Akwasi Agyeman

Hedgehog Signaling Drives Cellular Survival in Human Colon Carcinoma Cells

Aberrant activation of Hedgehog (HH) signaling is implicated in many human cancers. Classical HH signaling is characterized by Smoothened (Smo)-dependent activation of Gli1 and Gli2, which transcriptionally regulate target genes. A small molecule inhibitor of Gli1 and Gli2, GANT61, was used to block HH signaling in human colon carcinoma cell lines that express HH signaling components. GANT61 administration induced robust cytotoxicity in 5 of 6 cell lines and moderate cytotoxicity in the remaining 1 cell line. In comparison, the classical Smo inhibitor, cyclopamine, induced modest cytotoxicity. Further, GANT61 treatment abolished the clonogenicity of all six human colon carcinoma cell lines. Analysis of the molecular mechanisms of GANT61-induced cytotoxicity in HT29 cells showed increased Fas expression and decreased expression of PDGFRα, which also regulates Fas. Furthermore, DR5 expression was increased whereas Bcl-2 (direct target of Gli2) was downregulated following GANT61 treatment. Suppression of Gli1 by shRNA mimicked the changes in gene expression observed in GANT61-treated cells. Overexpression of dominant-negative FADD (to abrogate Fas/DR5-mediated death receptor signaling) and/or Bcl-2 (to block mitochondria-mediated apoptosis) partially rescued GANT61-induced cytotoxicity in HT29 cells. Thus, activated GLI genes repress DR5 and Fas expressions while upregulating Bcl-2 and PDGFRα expressions to inhibit Fas and facilitate cell survival. Collectively, these results highlight the importance of Gli activation downstream of Smo as a therapeutic target in models of human colon carcinoma.

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

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 G(1)-S (24 hours) and in early S-phase (32 hours), with elevated p21(Cip1), cyclin E, and cyclin A in HT29 cells. GANT61 induced DNA damage within 24 hours, 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 and induced γH2AX, PARP cleavage, caspase-3 activation, and cell death. GANT61 induced γH2AX nuclear foci, while transient transfection of Gli3R showed 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 (i) direct binding of Gli1 and Gli2 to the promoters of target genes HIP1 and BCL-2, (ii) Gli-luciferase activity, and (iii) 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.

Inhibition of NF-κB Signaling by Quinacrine Is Cytotoxic to Human Colon Carcinoma Cell Lines and Is Synergistic in Combination with Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL) or Oxaliplatin

Colorectal cancer is the third most common malignancy in the United States. Modest advances with therapeutic approaches that include oxaliplatin (l-OHP) have brought the median survival rate to 22 months, with drug resistance remaining a significant barrier. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is undergoing clinical evaluation. Although human colon carcinomas express TRAIL receptors, they can also demonstrate TRAIL resistance. Constitutive NF-κB activation has been implicated in resistance to TRAIL and to cytotoxic agents. We have demonstrated constitutive NF-κB activation in five of six human colon carcinoma cell lines; this activation is inhibited by quinacrine. Quinacrine induced apoptosis in colon carcinomas and potentiated the cytotoxic activity of TRAIL in RKO and HT29 cells and that of l-OHP in HT29 cells. Similarly, overexpression of IκBα mutant (IκBαM) or treatment with the IKK inhibitor, BMS-345541, also sensitized these cells to TRAIL and l-OHP. Importantly, 2 h of quinacrine pretreatment resulted in decreased expression of c-FLIP and Mcl-1, which were determined to be transcriptional targets of NF-κB. Extended exposure for 24 h to quinacrine did not further sensitize these cells to TRAIL- or l-OHP-induced cell death; however, exposure caused the down-regulation of additional NF-κB-dependent survival factors. Short hairpin RNA-mediated knockdown of c-FLIP or Mcl-1 significantly sensitized these cells to TRAIL and l-OHP. Taken together, data demonstrate that NF-κB is constitutively active in colon cancer cell lines and NF-κB, and its downstream targets may constitute an important target for the development of therapeutic approaches against this disease.

cDNA Microarray Gene Expression Profiling of Hedgehog Signaling Pathway Inhibition in Human Colon Cancer Cells

