Glioma-associated Oncogene Homolog Research Articles

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.

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.

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.

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.

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.

Salvianolic acid B inhibits the progression of liver fibrosis in rats via modulation of the Hedgehog signaling pathway.

Salvianolic acid B (Sal B) has previously reported anti-hepatic fibrosis effects, though it is not clear if it can inhibit hepatic fibrosis by regulating the hedgehog (Hh) signaling pathway. The aim of the present study was to explore the roles and mechanism of Sal B in preventing and treating liver fibrosis in rats. The study also aimed to determine the role of the Hh signaling pathway in this process. A rat model of liver fibrosis was induced through the subcutaneous injection of 50% carbon tetrachloride, followed by treatment with Sal B. After gavage, blood was collected to detect serum markers of liver injury. The degree of liver fibrosis and tissue damage was assessed using histopathological analysis. Western blotting and reverse transcription-quantitative PCR were used to detect the expression levels of TGF-β1 and Hh signaling pathway-related genes, including Sonic hedgehog (Shh) protein, membrane protein receptor protein patched homolog 1 (Ptch1), membrane protein receptor Smoothened (Smo) and transcription factor glioma-associated oncogene homolog 1 (Gli1). Serum alanine aminotransferase, aspartate aminotransferase and total bilirubin levels were decreased, whilst levels of albumin were increased in rats with liver fibrosis that were treated with Sal B (P<0.05). Additionally, significant increases in TGF-β1, Shh, Ptch1, Smo, Gli1 and α-smooth muscle actin expression levels were observed in the liver tissues of rats with hepatic fibrosis (P<0.05). However, Sal B treatment significantly reduced the expression levels of these proteins (P<0.05). In conclusion, the results of the present study suggested that the Hh signaling pathway may be activated during the process of rat liver fibrosis. Thus, Sal B may exert its anti-hepatic fibrosis effects, at least in part, by inhibiting the activation of the Hh signaling pathway.

G-protein coupled receptor Smo positively regulates proliferation and migration of adult neural stem cells in vitro

To investigate the role of G-protein coupled receptor Smoothened (Smo) in regulating proliferation and migration of adult neural stem cells (ANSCs) and explore the underlying mechanism. Cultured ANSCs were treated with purmorphamine (PM, an agonist of Smo) or cyclopamine (CPM, an inhibitor of Smo), and the changes in cell proliferation migration abilities were assessed using cell counting kit-8 (CCK8) assay and wound healing assay, respectively. The mRNA expressions of membrane receptor Patched 1 (Ptch1), Smo, glioma-associated oncogene homolog 1 (Gli1), axon guidance cue slit1 (Slit1) and brain-derived neurotrophic factor (BDNF) in the treated cells were detected using real-time quantitative PCR (RT-PCR). PM significantly promoted the proliferation (P < 0.01) and migration of ANSCs (P < 0.01), and up-regulated the mRNA expressions of Ptch1, Smo, Gli1, Slit1 and BDNF. Treatment with CPM significantly inhibited the proliferation and migration of ANSCs. Modulating Smo activity can positively regulate the proliferation and migration of ANSCs possibly by regulating the expressions of BDNF and Slit1.

Silencing GS Homeobox 2 Alleviates Gemcitabine Resistance in Pancreatic Cancer Cells by Activating SHH/GLI1 Signaling Pathway.

