PMC0
summary and future directions
Chromosome instability is found in the majority of colon cancers, resulting primarily from deregulation of the DNA replication and mitotic spindle checkpoints (reviewed in [>>45<<]). Genes involved in canonical HH signaling have been linked to genomic instability, involving inactivation of DNA repair mechanisms, defects in checkpoint activation, and predisposition to development of cancers[46-49]. It therefore
Genes involved in canonical HH signaling have been linked to genomic instability, involving inactivation of DNA repair mechanisms, defects in checkpoint activation, and predisposition to development of cancers[>>46<<-49]. It therefore follows that termination of HH signaling at the level of GLI may constitute a critical event in determining the balance between cell survival and cell death. The combination of molecular and cellular approaches that are
canonical hedgehog signaling in cancer
Canonical HH signaling engages PTCH, SMO and the GLI family of transcription factors (Figure 1), and in normal cellular processes is involved in embryogenesis, tissue patterning, stem cell function, and differentiation[>>1<<, 2]. Several types of human cancers have demonstrated aberrant activation of the HH pathway by ligand-independent signaling such as, amplification of GLI1 or GLI2, mutations in PTCH or SMO, or dysregulated gene expression[1, 3].
Canonical HH signaling engages PTCH, SMO and the GLI family of transcription factors (Figure 1), and in normal cellular processes is involved in embryogenesis, tissue patterning, stem cell function, and differentiation[1, >>2<<]. Several types of human cancers have demonstrated aberrant activation of the HH pathway by ligand-independent signaling such as, amplification of GLI1 or GLI2, mutations in PTCH or SMO, or dysregulated gene expression[1, 3].
Several types of human cancers have demonstrated aberrant activation of the HH pathway by ligand-independent signaling such as, amplification of GLI1 or GLI2, mutations in PTCH or SMO, or dysregulated gene expression[>>1<<, 3]. In colon cancer, aberrant HH signaling progresses during carcinogenesis and in metastatic disease[4-6], and is also activated in human colon carcinoma cell lines[7-9] and xenograft models[4], by ligand-dependent activation, that
Several types of human cancers have demonstrated aberrant activation of the HH pathway by ligand-independent signaling such as, amplification of GLI1 or GLI2, mutations in PTCH or SMO, or dysregulated gene expression[1, >>3<<]. In colon cancer, aberrant HH signaling progresses during carcinogenesis and in metastatic disease[4-6], and is also activated in human colon carcinoma cell lines[7-9] and xenograft models[4], by ligand-dependent activation, that occurs
In colon cancer, aberrant HH signaling progresses during carcinogenesis and in metastatic disease[>>4<<-6], and is also activated in human colon carcinoma cell lines[7-9] and xenograft models[4], by ligand-dependent activation, that occurs in GI cancers[1, 10].
In colon cancer, aberrant HH signaling progresses during carcinogenesis and in metastatic disease[4-6], and is also activated in human colon carcinoma cell lines[>>7<<-9] and xenograft models[4], by ligand-dependent activation, that occurs in GI cancers[1, 10].
In colon cancer, aberrant HH signaling progresses during carcinogenesis and in metastatic disease[4-6], and is also activated in human colon carcinoma cell lines[7-9] and xenograft models[>>4<<], by ligand-dependent activation, that occurs in GI cancers[1, 10].
aberrant HH signaling progresses during carcinogenesis and in metastatic disease[4-6], and is also activated in human colon carcinoma cell lines[7-9] and xenograft models[4], by ligand-dependent activation, that occurs in GI cancers[>>1<<, 10]. However, the role of HH signaling and its importance in driving cellular survival in colon cancer are not well defined.
aberrant HH signaling progresses during carcinogenesis and in metastatic disease[4-6], and is also activated in human colon carcinoma cell lines[7-9] and xenograft models[4], by ligand-dependent activation, that occurs in GI cancers[1, >>10<<]. However, the role of HH signaling and its importance in driving cellular survival in colon cancer are not well defined.
Small molecule inhibitors of SMO have been studied in preclinical models, and applied to the treatment of various types of cancers in humans[>>4<<, 9, 11-14].
Small molecule inhibitors of SMO have been studied in preclinical models, and applied to the treatment of various types of cancers in humans[4, >>9<<, 11-14]. Those tumors sensitive to SMO inhibitors, which include basal cell carcinoma[15, 16] and medulloblastoma[11, 17], rely on canonical HH signaling for cellular survival. In other cancer types, SMO inhibitors including GDC-0449,
Small molecule inhibitors of SMO have been studied in preclinical models, and applied to the treatment of various types of cancers in humans[4, 9, >>11<<-14]. Those tumors sensitive to SMO inhibitors, which include basal cell carcinoma[15, 16] and medulloblastoma[11, 17], rely on canonical HH signaling for cellular survival. In other cancer types, SMO inhibitors including GDC-0449, IPI-926
Those tumors sensitive to SMO inhibitors, which include basal cell carcinoma[>>15<<, 16] and medulloblastoma[11, 17], rely on canonical HH signaling for cellular survival.