Background Hedgehog (HH) signaling plays a critical role in normal cellular processes, in normal mammalian gastrointestinal development and differentiation, and in oncogenesis and maintenance of the malignant phenotype in a variety of human cancers. Increasing evidence further implicates the involvement of HH signaling in oncogenesis and metastatic behavior of colon cancers. However, genomic approaches to elucidate the role of HH signaling in cancers in general are lacking, and data derived on HH signaling in colon cancer is extremely limited. Methodology/Principal Findings To identify unique downstream targets of the GLI genes, the transcriptional regulators of HH signaling, in the context of colon carcinoma, we employed a small molecule inhibitor of both GLI1 and GLI2, GANT61, in two human colon cancer cell lines, HT29 and GC3/c1. Cell cycle analysis demonstrated accumulation of GANT61-treated cells at the G1/S boundary. cDNA microarray gene expression profiling of 18,401 genes identified Differentially Expressed Genes (DEGs) both common and unique to HT29 and GC3/c1. Analyses using GenomeStudio (statistics), Matlab (heat map), Ingenuity (canonical pathway analysis), or by qRT-PCR, identified p21Cip1 (CDKN1A) and p15Ink4b (CDKN2B), which play a role in the G1/S checkpoint, as up-regulated genes at the G1/S boundary. Genes that determine further cell cycle progression at G1/S including E2F2, CYCLIN E2 (CCNE2), CDC25A and CDK2, and genes that regulate passage of cells through G2/M (CYCLIN A2 [CCNA2], CDC25C, CYCLIN B2 [CCNB2], CDC20 and CDC2 [CDK1], were down-regulated. In addition, novel genes involved in stress response, DNA damage response, DNA replication and DNA repair were identified following inhibition of HH signaling. Conclusions/Significance This study identifies genes that are involved in HH-dependent cellular proliferation in colon cancer cells, and following its inhibition, genes that regulate cell cycle progression and events downstream of the G1/S boundary.

Hedgehog Signaling Regulates Telomerase Reverse Transcriptase in Human Cancer Cells

The Hedgehog (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 previously reported that HH signaling is essential for human colon cancer cell survival and inhibition of this signal induces DNA damage and extensive cell death. Here we report that the HH/GLI axis regulates human telomerase reverse transcriptase (hTERT), which determines the replication potential of cancer cells. Suppression of GLI1/GLI2 functions by a C-terminus truncated GLI3 repressor mutant (GLI3R), or by GANT61, a pharmacological inhibitor of GLI1/GLI2, reduced hTERT protein expression in human colon cancer, prostate cancer and Glioblastoma multiforme (GBM) cell lines. Expression of an N-terminus deleted constitutively active mutant of GLI2 (GLI2ΔN) increased hTERT mRNA and protein expression and hTERT promoter driven luciferase activity in human colon cancer cells while GANT61 inhibited hTERT mRNA expression and hTERT promoter driven luciferase activity. Chromatin immunoprecipitation with GLI1 or GLI2 antibodies precipitated fragments of the hTERT promoter in human colon cancer cells, which was reduced upon exposure to GANT61. In contrast, expression of GLI1 or GLI2ΔN in non-malignant 293T cells failed to alter the levels of hTERT mRNA and protein, or hTERT promoter driven luciferase activity. Further, expression of GLI2ΔN increased the telomerase enzyme activity, which was reduced by GANT61 administration in human colon cancer, prostate cancer, and GBM cells. These results identify hTERT as a direct target of the HH signaling pathway, and reveal a previously unknown role of the HH/GLI axis in regulating the replication potential of cancer cells. These findings are of significance in understanding the important regulatory mechanisms that determine the functions of HH/GLI signaling in cancer cells.

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

Abstract 467: New molecular model identifying the critical role of the GLI2 oncogene in human colonic epithelial cell (HCEC) transformation

