Glioma-associated Oncogene Research Articles

Curcumin analogue BDDD-721 exhibits more potent anticancer effects than curcumin on medulloblastoma by targeting Shh/Gli1 signaling pathway.

Medulloblastoma (MB) is a malignant tumor in the fourth ventricle of children. The clinical treatment is mainly surgical resection combined with radiotherapy and chemotherapy, but the curative effect is not ideal, and the 3-year survival rate is very low. Previous study confirmed that curcumin attenuated the proliferation of medulloblastoma both in vitro and in vivo. In present study, we found a curcumin analogue named BDDD-721, exhibited more potent anti-tumor activity than curcumin. Compared with curcumin, BDDD-721 more effectively inhibited the proliferation, migration, invasion, and increased apoptosis of medulloblastoma cells. Furthermore, BDDD-721 treatment led to activation of glioma-associated oncogene homolog (Gli), reduced expression of Shh and its downstream target Smo, Gli1 and Ptch1. In addition, SAG (Shh signaling pathway agonist) antagonized the pro-apoptotic effects of BDDD-721 on medulloblastomas as confirmed by CCK8 assays and flow cytometry; while cyclopamine (Shh signaling pathway inhibitor) enhanced its effects on medulloblastomas. In conclusion, these results indicate that curcumin analogue BDDD-721 has more potent anticancer effects than curcumin on medulloblastomas by targeting Shh/Gli1 signaling pathway.

Bone Marrow-Derived Mesenchymal Stem Cell-Derived Exosomes Containing Gli1 Alleviate Microglial Activation and Neuronal Apoptosis In Vitro and in a Mouse Parkinson Disease Model by Direct Inhibition of Sp1 Signaling.

This study investigated possible therapeutic effect mechanisms of exosomes from bone marrow-derived mesenchymal stem cells (BMSC) in neuronal and microglial cells and in a Parkinson disease (PD) model. Neuronal SH-SY5Y cells and microglial HMC3 cells were subjected to 1-methyl-4-phenylpyridinium (MPP+) or LPS, respectively. The mRNA and protein expression was assessed using qRT-PCR, Western blotting, and enzyme-linked immunosorbent assay. Cell viability and apoptosis of SH-SY5Y cells were examined using the MTT assay and flow cytometry. Chromatin immunoprecipitation assays were performed to assess the binding relationship between glioma-associated oncogene homolog 1 (Gli1) and the Sp1 transcription factor promoter. BMSC-derived exosomes promoted cell proliferation and inhibited apoptosis in MPP+-treated SH-SY5Y cells and suppressed inflammatory markers in LPS-treated HMC3 cells. Sp1 knockdown decreased SH-SY5Y cell damage and HMC3 immune activation. Gli1 carried by BMSC exosomes directly bound with Sp1 to inhibit Sp1-mediated LRRK2 activation whereas exosomes secreted by Gli1-knockdown in BMSC did not. In a PD mouse model induced with MPTP, BMSC exosomes decreased neuron loss injury and the inflammatory response by inhibiting Sp1 signaling. Thus, BMSC-derived exosomal Gli1 alleviates inflammatory damage and neuronal apoptosis by inhibiting Sp1 in vitro and in vivo. These findings provide the basis for the potential clinical use of BMSC-derived exosomes in PD.

Ursolic acid inhibits the activation of smoothened-independent non-canonical hedgehog pathway in colorectal cancer by suppressing AKT signaling cascade.

It is being brought to light that smoothened (SMO)-independent non-canonical Hedgehog signaling is associated with the pathogenesis of various cancers. Ursolic acid (UA), a pentacyclic triterpenoid present in many medicinal herbs, manifests potent effectiveness against multiple malignancies including colorectal cancer (CRC). In our previous study, UA was found to protect against CRC in vitro by suppression of canonical Hedgehog signaling cascade. Here, the influence of UA on SMO-independent non-canonical Hedgehog signaling in CRC was investigated in the present study, which demonstrated that UA hampered the proliferation and migration, induced the apoptosis of HCT-116hSMO- cells with SMO gene knockdown, accompanied by the augmented expression of the suppressor of fused (SUFU), and lessened levels of MYC (c-Myc), glioma-associated oncogene (GLI1) and Sonic Hedgehog (SHH), and lowered phosphorylation of protein kinase B (PKB, AKT), suggesting that UA diminished non-canonical Hedgehog signal transduction in CRC. In HCT-116hSMO- xenograft tumor, UA ameliorated the symptoms, impeded the growth and caused the apoptosis of CRC, with heightened SUFU expression, and abated levels of MYC, GLI1, and SHH, and mitigated phosphorylation of AKT, indicating that UA down-regulated non-canonical Hedgehog signaling cascade in CRC. Taken together, UA may alleviate CRC by suppressing AKT signaling-dependent activation of SMO-independent non-canonical Hedgehog pathway.

