Dominant-negative inhibition of Ets 1 suppresses tumor growth, invasion and migration in rat C6 glioma cells and reveals differentially expressed Ets 1 target genes.

We previously reported that the inactivation of the Ets 1 transcription factor by a specific decoy strategy reduces rat C6 glioma cell proliferation and mmp-9 expression. In the present study, we analysed the effects of the dominant-negative form of Ets 1 (Ets-DB) on rat C6 glioma cell proliferation, migration, invasion, in vivo tumor growth on the chicken chorioallantoic membrane (CAM) and mmp-9 expression. In addition, we examined differences in gene expression between Ets-DB expressing and control cells using suppression subtractive hybridization (SSH). We found that retrovirus mediated expression of Ets-DB inhibited cellular proliferation, migration, invasion, mmp-9 expression, cellular growth in soft agar, and in vivo growth in the chicken chorioallantoic membrane assay. SSH analysis revealed expression of different genes in Ets-DB expressing cells involved in basic cellular processes. Each of these genes contained binding sites for different Ets-factors within their promoters. Finally, we found that, in addition to Ets 1, Elk-1, Elf-1, Fli-1 and Etv-1 are further Ets family members expressed in rat C6 glioma cells. Our results indicate that Ets transcription factors play important roles for basic properties of rat C6 glioma cells. Targeting of these factors might therefore become a useful experimental tool for therapeutic strategies against malignant gliomas.

Myc confers androgen-independent prostate cancer cell growth.

Prostate cancer is one of the most diagnosed and mortal cancers in western countries. A major clinical problem is the development of androgen-independent prostate cancer (AIPC) during antihormonal treatment. The molecular mechanisms underlying the change from androgen dependence to independence of these tumors are poorly understood and represent a challenge to develop new therapies. Based on genetic data showing amplification of the c-myc gene in AIPC, we studied the ability of c-myc to confer AIPC cell growth. Human androgen-dependent prostate cancer cells overexpressing c-myc grew independently of androgens and presented tumorigenic properties in androgen-depleted conditions. Analysis of signalling pathways by pharmacological inhibitors of the androgen receptor (AR) or by RNA interference directed against AR or c-myc showed that c-myc acted downstream of AR through multiple growth effectors. Thus c-myc is required for androgen-dependent growth and following ectopic expression can induce androgen-independent growth. Moreover, RNA interference directed against c-myc showed that growth of human AIPC cells, AR-positive or -negative, required c-myc expression. Furthermore, we showed that c-myc-overexpressing cells retain a functional p53 pathway and thus respond to etoposide.

Expression of an Ets-1 dominant-negative mutant perturbs normal and tumor angiogenesis in a mouse ear model.

We and others have shown that members of the Ets family of transcription factors are involved in morphogenic properties of endothelial cells in vitro. To investigate the role of these factors in the transcriptional regulation of angiogenesis in vivo, we set up a nontraumatic model that allows daily macroscopic examination of both growth factor- and tumor-induced angiogenesis in mouse ears. In the same animal, we were thus able to record variations in the patterns of neovessels induced and cell populations recruited by the angiogenic factors FGF-2 and VEGF. In this model, inhibition of FGF-2-induced angiogenesis by the pharmacological compound TNP-470 was readily observed, demonstrating that the mouse ear model is also useful in the evaluation of antiangiogenic strategies. Our functional analysis of Ets transcription factors activity utilized a competitor protein, Ets1-DB, a dominant negative Ets1 mutant lacking the transactivation domain. Retrovirus-mediated expression of Ets1-DB inhibited FGF-2-induced angiogenesis, while the expression of Ets1-DB in cancerous and stromal cells disturbed tumor-induced angiogenesis. These results illustrate the value of the ear model and highlight the role of Ets family members in the transcriptional regulation of tumor angiogenesis.