Those tumors sensitive to SMO inhibitors, which include basal cell carcinoma[15, >>16<<] and medulloblastoma[11, 17], rely on canonical HH signaling for cellular survival.
Those tumors sensitive to SMO inhibitors, which include basal cell carcinoma[15, 16] and medulloblastoma[>>11<<, 17], rely on canonical HH signaling for cellular survival.
Those tumors sensitive to SMO inhibitors, which include basal cell carcinoma[15, 16] and medulloblastoma[11, >>17<<], rely on canonical HH signaling for cellular survival.
In other cancer types, SMO inhibitors including GDC-0449, IPI-926 or LDE225, have demonstrated limited clinical activity (reviewed in [>>11<<, 12]). Intrinsic resistance to SMO inhibitors is frequent[11-14, 18, 19], and acquired resistance to GDC-0449 following initial response has been reported in medulloblastoma (heterozygous mutation, Asp->His at aa 473 in SMO)[20]. Thus
Intrinsic resistance to SMO inhibitors is frequent[>>11<<-14, 18, 19], and acquired resistance to GDC-0449 following initial response has been reported in medulloblastoma (heterozygous mutation, Asp->His at aa 473 in SMO)[20].
Intrinsic resistance to SMO inhibitors is frequent[11-14, >>18<<, 19], and acquired resistance to GDC-0449 following initial response has been reported in medulloblastoma (heterozygous mutation, Asp->His at aa 473 in SMO)[20].
Intrinsic resistance to SMO inhibitors is frequent[11-14, 18, >>19<<], and acquired resistance to GDC-0449 following initial response has been reported in medulloblastoma (heterozygous mutation, Asp->His at aa 473 in SMO)[20].
Intrinsic resistance to SMO inhibitors is frequent[11-14, 18, 19], and acquired resistance to GDC-0449 following initial response has been reported in medulloblastoma (heterozygous mutation, Asp->His at aa 473 in SMO)[>>20<<]. Thus targeting the GLI genes downstream of SMO, that constitute the core of HH-dependent gene regulation, may provide a significant advantage in eliminating HH signaling.
activation of gli by oncogenic, non-canonical signaling pathways
Non-canonical, oncogene-driven signaling pathways converge on the activation of GLI genes and further converge on their specific downstream targets[>>3<<, 18, 21, 22] (see Figure 1). The RAS/RAF/MEK/ERK pathway, with activating mutations in K-RAS or B-RAF that occur in high frequency in colon cancers[23-25], activates GLI function[18, 19, 21].
Non-canonical, oncogene-driven signaling pathways converge on the activation of GLI genes and further converge on their specific downstream targets[3, >>18<<, 21, 22] (see Figure 1). The RAS/RAF/MEK/ERK pathway, with activating mutations in K-RAS or B-RAF that occur in high frequency in colon cancers[23-25], activates GLI function[18, 19, 21].
Non-canonical, oncogene-driven signaling pathways converge on the activation of GLI genes and further converge on their specific downstream targets[3, 18, >>21<<, 22] (see Figure 1). The RAS/RAF/MEK/ERK pathway, with activating mutations in K-RAS or B-RAF that occur in high frequency in colon cancers[23-25], activates GLI function[18, 19, 21].
Non-canonical, oncogene-driven signaling pathways converge on the activation of GLI genes and further converge on their specific downstream targets[3, 18, 21, >>22<<] (see Figure 1). The RAS/RAF/MEK/ERK pathway, with activating mutations in K-RAS or B-RAF that occur in high frequency in colon cancers[23-25], activates GLI function[18, 19, 21].
The RAS/RAF/MEK/ERK pathway, with activating mutations in K-RAS or B-RAF that occur in high frequency in colon cancers[>>23<<-25], activates GLI function[18, 19, 21].