Sonic hedgehog (SHH) signaling pathway and glioma-associated oncogene homolog 1 (GLI1) play important roles in the initiation and progression of pancreatic ductal adenocarcinoma (PDAC). GS homeobox 2 (GSX2, formerly GSH2) is a downstream target of SHH signaling, but its role in pancreatic cancer remains unclear. This study evaluates the role of GSH2 in the development and drug resistance of pancreatic cancer. Both cell culture and xenograft mouse model were used. Immunohistochemistry, Western blotting and quantitative RT-PCR were used to examine the expression of GSH2 and other related molecules. CCK8 assay was used to test the cell proliferation, and flow cytometry used to examine cell apoptosis upon gemcitabine treatment. It was found that GSH2 is overexpressed in human pancreatic cancer tissues and cells. The expression of SHH and GLI1 was reversely correlated with GSH2 in pancreatic cancer cells. SHH and GLI1 have protein-protein interactions with GSH2. GSH2 silencing in pancreatic cancer cells inhibited cell proliferation, migration and invasion, increased cell apoptosis and sensitized pancreatic cancer cells to gemcitabine treatment. Furthermore, in vivo study demonstrated that silencing GSH2 increased the efficacy of gemcitabine-based treatment. Our results indicate that GSH2 is overexpressed in pancreatic cancer. GSH2 silencing in pancreatic cancer alleviates gemcitabine resistance by activating SHH/GLI1 pathway. Thus, targeting GSH2 in PDAC could be a novel cancer therapeutic strategy.

Predictive factors for the recurrence of surgically excised basal cell carcinomas: A retrospective clinical and immunopathological pilot study.

Basal cell carcinoma (BCC) is the most frequent form of skin cancer and is not a tumor with a lethal outcome if diagnosed and treated adequately. The gold standard for treatment is surgical excision with histologically safe margins. Even so, tumors excised with free margins may recur after a period of time. The identification of predictive factors for the recurrence of BCCs besides the localization, size and aggressive histology may be useful for the clinician. The aim of the present study was to identify clinical and pathological factors associated with recurrence in tumors with histologically free margins and assess via immunohistochemical staining, the expression of glioma-associated oncogene homolog 1 (GLI1), yes-associated protein (YAP), connective tissue growth factor (CTGF) and E-cadherin as they are involved in the development of BCCs, in the hope of identifying markers that are predictive for recurrence. In total, 8 recurrent BCCs and 38 non-recurrent tumors were analyzed. A Breslow index >2 (Se 100.0%, Sp 67.5%, P=0.008), Clark level >3 (Se 100.0%, Sp 47.5%, P<0.001), and excision margins both lateral (Se 87.5%, Sp 60.0%, P=0.04) and deep (Se 75.0%, Sp 82.5%, P<0.001) free from tumoral cells ≤1 mm proved to be predictive for recurrence in the present study. Recurrence may appear even after more than 3 years since the initial excision (Se 87.50%, Sp 70.0%, P<0.001). The expression levels of GLI1, YAP and E-cadherin were not different in the recurrent vs. non-recurrent BCCs. However, the low expression of CTGF may indicate a tumor with a higher aggressiveness. In conclusion, close follow-up of patients with excised BCCs at least annually is recommended and re-excision should be taken into consideration for locally advanced tumors especially if they are located in high-risk areas or those with histologically free margins <1 mm.

The Role of Smoothened-Dependent and -Independent Hedgehog Signaling Pathway in Tumorigenesis.

The Hedgehog (Hh)-glioma-associated oncogene homolog (GLI) signaling pathway is highly conserved among mammals, with crucial roles in regulating embryonic development as well as in cancer initiation and progression. The GLI transcription factors (GLI1, GLI2, and GLI3) are effectors of the Hh pathway and are regulated via Smoothened (SMO)-dependent and SMO-independent mechanisms. The SMO-dependent route involves the common Hh-PTCH-SMO axis, and mutations or transcriptional and epigenetic dysregulation at these levels lead to the constitutive activation of GLI transcription factors. Conversely, the SMO-independent route involves the SMO bypass regulation of GLI transcription factors by external signaling pathways and their interacting proteins or by epigenetic and transcriptional regulation of GLI transcription factors expression. Both routes of GLI activation, when dysregulated, have been heavily implicated in tumorigenesis of many known cancers, making them important targets for cancer treatment. Hence, this review describes the various SMO-dependent and SMO-independent routes of GLI regulation in the tumorigenesis of multiple cancers in order to provide a holistic view of the paradigms of hedgehog signaling networks involving GLI regulation. An in-depth understanding of the complex interplay between GLI and various signaling elements could help inspire new therapeutic breakthroughs for the treatment of Hh-GLI-dependent cancers in the future. Lastly, we have presented an up-to-date summary of the latest findings concerning the use of Hh inhibitors in clinical developmental studies and discussed the challenges, perspectives, and possible directions regarding the use of SMO/GLI inhibitors in clinical settings.

