Abstract 5459: Molecular mechanisms of dysregulated GLI activation in Hedgehog (HH)-dependent survival in human colon cancer.

In normal cellular processes the canonical Hedgehog (HH) pathway signals via the transmembrane receptor PTCH, intermediary molecule SMO, and activates the proteins GLI1 and GLI2, which transcriptionally regulate HH target genes. In colon cancer, HH signaling is aberrantly regulated, driving cell survival and genomic instability in oncogenesis, progression and metastasis. Oncogene-driven signaling pathways, specifically KRAS/BRAF in colon cancer, circumvent the HH/SMO axis to converge on and activate GLI. Using a small molecule inhibitor of GLI1/GLI2 transcription, GANT61, we have demonstrated: 1) inhibition of HH signaling at the level of GLI1 and GLI2 by GANT61 induces extensive cell death in human colon carcinoma cell lines in contrast to targeting SMO; 2) cells with acquired resistance to SMO inhibitors remain highly sensitive to GANT61; 3) oncogenic KRAS/BRAF signaling activates GLI transcription, inhibited by GANT61. These findings thus delineate GLI as a critical target in colon cancer. HT29 (BRAFV600E mutant), SW480 (KRASG12V mutant) and HCT116 (KRASG13D mutant) cells are sensitive to the MEK inhibitor, AZD6244, a novel, selective, ATP-uncompetitive inhibitor of MEK1/2 currently in Phase II clinical trial. AZD6244 inhibited transcription of GLI2, but not GLI1 (mRNA expressed at low level), suggesting integration of oncogenic KRAS/BRAF signals at the level of GLI2 transcription. In experiments with the protein synthesis inhibitor cycloheximide, MG132 (proteasome inhibitor), and AZD6244, stabilization of GLI1 and GLI2 was determined not to be a mechanism of MEK/ERK-dependent GLI activation in HT29 cells. The ZIC2 transcription factor effects translocation of GLI proteins to the nucleus and regulates GLI transcriptional activity. GLI1 and GLI2 proteins co-immunoprecipitated with ZIC2 in HT29 cells. Further, ZIC2 was extensively bound to the promoters of GLI1 and GLI2, and binding was significantly reduced by AZD6244 (ChIP analysis). ZIC2-dependent transcriptional regulation requires phosphorylation (p-ZIC2) by DNA-dependent protein kinase catalytic subunit (DNA-PKcs). The DNA-PKcs inhibitor, NU7441, inhibited GLI2-luciferase reporter activity. How GLI activation is regulated in cancer cells remains poorly understood, yet GLI marks an important target in cancer biology. We propose a model in which oncogenic KRAS/BRAF regulates GLI2 transcription involving ZIC2, which is phosphorylated by DNA-PKcs and regulated by ERK. Using drugs that inhibit GLI1/GLI2 transcription and studies of GLI activation in oncogenesis, these findings will provide new insights into critical targets that determine HH-dependent survival, and for the translation of drugs that target dysregulated HH signaling in cancer. Citation Format: Akwasi Agyeman, Tapati Mazumdar, Janet A. Houghton. Molecular mechanisms of dysregulated GLI activation in Hedgehog (HH)-dependent survival in human colon cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5459. doi:10.1158/1538-7445.AM2013-5459