The RAS/RAF/MEK/ERK pathway, with activating mutations in K-RAS or B-RAF that occur in high frequency in colon cancers[23-25], activates GLI function[>>18<<, 19, 21]. In HT29 cells (mutated B-RAF V600E[25]), we demonstrated inhibition of GLI-luciferase reporter activity, reduced expression of GLI1 mRNA and protein, and of p-ERK in response to the MEK/ERK and RAS/RAF signaling inhibitor
The RAS/RAF/MEK/ERK pathway, with activating mutations in K-RAS or B-RAF that occur in high frequency in colon cancers[23-25], activates GLI function[18, >>19<<, 21]. In HT29 cells (mutated B-RAF V600E[25]), we demonstrated inhibition of GLI-luciferase reporter activity, reduced expression of GLI1 mRNA and protein, and of p-ERK in response to the MEK/ERK and RAS/RAF signaling inhibitor U0126[26,
The RAS/RAF/MEK/ERK pathway, with activating mutations in K-RAS or B-RAF that occur in high frequency in colon cancers[23-25], activates GLI function[18, 19, >>21<<]. In HT29 cells (mutated B-RAF V600E[25]), we demonstrated inhibition of GLI-luciferase reporter activity, reduced expression of GLI1 mRNA and protein, and of p-ERK in response to the MEK/ERK and RAS/RAF signaling inhibitor U0126[26, 27]
In HT29 cells (mutated B-RAF V600E[>>25<<]), we demonstrated inhibition of GLI-luciferase reporter activity, reduced expression of GLI1 mRNA and protein, and of p-ERK in response to the MEK/ERK and RAS/RAF signaling inhibitor U0126[26, 27] (Figure 2).
In HT29 cells (mutated B-RAF V600E[25]), we demonstrated inhibition of GLI-luciferase reporter activity, reduced expression of GLI1 mRNA and protein, and of p-ERK in response to the MEK/ERK and RAS/RAF signaling inhibitor U0126[>>26<<, 27] (Figure 2).
In HT29 cells (mutated B-RAF V600E[25]), we demonstrated inhibition of GLI-luciferase reporter activity, reduced expression of GLI1 mRNA and protein, and of p-ERK in response to the MEK/ERK and RAS/RAF signaling inhibitor U0126[26, >>27<<] (Figure 2).
While loss-of-function mutations in PTCH and gain-of-function mutations in SMO activate HH signaling[>>1<<], acquired mutations in SMO or non-canonical GLI activation render cancer cells resistant to SMO antagonists.
targeting gli1 and gli2 with gant61
GLI1 and GLI2 are the primary activators of HH signaling; further, the cooperative roles of GLI1 and GLI2 are critical in the transcriptional regulation of HH target genes[>>1<<, 28-30]. While SMO has been extensively investigated as a therapeutic target[11-15, 17], few agents are available that target the GLI genes[31]. GANT61 was identified in a cell-based screen for small molecule inhibitors of GLI1-mediated
GLI1 and GLI2 are the primary activators of HH signaling; further, the cooperative roles of GLI1 and GLI2 are critical in the transcriptional regulation of HH target genes[1, >>28<<-30]. While SMO has been extensively investigated as a therapeutic target[11-15, 17], few agents are available that target the GLI genes[31]. GANT61 was identified in a cell-based screen for small molecule inhibitors of GLI1-mediated
While SMO has been extensively investigated as a therapeutic target[>>11<<-15, 17], few agents are available that target the GLI genes[31].
While SMO has been extensively investigated as a therapeutic target[11-15, >>17<<], few agents are available that target the GLI genes[31].
While SMO has been extensively investigated as a therapeutic target[11-15, 17], few agents are available that target the GLI genes[>>31<<]. GANT61 was identified in a cell-based screen for small molecule inhibitors of GLI1-mediated transcription. In the original study[31], GANT61 1) functions in the nucleus to abrogate GLI function; 2) blocks both GLI1- and GLI2- mediated
GANT61 was identified in a cell-based screen for small molecule inhibitors of GLI1-mediated transcription. In the original study[>>31<<], GANT61 1) functions in the nucleus to abrogate GLI function; 2) blocks both GLI1- and GLI2- mediated transcription; 3) in SUFU-null MEFS with constitutively active HH signaling, reduces expression of GLI1 and HIP1 mRNA in contrast to
We further demonstrated the specificity of GANT61 in targeting GLI1 and GLI2 from reduced GLI1 and GLI2 protein expression[>>8<<], inhibition of the binding of GLI1 and GLI2 to the promoter regions of HH target genes, specificity of reduction in GLI-luciferase reporter activity, and rapid inhibition of the transcriptional regulation of the GLI target gene BCL-2,
inhibition of gli induces greater cytotoxicity than targeting smo
In time course studies, 24 hr exposure to GANT61 or 72 hr exposure to cyclopamine was required to initiate cell death[>>9<<]. In addition the SMO inhibitor GDC-0449, like cyclopamine, demonstrated limited cytotoxic activity in HT29 cells at equimolar concentrations (unpublished data). Collectively, the data demonstrate increased sensitivity of human colon
These concentrations and time frames for the induction of cellular effects are similar to those determined in other model systems for inhibitors of HH signaling[>>4<<, 19, 33].
