cell xenografts was confirmed by Western blot analysis and immunohistochemistry. After a single i.p. application of a therapeutic 131 I dose (3 mCi), significant tumor reduction was achieved in NP-1 tumors in the therapy group compared with P-1 tumors and tumors in the control group. In conclusion, a ther- apeutic effect of 131 I has been demonstrated in prostate cancer cells after induction of tissue-specific iodide uptake activity by prostate-specific antigen promoter-directed NIS expression in vitro and in vivo. This study demonstrates the potential of NIS as a novel therapeutic gene for nonthy- roidal cancers, in particular prostate cancer. INTRODUCTION Prostate cancer is an important health issue in American men, representing the second leading cause of cancer death (1). Because no curative therapy for metastatic prostate cancer exists, novel therapeu- tic strategies are urgently needed. In marked contrast, metastatic thyroid cancer can be effectively managed, even in advanced cases, because of the ability of thyroidal cells to trap and concentrate iodine, making therapy with radioactive iodine possible and highly effective (2). Recently, the mechanism mediating iodide uptake across the basolateral membrane of thyroid follicular cells has been clarified by the cloning and characterization of the sodium iodide symporter, NIS (3–5). NIS is an intrinsic membrane glycoprotein with 13 putative transmembrane domains and is responsible for the ability of the thyroid gland to transport and concentrate iodide approximately 20 – 40-fold above plasma concentration (6). A novel form of gene therapy, using NIS gene transfer to induce iodide accumulation activity in prostate cancer cells by expression of functionally active NIS, would therefore extend the utility of radio- iodine therapy to the treatment of prostate cancer. To minimize extratumoral toxicity, a tissue-specific promoter, such as the PSA 3 promoter, may be used to provide selective, prostate-specific NIS gene expression (7–9). The PSA promoter has been extensively character- ized in recent years and has been shown to be responsible for prostate- specific and androgen-regulated expression of PSA, a serine protease of 237 amino acids, that is mainly expressed within the epithelial lining and acini of the prostate gland (10 –13). We reported recently the induction of tissue-specific, androgen- dependent iodide uptake activity in prostate cancer cells in vitro by PSA promoter-directed NIS gene delivery (14). The androgen-sensi- tive human prostatic adenocarcinoma cell line LNCaP was stably transfected with an expression vector in which full-length NIS cDNA had been coupled to a 6-kb PSA promoter fragment (14, 15). The stably transfected LNCaP cell line NP-1 showed perchlorate-sensitive, androgen-dependent iodide uptake activity, whereas no iodide uptake activity was detected in LNCaP cells transfected with the control vectors. The magnitude of iodide uptake in NP-1 cells concentrating 125 I 50-fold was highly encouraging and suggested that a therapeu- tic effect of accumulated radioiodine (14) could be achieved. Al- though these in vitro data suggested the feasibility of the concept of NIS gene transfer as a first step toward radioiodine therapy of prostate cancer, its utility required direct demonstration. Therefore, the aim of our current study was to investigate radioiodine accumulation in NIS-transfected LNCaP cell xenografts in vivo and to examine the therapeutic effectiveness of 131 I in vitro and in vivo for prostate cancer cells. MATERIALS AND METHODS Plasmid Constructs. The expression and control vectors have been gen- erated as described previously (14). The resulting expression plasmid construct contained full-length NIS cDNA coupled to the 6-kb PSA promoter fragment (Ref. 15; NIS/PSA-pEGFP-1). Two control vectors were designed containing NIS cDNA without the PSA promoter (NIS-pEGFP-1) and the PSA promoter without NIS cDNA (PSA-pEGFP-1). Establishment of Stable Transfected LNCaP Cell Lines. Stable trans- fection of LNCaP cells was performed as described previously (14). In brief, the androgen-sensitive human prostatic adenocarcinoma cell line LNCaP was transfected with NIS/PSA-pEGFP-1 and the control vectors NIS-pEGFP-1 and PSA-pEGFP-1, respectively, using LipofectAMINE Plus Reagent (Life Tech- nologies, Inc., Gaithersburg, MD). Selection was performed with geneticin, and surviving clones were isolated and subjected to screening for androgen- dependent iodide uptake activity. NP-1, the clone with the highest androgen- dependent iodide uptake activity, was chosen for the following studies, as well as the stably transfected (PSA-pEGFP-1) control cell line P-1 (14). In Vitro Clonogenic Assay. LNCaP cells stably transfected with the ex- pression vector (NP-1) or the control vector (P-1) were incubated for 7 h with 0.8 mCi Na 131 I in HBSS supplemented with 10 M NaI and 10 mM HEPES at pH 7.3. After incubation with radioiodine, cells were trypsinized and plated in quadruplicates at cell densities of 1000, 2000, 3000, 5000, and 7000 cells/well in 12-well plates. Four weeks later, after colony development, cells were fixed with methanol and stained with crystal violet, and colonies containing 50 cells were counted. Parallel experiments were performed for each cell line Received 4/21/00; accepted 9/20/00. 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 This study was supported in part by Grant Sp 581/1-1 (to C. S.) from the German Research Council (Deutsche Forschungsgemeinschaft, Bonn, Germany), by NIH Grants DK 41995 and CA 70892 (to D. J. T. and C. Y. F. Y.), by a CaPCURE research award (to J. C. M.), and by the Mayo Foundation. 2 To whom requests for reprints should be addressed, at Division of Endocrinology, Mayo Clinic, Guggenheim 625, 200 First Street SW, Rochester, MN 55905. Phone: (507) 284-2324; Fax: (507) 284-4521; E-mail: spitzweg.christine@mayo.edu. 3 The abbreviations used are: PSA, prostate-specific antigen; hNIS, human sodium iodide symporter. 6526 Research. on December 28, 2015. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from