Gli-binding Site Research Articles

The Hedgehog/GLI signaling pathway activates transcription of Slug (Snail2) in melanoma cells.

In melanoma and other cancers, invasion, epithelial-to-mesenchymal transition, metastasis and cancer stem cell maintenance are regulated by transcription factors including the Snail family. Slug (Snail2) protein generally supports migration and apoptosis resistance. However, its role in melanoma is not completely understood. The present study investigated the transcriptional regulation of the SLUG gene in melanoma. It demonstrated that SLUG is under the control of the Hedgehog/GLI signaling pathway and is activated predominantly by the transcription factor GLI2. The SLUG gene promoter contains a high number of GLI-binding sites. Slug expression is activated by GLI factors in reporter assays and inhibited by GANT61 (GLI inhibitor) and cyclopamine (SMO inhibitor). SLUG mRNA levels are lowered by GANT61 as assessed by reverse transcription-quantitative PCR. Chromatin immunoprecipitation revealed abundant binding of factors GLI1-3 in the four subregions of the proximal SLUG promoter. Notably, melanoma-associated transcription factor (MITF) is an imperfect activator of the SLUG promoter in reporter assays, and downregulation of MITF had no effect on endogenous Slug protein levels. Immunohistochemical analysis confirmed the above findings and showed MITF-negative regions in metastatic melanoma that were positive for GLI2 and Slug. Taken together, the results demonstrated a previously unrecognized transcriptional activation mechanism of the SLUG gene, which may represent its main regulation of expression in melanoma cells.

GLI1 interaction with p300 modulates SDF1 expression in cancer-associated fibroblasts to promote pancreatic cancer cells migration.

Carcinoma-associated fibroblasts (CAFs) play an important role in the progression of multiple malignancies. Secretion of cytokines and growth factors underlies the pro-tumoral effect of CAFs. Although this paracrine function has been extensively documented, the molecular mechanisms controlling the expression of these factors remain elusive. In this study, we provide evidence of a novel CAF transcriptional axis regulating the expression of SDF1, a major driver of cancer cell migration, involving the transcription factor GLI1 and histone acetyltransferase p300. We demonstrate that conditioned media from CAFs overexpressing GLI1 induce the migration of pancreatic cancer cells, and this effect is impaired by an SDF1-neutralizing antibody. Using a combination of co-immunoprecipitation, proximity ligation assay and chromatin immunoprecipitation assay, we further demonstrate that GLI1 and p300 physically interact in CAFs to co-occupy and drive SDF1 promoter activity. Mapping experiments highlight the requirement of GLI1 N-terminal for the interaction with p300. Importantly, knockdowns of both GLI1 and p300 reduce SDF1 expression. Further analysis shows that knockdown of GLI1 decreases SDF1 promoter activity, p300 recruitment, and levels of its associated histone marks (H4ac, H3K27ac, and H3K14ac). Finally, we show that the integrity of two GLI binding sites in the SDF1 promoter is required for p300 recruitment. Our findings define a new role for the p300-GLI1 complex in the regulation of SDF1, providing new mechanistic insight into the molecular events controlling pancreatic cancer cells migration.

Hedgehog and Platelet-derived Growth Factor Signaling Intersect during Postnatal Lung Development.

Normal lung development critically depends on HH (Hedgehog) and PDGF (platelet-derived growth factor) signaling, which coordinate mesenchymal differentiation and proliferation. PDGF signaling is required for postnatal alveolar septum formation by myofibroblasts. Recently, we demonstrated a requirement for HH in postnatal lung development involving alveolar myofibroblast differentiation. Given shared features of HH signaling and PDGF signaling and their impact on this key cell type, we sought to clarify their relationship during murine postnatal lung development. Timed experiments revealed that HH inhibition phenocopies the key lung myofibroblast phenotypes of Pdgfa (platelet-derived growth factor subunit A) and Pdgfra (platelet-derived growth factor receptor alpha) knockouts during secondary alveolar septation. Using a dual signaling reporter, Gli1lZ;PdgfraEGFP, we show that HH and PDGF pathway intermediates are concurrently expressed during alveolar septal myofibroblast accumulation, suggesting pathway convergence in the generation of lung myofibroblasts. Consistent with this hypothesis, HH inhibition reduces Pdgfra expression and diminishes the number of Pdgfra-positive and Pdgfra-lineage cells in postnatal lungs. Bulk RNA sequencing data of Pdgfra-expressing cells from Postnatal Day 8 (P8) lungs show that HH inhibition alters the expression not only of well-established HH targets but also of several putative PDGF target genes. This, together with the presence of Gli-binding sites in PDGF target genes, suggests HH input into PDGF signaling. We identified these HH/PDGF targets in several postnatal lung mesenchymal cell populations, including myofibroblasts, using single-cell transcriptomic analysis. Collectively, our data indicate that HH signaling and PDGF signaling intersect to support myofibroblast/fibroblast function during secondary alveolar septum formation. Moreover, they provide a molecular foundation relevant to perinatal lung diseases associated with impaired alveolarization.

RNA-seq and ChIP-seq Identification of Unique and Overlapping Targets of GLI Transcription Factors in Melanoma Cell Lines.