Abstract 2433: Targeting tGLI1 pharmacologically as a new therapeutic strategy for breast cancer brain metastases

Abstract Breast cancer is the second leading cause of brain metastases in women; patients with breast cancer brain metastasis (BCBM) survive only 6-18 months after diagnosis. Novel agents that act on targetable driver mutations (EGFR, PI3K, etc.) and penetrate the blood-brain barrier (BBB) have shown promise as systemic options for patients with distant lung and melanoma metastases. Nevertheless, brain metastases represent an area of unmet clinical need for which new pathways, novel mechanisms, and innovative therapies are needed. Cancer stem cells are thought to be a driving force behind not only distant metastasis, but also late-stage recurrence. The hedgehog pathway is an important mediator of breast cancer stem cells (BCSC); however, FDA-approved therapies targeting this pathway have demonstrated limited efficacy in breast cancer clinical trials. Despite advances made in understanding BCSC, it remains challenging to effectively target BCSC underscoring the need to identify and inhibit novel mediators of BCSC to treat BCBM. Our laboratory recently reported that truncated glioma-associated oncogene homolog 1 (tGLI1) promotes preferential breast cancer metastasis to the brain by activating BCSC and astrocytes in the tumor microenvironment (Oncogene 39:64-78, 2020). tGLI1 is an alternatively spliced GLI1 variant that functions as a tumor-specific gain-of-function transcription factor and terminal effector of the hedgehog pathway. tGLI1 knockdown abrogated BCBM, providing the rationale to therapeutically target tGLI1. In this study, we conducted cell-based chemical screens followed by validations and found that ketoconazole (KCZ), an FDA-approved antifungal, selectively targets tGLI1-expressing cancer cells, leading to suppression of BCSC in vitro and BCBM in vivo. Modification of KCZ moieties produced derivatives that retained tGLI1-selectivity against both BCSC in vitro and BCBM in vivo with increased BBB penetrance. Mechanistic studies suggest that KCZ-dependent cell kill is mediated through reducing tGLI1’s ability to bind and transactivate its target gene promoters, reducing expression of target genes Nanog, OCT4, and VEGFA. Based on these data, we opened an early Phase I clinical trial to determine if KCZ penetrates the BBB and modulates tGLI1 signaling in BCBM (NCT03796273). Preliminary analysis confirmed KCZ accumulation in collected brain biospecimens. We further investigated structure-activity relationship by synthesizing and testing 22 novel compounds, and identified two tGLI1-selective compounds that no longer inhibit CYP3A4. Since KCZ-mediated CYP3A4 antagonism can result in adrenal insufficiency and drug-drug interactions, these two novel tGLI1 inhibitors could be promising agents superior to KCZ. Collectively, these data establish tGLI1 as an actionable target for BCBM and validate KCZ and its novel derivatives as promising treatment options for patients with BCBM. Citation Format: Daniel Doheny, Sara Manore, Grace L. Wong, Dongqin Zhu, Sherona Sirkisoon, Marlyn Anguelov, Noah R. Augayo, Angelina T. Regua, Anderson Cox, Cristina Furdui, Terrence Smalley, Alexandra Thomas, Adrianna Masters, Roy E. Strowd, Hui-Wen Lo. Targeting tGLI1 pharmacologically as a new therapeutic strategy for breast cancer brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2433.

Abstract 92: tGLI1 and IL-6/IL-6R/GP130/STAT3 pathways cooperate to promote breast cancer stem cells and are co-activated in HER2-enriched and triple-negative breast cancers

