© 2000 Nature America, Inc. 0929-1903/00/$15.00/ϩ0 www.nature.com/cgt The cellular oncogene EWS/activating transcription factor 1 is unable to activate adenovirus-borne promoters: Implications for cytotoxic prodrug therapy of malignant melanoma of soft parts Raymond W. M. Lung and Kevin A. W. Lee Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, Special Administrative Region, China. The cellular oncoprotein Ewing’s sarcoma oncogene (EWS)/activating transcription factor 1 (ATF1) is a highly specific marker for malignant melanoma of soft parts (MMSP) and is a potent activator of several cAMP-inducible promoters, including the somatostatin promoter. Here we explored the potential for using the somatostatin promoter to direct toxic gene expression in MMSP cells. When introduced into MMSP cells, a somatostatin-herpes simplex virus thymidine kinase fusion gene confers strong and cell-specific sensitivity to the cytotoxic prodrug ganciclovir. Ganciclovir sensitivity requires the ATF-binding site present in the somatostatin promoter, indicating that toxic gene expression is caused by EWS/ATF1. We also tested the efficacy of recombinant adenoviruses adenoviruses for gene delivery and expression in two MMSP cell lines (DTC1 and Su-ccs-1). Surprisingly, several promoters (including somatostatin) that are strongly activated by EWS/ATF1 in transient assays are not activated in DTC1 and Su-ccs-1 cells when present in an adenovirus vector. In summary, our findings demonstrate the potential for using the somatostatin promoter for cytotoxic prodrug therapy for MMSP. However, first-generation adenovirus vectors cannot be used as promoter delivery vehicles for toxic gene expression in MMSP cells. Cancer Gene Therapy (2000) 7, 396–406 Key words: EWS/ATF1 oncogene; malignant melanoma of soft parts; adenovirus; gene therapy. hromosomal translocations of the N-terminal region combined with chemotherapy and radiotherapy,10,13 but Cof the Ewing’s sarcoma oncogene (EWS) to a recurrence is high and mortality is ϳ45% due to meta- variety of cellular transcription factors produces domi- static disease. The need for more effective therapy of nant oncogenes (EWS fusion proteins (EFPs)) that MMSP and other EFP-related tumors is apparent and cause distinct sarcomas.1,2 In malignant melanoma of emerging gene therapy techniques may offer new ap- soft parts (MMSP), EWS is fused to activating transcrip- proaches. 3 tion factor 1 (ATF1); in Ewing’s sarcoma, EWS is fused In the case of Ewing’s sarcoma5 and desmoplastic 4–8 to the ETS domain family members; and in desmo- small round cell tumors14 the oncogenic properties of plastic small round cell tumors, EWS is fused to the EFPs have been demonstrated directly and deregulated 9 Wilm’s tumor oncogene, WT1. Because EFPs are target genes have been identified.15–20 Furthermore, in strong transcriptional activators, it is thought that EFP- Ewing’s sarcoma, agents that antagonize EWS-Fli1 fu- induced malignancies arise via aberrant gene activation, sion proteins can also inhibit cellular proliferation,22–24 with the tumor type being specified by the EWS fusion indicating the potential for the development of thera- partner. peutic agents that target EWS/Fli. In contrast to EWS/ MMSP (also known as clear cell sarcoma) is typically Fli, the role that EWS/ATF1 plays in tumorigenesis is associated with tendons and aponeuroses and is thought 10,11 poorly characterized. Because ATF1 is a cAMP-induc- to be of neuroectodermal origin. MMSP is a very ible activator,25,26 it is predicted that EWS/ATF1 acts via aggressive, early onset tumor that initially grows slowly, constitutive activation of cAMP-inducible genes. How- resulting in 20% of patients having metastases at the 12 ever, the critical target genes have not yet been identi- time of diagnosis. Current treatment involves surgery fied. In addition, a role for EWS/ATF1 in tumor main- tenance has not yet been established. Consequently, it is Received April 30, 1999; accepted July 8, 1999. not known whether therapeutic approaches aimed at adenovirusdress correspondence and reprint requests to Dr. Kevin A. W. antagonizing EWS/ATF1 will be applicable to MMSP. Lee, Hong Kong University of Science and Technology, Clear Water Bay, An alternative therapeutic approach (cytotoxic pro- Kowloon, Hong Kong, S. A. R. China. E-mail address: bokaw@ drug therapy) for MMSP involves the exploitation of usthk.ust.hk tumor-specific promoters to target tumor cells for the 396 Cancer Gene Therapy, Vol 7, No 3, 2000: pp 396–406 LUNG AND LEE: GENE THERAPY FOR MMSP 397 action of cytotoxic agents.27–29 Previous studies have inserting a BamHI/AatII fragment from p⌬(Ϫ71A)SomCAT characterized EWS/ATF1 as a tumor-specific activator and an oligonucleotide to reconstruct the somatostatin pro- 30–32 moter to position Ϫ71 into the multiple cloning site of of promoters