Repair of spinal cord injury in rats via exosomes from bone mesenchymal stem cells requires sonic hedgehog.

ObjectiveThe loss of neural ability leading to subsequent diminishing of motor function and the impairment below the location of the injury is a result of the SCI (Spinal Cord Injury). Among the many therapeutic agents for SCI, the exosomes considered as extracellular vesicles seem to be the most promising. Sonic Hedgehog (Shh) is an exosome-carrying protein. This Study's purpose was to identify whether Shh is required for exosomes from BMSCs (mesenchymal stem cells of the bone) and plays a protective effect on SCI.MethodsSpinal cord injection with shRNA Shh-adeno associated virus (sh-Shh-AAV) were used to silence Shh. Exosomes were extracted from BMSCs. Rats that had suffered SCI were given intravenous injections of exosomes through the veins of the tail. Immunohistochemistry was used to identify the expression of Shh glycoprotein molecule as well as the expression of Gli-1 (glioma-associated oncogene homolog 1) in the rat spinal cord tissues. Western blot was performed to measure the levels of growth associated protein-43 (GAP-43). The BBB (Basso Beattie Bresnahan) score was used to assess the motor functions of the hind legs. In the same manner, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling or TUNEL and Nissl Staining was deployed to assess the level of regeneration of neurons and assess the level of histopathological damage in the tissues of the Spinal Cord.ResultsIn the case of the rats with SCI, the levels of display of Gli-1 and Shh showed dramatic improvement after the BMSCs exosome injections. In comparison to rats with SCI, the subjects of BMSCs exosomes group showed an improvement in their SCI, including a higher BBB score and Nissl body count, increasing GAP-43 expression, along with a much-decreased number of cells that suffered apoptosis. While the exosome effect on Spinal Cord Injury was completely ineffective in rats that had Shh silencing.ConclusionsExosomes secreted from BMSCs showed great effectiveness in the SCI healing with a vital involvement of Shh in this repair.

BSCI-14. tGLI1 is an actionable therapeutic target in breast cancer brain metastases

Breast cancer is the second leading cause of brain metastases in women; patients with breast cancer brain metastasis (BCBM) survive a median of 14.1 months following diagnosis. Cancer stem cells are thought to be one of the driving forces behind distant metastasis, treatment resistance, and late-stage recurrence. Despite advances made in understanding breast cancer stem cells (BCSC), it remains challenging to effectively target BCSC underscoring the need to identify and inhibit novel mediators of BCSC for treating BCBM patients. The hedgehog-smoothened pathway is an important mediator of breast cancer stem cells (BCSC); however, FDA-approved therapies targeting smoothened have demonstrated limited clinical efficacy in breast cancer. Truncated glioma-associated oncogene homolog 1 (tGLI1) was discovered in our laboratory as an alternative GLI1 splice variant that functions as a tumor-specific gain-of-function transcription factor and terminal effector of the hedgehog pathway. Our laboratory recently reported that tGLI1 promotes preferential metastasis to the brain in breast cancer by activating BCSC and astrocytes in the tumor microenvironment (Oncogene 39:64–78, 2020). tGLI1 knockdown abrogated BCBM, providing the rationale to therapeutically target tGLI1. This study aimed to determine if tGLI1 can be therapeutically targeted. Cell-based chemical screens followed by validations demonstrated that ketoconazole, an FDA-approved azole antifungal, and novel derivatives specifically inhibit tGLI1 leading to suppression of BCSC in vitro and BCBM in vivo. Mechanistic studies suggest that KCZ-dependent cell kill is, in part, mediated through downregulation of tGLI1 target genes OCT4, Nanog, and VEGFA. Based on these data, we opened a window-of-opportunity study in patients with BCBM to determine if ketoconazole penetrates the blood-brain barrier (BBB) and alters tGLI1 signaling in humans (NCT03796273). Preliminary sample analysis demonstrates ketoconazole crosses the blood-tumor barrier and that tGLI1 expression correlates with tGLI1 signaling in resected samples. Collectively, these data establish tGLI1 as an actionable target for BCBM.

