Glioma-associated Oncogene Homolog Research Articles

Sonic Hedgehog Signaling in Organogenesis, Tumors, and Tumor Microenvironments.

During mammalian embryonic development, primary cilia transduce and regulate several signaling pathways. Among the various pathways, Sonic hedgehog (SHH) is one of the most significant. SHH signaling remains quiescent in adult mammalian tissues. However, in multiple adult tissues, it becomes active during differentiation, proliferation, and maintenance. Moreover, aberrant activation of SHH signaling occurs in cancers of the skin, brain, liver, gallbladder, pancreas, stomach, colon, breast, lung, prostate, and hematological malignancies. Recent studies have shown that the tumor microenvironment or stroma could affect tumor development and metastasis. One hypothesis has been proposed, claiming that the pancreatic epithelia secretes SHH that is essential in establishing and regulating the pancreatic tumor microenvironment in promoting cancer progression. The SHH signaling pathway is also activated in the cancer stem cells (CSC) of several neoplasms. The self-renewal of CSC is regulated by the SHH/Smoothened receptor (SMO)/Glioma-associated oncogene homolog I (GLI) signaling pathway. Combined use of SHH signaling inhibitors and chemotherapy/radiation therapy/immunotherapy is therefore key in targeting CSCs.

Gli1 interacts with YAP1 to promote tumorigenesis in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is the predominant esophageal cancer type in China. The aberrant activation of glioma-associated oncogene homolog1 (Gli1), a key factor in Hedgehog (Hh) signaling pathway, has been found in esophageal carcinoma. Moreover, Yes-associated protein 1 (YAP1), the major mediator of Hippo signaling pathway, has been linked to esophageal carcinoma progression. However, the precise roles and the underlying mechanism of both Gli1 and YAP1 in ESCC are unclear. Here, we found that Gli1 and YAP1 are overexpressed in ESCC and are associated with poor prognosis. In addition, we confirmed that knockdown of Gli1 or YAP1 suppresses ESCC cell growth, migration, and invasion in ESCC TE1 and EC109 cells. Significantly, Gli1 interacts with YAP1 in ESCC cells. Both Gli1 and YAP1 proteins are closely correlated with each other in human ESCC samples. Mechanistically, Gli1 upregulates YAP1 in a LATS1-independent manner. Conversely, YAP1 induces Gli1 by regulating phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. Most importantly, we demonstrated that the interaction between Gli1 and YAP1 promotes ESCC tumor growth in vitro and in vivo. Our findings established a novel signaling mechanism by which the interaction between Gli1 and YAP1 promotes ESCC cell growth. This signaling regulation of the tumorigenesis provides a new therapeutic strategy for highly lethal ESCC.

"Hedgehog pathway": a potential target of itraconazole in the treatment of cancer.

Itraconazole is an antifungal drug that has been clinically used for over 30years. In recent years, scholars have discovered that it possesses an anticancer effect. Moreover, its mechanism has been clarified to some degree. What deserves to be mentioned is that itraconazole acting on the Hedgehog pathway has made a new progress in the treatment of cancers. While interestingly, studies have demonstrated that the Hedgehog pathway is largely activated in different cancer cells. This review tries to highlight the effect of itraconazole on smoothened receptor (SMO) in the Hedgehog pathway, thereby reducing the glioma-associated oncogene homolog (GLI) release and finally exhibiting a range of anticancer effects, promoting apoptosis of cancer cells, and inhibiting proliferation by indirect inhibition of NF-κB pathway and inflammation, moreover, promoting the expression of cyclin-dependent kinase inhibitors, inhibiting the expression of target genes transcribed by GLI such as BCL-2 and Cyclin-D1. Besides, itraconazole increases the number of Bnip3, subsequently, inducing the dissociation of the Beclin-1/BCL-2 binding complex, as a result of ultimately promoting autophagy of cancer cells. As a new anticancer drug, whether itraconazole eventually entering clinical application requires the joint eforts of all scholars. In any case, an in-depth study on itraconazole will bring new hope for cancer patients in the near future.

Control of Muscle Fibro-Adipogenic Progenitors by Myogenic Lineage is Altered in Aging and Duchenne Muscular Dystrophy.

Fibro-adipogenic progenitors (FAPs), a muscle-resident stem cell population, have recently emerged as important actors of muscle regeneration by interacting with myogenic progenitors (MPs) to promote the formation of new muscle fibers. However, FAPs are also considered as main contributors of intramuscular fibrotic and fat depositions, resulting in a poor quality of muscles and a defective regeneration in aging and Duchenne Muscular Dystrophy disease (DMD). Therefore, the understanding of the control of FAP fate is an important aspect of muscle repair and homeostasis, but little is known in humans. We wondered the extent to which human FAP proliferation, adipogenesis and fibrogenesis can be regulated by human myogenic progenitors (MPs) in physiological and pathological contexts. FAPs and MPs were isolated from skeletal muscles of healthy young or old donors and DMD patients. FAP/MP contact co-cultures and conditioned-media from undifferentiated MPs or differentiated myotubes were assessed on both proliferation and fibro-adipogenic differentiation of FAPs. We showed that soluble molecules released by MPs activate the phosphoinositide 3-kinase (PI3Kinase)/Akt pathway in FAPs, resulting in the stimulation of FAP proliferation. FAP differentiation was regulated by MP-derived myotubes through the secretion of pro-fibrogenic factors and anti-adipogenic factors. Importantly, the regulation of FAP adipogenic and fibrogenic fates by myotubes was found to be mediated by Smad2 phosphorylation and the gene expression of glioma-associated oncogene homolog 1 (GLI1). Surprisingly, the regulations of proliferation and differentiation were disrupted for FAPs and MPs derived from aged individuals and patients with DMD. Our results highlight a novel crosstalk between FAPs and the myogenic lineage in humans that could be crucial in the formation of adipocyte and myofibroblast accumulation in dystrophic and aged skeletal muscle.