Simple SummaryThe majority of melanomas show hyperactivation of the MAPK signaling pathway, most often through mutations in BRAF and NRAS. Despite significant progress in therapy, targeting this signaling pathway solely has not been the solution for stopping the progression of this disease. Recently, researchers recognized the involvement of the Hedgehog-GLI (HH-GLI) signaling pathway in melanoma and its crosstalk with the MAPK pathway. In order to identify new HH-GLI-regulated targets that could be involved in the crosstalk with the MAPK pathway, we performed RNA sequencing and ChIP sequencing on three melanoma cell lines. By combining RNA-seq and ChIP-seq results, we successfully validated 15 novel targets of GLI proteins in melanoma cell lines. These findings will contribute to a better understanding of the GLI code and its role in melanoma.Background: Despite significant progress in therapy, melanoma still has a rising incidence worldwide, and novel treatment strategies are needed. Recently, researchers have recognized the involvement of the Hedgehog-GLI (HH-GLI) signaling pathway in melanoma and its consistent crosstalk with the MAPK pathway. In order to further investigate the link between the two pathways and to find new target genes that could be considered for combination therapy, we set out to find transcriptional targets of all three GLI proteins in melanoma. Methods: We performed RNA sequencing on three melanoma cell lines (CHL-1, A375, and MEL224) with overexpressed GLI1, GLI2, and GLI3 and combined them with the results of ChIP-sequencing on endogenous GLI1, GLI2, and GLI3 proteins. After combining these results, 21 targets were selected for validation by qPCR. Results: RNA-seq revealed a total of 808 differentially expressed genes (DEGs) for GLI1, 941 DEGs for GLI2, and 58 DEGs for GLI3. ChIP-seq identified 527 genes that contained GLI1 binding sites in their promoters, 1103 for GLI2 and 553 for GLI3. A total of 15 of these targets were validated in the tested cell lines, 6 of which were detected by both RNA-seq and ChIP-seq. Conclusions: Our study provides insight into the unique and overlapping transcriptional output of the GLI proteins in melanoma. We suggest that our findings could provide new potential targets to consider while designing melanoma-targeted therapy.

Loss of Hilnc prevents diet-induced hepatic steatosis through binding of IGF2BP2.

The Hedgehog (Hh) signalling pathway plays a critical role in regulating liver lipid metabolism and related diseases. However, the underlying mechanisms are poorly understood. Here, we show that the Hh signalling pathway induces a previously undefined long non-coding RNA (Hilnc, Hedgehog signalling-induced long non-coding RNA), which controls hepatic lipid metabolism. Mutation of the Gli-binding sites in the Hilnc promoter region (HilncBM/BM) decreases the expression of Hilnc in vitro and in vivo. HilncBM/BM and Hilnc-knockout mice are resistant to diet-induced obesity and hepatic steatosis through attenuation of the peroxisome proliferator-activated receptor signalling pathway, as Hilnc directly interacts with IGF2BP2 to enhance Pparγ mRNA stability. Furthermore, we identify a potential functional human homologue of Hilnc, h-Hilnc, which has a similar function in regulating cellular lipid metabolism. These findings uncover a critical role of the Hh-Hilnc-IGF2BP2 signalling axis in lipid metabolism and suggest a potential therapeutic target for the treatment of diet-induced hepatic steatosis.

CRISPR editing of the GLI1 first intron abrogates GLI1 expression and differentially alters lineage commitment.

GLI1 is one of three GLI family transcription factors that mediate Sonic Hedgehog signaling, which plays a role in development and cell differentiation. GLI1 forms a positive feedback loop with GLI2 and likely with itself. To determine the impact of GLI1 and its intronic regulatory locus on this transcriptional loop and human stem cell differentiation, we deleted the region containing six GLI binding sites in the human GLI1 intron using CRISPR/Cas9 editing to produce H1 human embryonic stem cell (hESC) GLI1‐edited clones. Editing out this intronic region, without removing the entire GLI1 gene, allowed us to study the effects of this highly complex region, which binds transcription factors in a variety of cells. The roles of GLI1 in human ESC differentiation were investigated by comparing RNA sequencing, quantitative‐real time PCR (q‐rtPCR), and functional assays. Editing this region resulted in GLI1 transcriptional knockdown, delayed neural commitment, and inhibition of endodermal and mesodermal differentiation during spontaneous and directed differentiation experiments. We found a delay in the onset of early osteogenic markers, a reduction in the hematopoietic potential to form granulocyte units, and a decrease in cancer‐related gene expression. Furthermore, inhibition of GLI1 via antagonist GANT‐61 had similar in vitro effects. These results indicate that the GLI1 intronic region is critical for the feedback loop and that GLI1 has lineage‐specific effects on hESC differentiation. Our work is the first study to document the extent of GLI1 abrogation on early stages of human development and to show that GLI1 transcription can be altered in a therapeutically useful way.