Abstract Breast cancer is the most frequently diagnosed cancer and second leading cause of cancer-related deaths in women; 30% of primary breast cancer cases will develop distant metastases. The majority (90%) of breast cancer-related deaths are due to distant metastases. HER2-enriched breast cancer and triple-negative breast cancer (TNBC) have the highest propensity to metastasize and have limited to no treatment options. Metastatic HER2-enriched breast cancer and TNBC patients commonly develop therapeutic resistance and, therefore, lack efficacious therapies. Due to dismal prognoses in metastatic HER2-enriched breast cancer and TNBC patients, there remains an urgent need to identify novel molecular pathways for actionable therapeutic targets. Truncated glioma-associated oncogene homolog 1 (tGLI1), an alternatively spliced variant of GLI1, is a gain-of-function tumor-specific terminal effector in the Hedgehog pathway. tGLI1 promotes breast cancer growth and circulating tumor cells to the brain. More recently, our lab has reported tGLI1 physically interacts with Signal Transducer and Activator of Transcription 3 (STAT3); tGLI1 and STAT3 are co-overexpressed in node-positive HER2-enriched and triple-negative breast carcinomas. The Interleukin-6 (IL-6) signaling axis is a primary regulator of STAT3 and is frequently dysregulated and hyperactivated in multiple cancers, including breast cancer. IL-6 functions through a hexameric receptor complex comprised of IL-6 receptor α (IL-6R) and glycoprotein 130 (GP130) to recruit Janus tyrosine kinases (JAK). In turn, JAKs phosphorylate STAT3 resulting in homodimerization, nuclear translocation, and transcriptional activity. Independently, tGLI1 and IL-6/IL-6R/GP130/STAT3 pathways have been previously investigated as mediators for breast cancer metastases; however, whether they cooperate to promote breast cancer progression remains unknown. Herein, we report tGLI1 and IL-6/IL-6R/GP130/STAT3 pathways to be frequently co-expressed and co-activated in HER2-enriched breast cancer and TNBC tumors and that the co-activation is associated with a shortened metastasis-free survival (MFS). Additionally, when stratified by metastatic site, breast tumors with high tGLI1 and IL-6/IL-6R/GP130/STAT3 co-activation are associated with a worse bone, brain, and lung MFS compared to tumors with low co-activation. Ectopic co-overexpression of tGLI1 and GP130 upregulates mRNA expression of stemness genes, Nanog, Sox2, and Oct4, leading to the subsequent promotion of the breast cancer stem cell (CSC) subpopulation in vitro. Taken together, our findings revealed a novel crosstalk between tGLI1 and the IL-6/IL-6R/GP130/STAT3 signaling axis to enhance the CSC subpopulation, suggesting the importance of the crosstalk in mediating metastasis of HER2-enriched breast cancer and TNBC. Citation Format: Sara G. Manore, Daniel L. Doheny, Dongqin Zhu, Grace L. Wong, Angelina T. Regua, Austin Arrigo, Calvin J. Wagner, Mariana Najjar, Hui-Wen Lo. tGLI1 and IL-6/IL-6R/GP130/STAT3 pathways cooperate to promote breast cancer stem cells and are co-activated in HER2-enriched and triple-negative breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 92.

Abstract 2354: Astrocytes activated by tGLI1-expressing breast cancer brain metastases upregulate CNTF to activate astrocytes and promote breast cancer stem cells

Abstract Breast cancer is the most frequently diagnosed cancer, and second most common source of brain metastasis in women. Despite recent advances in detection and therapeutics, patients with breast cancer brain metastases (BCBM) survive only 6-18 months after diagnosis. HER2-enriched breast cancer and triple-negative breast cancer (TNBC) subtypes have the highest propensity to metastasize to the brain. Mechanisms that drive BCBM remain unclear, contributing to limited effective treatments and poor prognoses for HER2-enriched breast cancer and TNBC patients. Truncated glioma-associated oncogene homolog 1 (tGLI1), a gain-of-function GLI1 transcription factor discovered in our lab, promotes BCBM of circulating tumor cells in vivo. We previously reported that tGLI1 enriches the breast cancer stem cell (BCSC) subpopulation, and that tGLI1-positive BCSCs strongly activate astrocytes, the most abundant glial cell type in the brain. The mechanisms by which tGLI1-activated astrocytes promote BCBM have not been investigated. Since tumor-associated astrocytes have been reported to secrete specific cytokines, such as ciliary neurotrophic factor (CNTF), to activate astrocytes and cancer cells by binding to IL-6R and CNTF receptor-alpha (CNTFRα), we determined whether astrocytes activated by tGLI1-positive breast cancer cells secrete elevated levels of CNTF. Results of CNTF ELISA revealed that conditioned media from tGLI1-positive breast cancer cells increases the ability of astrocytes to secrete CNTF compared to GLI1-positive or control cells. Furthermore, we found that exogenous CNTF significantly activates astrocytes and promotes BCSCs, important mediators of tumor progression and metastasis in breast cancer. Analysis of patient datasets reveals CNTFRα is more highly expressed in brain metastases compared to breast cancer and normal breast tissues, suggesting that CNTF secreted by activated astrocytes can promote the growth of BCSCs in the brain. We further found that CNTF and CNTFRα gene signatures are significantly associated with worse brain metastasis-free survival, suggesting an important role for the tGLI1-CNTF-CNTFRα pathway in BCBM development. In summary, our study demonstrates for the first time that astrocytes activated by tGLI1-expressing BCBM secrete CNTF, CNTF further activates astrocytes and promotes BCSCs, and that this tumor-astrocyte interaction could be a novel mechanism for BCBM development and progression. Citation Format: Grace L. Wong, Sherona R. Sirkisoon, Noah R. Aguayo, Daniel L. Doheny, Dongqin Zhu, Angelina T. Regua, Austin Arrigo, Sara G. Manore, Calvin J. Wagner, Alexandra Thomas, Ravi Singh, Fei Xing, Guangxu Jin, Kounosuke Watabe, Hui-Wen Lo. Astrocytes activated by tGLI1-expressing breast cancer brain metastases upregulate CNTF to activate astrocytes and promote breast cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2354.

Gli1 promotes epithelial-mesenchymal transition and metastasis of non-small cell lung carcinoma by regulating snail transcriptional activity and stability.