containing ATF-binding sites. Tran- ⌬ ⌬ Ϫ scriptional activation is dependent upon the EWS acti- p E1SP1A. p E1( 42A) (used to construct AD42) was ob- 30,31 tained by insertion of an AatII/BamHI blunt fragment from vation domain (EAD) and the DNA-binding domain ⌬ Ϫ 30–32 p ( 71)SomCAT into the EcoRI/BamHI blunt sites of the of ATF1. Most significantly, promoters that can be multiple cloning region of p⌬E1sp1A. p⌬E1(25A) (used to activated by EWS/ATF1 (e.g., the somatostatin pro- construct AD25V) was obtained by inserting a BamHI/AflIII moter) are constitutively active when transiently intro- blunt fragment from pVIP25CAT into the EcoRI/BamHI duced into tumor-derived cell lines (DTC1 and Su-ccs-1, blunt sites of p⌬E1sp1A. p⌬E1(VIPB) (used to construct hereafter referred to as MMSP cells) containing endog- ADVIP) was obtained by inserting a SacI/BamHI blunt frag- enous EWS/ATF1.30 In addition to a cell-specific target- ment from the pVIP4CAT sequence into the EcoRI/BamHI ing agent (such as a tumor-specific promoter), cytotoxic blunt sites of p⌬E1sp1B. prodrug treatment requires a safe and efficient vector to Construction and propagation of recombinant introduce the therapeutic gene and an effective prodrug. adenoviruss Among many available systems, recombinant adenovi- ruses (adenoviruss)33,34 and the herpes simplex virus Recombinant adenoviruss (adenovirus type 5) were con- thymidine kinase (hsvtk) gene have been well studied. structed by in vivo recombination of plasmids in 293 cells using adenovirus vector construction kit C (Microbix Biosystems, The combined use of hsvtk as a toxic gene and adeno- ⌬ ⌬ virus as a gene delivery vehicle has proven effective for Toronto, Canada). p E1SP1A and p E1SP1B contain the prodrug sensitization of other tumor cell lines.35–37 left-hand end of adenovirus type 5 (from base pair 22 (0 map units) (m.u.) to base pair 5790 (16.1 m.u.)), with a deletion of In the current study, we tested the use of a somatosta- E1 sequences from base pair 342 to base pair 3523 (1.0–9.8 tin promoter-hsvtk fusion gene and adenovirus vectors m.u.), but containing the entire packaging signal. A multiclon- for cytotoxic prodrug treatment of MMSP cell lines. Our ing site is present in the E1 region, allowing for insertion of the results demonstrate the potential of the somatostatin foreign gene. p⌬E1SP1A and p⌬E1SP1B are identical except promoter and hsvtk as cytotoxic agents for MMSP cells. for the orientation of the multicloning site between ClaI and Surprisingly however, several promoters (including so- BglII. The pBHG10 plasmid contains the rest of the adenovirus matostatin) that are strongly activated in MMSP cells type 5 genome and a small overlapping region with both p⌬E1sp1A and p⌬E1sp1B to allow recombination after co- using plasmid-based transient assays are not activated 42 when present in an adenovirus vector. Thus, first-gener- transfection into 293 cells. pBHG10 is noninfectious due to ation adenovirus vectors cannot be used as promoter lack of a packaging signal needed for encapsidation of viral DNA;43 in addition, to allow a bigger insert, the nonessential delivery vehicles for EWS/ATF1-dependent toxic gene E3 region (78.3–85.8m.u.) is deleted. Low-passage 293 cells expression in MMSP cells. (Ͻ40 passages) in a 60-mm dish were set up at 50% conflu- ence. A total of 10 ␮gofp⌬E1(Ϫ71A), p⌬E1(Ϫ42A), MATERIALS AND METHODS p⌬E1(25A), or p⌬E1(VIPB) and 10 ␮g of pBHG10 were then cotransfected into the cells by calcium phosphate precipitation. Plasmids Medium was renewed the following day and on reaching confluence, the cells were trypsinized and diluted. On release p⌬(Ϫ71)SomCAT contains the somatostatin promoter at po- from the cells, recombinant viruses were confirmed by poly- sition Ϫ71, fused to the chloramphenicol acetyl transferase 38 merase chain reaction analysis of isolated viral DNA and (CAT) coding sequences. p⌬(Ϫ71)SomCAT contains a sin- titrated on 293 cells. Purification of viral DNA was performed gle ATF1-binding site, which is required for activation by both 38 30 as follows. Infected 293 cells were harvested and lysed by the cAMP and EWS/ATF1. p⌬(Ϫ42)SomCAT is the same as addition of 400 ␮ p⌬(Ϫ71)SomCAT, except that sequences from position Ϫ71 to L of lysis buffer (20 mM of Tris-Cl (pH 7.5), 30 0.5% Nonidet P-40, 500 mM NaCl, and 1 mM dithiothreitol) Ϫ42 of the somatostatin promoter are deleted. Therefore, on ice for 5 minutes. After centrifugation for 5 minutes in a p⌬(Ϫ42)SomCAT lacks the ATF1-binding site of the soma- 30 ⌬ Ϫ ⌬ Ϫ microfuge, the supernatant was adjusted by the addition of 15 tostatin promoter. p ( 42)SomTK and p ( 71)SomTK are ␮ ␮ identical with p⌬(Ϫ42)SomCAT and p⌬(Ϫ71)SomCAT, re- L of 20% sodium dodecyl sulfate and 150 g of proteinase K spectively, except that the CAT sequence is replaced by the and incubated at 55°C for 30 minutes.