Abstract 1656: Termination of hedgehog (HH) survival signaling at the level of the GLI genes induces DNA damage and extensive cell death in human colon carcinoma cells

Abstract

Abstract Temporally and spatially regulated activity of the HH signaling pathway is important for normal embryonic development and organ patterning while aberrant activation of this pathway is involved in the development of genomic instability and dysregulated proliferation in several human cancers. There is emerging evidence that HH signaling progresses during colon carcinogenesis and in metastatic disease, however little is known functionally about this signaling pathway and how it affects the survival and pathogenesis of colon cancer. Canonical HH signaling occurs via Ptch1, Smo, and activation of Gli1 and Gli2, which transcriptionally regulate HH target genes. We demonstrate that a small molecule inhibitor of Gli1 and Gli2, GANT61, in contrast to cyclopamine (Smo inhibitor), induced extensive cell death in six human colon carcinoma cell lines. Detailed mechanistic studies in HT29 cells demonstrated that GANT61 induced transient accumulation of cells at the G1/S boundary and in early S at 24 hr and 32 hr, respectively, along with upregulation of p21Cip1, cyclin E and cyclin A in G1- and S- phase cells. By 40 hr, GANT61-treated cells (but not cells treated with cyclopamine) became subG1. shRNA-mediated knockdown of p21Cip1 failed to alter the cytotoxicity of GANT61. cDNA microarray gene profiling in GANT61-treated cells identified significant changes in expression of genes involved in DNA damage signaling, DNA replication and DNA repair. In single cell assays (Comet), GANT61 induced DNA damage by 24 hr with increased Tail Moment and in particular Tail Length; within 4 hr, γH2AX foci were detected at sites of DNA breaks (confocal microscopy). Upregulation of activated forms of ATM and Chk2 but not ATR or Chk1 at 4 hr, that were sustained (24 hr), was observed by western analysis following GANT61 exposure. Further, by 4 hr, p-Chk2 foci co-localized with γH2AX foci in nuclei. Partial knockdown of GLI1 and GLI2 using shRNA partially protected HT29 cells from GANT61-induced cell death, and reduced nuclear γH2AX foci. Studies of the extrinsic and intrinsic pathways of apoptosis demonstrated increased expression of Fas and DR5 receptors, decreased expression of PDGFRα (GLI1 target which regulates Fas), and decreased Bcl-2 expression (GLI2 target) following GANT61 treatment. Expression of DNFADD (to abrogate death receptor signaling) and/or over-expression of Bcl-2 (to block mitochondria-mediated apoptosis) partially rescued from GANT61-induced cell death. Data demonstrate the critical role of HH signaling in colon cancer cell survival, and that inhibition of this pathway at the level of the GLI genes in contrast to Smo induces 1) DNA damage, 2) transient accumulation of cells at G1/S and early S, 3) ineffective checkpoint activation, and 4) extensive cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1656. doi:10.1158/1538-7445.AM2011-1656