Colorectal cancer, comprising 85% sporadic non-hereditary tumors, is the third in cancer incidence and the second leading cause of cancer mortality in the United States. Initiating mutations in key genes, APC, KRAS and p53, determine transformation and early oncogenesis in the colon. The initiating mutation in the APC tumor suppressor gene activates Wnt signaling. Progression mostly requires mutation in the KRAS oncogene, facilitating cytoplasmic->nuclear translocation of β-catenin. Mutation in p53 coincides with the transition from adenoma->carcinoma. While the specific genetic mutations that determine oncogenesis in colon cancer are well documented, the fundamental biologic mechanisms that determine synergism in effecting transformation remain poorly understood. The GLI genes are transcription factors that regulate Hedgehog signaling, which is activated in colon cancer. GLI1 and GLI2 are oncogenes, induce transformation, and determine oncogenesis in glioma, medulloblastoma (GLI1), and basal cell carcinoma (GLI2). We have demonstrated in colon carcinoma cells that GLI2 is activated by oncogenic KRAS, activates GLI1, and determines cell survival. In immortalized HCEC cells with defined genetic mutations, we discovered that GLI2 is activated during transformation, dependent on synergistic interactions between mAPC and KRAS[G12V] pathways in the presence of inactive p53. This renders cells dependent on GLI for survival. In isogenic HCEC models, KRAS[G12V] determines β-catenin subcellular trafficking between membrane adherens junctions (AJ), cytoplasm and nucleus. This is apparent following inhibition of ERK (AJ->cytoplasm) and/or GSK3β (AJ->nucleus) that determine β-catenin subcellular localization. Further, we have identified GLI2 as a new Wnt target gene, transcriptionally regulated by Wnt-dependent TCF4, which binds to consensus sequences in the GLI2 promoter. Oncogenic KRAS signaling channels through and converges on GLI2 to drive GLI2 to a higher activating state. GLI2 is transcriptionally regulated by an ERK-dependent mechanism that requires the GLI cofactor, ZIC2. Thus, GLI2 is activated by both Wnt and oncogenic KRAS signaling pathways. In summary, we present a new molecular model that identifies the GLI2 oncogene as a critical determinant of human colonic epithelial cell transformation. These studies will: 1) define the key mutational events and biologic mechanisms that functionally activate the oncogene GLI2 during colonic epithelial cell transformation; 2) define the role of GLI2 in early oncogenesis; 3) identify key molecules and potential new targets that determine transformation; 4) lead to improved therapeutic strategies specific to colorectal cancer. Citation Format: Tapati Mazumdar, Akwasi Agyeman, Jerry W. Shay, Janet A. Houghton. New molecular model identifying the critical role of the GLI2 oncogene in human colonic epithelial cell (HCEC) transformation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 467. doi:10.1158/1538-7445.AM2014-467

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

Abstract 4699A: Hedgehog signaling (HH/Gli) transcriptionally regulates hTERT gene expression in human cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Tightly coordinated Hedgehog (HH) signaling is critical during normal embryonic development. Aberrant HH signaling is involved in driving cell proliferation in multiple human cancers. Classical HH signaling involves the interaction of the soluble HH ligands with their receptor, Patched (Ptch), thereby releasing a transmembrane protein Smoothened (Smo) from Ptch-mediated inhibition. Smo then activates the Gli family of transcription factors that regulate HH target genes. We have previously reported that HH signaling provides essential survival support to human colon cancer cells and inhibition of this signal induces DNA damage and extensive cell death. However the downstream targets that govern the regulation of cell survival by HH/Gli in cancer cells are not completely defined. This study demonstrates that the Gli proteins transcriptionally regulate the expression of human telomerase reverse transcriptase (hTERT) gene in human cancer cells. hTERT is a known regulator of telomere homeostasis, which determines the replicative potential and hence the life span of cells. Suppression of both Gli1 and Gli2 functions by exogenous expression of a C-terminus truncated Gli3 repressor mutant (Gli3R), or by GANT61, a pharmacological inhibitor of Gli1 and Gli2 activity, reduced hTERT protein expression over a period of 72 hr in human colon, prostate and brain cancer cell lines. Further, exogenously expressed Gli2 significantly increased hTERT protein expression in human colon cancer cell lines. Exposure to GANT61 also inhibited hTERT mRNA expression within 24 hr in human colon cancer cell lines. Insilico analysis of the hTERT promoter revealed 7 putative Gli binding sites suggesting a transcriptional mode of regulation of hTERT expression by Gli2. Chromatin immunoprecipitation with Gli2 antibody precipitated fragments of the hTERT promoter in human colon cancer cell lines, indicating a direct interaction between Gli2 and the hTERT promoter. The binding between Gli2 and hTERT promoter was significantly reduced upon exposure to GANT61. Further, overexpression of the wild type hTERT cDNA in cancer cells prevented the cytotoxic effects of blocking the HH signaling pathway with GANT61. These findings demonstrate hTERT to be a direct transcriptional target and a critical mediator of the HH/Gli signaling pathway, 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: {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 4699A. doi:1538-7445.AM2012-4699A

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

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