Hedgehog-GLI signalling promotes chemoresistance through the regulation of ABC transporters in colorectal cancer cells

Colorectal cancer (CRC) is a leading cause of cancer death. Chemoresistance is a pivotal feature of cancer cells leading to treatment failure and ATP-binding cassette (ABC) transporters are responsible for the efflux of several molecules, including anticancer drugs. The Hedgehog-GLI (HH-GLI) pathway is a major signalling in CRC, however its role in chemoresistance has not been fully elucidated. Here we show that the HH-GLI pathway favours resistance to 5-fluorouracil and Oxaliplatin in CRC cells. We identified potential GLI1 binding sites in the promoter region of six ABC transporters, namely ABCA2, ABCB1, ABCB4, ABCB7, ABCC2 and ABCG1. Next, we investigated the binding of GLI1 using chromatin immunoprecipitation experiments and we demonstrate that GLI1 transcriptionally regulates the identified ABC transporters. We show that chemoresistant cells express high levels of GLI1 and of the ABC transporters and that GLI1 inhibition disrupts the transporters up-regulation. Moreover, we report that human CRC tumours express high levels of the ABCG1 transporter and that its expression correlates with worse patients’ prognosis. This study identifies a new mechanism where HH-GLI signalling regulates CRC chemoresistance features. Our results indicate that the inhibition of Gli1 regulates the ABC transporters expression and therefore should be considered as a therapeutic option in chemoresistant patients.

Abstract 6336: Non-canonical activation of Gli-2 by TGF-β/Smad3 signaling pathway in modulation of chemoresistance of hepatocellular carcinoma

Abstract BACKGROUND: It has been demonstrated in our previous study that poorly-differentiated hepatoma cells with low α-feto-protein (AFP) expression and a CD133-/EpCAM- surface profile exhibited aberrant hedgehog signaling (Hh) activity, epithelial-mesenchymal transition and chemoresistance. However, expression of canonical Hh molecules (Sonic ligand and SMO) was not increased in poorly-differentiated hepatoma cells in contrast to well-differentiated hepatoma cells, indicating that Hh transcription factors Gli-1/2 may be activated through a non-classic pathway. The present study aims to investigate whether the TGF-β/Smad3 pathway is responsible for Gli-2 activation through a non-canonical pathway. METHODS: Expression of Sonic Hh (Shh) and Smad3 was assessed in both hepatoma cells and HCC specimens. Gli-2 and Smad3 expression was suppressed by lentiviral vector-mediated RNA interference in poorly-differentiated hepatoma Huh-7-DN and HLE cells. Chemosensitivity was evaluated in hepatoma cells with a Gli-2 or Smad3 inhibitor. RESULTS: Significantly increased Gli-1 and Gli-2 protein levels were observed in 78% (11 out of 14) and 50% (7 out of 14) HCC specimens. In 7 HCC patients with a normal serum AFP value (<20 μg/L), Gli-1/2 protein levels were obviously higher than their pericancerous tissue. Moreover, increased Gli-2 and ABCC1 protein levels were accompanied with increased TGF-β1 and p-Smad3 protein levels in 5 out 7 HCC specimens; whereas Shh protein levels were similar to paired pericancerous tissue. Consistent with the findings from HCC specimens, significantly higher Gli-2 and p-Smad3 nuclear protein levels were associated with increased ABCC1 protein levels in poorly-differentiated hepatoma cells (Huh-7-trans, Huh-7-DN, HLE and HLF); whereas Shh and SMO protein levels were not significantly different from well-differentiated hepatoma Huh-7 and Hep3B cells. Suppressing Gli-2 expression by specific shRNA caused marked decreased expression of Gli-2 and ABCC1 in Huh-7-DN, Huh-7-trans and HLE cells. In addition, expression of Smad3, Gli-2 and ABCC1 was significantly down-regulated in HLE cells after infected with a lentiviral vector harboring shSmad3, indicating that Gli-2 is a downstream target of Smad3. Sorafenib IC50 was significantly reduced from 35.76 to 17.45 μM or 14.04 μM in HLE cells when treated with a GLI inhibitor (GANT-61) or a Smad3 inhibitor (SIS3) at 5 μM. A potential GLI-binding site in the ABCC1 promoter was identified. ABCC1-Gli-luciferase reporter activity was increased up to 39-fold in HLE cells when transfected with constructs containing the potential GLI-binding site, compared to the construct that lacked a GLI-binding site. CONCLUSION: TGF-β/Smad3 activated Gli-2 and its direct transcriptional target ABCC1 in poorly-differentiated and chemoresistant hepatoma cells. Suppressing TGF-β/Smad3/Gli-2 signaling activity confers a potential molecular target in partially alleviating Sorafenib resistance for the treatment of refractory hepatoma. Citation Format: Jia Ding, Hui-yan Li, Li Zhang, Xiao-tian Zhou, Jian Wu. Non-canonical activation of Gli-2 by TGF-β/Smad3 signaling pathway in modulation of chemoresistance of hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6336.

