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. 2014 Nov 7;289(45):31513-25.
doi: 10.1074/jbc.M114.606483. Epub 2014 Sep 24.

Gli1 protein regulates the S-phase checkpoint in tumor cells via Bid protein, and its inhibition sensitizes to DNA topoisomerase 1 inhibitors

Kaushlendra Tripathi  1 , Chinnadurai Mani  1 , Reagan Barnett  1 , Sriram Nalluri  1 , Lavanya Bachaboina  1 , Rodney P Rocconi  1 , Mohammed Athar  2 , Laurie B Owen  1 , Komaraiah Palle  3
Affiliations

Gli1 protein regulates the S-phase checkpoint in tumor cells via Bid protein, and its inhibition sensitizes to DNA topoisomerase 1 inhibitors

Kaushlendra Tripathi et al. J Biol Chem. .

Abstract

Aberrant expression of hedgehog molecules, particularly Gli1, is common in cancers of many tissues and is responsible for their aggressive behavior and chemoresistance. Here we demonstrate a novel and tumor-specific role for aberrant Gli1 in the regulation of the S-phase checkpoint that suppresses replication stress and resistance to chemotherapy. Inhibition of Gli1 in tumor cells induced replication stress-mediated DNA damage response, attenuated their clonogenic potential, abrogated camptothecin (CPT)-induced Chk1 phosphorylation, and potentiated its cytotoxicity. However, in normal fibroblasts, Gli1 siRNAs showed no significant changes in CPT-induced Chk1 phosphorylation. Further analysis of ataxia telangiectasia and Rad3-related protein (ATR)/Chk1 signaling cascade genes in tumor cells revealed an unexpected mechanism whereby Gli1 regulates ATR-mediated Chk1 phosphorylation by transcriptional regulation of the BH3-only protein Bid. Consistent with its role in DNA damage response, Bid down-regulation in tumor cells abolished CPT-induced Chk1 phosphorylation and sensitized them to CPT. Correspondingly, Gli1 inhibition affected the expression of Bid and the association of replication protein A (RPA) with the ATR- interacting protein (ATRIP)-ATR complex, and this compromised the S-phase checkpoint. Conversely, complementation of Bid in Gli1-deficient cells restored CPT-induced Chk1 phosphorylation. An in silico analysis of the Bid promoter identified a putative Gli1 binding site, and further studies using luciferase reporter assays confirmed Gli1-dependent promoter activity. Collectively, our studies established a novel connection between aberrant Gli1 and Bid in the survival of tumor cells and their response to chemotherapy, at least in part, by regulating the S-phase checkpoint. Importantly, our data suggest a novel drug combination of Gli1 and Top1 inhibitors as an effective therapeutic strategy in treating tumors that expresses Gli1.

Keywords: Cancer Biology; Cell Cycle; Checkpoint Control; Chemoresistance; DNA Damage; DNA Damage Response; DNA Repair; DNA Replication; DNA Topoisomerase; Hedgehog Signaling Pathway.

