A genomic-scale view of the cAMP response element-enhancer decoy: a tumor target-based genetic tool

Abstract

Enhancer DNA decoy oligodeoxynucleotides (ODNs) inhibit transcription by competing for transcription factors. A decoy ODN composed of the cAMP response element (CRE) inhibits CRE-directed gene transcription and tumor growth without affecting normal cell growth. Here, we use DNA microarrays to analyze the global effects of the CRE-decoy ODN in cancer cell lines and in tumors grown in nude mice. The CRE-decoy up-regulates the AP-2beta transcription factor gene in tumors but not in the livers of host animals. The up-regulated expression of AP-2beta is clustered with the up-regulation of other genes involved in development and cell differentiation. Concomitantly, another cluster of genes involved in cell proliferation and transformation is down-regulated. The observed alterations indicate that CRE-directed transcription favors tumor growth. The CRE-decoy ODN, therefore, may serve as a target-based genetic tool to treat cancer and other diseases in which CRE-directed transcription is abnormally used.

Fig 1.

Reverted phenotype of PC3M tumors treated with the CRE-decoy. PC3M human prostate carcinoma cells (2.0 × 10⁶) were inoculated s.c. into the left flank of nude mice. When tumors became palpable, the CRE-decoy or control ODN (0.1 mg/0.1 ml of saline per mouse, daily) or saline (0.1 ml per mouse) was injected i.p. into the mice. Tumor volumes were obtained from daily measurement and calculation (26). At the end of the experiment, animals were killed, and tumors, livers, and spleens were removed, weighed, immediately frozen in liquid N₂, and stored at −80°C until used. Lanes: 1–4, untreated control tumors; 5–8, control ODN-treated tumors; 9–12, CRE-decoy-treated tumors; 13–16, CRE-decoy-treated livers; 17–20, control ODN-treated livers; and 21–24, untreated control livers. The entire cluster image is shown in A. Full gene names are shown for representative clusters containing functionally related genes involved in reverse transformation (B), cell cycle (C), metabolism (D), and proliferation (E). These clusters also contain uncharacterized genes and named genes not involved in these processes. The size of the tumors compared with untreated (saline-injected) control tumors were 50% and 95% of their original size after 4 days of treatment with CRE-decoy and control ODN, respectively. There was no sign of systemic toxicity in treated animals, and the sizes of the liver and spleen remained unchanged.

Fig 2.

CRE-decoy inhibition is unrelated to PS-ODN or CpG side effects. Data are the same as in Fig. 1, except that data from mouse RIα antisense PS-ODN (mPSAS)-treated tumors and livers were included in the cluster analysis. Lanes: 1–4, untreated control tumors; 5–8, mPSAS-treated tumors; 9–12, control ODN-treated tumors; 13–16, CRE-decoy-treated tumors; 17–20, CRE-decoy-treated livers; 21–24, control ODN-treated livers; 25–28, mPSAS-treated livers; and 29–32, untreated control livers.

Fig 3.

Cells overexpressing KCREB fail to exhibit the CRE-decoy expression signatures. Data from untreated control cells, control ODN-treated, CRE-decoy-treated cells of untransfected PC3M parental cells, and wild-type CREB or mutant KCREB-transfected cells (see Experimental Procedures) were combined and clustered. Lanes: 1 and 2, untreated; 3 and 4; control ODN-treated; 5 and 6, CRE-decoy-treated KCREB-overexpressing cells; 7 and 8, untreated; 9 and 10, control ODN-treated; 11 and 12; CRE-decoy-treated parental PC3M cells; 13 and 14, untreated; 15 and 16, control ODN-treated; 17 and 18, CRE-decoy-treated CREB-overexpressing cells. (A) Entire cluster image. (B) Cell-cycle signature. (C) Proliferation signature. These clusters also contain unknown genes and named genes not involved in defined functions.

Fig 4.

Inhibition of MCF-7 tumor growth in nude mice by CRE-decoy ODN in a time-release pellet. Tumor cells (1–2 × 10⁶ per mouse) were inoculated into mammary gland fat pad of athymic mice. For estrogen-dependent tumor growth, a 17β–estradiol pellet (90-day release) was implanted s.c. 1 day before cell inoculation. Drug administration was started 14–21 days after cell inoculation, when tumors became palpable. Time-release (60-day) pellets containing 2.5 mg of CRE (○, CRE-decoy), 5.0 mg of CRE-decoy (•), or 5.0 mg of CRE (⋄, control ODN) at 1 kg/day were implanted interscapularly. CRE was also injected i.p. at 5.0 mg/kg daily, 5 times per week, for 8 weeks (▵). A tamoxifen pellet (▪, 60-day release, 4.2 mg/kg/day) was implanted interscapularly. A placebo (♦, vehicle pellet) was implanted interscapularly. Tumor volumes were obtained from daily measurements and calculations (26). Data represent means ± SD of five to seven tumors in each group.