Regulation of GLI1 by cis DNA elements and epigenetic marks

GLI1 is one of three transcription factors (GLI1, GLI2 and GLI3) that mediate the Hedgehog signal transduction pathway and play important roles in normal development. GLI1 and GLI2 form a positive-feedback loop and function as human oncogenes. The mouse and human GLI1 genes have untranslated 5′ exons and large introns 5′ of the translational start. Here we show that Sonic Hedgehog (SHH) stimulates occupancy in the introns by H3K27ac, H3K4me3 and the histone reader protein BRD4. H3K27ac and H3K4me3 occupancy is not significantly changed by removing BRD4 from the human intron and transcription start site (TSS) region. We identified six GLI binding sites (GBS) in the first intron of the human GLI1 gene that are in regions of high sequence conservation among mammals. GLI1 and GLI2 bind all of the GBS in vitro. Elimination of GBS1 and 4 attenuates transcriptional activation by GLI1. Elimination of GBS1, 2, and 4 attenuates transcriptional activation by GLI2. Eliminating all sites essentially eliminates reporter gene activation. Further, GLI1 binds the histone variant H2A.Z. These results suggest that GLI1 and GLI2 can regulate GLI1 expression through protein-protein interactions involving complexes of transcription factors, histone variants, and reader proteins in the regulatory intron of the GLI1 gene. GLI1 acting in trans on the GLI1 intron provides a mechanism for GLI1 positive feedback and auto-regulation. Understanding the combinatorial protein landscape in this locus will be important to interrupting the GLI positive feedback loop and providing new therapeutic approaches to cancers associated with GLI1 overexpression.

P.E95K mutation in Indian hedgehog causing brachydactyly type A1 impairs IHH/Gli1 downstream transcriptional regulation

BackgroundBrachydactyly type A1 (BDA1, OMIM 112500) is a rare inherited malformation characterized primarily by shortness or absence of middle bones of fingers and toes. It is the first recorded disorder of the autosomal dominant Mendelian trait. Indian hedgehog (IHH) gene is closely associated with BDA1, which was firstly mapped and identified in Chinese families in 2000. Previous studies have demonstrated that BDA1-related mutant IHH proteins affected interactions with its receptors and impaired IHH signaling. However, how the altered signaling pathway affects downstream transcriptional regulation remains unclear.ResultsBased on the mouse C3H10T1/2 cell model for IHH signaling activation, two recombinant human IHH-N proteins, including a wild type protein (WT, amino acid residues 28–202) and a mutant protein (MT, p.E95k), were analyzed. We identified 347, 47 and 4 Gli1 binding sites in the corresponding WT, MT and control group by chromatin immunoprecipitation and the overlapping of these three sets was poor. The putative cis regulated genes in WT group were enriched in sensory perception and G-protein coupled receptor-signaling pathway. On the other hand, putative cis regulated genes were enriched in Runx2-related pathways in MT group. Differentially expressed genes in WT and MT groups indicated that the alteration of mutant IHH signaling involved cell-cell signaling and cellular migration. Cellular assay of migration and proliferation validated that the mutant IHH signaling impaired these two cellular functions.ConclusionsIn this study, we performed integrated genome-wide analyses to characterize differences of IHH/Gli1 downstream regulation between wild type IHH signaling and the E95K mutant signaling. Based on the cell model, our results demonstrated that the E95K mutant signaling altered Gli1-DNA binding pattern, impaired downstream gene expressions, and leaded to weakened cellular proliferation and migration. This study may help to deepen the understanding of pathogenesis of BDA1 and the role of IHH signaling in chondrogenesis.

The role of GLI-SOX2 signaling axis for gemcitabine resistance in pancreatic cancer

Pancreatic cancer, mostly pancreatic ductal adenocarcinomas (PDAC), is one of the most lethal cancers, with a dismal median survival around 8 months. PDAC is notoriously resistant to chemotherapy. Thus far, numerous attempts using novel targeted therapies and immunotherapies yielded limited clinical benefits for pancreatic cancer patients. It is hoped that delineating the molecular mechanisms underlying drug resistance in pancreatic cancer may provide novel therapeutic options. Using acquired gemcitabine resistant pancreatic cell lines, we revealed an important role of the GLI-SOX2 signaling axis for regulation of gemcitabine sensitivity in vitro and in animal models. Down-regulation of GLI transcriptional factors (GLI1 or GLI2), but not SMO signaling inhibition, reduces tumor sphere formation, a characteristics of tumor initiating cell (TIC). Down-regulation of GLI transcription factors also decreased expression of TIC marker CD24. Similarly, high SOX2 expression is associated with gemcitabine resistance whereas down-regulation of SOX2 sensitizes pancreatic cancer cells to gemcitabine treatment. We further revealed that elevated SOX2 expression is associated with an increase in GLI1 or GLI2 expression. Our ChIP assay revealed that GLI proteins are associated with a putative Gli binding site within the SOX2 promoter, suggesting a more direct regulation of SOX2 by GLI transcription factors. The relevance of our findings to human disease was revealed in human cancer specimens. We found that high SOX2 protein expression is associated with frequent tumor relapse and poor survival in stage II PDAC patients (all of them underwent gemcitabine treatment), indicating that reduced SOX2 expression or down-regulation of GLI transcription factors may be effective in sensitizing pancreatic cancer cells to gemcitabine treatment.