These concentrations and time frames for the induction of cellular effects are similar to those determined in other model systems for inhibitors of HH signaling[4, >>19<<, 33].
These concentrations and time frames for the induction of cellular effects are similar to those determined in other model systems for inhibitors of HH signaling[4, 19, >>33<<].
genetic downregulation of gli1 and gli2 by gli3r parallels cellular effects mediated by pharmacologic inhibition of gli1 and gli2 by gant61
A cleaved C-terminally truncated form of GLI3 (GLI3R) demonstrates repressor activity for GLI1 and GLI2 transcriptional regulation, and thereby silences HH-GLI target genes[>>4<<, 34]. We confirmed the critical role of GLI1 and GLI2 in cell survival by genetic downregulation of GLI1 and GLI2 following transient transfection of GLI3R[4, 34] in HT29 cells. Transient transfection and expression of GLI3R
A cleaved C-terminally truncated form of GLI3 (GLI3R) demonstrates repressor activity for GLI1 and GLI2 transcriptional regulation, and thereby silences HH-GLI target genes[4, >>34<<]. We confirmed the critical role of GLI1 and GLI2 in cell survival by genetic downregulation of GLI1 and GLI2 following transient transfection of GLI3R[4, 34] in HT29 cells. Transient transfection and expression of GLI3R (GLI3R-pCS2-MT,
We confirmed the critical role of GLI1 and GLI2 in cell survival by genetic downregulation of GLI1 and GLI2 following transient transfection of GLI3R[>>4<<, 34] in HT29 cells.
We confirmed the critical role of GLI1 and GLI2 in cell survival by genetic downregulation of GLI1 and GLI2 following transient transfection of GLI3R[4, >>34<<] in HT29 cells.
To begin to explore the cellular mechanisms downstream of GLI1/GLI2 inhibition that lead to cell death, we demonstrated that γH2AX, a marker of DNA DSBs[>>35<<], was expressed in GANT61-treated cells (Figure 4).
One of the critical regulators of DNA damage response is p53, which is stabilized upon DNA damage and modulates multiple components of the DNA damage response pathway[>>36<<, 37]. Expression of Gli3R-myc in human colon carcinoma cell lines harboring mutant p53 (functionally inactive) demonstrated DNA damage response suggesting a p53 independent mechanism[[32]]. Further, GANT61 treatment of HT29 cells
One of the critical regulators of DNA damage response is p53, which is stabilized upon DNA damage and modulates multiple components of the DNA damage response pathway[36, >>37<<]. Expression of Gli3R-myc in human colon carcinoma cell lines harboring mutant p53 (functionally inactive) demonstrated DNA damage response suggesting a p53 independent mechanism[[32]]. Further, GANT61 treatment of HT29 cells demonstrated
Figure 4Treatment of HT29 cells with vehicle alone (0.2% DMSO) or GANT61 (20 μM; upper panel), or transient transfection of vector alone, or GLI3R-pCS2-MT[>>4<<, 34](lower panel) for 72
Figure 4Treatment of HT29 cells with vehicle alone (0.2% DMSO) or GANT61 (20 μM; upper panel), or transient transfection of vector alone, or GLI3R-pCS2-MT[4, >>34<<](lower panel) for 72
inhibition of gli1/gli2 by gant61 induces arrest in early s
In contrast 5-fluorouracil (FUra) combined with leucovorin (1 μM), which targets thymidylate synthase in the inhibition of DNA replication and induction of DNA damage, arrested HT29 cells in mid S-phase[>>38<<] (67.0% by FACS/PI staining; Figure 6A), thereby demonstrating the difference in S-phase target location of the mechanism of GANT61-induced inhibition of DNA replication.
The cell cycle checkpoints constitute a regulatory mechanism to arrest the cell cycle in response to DNA damage so that cell cycle progression and repair may be temporally coordinated[>>39<<-42]. In contrast to the checkpoints at the G1/S and G2/M transitions, the S-phase checkpoint can only delay the progression of S-phase. ATM is the master transducer of the S-phase checkpoint, responds to DNA DSBs, and together with its
Activation of Cdk2 and the loading of Cdc45 onto replication origins are inhibited, DNA replication is inhibited, the intra-S-phase checkpoint is activated, and cyclin E accumulates[>>43<<-45], as demonstrated in GANT61-treated HT29 cells at 24 hr (Figure 6B).
Accumulation of HT29 cells in early S[>>9<<](Figure 6A), and the data of Figure 6B, suggest activation of an intra-S-phase checkpoint, which is not sustained.
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