Background: Activating transcription factor-3 (ATF3) is a member of the ATF/cAMP-responsive element binding protein (CREB) transcription factor family and is involved in the cellular response to a large variety of stresses including DNA damage. Importantly, ATF3 elicits tumor suppressive functions and its dysfunction has been associated with an impaired p53-mediated cellular response to DNA damage, thus allowing cells to be transformed by oncogenes. In preliminary experiments work we found that blocking Hsp90, which represents a novel target for molecular cancer therapies, alters ATF3 expression in cancer cells. Hence, we sought to investigate the regulation and role of ATF3 in colon cancer cells, as Hsp90 inhibitors may induce this transcription factor. Methods: Human colon cancer cell lines (HCT116, SW620, HT29) and the Hsp90 inhibitor 17-DMAG were used for experiments. HCT116 colon cancer cells were transfected with either a ATF3-shRNA, or Luciferase-shRNA expression plasmid, and stable clones selected and screened. Changes in signaling pathway activation and ATF3 expression were determined by Western blotting and real-time PCR. The effects of ATF3 inhibition on tumor growth in vivo were determined in a subcutaneous tumor model, in a liver metastasis model and in a peritoneal carcinomatosis model in athymic nude mice (BALB/cnu/nu). Results: ATF3 expression was slightly detectable in all colon cancer cells. Surprisingly we found that blocking Hsp90 substantially up-regulated ATF3 expression in colon cancer cells on both protein and mRNA levels, in addition to a 17-DMAG-associated inhibition of multiple oncogenic signaling pathways, suggesting that anti-metastatic effects of Hsp90 inhibitors may be due to an ATF3 up-regulation. Using selective pathway inhibitors, we identified that blocking MAPK/Erk up-regulated ATF3, whereas inhibition of PI-3K/Akt, SAPK, and p38 did not. Stable knock-down of ATF3 in HCT116 not only decreased ATF3 mRNA, but also markedly diminished the Hsp90 inhibitor mediated induction of ATF3 protein. In vivo, suppression of ATF3 by RNAi (2 clones tested) significantly accelerated subcutaneous tumor growth of HCT116 cells, as compared to Luciferase-shRNA transfected controls (P<0.05 for both), suggesting that ATF3 indeed functions as a tumor suppressor in colon cancer. In addition, ATF3 suppression significantly enhanced growth of liver metastases and peritoneal carcinomatosis (P<0.05). Conclusions: The transcription factor ATF3 functions as a tumor suppressor in colon cancer and is inducible by blocking Hsp90. Hence, Hsp90 inhibitors appear to elicit their anti-tumor efficacy at least in part through up-regulating ATF3, suggesting that ATF3 could be an important modulator of the efficacy of Hsp90 inhibitors.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4975.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

American Association for Cancer Research
2009