Resveratrol increases the sensitivity of multiple myeloma cells against bortezomib via Hedgehog signaling pathway

Purpose: To investigate the effect of resveratrol (RSV) on bortezomib (BTZ)-resistant multiple myeloma (MM) cells, and to elucidate the underlying mechanism of action.&#x0D; Methods: H929 cell lines were exposed to BTZ for 8 months to establish BTZ-resistant MM cell model. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was measured using annexin V/propidium iodide (PI) staining while cell cycle analysis was evaluated by flow cytometry. The expression of Hedgehog (Hh) signaling proteins (sonic hedgehog (SHH), smoothened (SMO), and glioma-associated oncogene homolog (GLI)) was analyzed by western blot.&#x0D; Results: H929R was confirmed as a MM cell line that is resistant to BTZ. RSV enhanced the sensitivity of H929R cells against BTZ via inhibition of cell viability and colony formation, induction of cell apoptosis and regulation of expression of apoptosis-related proteins. Furthermore, RSV inhibited the expression of Hh signaling proteins (p &lt; 0.05.&#x0D; Conclusion: RSV enhances the sensitivity of MM cells to BTZ, partly via Hh signaling pathway. Thus, Hh pathway is a probable target for MM treatment, and RSV has potentials for use in the clinical management of MM.

Abstract 2866: Truncated glioma-associated oncogene homolog 1 (tGLI1) is an actionable therapeutic target in 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 following diagnosis. Current standard of care options for BCBM include stereotactic radiosurgery, whole-brain radiotherapy, and surgical resection. Novel agents that act on targetable driver mutations (EGFR, ALK, PI3K, etc) and penetrate the blood-brain barrier have recently shown increasing 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 one of the driving forces behind not only distant metastasis, but also late-stage recurrence. The hedgehog pathway has been identified as 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 is still challenging to effectively target BCSC underscoring the need to identify and inhibit novel mediators of BCSC for treating BCBM patients. Our laboratory recently reported that truncated glioma-associated oncogene homolog 1 (tGLI1) promotes preferential metastasis to the brain in breast cancer 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. We found that tGLI1 knockdown abrogated BCBM, providing the rationale to therapeutically target tGLI1. Cell-based chemical screens followed by validations demonstrated that ketoconazole, an FDA-approved azole antifungal, specifically inhibits tGLI1 leading to suppression of BCSC in vitro and BCBM in vivo. Modification of KCZ side chains produced derivative compounds that retained tGLI1-selectivity in both in vitro models of BCSC and in vivo models of BCBM with increased blood-brain barrier penetrance. Mechanistic studies suggest that KCZ-dependent cell kill is, in part, mediated through downregulation of tGLI1 target genes OCT4, Nanog, and VEGFA. Based on these data, we opened a window-of-opportunity study in patients with BCBM to determine if ketoconazole penetrates the blood-brain barrier and alters tGLI1 signaling in humans (NCT03796273). Preliminary sample analysis has confirmed tGLI1 expression in collected BCBM and glioma samples. To help identify more effective tGLI1 inhibitors, we screened 63 azole compounds for tGLI1-selectivity and identified four additional compounds as potential tGLI1 inhibitors. Collectively, these data establish tGLI1 as an actionable target for BCBM. Citation Format: Daniel L. Doheny, Sherona R. Sirkisoon, Tadas Rimkus, Dongqin Zhu, Noah R. Aguayo, Marlyn Anguelov, Sara Manore, Fei Xing, Linda Metheny-Barlow, Kounosuke Watabe, Alexandra Thomas, Adrianna Henson Masters, Roy E. Strowd, Hui-Wen Lo. Truncated glioma-associated oncogene homolog 1 (tGLI1) is an actionable therapeutic target in breast cancer brain metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2866.