Metastasis is crucial for the mortality of non-small cell lung carcinoma (NSCLC) patients. The epithelial–mesenchymal transition (EMT) plays a critical role in regulating tumor metastasis. Glioma-associated oncogene 1 (Gli1) is aberrantly active in a series of tumor tissues. However, the molecular regulatory relationships between Gli1 and NSCLC metastasis have not yet been identified. Herein, we reported Gli1 promoted NSCLC metastasis. High Gli1 expression was associated with poor survival of NSCLC patients. Ectopic expression of Gli1 in low metastatic A549 and NCI-H460 cells enhanced their migration, invasion abilities and facilitated EMT process, whereas knock-down of Gli1 in high metastatic NCI-H1299 and NCI-H1703 cells showed an opposite effect. Notably, Gli1 overexpression accelerated the lung and liver metastasis of NSCLC in the intravenously injected metastasis model. Further research showed that Gli1 positively regulated Snail expression by binding to its promoter and enhancing its protein stability, thereby facilitating the migration, invasion and EMT of NSCLC. In addition, administration of GANT-61, a Gli1 inhibitor, obviously suppressed the metastasis of NSCLC. Collectively, our study reveals that Gli1 is a critical regulator for NSCLC metastasis and suggests that targeting Gli1 is a prospective therapy strategy for metastatic NSCLC.

Inhibition of Gli1 suppressed hyperglycemia-induced meibomian gland dysfunction by promoting pparγ expression

Diabetes is one of the risk factors for meibomian gland dysfunction (MGD); however, the underlying molecular mechanism remains unknown. The current study aims to examine the effects of glioma-associated oncogene homolog 1 (Gli1), a transcription factor of the sonic hedgehog (Shh) pathway, in the modulation of diabetic-related MGD. Here, using RNA sequencing and qRT-PCR, we examined the mRNA changes of Shh pathway involving genes. mRNA sequencing analysis showed that the Shh pathway involving genes Shh and Gli1 were markedly upregulated in diabetic MG, and qRT-PCR detection of Shh pathway-associated genes found that Gli1 expression increased most significantly. Contrary to the elevation of Gli1 level, the expression of pparγ was downregulated in diabetic MG and in high glucose treated organotypic cultured mouse MG. GANT61, an inhibitor of Gli1, effectively inhibited the reduction of pparγ expression and lipid accumulation induced by high glucose, which was suppressed by pparγ inhibitor T0070907. We further demonstrated that advanced glycation end products (AGEs) treatment also promoted the expression of Gli1 and pparγ in organotypic cultured mouse MG. AGEs inhibitor Aminoguanidine suppressed high glucose caused Gli1 upregulation in organotypic cultured mouse MG. These results suggest that suppression of Gli1 may be a potentially useful therapeutic option for diabetic-related MGD.

Gliko BMSC: A potential strategy of treatment for renal fibrosis.

ObjectiveThere are many researches on using bone marrow mesenchymal stem cells (BMSCs) in the treatment of acute kidney injury (AKI), which has certain effects, but the mechanism of action is still unclear. Previous researches show that glioma-associated oncogene homolog 1 (Gli 1) can promote the proliferation and migration of cells, which can also promote renal fibrosis. Therefore, we investigate the influence of Gli-regulated BMSCs on repairing AKI and renal fibrosis induced by limb Ischemia-Reperfusion (I/R).MethodsThe Crispr-Cas9 technique was adopted to knock out the Gli1 gene from the mouse BMSCs according to green fluorescent tracing, and the BMSCs (BMSCs-Gliko) with Gli1 gene knocked out and the BMSCs as control group were obtained. The cell proliferation, apoptosis, cycle and SHH signal pathway gene level were tested. The mice were built to the AKI model with inducing I/R injury, then the BMSCs-Gliko and BMSCs cells were injected into the mice, and their IL-1, IL-1B, TNF-a, serum creatinine (Scr) and blood urea nitrogen (BUN) levels were tested; Western blot was employed to test the expression of α-SMA, SMAD2 and SMAD4 in the renal tissues of mice. Finally, flow cytometry was used to test the content of BMSCs containing green fluorescence in the blood of mice.ResultsThe BMSCs-Gliko containing green fluorescence and the mouse AKI model were built; both BMSCs and BMSCs-Gliko can reduce the damage level, and BMSCs-Gliko outperformed BMSCs in protecting renal tubules and anti-fibrosis. Our study also shows that BMSCs-Gliko stayed longer in the blood of mice, which might also be one of the reasons why BMSCs-Gliko outperformed BMSCs in preventing renal tubules and fibrosis. To sum it up, could be key target of using.