A novel ZIC3 gene mutation identified in patients with heterotaxy and congenital heart disease

Heterotaxy syndrome (HTX) is characterized by left-right (LR) asymmetry disturbances associated with severe heart malformations. However, the exact genetic cause of HTX pathogenesis remains unclear. The aim of this study was to investigate the pathogenic mechanism underlying heterotaxy syndrome. Targeted next-generation sequencing (NGS) was performed for twenty-two candidate genes correlated with LR axis development in sixty-six HTX patients from unrelated families. Variants were filtered from databases and predicted in silico using prediction programs. A total of twenty-one potential disease-causing variants were identified in seven genes. Next, we used Sanger sequencing to confirm the identified variants in the family pedigree and found a novel hemizygous mutation (c.890G > T, p.C297F) in the ZIC3 gene in a male patient that was inherited from his mother, who was a carrier. The results of functional indicated that this ZIC3 mutation decreases transcriptional activity, affects the affinity of the GLI-binding site and results in aberrant cellular localization in transfected cells. Moreover, morpholino-knockdown experiments in zebrafish demonstrated that zic3 mutant mRNA failed to rescue the abnormal phenotype, suggesting a role for the novel ZIC3 mutation in heterotaxy syndrome.

PO-171 Identifying IFITM1-dependent synthetized proteins in interferon gamma stimulated cells

IntroductionSignalling pathway Hedgehog-Gli (Hh-Gli) is involved in embryonal ovarian development, but its atypical activation can lead to different types of ovarian tumours. Our previous studies showed aberrant Hh-Gli activity in some ovarian tumour types and hypermethylation in a promoter of tumour suppressor gene PTCH1 in the CpG islands near the GLI-binding site (Sabol et al. Int J Oncol 2012, 2017;Musani et al. Gene 2013; Maurac et al. Int J Gynecol Pathol 2012). We and other are aware that various genetic and epigenetic alterations contribute to aberrant Hh-Gli pathway activity in ovarian cancer, which could serve as interesting targets for cancer treatment and therapy, especially if can be used to bypass resistance and to target cancer in more efficient way. We hypothesise that changes in the expression of miRNA molecules related to the Hh-Gli signalling pathway genes contribute to the development of high-grade serous ovarian carcinoma (HGSOC).Material and methodsWe conducted a miRNA profiling of HGSOC and healthy Fallopian tube control samples with Agilent SurePrint G3 Human miRNA 8 × 60K Microarray Kit containing probes for 2549 human miRNAs. Gene expression profiling was preformed using Agilent SurePrint G3 Human Gene Expression v3 8 × 60K Microarray Kit, which covers 37 756 known RefSeq coding transcripts. Data were analysed using R/Bioconductor packages AgiMicroRna and limma. On-line DIANA Tools (TarBase, microT-CDS and miRPath) were used to find which Hh-Gli genes are targets of miRNAs differentially expressed in HGSOC.Results and discussionsData filtration gave 55 miRNAs: 32 up- and 23 down-regulated in HGSOC. Out of 47 genes involved in Hh-Gli pathway in humans (KEGG Pathway hsa04340), 35 are known targets for 27 over-expressed miRNAs, 22 are known targets for 16 under-expressed miRNAs, while 28 genes are potential targets for 26 up-regulated miRNAs and 24 are potential targets for 19 down-regulated miRNAs in HGSOC. In addition, 1090 genes were significantly over-expressed and 1692 were under-expressed in HGSOC. Further analysis revealed a couple of potential combinations of miRNAs and their target genes which are members of Hh-Gli pathway: ADRBK2/hsa-miR-96–5 p, BCL2/hsa-miR-16–5 p and hsa-miR-96–5 p, BOC/hsa-miR-224–5 p, and IHH/hsa-miR-16–5 p, hsa-miR-107 and hsa-miR-103a-3p.ConclusionOur results highlighted several candidate miRNAs targeting Hh-Gli signalling pathway genes, which, when additionally verified, could be potentially used for better therapeutic and early prevention approaches for HGSOC.

PO-155 Therapeutic evaluation of HEDGEHOG-GLI signalling pathway through miRNA and mRNA profiling of ovarian carcinoma

IntroductionSignalling pathway Hedgehog-Gli (Hh-Gli) is involved in embryonal ovarian development, but its atypical activation can lead to different types of ovarian tumours. Our previous studies showed aberrant Hh-Gli activity in some ovarian tumour types and hypermethylation in a promoter of tumour suppressor gene PTCH1 in the CpG islands near the GLI-binding site (Sabol et al. Int J Oncol 2012, 2017;Musani et al. Gene 2013; Maurac et al. Int J Gynecol Pathol 2012). We and other are aware that various genetic and epigenetic alterations contribute to aberrant Hh-Gli pathway activity in ovarian cancer, which could serve as interesting targets for cancer treatment and therapy, especially if can be used to bypass resistance and to target cancer in more efficient way. We hypothesise that changes in the expression of miRNA molecules related to the Hh-Gli signalling pathway genes contribute to the development of high-grade serous ovarian carcinoma (HGSOC).Material and methodsWe conducted a miRNA profiling of HGSOC and healthy Fallopian tube control samples with Agilent SurePrint G3 Human miRNA 8 × 60K Microarray Kit containing probes for 2549 human miRNAs. Gene expression profiling was preformed using Agilent SurePrint G3 Human Gene Expression v3 8 × 60K Microarray Kit, which covers 37 756 known RefSeq coding transcripts. Data were analysed using R/Bioconductor packages AgiMicroRna and limma. On-line DIANA Tools (TarBase, microT-CDS and miRPath) were used to find which Hh-Gli genes are targets of miRNAs differentially expressed in HGSOC.Results and discussionsData filtration gave 55 miRNAs: 32 up- and 23 down-regulated in HGSOC. Out of 47 genes involved in Hh-Gli pathway in humans (KEGG Pathway hsa04340), 35 are known targets for 27 over-expressed miRNAs, 22 are known targets for 16 under-expressed miRNAs, while 28 genes are potential targets for 26 up-regulated miRNAs and 24 are potential targets for 19 down-regulated miRNAs in HGSOC. In addition, 1090 genes were significantly over-expressed and 1692 were under-expressed in HGSOC. Further analysis revealed a couple of potential combinations of miRNAs and their target genes which are members of Hh-Gli pathway: ADRBK2/hsa-miR-96–5 p, BCL2/hsa-miR-16–5 p and hsa-miR-96–5 p, BOC/hsa-miR-224–5 p, and IHH/hsa-miR-16–5 p, hsa-miR-107 and hsa-miR-103a-3p.ConclusionOur results highlighted several candidate miRNAs targeting Hh-Gli signalling pathway genes, which, when additionally verified, could be potentially used for better therapeutic and early prevention approaches for HGSOC.

