Abstract 1790A: Hedgehog signaling at the level of GLI transcriptionally regulates hTERT in human cancer cells.

The HH signaling pathway is critical for normal embryonic development, tissue patterning and cell differentiation. Aberrant HH signaling is involved in multiple human cancers. HH signaling involves a multi-protein cascade activating the GLI proteins that transcriptionally regulate HH target genes. We have recently reported that HH signaling is essential for human colon cancer cell survival, and that inhibition of this signal induces DNA damage and extensive cell death. The current study demonstrates that HH signaling regulates human telomerase reverse transcriptase (hTERT), which determines the limitless replication potential of cancer cells. Suppression of both GLI1 and GLI2 functions by exogenous expression of a C-terminus truncated GLI3 repressor mutant (GLI3R), or by GANT61, a pharmacologic inhibitor of GLI1 and GLI2 transcriptional activity, reduced hTERT protein expression in human colon, prostate and brain cancer (glioblastoma, GBM) cell lines. Exogenous expression of a constitutively active mutant of GLI2, GLI2m, significantly increased hTERT transcription, protein expression, and hTERT promoter-luciferase activity, in human colon cancer cells. Exposure to GANT61 inhibited hTERT mRNA expression in human colon cancer cells. Insilico analysis of the hTERT promoter revealed 7 putative GLI binding sites suggesting a direct transcriptional mode of regulation of hTERT by GLI. Chromatin immunoprecipitation (ChIP) analysis with GLI1 or GLI2 antibodies precipitated fragments of the hTERT promoter in human colon cancer cells, indicating a direct interaction between GLI proteins and the hTERT promoter. The binding between GLI2 and the promoter of hTERT was significantly reduced upon exposure to GANT61. Of interest, exogenous expression of GLI1 or GLI2m in non-cancerous 293T cells failed to alter the levels of hTERT mRNA and protein, or hTERT promoter-luciferase activity. Further, ChIP analysis of GFP-tagged GLI2 did not precipitate the hTERT promoter in 293T cells, in contrast to events in malignant cells. GLI2m also increased telomerase activity in human colon cancer cells, while GANT61 reduced the telomerase activity in human colon, prostate and GBM cells. These results demonstrate that the HH signaling pathway directly regulates hTERT by direct interaction with GLI in cancer cells in contrast to non-transformed cells, and identify a previously unknown role of the HH/GLI axis in regulating the replication potential of cancer cells. These findings are of significance in understanding important regulatory mechanisms that determine the role of HH/GLI signaling in cancer cell survival. Citation Format: Tapati Mazumdar, Ranjodh Sandhu, Maha Qadan, Victoria Magloire, Akwasi Agyeman, Bibo Li, Janet A. Houghton. Hedgehog signaling at the level of GLI transcriptionally regulates hTERT in human cancer cells. [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 1790A. doi:10.1158/1538-7445.AM2013-1790A