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Figures

FIGURE 1.
FIGURE 1.
Inhibition of Gli1 in cancer cells induces DDR and attenuates their clonogenic potential. A, A549 cells were transfected with control (siCon) or Gli1-specific (siGli1) siRNAs, and, after 48 h post-transfection, cells were analyzed for γH2AX foci. B and C, histograms showing the percentage of cells positive for γH2AX foci (B) and clonogenic cell survival (C). Each data point in B represents the mean of at least ten fields for γH2AX focus-positive cells, and the data presented in C are mean ± S.D. of three replicates. D, SCEs were performed as described under “Experimental Procedures,” and the histogram shows rate of SCE in control and Gli1 siRNAs transfected cells. 50 metaphases were scored, and their SCEs are presented as mean ± S.E. (*** indicates that the data are significant at p < 0.001).
FIGURE 2.
FIGURE 2.
Gli1 inhibition abrogates Chk1 phosphorylation and sensitizes cancer cells to CPT. A and B, after 48 h of siRNA transfection, cells were treated with DMSO or 500 nm CPT for 2 h. Cell lysates were normalized for protein concentrations and analyzed by SDS-PAGE and immunoblotted for various DDR proteins in A549 cells (A) and HT29 cells (B). C and D, Gli1 down-regulation potentiated CPT-induced cytotoxicity in A549 (C) and HT29 (D) cells in clonogenic survival assays. Data are mean ± S.D. of triplicates. E and F, the Gli1 inhibition-mediated Chk1 phosphorylation defect is specific to tumor cells and has no significant effects on Chk1 phosphorylation in normal fibroblasts, BJ cells (E), and HDF cells (F).
FIGURE 3.
FIGURE 3.
Gli1 inhibition-mediated abrogation of Chk1 phosphorylation is not due to the defects in DNA synthesis. A, control and Gli1 down-regulated A549 cells were treated with 10 μm BrdU for 30 min, and cells were labeled with FITC-conjugated anti-BrdU antibodies and propidium iodide and analyzed by flow cytometry to assess the number of cells actively synthesizing DNA. Top panel, BrdU versus propidium iodide staining. Bottom panel, FITC versus count. Data are representative of two independent experiments. B, A549 cells transfected with multiple Gli1 siRNAs targeting to different regions of Gli1 exhibit similar defect in CPT-induced Chk1 phosphorylation.
FIGURE 4.
FIGURE 4.
Gli1 down-regulation inhibits the S-phase checkpoint, induces CPT-resistant DNA synthesis, and increases the number of replication-coupled DSBs. A, control and Gli1 down-regulated A549 cells were treated with the indicated concentrations of CPT or DMSO, and the rate of DNA synthesis was assessed by [3H]thymidine incorporation. Data mean ± S.D. of triplicates (the error bars are too small to be seen). B, control and Gli1 down-regulated A549 cells were exposed to 500 nm CPT and assessed for phospho-RPA (S4/8) foci or added to EdU for 30 min and exposed to CPT to assess the replication-coupled DSBs. C, γH2AX foci (green) colocalized with EdU (red) as a replication marker. D, A549 cells transfected with control and Gli1 siRNAs were exposed to 500 nm CPT for 2 h and allowed to recover for another 24 h in drug-free medium and assessed by flow cytometry. An increased number of sub-G0 cells was observed in CPT-exposed Gli1 knockdown cells after 24 h (red line).
FIGURE 5.
FIGURE 5.
Gli1 inhibition affects the S-phase checkpoint by down-regulation of Bid. A and B, Gli1-proficient and knockdown tumor cells were exposed to DMSO or 500 nm CPT and assessed for different DDR proteins involved in the ATR/Chk1 signaling cascade in A549 (A) and H1299 (B) cells. C–F, similarly, control and Bid down-regulated A549 cells were assessed for CPT sensitivity (C) and CPT-induced Chk1 phosphorylation in different cancer cells: A549 (D), H1299 (E) and A2780/CP70 (F). The data in C are mean ± S.D. of triplicates.
FIGURE 6.
FIGURE 6.
Gli1 knockdown affects the interaction of RPA70 with ATR-ATRIP protein complexes and attenuates the CPT-induced S-phase checkpoint response via Bid. A, control and Gli1 down-regulated A549 cells were exposed to CPT or DMSO. The protein lysates were used for immunoprecipitation assays with RPA70 antibodies and analyzed by immunoblotting for indicated proteins, and the levels of represented proteins are shown as inputs (bottom panel). B, complementation of Bid in Gli1 down-regulated cells corrects the CPT-induced Chk1 phosphorylation defect.
FIGURE 7.
FIGURE 7.
Gli1 regulates Bid expression in tumor cells through its promoter activity. A, in silico analysis of the BID promoter region (5′ UTR) identified a consensus Gli1 binding site (boldface). B, luciferase reporter assay were performed as described under “Experimental Procedures,” and the results demonstrate the Gli1 status-dependent activity of the Bid promoter (Bid) in A549 cells. Data are mean ± S.D. from triplicates
FIGURE 8.
FIGURE 8.
Pharmacological inhibition of Gli using GANT61 abrogates Chk1 phosphorylation and sensitizes cancer cells to CPT. A, HT29 cells were treated with 20 μm GANT61 and assessed for γH2AX foci. B and C, HT29 cells were pretreated with GANT61 and assessed for CPT-induced DDR proteins by immunoblotting (B) and cell sensitivity to CPT by clonogenic survival assays (C). Data are mean ± S.D. of triplicates. D, similar DDRs were observed in GANT61-pretreated A2780/CP70 cells.
FIGURE 9.
FIGURE 9.
A putative model showing the mechanism of Gli1 inhibition-mediated S-phase checkpoint abrogation. In response to CPT-induced replication stress or stalled replication forks, Bid binding to RPA facilitates its interaction with the ATRIP-ATR complex, which subsequently phosphorylates Chk1 to relay the S-phase checkpoint response. In tumor cells, aberrant Gli1 controls Bid expression through its promoter activity to promote their survival and resistance to the drugs that interfere with replication, such as the Top1 poison CPT.
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References

    1. Vokes S. A., Ji H., McCuine S., Tenzen T., Giles S., Zhong S., Longabaugh W. J. R., Davidson E. H., Wong W. H., McMahon A. P. (2007) Genomic characterization of Gli-activator targets in sonic hedgehog-mediated neural patterning. Dev. Camb. Engl. 134, 1977–1989 - PubMed
    1. Lum L., Beachy P. A. (2004) The Hedgehog response network: sensors, switches, and routers. Science 304, 1755–1759 - PubMed
    1. Dahmane N., Ruiz i Altaba A. (1999) Sonic hedgehog regulates the growth and patterning of the cerebellum. Dev. Camb. Engl. 126, 3089–3100 - PubMed
    1. Gorlin R. J. (2004) Nevoid basal cell carcinoma (Gorlin) syndrome. Genet. Med. 6, 530–539 - PubMed
    1. Johnson R. L., Rothman A. L., Xie J., Goodrich L. V., Bare J. W., Bonifas J. M., Quinn A. G., Myers R. M., Cox D. R., Epstein E. H., Jr., Scott M. P. (1996) Human homolog of patched, a candidate gene for the basal cell nevus syndrome. Science 272, 1668–1671 - PubMed

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