Abstract 5415: STAT3 and GLI1/tGLI1 oncogenic transcription factors interact to promote the aggressiveness of triple-negative and HER2-enriched breast cancers

Abstract Patients with triple-negative (TN) and HER2-enriched (HER2) breast cancers have high rates of metastasis, which accounts for 90% mortality for these patients, highlighting the need for improved therapeutic targets. The oncogenic transcription factors, STAT3, GLI1, and truncated GLI1 (tGLI1) are associated with poor clinical outcomes for breast cancer patients. However, physical or functional interactions between STAT3 and GLI1/tGLI1 have never been investigated. The purpose of this study is to determine whether the JAK2/STAT3 and GLI1/tGLI1 pathways are concurrently activated in TN and HER2 breast cancers. We examined 47 node-positive breast tumor samples using immunohistochemical staining and found that p-STAT3 (Y705), GLI1, and tGLI1 were co-overexpressed in 64% of TN breast cancers, 68% of HER2 breast cancers, and in 65% of lymph node metastases. Gene Set Enrichment Analysis of 710 breast tumors revealed that the activation signatures for STAT3 and GLI1/tGLI1 are co-enriched in TN and HER2 subtypes, but not in the luminal subtypes. Furthermore, Kaplan-Meier and log-rank analyses revealed that breast tumors with high levels of STAT3 and GLI1/tGLI1 co-activation were associated with shortened metastasis-free survival compared to those with lower levels. To determine whether these two pathways functionally cooperate, we examined whether STAT3 and GLI1/tGLI1 formed complexes by IP-WB and observed that STAT3 interacted with both GLI1 and tGLI1. ChIP assay showed that these complexes bind to both consensus GLI1- and STAT3-binding sites. We further observed that co-overexpression of STAT3 and GLI1/tGLI1 resulted in significant activation of a promoter controlled by GLI1-binding sites. We next analyzed three ChIP-Seq datasets to identify gene promoters that can be occupied by the STAT3-GLI1/tGLI1 complexes, and found 36 potential target genes. Validation of these target genes by RT-qPCR and Kaplan-Meier analysis resulted in three genes that are upregulated by STAT3-GLI1 and/or STAT3-tGLI1, namely, R-Ras2, Cep70, and UPF3A, and correlated with poor survival. These novel transcriptional targets of STAT3 and GLI1/tGLI1 are implicated in breast tumorigenesis, angiogenesis, and metastasis suggesting that co-activation of STAT3 and GLI1/tGLI1 may promote aggressive breast cancers. Lastly, co-overexpression of STAT3 and GLI1/tGLI1 increased the mammosphere-forming ability of breast cancer cells and interestingly, only the STAT3-tGLI1 complex allowed mammosphere formation of immortalized mammary epithelial cells. In conclusion, our study reports, for the first time, that the physical and functional interactions between STAT3 and GLI1/tGLI1 oncogenic transcription factors lead to gene co-activation, stem-like phenotype of breast cancer cells, and unfavorable prognosis for patients with triple-negative and HER2 breast cancers. Citation Format: Sherona R. Sirkisoon, Richard L. Carpenter, Rimkus Tadas, Anderson Ashley, Alexandria Harrison, Allison M. Lange, Guangxu Jin, Kounosuke Watabe, Hui-Wen Lo. STAT3 and GLI1/tGLI1 oncogenic transcription factors interact to promote the aggressiveness of triple-negative and HER2-enriched breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5415.