GLI1 activates pro-fibrotic pathways in myelofibrosis fibrocytes

Bone marrow (BM) fibrosis was thought to be induced exclusively by mesenchymal stromal cells (MSCs). However, we and others found that neoplastic fibrocytes induce BM fibrosis in myelofibrosis (MF). Because glioma-associated oncogene-1 (GLI1), an effector of the Hedgehog pathway, plays a role in the induction of BM fibrosis, we wondered whether GLI1 affects fibrocyte-induced BM fibrosis in MF. Multiplexed fluorescence immunohistochemistry analysis of MF patients’ BM detected high levels of GLI1 in MF fibrocytes compared to MSCs or normal fibrocytes. Immunostaining, RNA in situ hybridization, gene expression analysis, and western immunoblotting detected high levels of GLI1 and GLI1-induced matrix metalloproteases (MMP) 2 and 9 in MF patients BM-derived cultured fibrocytes. Similarly, MF patients’ BM-derived GLI1+ fibrocytes were found in BMs and spleens of MF xenograft mice. GLI1 silencing reduced the levels of MMP2/9, phosphorylated SMAD2/3, and procollagen-I, and knockdown or inhibition of GLI1 decreased fibrocyte formation and induced apoptosis of both fibrocytes and fibrocyte progenitors. Because Janus kinase (JAK)2-induced STAT3 is constitutively activated in MF and because STAT3 induces GLI1 expression, we sought to determine whether STAT3 activates GLI1 in MF fibrocytes. Imaging analysis detected phosphotyrosine STAT3 in MF patients’ BM fibrocytes, and transfection of fibrocytes with STAT3-siRNA or treatment with a JAK1/2 inhibitor ruxolitinib reduced GLI1 and MMP2/9 levels. Chromatin immunoprecipitation and a luciferase assay revealed that STAT3 induced the expression of the GLI1 gene in both MF BM fibrocytes and fibrocyte progenitors. Together, our data suggest that STAT3-activated GLI1 contributes to the induction of BM fibrosis in MF.

GANT61 suppresses cell survival, invasion and epithelial-mesenchymal transition through inactivating AKT/mTOR and JAK/STAT3 pathways in anaplastic thyroid carcinoma

ABSTRACT Glioma-associated oncogene (Gli) antagonist-61 (GANT61) not only suppresses the malignant behavior of several cancers but also presents synergistic effects with other anticancer agents on suppressing the progression of cancers, while relevant information is rare in anaplastic thyroid carcinoma (ATC). This study aimed to explore the therapeutic effect of GANT61 in ATC and its molecular mechanism. ATC cells (8505C and CAL-62) were treated with GANT61, followed by detection of cell proliferation, apoptosis, invasion and epithelial-mesenchymal transition (EMT) markers. Subsequently, RNA sequencing was performed to explore the potential downstream pathway. Following that, rescue experiments were conducted by SC79 (AKT activator) or colivelin (STAT3 activator) monotreatment or combined with GANT61 in ATC cells. GANT61 reduced Gli1 expression, suppressed proliferation at several time settings, promoted apoptosis, inhibited invasion and increased E-cadherin while decreased Vimentin and Snail expressions (EMT markers) in ATC cells. The subsequent RNA sequence identified 85 upregulated differentially expressed genes (DEGs) and 71 downregulated DEGs in GANT61-treated ATC cells, which were mainly enriched in PI3K/AKT, JAK/STAT, Hedgehog and mTOR pathways. Next, the inactivation of AKT/mTOR and JAK/STAT3 pathways by GANT61 treatment was verified by western blot. The following rescue experiments showed that SC79 or colivelin treatment promoted the malignant behaviors of ATC cells. More importantly, SC79 or colivelin treatment compensated the effect of GANT61 treatment on cell proliferation at several time settings and apoptosis, invasion, and part of that on EMT in ATC cells. GANT61 suppresses cell survival, invasion and EMT through inactivating AKT/mTOR or JAK/STAT3 pathways in ATC.

Deleterious Rare Mutations of GLI1 Dysregulate Sonic Hedgehog Signaling in Human Congenital Heart Disease.

The Glioma-associated oncogene (Gli) family members of zinc finger DNA-binding proteins are core effectors of Sonic hedgehog (SHH) signaling pathway. Studies in model organisms have identified that the Gli genes play critical roles during organ development, including the heart, brain, kidneys, etc. Deleterious mutations in GLI genes have previously been revealed in several human developmental disorders, but few in congenital heart disease (CHD). In this study, the mutations in GLI1-3 genes were captured by next generation sequencing in human cohorts composed of 412 individuals with CHD and 213 ethnically matched normal controls. A total of 20 patient-specific nonsynonymous rare mutations in coding regions of human GLI1-3 genes were identified. Functional analyses showed that GLI1 c.820G> T (p.G274C) is a gain-of-function mutation, while GLI1 c.878G>A (p.R293H) and c.1442T>A (p.L481X) are loss-of-function mutations. Our findings suggested that deleterious rare mutations in GLI1 gene broke the balance of the SHH signaling pathway regulation and may constitute a great contribution to human CHD, which shed new light on understanding genetic mechanism of embryo cardiogenesis regulated by SHH signaling.

