Abstract 3112: Molecular events in DNA damage induced downstream of blockade of the Hedgehog (HH) signaling axis in colon carcinoma cells

Abstract

Coordinated Hedgehog (HH) signaling actively regulates key processes including tissue patterning, differentiation and proliferation during embryonic development. Aberrant HH pathway activation has been identified in several human cancers including colon carcinoma. The GLI proteins downstream of signaling via PTCH and SMO transcriptionally regulate HH target genes. Pharmacologic inhibition of GLI1 and GLI2 by the small molecule antagonist GANT61, or genetic inhibition by a C-terminus deleted GLI3 repressor, GLI3R, induced DNA damage (marked by increased γH2AX, p-ATM and p-Chk2) and extensive cell death in human colon carcinoma cells. cDNA microarray gene profiling of human colon carcinoma cells exposed to GANT61 demonstrated changes in expression of genes involved in DNA damage and DNA repair pathways. We therefore investigated the kinetics of key regulatory proteins at the chromatin level. Exposure to GANT61 for 24 hr followed by washout induced DNA damage followed by DNA repair with reversal of cytotoxicity. Employing this model, continuous exposure to GANT61 (DNA damage) resulted in increased chromatin-bound γH2AX (DNA DSBs); removal of GANT61 after 24 hr (DNA repair) led to decreased chromatin-bound γH2AX. ATM and Chk2 were chromatin-bound during the initial DNA damage phase and later were released. Of significance, HCT15 (a Chk2-deficient colon carcinoma cell line) was less sensitive to blockade of HH signaling, indicating the critical contribution of Chk2 in the DNA damage response. DNA repair is characterized by MDC1-dependent binding of the MRN (MRE11, RAD50 and NBS1) complex at DNA break sites. Following GLI1/GLI2 inhibition, NBS1 nuclear foci colocalized in single cells with MDC1 but not with γH2AX foci. During DNA damage, NBS1 demonstrated limited binding to chromatin, while MRE11 and MDC1 binding was induced and sustained. During DNA repair NBS1, MRE11 and MDC1 were highly chromatin-bound, while γH2AX was released. Gli3R-mediated inhibition of HH signaling demonstrated a similar chromatin-binding profile among γH2AX, MDC1 and NBS1. During DNA damage, MDC1 co-immunoprecipitated with reduced levels of NBS1 and increased levels of γH2AX; during DNA repair, increased levels of NBS1 and decreased levels of γH2AX co-immunoprecipitated with MDC1. Although chromatin-bound NBS1 was limited during DNA damage and abundant during DNA repair, shRNA-mediated knockdown of NBS1 did not prevent DNA repair following GANT61 exposure (24 hr) and washout, suggesting the existence of redundant DNA repair axes in this mode of DNA damage. The regulation of events upstream and downstream of DNA strand break induction following blockade of HH signaling will provide insight into the critical mechanisms that regulate HH/GLI-dependent survival, currently unknown in any type of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3112. doi:1538-7445.AM2012-3112