Abstract LB-091: Hedgehog signaling induces drug resistance in gastric cancer cells via upregulation of ABCG2

Abstract Gastric cancer is the third leading cause of cancer-related mortality worldwide. Currently, there are no specific therapeutic options to cure gastric cancer, and chemotherapy is frequently used. Some patients respond initially to chemotherapy but all patients eventually succumb to the disease. Thus, identifying molecular mechanisms responsible for chemotherapy resistance may help design novel strategies to sensitize cancer cells to chemotherapy. In this study, we revealed that treatment with chemotherapeutic reagents CDDP and 5Fu induced expression of GLI1, GLI2 and PTCH1 in gastric cancer cell lines, suggestive of hedgehog (Hh) signaling activation. GLI1 knockdown sensitized gastric cancer cells to CDDP and 5Fu treatment whereas ectopic GLI1 expression desensitized cancer cells to these treatments. Further analyses indicate elevated Hh signaling is associated with an increase in putative cancer stem cell marker expression, such as ABCG2. We found that GLI1 protein binds to the promoter of ABCG2 through a Gli-binding consensus site. Disruption of ABCG2 function, through ectopic expression of an ABCG2 dominant negative construct or a specific ABCG2 inhibitor, resulted in an increase of drug sensitivity both in culture and in mice. The relevance of our studies to gastric cancer patient care is reflected by our discovery that high ABCG2 expression was associated with poor survival in the gastric cancer patients who underwent chemotherapy. Taken together, we have identified a molecular mechanism by which gastric cancer cells gain chemotherapy resistance. Citation Format: BEIQIN YU, DONGSHENG GU, XIAOLI ZHANG, BINGYA LIU, JINGWU XIE. Hedgehog signaling induces drug resistance in gastric cancer cells via upregulation of ABCG2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-091.

Synergistic co-regulation and competition by a SOX9-GLI-FOXA phasic transcriptional network coordinate chondrocyte differentiation transitions.

The growth plate mediates bone growth where SOX9 and GLI factors control chondrocyte proliferation, differentiation and entry into hypertrophy. FOXA factors regulate hypertrophic chondrocyte maturation. How these factors integrate into a Gene Regulatory Network (GRN) controlling these differentiation transitions is incompletely understood. We adopted a genome-wide whole tissue approach to establish a Growth Plate Differential Gene Expression Library (GP-DGEL) for fractionated proliferating, pre-hypertrophic, early and late hypertrophic chondrocytes, as an overarching resource for discovery of pathways and disease candidates. De novo motif discovery revealed the enrichment of SOX9 and GLI binding sites in the genes preferentially expressed in proliferating and prehypertrophic chondrocytes, suggesting the potential cooperation between SOX9 and GLI proteins. We integrated the analyses of the transcriptome, SOX9, GLI1 and GLI3 ChIP-seq datasets, with functional validation by transactivation assays and mouse mutants. We identified new SOX9 targets and showed SOX9-GLI directly and cooperatively regulate many genes such as Trps1, Sox9, Sox5, Sox6, Col2a1, Ptch1, Gli1 and Gli2. Further, FOXA2 competes with SOX9 for the transactivation of target genes. The data support a model of SOX9-GLI-FOXA phasic GRN in chondrocyte development. Together, SOX9-GLI auto-regulate and cooperate to activate and repress genes in proliferating chondrocytes. Upon hypertrophy, FOXA competes with SOX9, and control toward terminal differentiation passes to FOXA, RUNX, AP1 and MEF2 factors.

Interaction between STAT3 and GLI1/tGLI1 oncogenic transcription factors promotes the aggressiveness of triple-negative breast cancers and HER2-enriched breast cancer.

STAT3, GLI1, and truncated GLI1 (tGLI1) are oncogenic transcription factors playing important roles in breast cancer. tGLI1 is a gain-of-function GLI1 isoform. Whether STAT3 physically and/or functionally interacts with GLI1/tGLI1 has not been explored. To address this knowledge gap, we analyzed 47 node-positive breast cancer specimens using immunohistochemical staining and found that p-STAT3 (Y705), GLI1, and tGLI1 are co-overexpressed in the majority of triple-negative (64%) and HER2-enriched (68%) breast carcinomas, and in lymph node metastases (65%). Using Gene Set Enrichment Analysis, we analyzed 710 breast tumors and found that STAT3- and GLI1/tGLI1-activation signatures are co-enriched in triple-negative and HER2-enriched, but not in luminal subtypes of breast cancers. Patients with high levels of STAT3 and GLI1/tGLI1 co-activation in their breast tumors had worse metastasis-free survival compared to those with low levels. Since these proteins co-overexpress in breast tumors, we examined whether they form complexes and observed that STAT3 interacted with both GLI1 and tGLI1. We further found that the STAT3-GLI1 and STAT3-tGLI1 complexes bind to both consensus GLI1- and STAT3-binding sites using chromatin immunoprecipitation (ChIP) assay, and that the co-overexpression markedly activated a promoter controlled by GLI1-binding sites. To identify genes that can be directly co-activated by STAT3 and GLI1/tGLI1, we analyzed three ChIP-Seq datasets and identified 36 potential target genes. Following validations using RT-PCR and survival analysis, we identified three genes as novel transcriptional targets of STAT3 and GLI1/tGLI1, R-Ras2, Cep70, and UPF3A. Finally, we observed that co-overexpression of STAT3 with GLI1/tGLI1 promoted the ability of breast cancer cells to form mammospheres and that STAT3 only cooperates with tGLI1 in immortalized mammary epithelial cells. In summary, our study identified novel physical and functional cooperation between two families of oncogenic transcription factors, and the interaction contributes to aggressiveness of breast cancer cells and poor prognosis of triple-negative and HER2-enriched breast cancers.