Expression and significance of GLI1 and Shh in the malignant transformation of ovarian endometriosis

Objective: To investigate the expression levels and clinical significance of glioma-associated oncogene homolog 1 (GLI1) and sonic hedgehog signaling molecule (Shh) in the malignant transformation of ovarian endometriosis (EM). Methods: The expressions of GLI1 and Shh were detected by real-time reverse transcription (RT)-polymerase chain reaction (PCR) and EnVision method in 50 cases of ovarian EM tissues, 35 cases of atypical endometriosis (aEM) and 50 cases of endometriosis-associated ovarian cancer (EAOC). The expression differences of two molecular markers in the malignant transformation of ovarian EM were compared, and the relationships between two molecular markers and the clinicopathological features and prognosis of EAOC were analyzed. Results: (1) RT-PCR showed that the expression levels of GLI1 mRNA in EM, aEM and EAOC group were 1.77±0.40, 3.54±0.44, and 7.80±0.24, respectively. The expression levels of Shh mRNA were 0.95±0.21, 3.14±0.35, and 5.41±0.31, respectively. GLI1 and Shh mRNA in EAOC group were significantly higher than those in EM and aEM group (all P<0.01), and there were statistically significant differences between EM and aEM group (all P<0.01). The percentages of GLI1 in ovarian EM, aEM and EAOC were 32% (16/50), 57% (20/35), and 66% (33/50), respectively, meanwhile, the positive expression rates of Shh were 20% (10/50), 49% (17/35), and 54% (27/50), respectively (all P<0.01). GLI1 mRNA expression was positively correlated with Shh mRNA expression in EAOC tissues (r=0.721, P<0.01). The expressions of GLI1 protein were proportionated to Shh protein in EAOC tissues (r=0.608,P=0.001). (2) The expression of GLI1 was significantly related to the International Federation of Gynecology and Obstetrics (FIGO) stage, cancer antigen 125 (CA125) levels, lymph node metastasis, and Platinum resistance in EAOC patients (all P<0.05). The expression of Shh were related to FIGO stage and lymph node metastasis in EAOC patients (all P<0.05). Logistic regression analysis showed that GLI1 expression was an independent risk factor for poor prognosis in EAOC patients (P<0.05). Kaplan-meier survival analysis showed that the overall survival rate of EAOC patients with high GLI1 expression and low GLI1 expression was 12.1% and 35.3%, respectively, with statistical significance (χ²=10.73, P<0.01). The overall survival rate of EAOC patients with high and low expression of Shh protein was 11.1% and 30.4%, in which there was statistically significant difference (χ²=3.96, P=0.047). Conclusion: GLI1 and Shh are highly associated with the malignant transformation of ovarian EM, which may play a role in promoting malignant degeneration of ovarian EM, and the high expression of GLI1 and Shh indicates a poor prognosis in EAOC patients.

A Critical Function for the Transcription Factors GLI1 and GLI2 in the Proliferation and Survival of Human Mast Cells

Mast cell hyperactivity and accumulation in tissues are associated with allergy and other mast cell-related disorders. However, the molecular pathways regulating mast cell survival in homeostasis and disease are not completely understood. As glioma-associated oncogene (GLI) proteins are involved in both tissue homeostasis and in the hematopoietic system by regulating cell fate decisions, we sought to investigate the role for GLI proteins in the control of proliferation and survival of human mast cells. GLI1 transcripts were present in primary human mast cells and mast cell lines harboring or not activating mutations in the tyrosine kinase receptor KIT (HMC-1.1 and HMC-1.2, and LAD2 cells, respectively), while GLI2 transcripts were only present in HMC-1.1 and HMC-1.2 cells, suggesting a role for oncogenic KIT signaling in the regulation of GLI2. Reduction in GLI activity by small molecule inhibitors, or by shRNA-mediated knockdown of GLI1 or GLI2, led to increases in apoptotic cell death in both cultured human and murine mast cells, and reduced the number of peritoneal mast cells in mice. Although GLI proteins are typically activated via the hedgehog pathway, steady-state activation of GLI in mast cells occurred primarily via non-canonical pathways. Apoptosis induced by GLI silencing was associated with a downregulation in the expression of KIT and of genes that influence p53 stability and function including USP48, which promotes p53 degradation; and iASPP, which inhibits p53-induced transcription, thus leading to the induction of p53-regulated apoptotic genes. Furthermore, we found that GLI silencing inhibited the proliferation of neoplastic mast cell lines, an effect that was more pronounced in rapidly growing cells. Our findings support the conclusion that GLI1/2 transcription factors are critical regulators of mast cell survival and that their inhibition leads to a significant reduction in the number of mast cells in vitro and in vivo, even in cells with constitutively active KIT variants. This knowledge can potentially be applicable to reducing mast cell burden in mast cell-related diseases.

Saikosaponin B1 and Saikosaponin D inhibit tumor growth in medulloblastoma allograft mice via inhibiting the Hedgehog signaling pathway.

