Blocking Hedgehog Survival Signaling at the Level of the GLI Genes Induces DNA Damage and Extensive Cell Death in Human Colon Carcinoma Cells

Abstract

Canonical Hedgehog (HH) signaling is characterized by Smoothened (Smo)-dependent activation of the transcription factors Gli1 and Gli2, which regulate HH target genes. In human colon carcinoma cells, treatment with the Gli small molecule inhibitor GANT61 induces extensive cell death, in contrast to the Smo inhibitor cyclopamine. Here we elucidate cellular events upstream of cell death elicited by GANT61, which reveal the basis for its unique cytotoxic activity in colon carcinoma cells. Unlike cyclopamine, GANT61 induced transient cellular accumulation at G1/S (24 hr) and in early S-phase (32 hr), with elevated p21Cip1, cyclin E and cyclin A in HT29 cells. GANT61 induced DNA damage within 24 hr, with the appearance of p-ATM and p-Chk2. Pharmacologic inhibition of Gli1 and Gli2 by GANT61 or genetic inhibition by transient transfection of the Gli3 repressor (Gli3R), downregulated Gli1 and Gli2 expression, induced γH2AX, PARP cleavage, caspase-3 activation and cell death. GANT61 induced γH2AX nuclear foci, while transient transfection of Gli3R demonstrated expression of Gli3R and γH2AX foci within the same nuclei in HT29, SW480 and HCT116. GANT61 specifically targeted Gli1 and Gli2 substantiated by specific inhibition of 1) direct binding of Gli1 and Gli2 to the promoters of target genes HIP1 and BCL-2, 2) Gli-luciferase activity, and 3) transcriptional activation of BCL-2. Taken together, these findings establish that inhibition of HH signaling at the level of the GLI genes downstream of Smo is critical in the induction of DNA damage in early S-phase, leading to cell death in human colon carcinoma cells.

PCAF ubiquitin ligase activity inhibits Hedgehog/Gli1 signaling in p53-dependent response to genotoxic stress

“…Interestingly, these observations are consistent with the enhanced growth arrest and cell death induced by the Gli1 antagonist GANT61. 37,38 Targeting the Hh pathway has a crucial role for the therapy of Hh-dependent tumors (e.g. Mb).…”

Gli1 Protein Regulates the S-phase Checkpoint in Tumor Cells via Bid Protein, and Its Inhibition Sensitizes to DNA Topoisomerase 1 Inhibitors

“…Inhibition of Gli1 has also been shown to induce DDR and to attenuate cell survival. However, the mechanism for Gli1 inhibition-mediated DDR is not known (53,54). To investigate the role of Gli1-mediated signaling in tumor cell survival and DDRs, we transiently down-regulated Gli1 in several cancer cells using siRNAs and evaluated for the formation of ␥H2AX foci (a surrogate marker for DSB and replication stress) and their clonogenic potential.…”

MicroRNA-338-3p Inhibits Colorectal Carcinoma Cell Invasion and Migration by Targeting Smoothened

“…Up-regulation of SMO in CRC was shown to correlate with a higher biological aggressiveness, advanced stage, poor differentiation, larger size and high proliferative activity (25). Furthermore, it is also well known that SMO regulation, both in physiological and pathological conditions, is exerted mostly at post-transcriptional level (26). Despite this central role of SMO as mediator of all Hh signaling, the mechanisms by which SMO activation is regulated and coupled to downstream components remain enigmatic.…”