Abstract 5495: GANT61-induced cell death involves inhibition of transcription and DNA licensing at GLI sites in the promoters of GLI-dependent target genes in human colon carcinoma cells

Abstract The GLI genes, GLI1 and GLI2, are transcriptional regulators of the Hedgehog signaling response, binding at promoters to GACCACCCA-like consensus sequences. From genetic and biochemical studies, GLI2 is the primary mediator of HH signaling, which activates GLI1 to transcriptionally regulate target genes. In cancers both GLI1 and GLI2 are oncogenes, aberrantly and constitutively activated by oncogene-driven signaling pathways, in particular KRAS/BRAF in colon cancer. GANT61, a specific GLI inhibitor, induced extensive cytotoxicity in human models of colon cancer, indicating GLI to be a critical target in cancer cell survival. We have determined FOXM1 to be a transcriptional target of GLI1 at a GCCCACCCA consensus sequence. In HT29 cells, inhibition of GLI binding to the FOXM1 promoter by GANT61 led to inhibition of binding of the pause-release factors DSIF, NELF, and p-TEFb in the region of the TSS, with inhibition of binding of Pol II at both GLI and TSS sites. In R-loop regions, RNA:DNA hybrid formation was reduced at both the GLI and TSS sites in the FOXM1 promoter by GANT61, sensitive to RNaseH. Bisulfite conversion/PCR also determined >50% reduction in ssDNA (C->T conversions) in the GLI binding region in GANT61-treated cells. Data support GANT61-induced inhibition of GLI-dependent transcription at the PIC before R-loops are formed. Pretreatment of HT29 cells with α-amanitin (Pol II inhibitor) reduced GANT61-induced γH2AX foci, indicating the importance of transcription in GANT61-induced DNA damage. Co-localization of GLI1 and BrdU foci, inhibited by GANT61, indicated GLI1 and DNA replication were linked. By co-immunoprecipitation, GLI1 bound the DNA replication licensing factors ORC4, CDT1, and MCM2, decreased in the presence of GANT61. By confocal microscopy, significant co-localization of GLI1 and ORC4 foci was inhibited by GANT61, and enrichment of ORC4 occurred at the GLI binding site in the FOXM1 promoter. In addition to non-transcriptional mechanisms of interaction between GLI1 and DNA licensing factors, CDT1 was found to be a transcription target of GLI1 at a putative GACCACCCG consensus sequence in the promoter. ChIP analysis determined significant enrichment of γH2AX at this site in GANT61-treated HT29 cells. Overexpression of CDT1 in HT29 cells reduced GANT61-induced cell death and cleavage of caspase-3, indicating a significant role of DNA replication licensing in the mechanism of induction of GANT61-induced cell death. In summary, we have demonstrated inhibition of GLI-dependent transcription by GANT61 at the PIC during the initiation of RNA synthesis, and the importance of inhibiting DNA replication licensing at sites in promoter regions that bind the transcription factor GLI1 by both non-transcriptional and transcriptional mechanisms. Supported by NCI Award 1 RO1 CA183921-01A1 and by Southern Research Institute Citation Format: Ruowen Zhang, Jiahui Wu, Sylvain Ferrandon, Katie J. Glowacki, Janet A. Houghton. GANT61-induced cell death involves inhibition of transcription and DNA licensing at GLI sites in the promoters of GLI-dependent target genes in human colon carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5495. doi:10.1158/1538-7445.AM2017-5495

GLI1-mediated regulation of side population is responsible for drug resistance in gastric cancer.

Gastric cancer is the third leading cause of cancer-related mortality worldwide. Chemotherapy is frequently used for gastric cancer treatment. Most patients with advanced gastric cancer eventually succumb to the disease despite some patients responded initially to chemotherapy. Thus, identifying molecular mechanisms responsible for cancer relapse following chemotherapy will help design new ways to treat gastric cancer. In this study, we revealed that the residual cancer cells following treatment with chemotherapeutic reagent cisplatin have elevated expression of hedgehog target genes GLI1, GLI2 and PTCH1, suggestive of hedgehog signaling activation. We showed that GLI1 knockdown sensitized gastric cancer cells to CDDP whereas ectopic GLI1 expression decreased the sensitivity. Further analyses indicate elevated GLI1 expression is associated with an increase in tumor sphere formation, side population and cell surface markers for putative cancer stem cells. We have evidence to support that GLI1 is critical for maintenance of putative cancer stem cells through direct regulation of ABCG2. In fact, GLI1 protein was shown to be associated with the promoter fragment of ABCG2 through a Gli-binding consensus site in gastric cancer cells. Disruption of ABCG2 function, through ectopic expression of an ABCG2 dominant negative construct or a specific ABCG2 inhibitor, increased drug sensitivity of cancer cells both in culture and in mice. The relevance of our studies to gastric cancer patient care is reflected by our discovery that high ABCG2 expression was associated with poor survival in the gastric cancer patients who underwent chemotherapy. Taken together, we have identified a molecular mechanism by which gastric cancer cells gain chemotherapy resistance.