Medulloblastoma (MB), accounting for nearly 10% of all childhood brain tumors, are implicated with aberrant activation of the Hedgehog (Hh) signaling pathway. Saikosaponin B1 (SSB1) and Saikosaponin D (SSD), two bioactive constituents of Radix Bupleuri, are reported to have many biological activities including anticancer activities. In our work, we evaluated the inhibition of SSB1 and SSD on MB tumor growth in allograft mice and explored the underlying mechanisms. The associated biological activity was investigated in Shh Light II cells, an Hh-responsive fibroblast cell line, using the Dual-Glo® Luciferase Assay System. First, SSB1 (IC50, 241.8nM) and SSD (IC50, 168.7nM) inhibited GLI-luciferase activity in Shh Light II cells stimulated with ShhN CM, as well as Gli1 and Ptch1 mRNA expression. In addition, both compounds suppressed the Hh signaling activity provoked by smoothened agonist (SAG) or excessive Smoothened (SMO) expression. Meanwhile, SSB1 and SSD did not inhibit glioma-associated oncogene homolog (GLI) luciferase activity activated by abnormal expression of downstream molecules, suppressor of fuse (SUFU) knockdown or GLI2 overexpression. Consequently, SSB1 (30mg/kg, ip) and SSD (10mg/kg, ip) displayed excellent in vivo inhibitory activity in MB allografts, and the tumor growth inhibition ratios were approximately 50% and 70%, respectively. Our findings, thus, identify SSB1 and SSD significantly inhibit tumor growth in MB models by inhibiting the Hedgehog pathway through targeting SMO.

Sequential stabilization of RNF220 by RLIM and ZC4H2 during cerebellum development and Shh-group medulloblastoma progression.

ABSTRACTSonic hedgehog (Shh) signaling is essential for the proliferation of cerebellar granule neuron progenitors (CGNPs), and its misregulation is linked to various disorders, including cerebellar cancer medulloblastoma (MB). During vertebrate neural development, RNF220, a ubiquitin E3 ligase, is involved in spinal cord patterning by modulating the subcellular location of glioma-associated oncogene homologs (Glis) through ubiquitination. RNF220 is also required for full activation of Shh signaling during cerebellum development in an epigenetic manner through targeting embryonic ectoderm development. ZC4H2 was reported to be involved in spinal cord patterning by acting as an RNF220 stabilizer. Here, we provided evidence to show that ZC4H2 is also required for full activation of Shh signaling in CGNP and MB progression by stabilizing RNF220. In addition, we found that the ubiquitin E3 ligase RING finger LIM domain-binding protein (RLIM) is responsible for ZC4H2 stabilization via direct ubiquitination, through which RNF220 is also thus stabilized. RLIM is a direct target of Shh signaling and is also required for full activation of Shh signaling in CGNP and MB cell proliferation. We further provided clinical evidence to show that the RLIM‒ZC4H2‒RNF220 cascade is involved in Shh-group MB progression. Disease-causative human RLIM and ZC4H2 mutations affect their interaction and regulation. Therefore, our study sheds light on the regulation of Shh signaling during cerebellar development and MB progression and provides insights into neural disorders caused by RLIM or ZC4H2 mutations.

A novel promoter-associated non-coding small RNA paGLI1 recruits FUS/P65 to transactivate GLI1 gene expression and promotes infiltrating glioma progression

Glioma-associated oncogene homolog 1 (GLI1) is a core component of the Hedgehog (HH) signalling pathway and is a transcription activator of numerous oncogenes, such as SOX9, VEGFA, BCL2, and CDK2. The complex regulation of GLI1 involves numerous pathways and molecules, including HH-dependent and independent, epigenetic and post-transcriptional mechanisms. Here, we report the discovery, characterization and function of a novel sense promoter-associated ncRNA, paGLI1 that is overexpressed in infiltrating glioma. We show that paGLI1 promotes GLI1 gene transcription through binding to and recruitment of the transcription factor complex FUS/P65 by interacting with paGLI1 DNA sequence. This interaction facilitates FUS/P65 binding to the GLI1 promoter to activate GLI1 transcription and hence its downstream oncogenes, which results in enhancement of glioma cell proliferation and invasiveness. Importantly, over-expression of paGLI1 is a significant unfavorable prognosticator for both disease-specific and progression-free survival in glioma patients, with relative risks being 2.932 (95% confidence interval: 1.280 to 6.713) (P < 0.05) and 2.284 (95% confidence interval: 1.051 to 4.966) (P < 0.05), respectively. The novel paGLI1/FUS/P65 regulatory mechanisms play important roles in infiltrating glioma progression and may serve as potential targets for future therapeutics.

Suppressive GLI2 fragment enhances liver metastasis in colorectal cancer.

Linoleic acid (LA) has been shown to cause inflammation and promote development of colorectal cancer (CRC). Moreover, many literatures show that LA is associated with cancer metastasis. Metastatic cancer cells have high stemness, suggesting that LA might affect the stemness of cancer cells. In this study, we examined the effect of LA on the hedgehog system, which affects cancer stemness. In CT26 cells, LA treatment induced the expression of sonic hedgehog (Shh); the signal transduction factor, and glioma-associated oncogene homolog (Gli) 2, whereas the expression of SRY-box transcription factor (Sox) 17 was suppressed. Furthermore, LA reduced GLI2 ubiquitination, resulting in an increase in the N-terminal fragment of GLI2, known as suppressive GLI2, produced by cleavage of GLI2. LA-induced cleaved GLI2 was also detected in Colo320 and HT29 human CRC cells. Knocking down Gli2 abrogated the LA-mediated suppression of Sox17 expression. These results suggest that LA promotes tumor cell stemness by increasing of suppressive GLI2 fragments via GLI2 modification. In mouse liver metastasis models, LA enhanced metastasis with production of the suppressive GLI2 fragments in CT26 and HT29 cells, whereas knockdown of GLI2 abrogated LA-induced metastatic activity. In human CRCs, the cases with liver metastasis showed the suppressive GLI2 fragments. This study provides mechanistic insights into LA-induced stemness in colon cancer cells. This finding suggests that dietary intake of LA might increase the stemness of cancer cells and enhance metastatic activity of the cancer.

Glycolytic Metabolic Remodeling by the Truncate of Glioma-Associated Oncogene Homolog 1 in Triple-Negative Breast Cancer Cells.

Hedgehog (Hh) signaling pathway plays an essential role in embryonic development, tissue regeneration, and stem cell renewal. In particular, terminal effectors of the Hh signaling pathway are associated with the regulation of glioma-associated oncogene homolog 1 (GLI1) transcription factors. Overexpression of GLI1 is closely associated with poor prognosis in breast cancer. The Hh-GLI1 signaling pathway is activated and participates in the tumorigenesis and progression of breast cancer, especially in the aggressive subtype of triple-negative breast cancer (TNBC). However, the role of GLI1 in regulating TNBC metabolism remains unclear. This study aimed to explore the functional role of GLI1 in glycolytic metabolism in TNBC. Immunohistochemical analysis of GLI1 expression in a tissue microarray revealed significant correlations between GLI1 expression and advanced tumor stage and grade. GLI1 expression levels were drastically increased in MDA-MB-231 cells compared to those in other cell lines. Inhibition of GLI1 expression using GLI1 small interfering RNA (siRNA) in MDA-MB-231 cells resulted in a significant reduction in cell proliferation and induced cell cycle arrest at the G1 phase. Furthermore, GLI1 downregulation significantly reduced the expression of glycolysis-regulated proteins. GLI1 knockdown resulted in reduced glycolytic rates and extracellular lactate levels. Moreover, metabolic stress after GLI1 knockdown activated the energy sensor, adenosine monophosphate-activated protein kinase, which subsequently resulted in autophagy induction. In conclusion, this study indicates that targeting GLI1 reprograms the tumor glucose metabolism to suppress breast cancer cell growth and proliferation.

The Role of Indian Hedgehog Signaling in Tendon Response to Subacromial Impingement: Evaluation Using a Mouse Model

Background: The underlying cellular and molecular mechanisms involved in the development of tendinopathy due to subacromial supraspinatus tendon (SST) impingement and the response to subsequent removal of impingement remain unknown. Purpose: To investigate the involvement of Indian hedgehog (IHH) signaling in the development of SST tendinopathy and the subsequent healing process after the relief of subacromial impingement in a novel mouse shoulder impingement model. Study Design: Controlled laboratory study. Methods: A total of 48 male wild-type C57BL/6 mice were used in this study. Supraspinatus tendinopathy was induced by inserting a microsurgical clip into the subacromial space bilaterally. Eleven mice were sacrificed at 4 weeks after surgery to establish impingement baseline; 24 mice underwent clip removal at 4 weeks after surgery and then were euthanized at 2 or 4 weeks after clip removal. Thirteen mice without surgical intervention were utilized as the control group. All SSTs were evaluated with biomechanical testing; quantitative histomorphometry after staining with hematoxylin and eosin, Alcian blue, and picrosirius red; and immunohistochemical staining (factor VIII, IHH, Patched1 [PTCH1], and glioma-associated oncogene homolog 1 [GLI1]). Results: The mean failure force and stiffness in the 4-week impingement group decreased significantly compared with the control group (P < .001) and gradually increased at 2 and 4 weeks after clip removal. Histological analysis demonstrated increased cellularity and disorganized collagen fibers in the SST, with higher modified Bonar scores at 4 weeks, followed by gradual improvement after clip removal. The IHH-positive area and PTCH1- and GLI1-positive cell percentages significantly increased after 4 weeks of clip impingement (20.64% vs 2.06%, P < .001; 53.9% vs 28.03%, P = .016; and 30% vs 12.19%, P = .036, respectively) and continuously increased after clip removal. Conclusion: The authors’ findings suggest that the hedgehog signaling pathway and its downstream signaling mediator and target GLI1 may play a role in the development and healing process of rotator cuff tendinopathy due to extrinsic rotator cuff impingement. Clinical Relevance: This study suggests the potential for the hedgehog pathway, together with its downstream targets, as candidates for further study as potential therapeutic targets in the treatment of supraspinatus tendinopathy.