MicroRNA-873 serves a critical role in human cervical cancer proliferation and metastasis via regulating glioma-associated oncogene homolog 1.

Cervical cancer is a common gynecological malignancy with high morbidity worldwide. MicroRNAs (miRNAs) serve critical roles in cervical cancer progression. Accumulating evidence indicates that miR-873 functions as a tumor suppressor in certain types of cancer. However, the function and mechanism of miR-873 in the progression of cervical cancer have not been completely elucidated. In the present study, the role and mechanism of miR-873 in the proliferation, migration and invasion of cervical cancer cells were investigated. miR-873 expression was markedly decreased in cervical cancer, while glioma-associated oncogene homolog 1 (GLI1) was found to be a direct target of miR-873 by conducting dual-luciferase reporter assays. Furthermore, miR-873 overexpression reduced the expression of GLI1, and decreased the proliferation, metastasis and epithelial-mesenchymal transition of cancer cells. In rescue experiments, overexpression of GLI1 in cervical cancer cells effectively reversed the inhibitory effect induced by miR-873 mimics. Therefore, the results of the present study suggested that miR-873 functions as a tumor suppressor miRNA, and future studies should address its potential application in the treatment of cervical cancer.

Abstract A16: A novel therapeutic approach to inhibit the bidirectional oncogenic crosstalk between pancreatic cancer cells and the surrounding stroma

Abstract Background: Pancreatic cancer (PaCa) is a leading cause of cancer-related deaths and is projected to be the second most common cancer in a decade. It is characterized by a dense stroma surrounding tumor cells, known as desmoplasia that is stimulated by Sonic Hedgehog (SHH) released by PaCa cells. Stromal cells, in turn, release mitogenic substances such as hepatocyte growth factor (HGF) and Insulin-like growth factor receptor (IGF-1) by glioma-associated oncogene homolog 1 (GLI1) transcription factor that stimulate tumor growth, invasion, and resistance to therapy. We investigated the potential of targeting the metastasis suppressor gene N-myc downstream regulated gene 1 (NDRG1) to interrupt the oncogenic crosstalk between cancer associated human pancreatic stellate cells (PSCs) and PaCa cells. Methods: We assessed novel thiosemicarbazone anti-cancer agents, Dp44mT and DpC, that upregulate NDRG1 and their effects on: (i) SHH production by PaCa cells and also HGF and IGF-1 production by PSCs; (ii) HGF and IGF-1 receptors and their downstream signaling in indirect 2D and direct 3D co-culture of PSCs and PaCa cells; (iii) GLI1 and its upstream mediators in PSCs; and (iv) proliferative and metastatic properties of PaCa cells co-cultured with PSCs. Results: Dp44mT and DpC upregulated NDRG1 and reduced SHH production by PaCa. These agents also inhibited downstream HGF and IGF-1 signaling pathways in PaCa cells, leading to reduced metastatic potential in co-culture 2D and 3D models. Further, these agents also inhibited GLI1 levels and activation in PSCs, leading to reduced HGF and IGF-1 production by these stromal cells. NDRG1 overexpression alone had similar effects, while silencing NDRG1 further potentiated the oncogenic crosstalk between PaCa and PSC cells. Conclusions: Novel agents targeting NDRG1 have the unique ability to block the oncogenic crosstalk between PaCa cells and the surrounding stroma. This presents a novel approach to PaCa treatment that can potentially overcome the major problems attributed to desmoplasia, including therapeutic resistance and metastasis. Citation Format: Bekesho Geleta, Kyung Chan Park, Patric J. Jansson, Minoti Apte, Des R. Richardson, Zaklina Kovacevic. A novel therapeutic approach to inhibit the bidirectional oncogenic crosstalk between pancreatic cancer cells and the surrounding stroma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr A16.

PRC1 promotes GLI1-dependent osteopontin expression in association with the Wnt/\u03b2-catenin signaling pathway and aggravates liver fibrosis

BackgroundPRC1 (Protein regulator of cytokinesis 1) regulates microtubules organization and functions as a novel regulator in Wnt/β-catenin signaling pathway. Wnt/β-catenin is involved in development of liver fibrosis (LF). We aim to investigate effect and mechanism of PRC1 on liver fibrosis.MethodsCarbon tetrachloride (CCl4)-induced mice LF model was established and in vitro cell model for LF was induced by mice primary hepatic stellate cell (HSC) under glucose treatment. The expression of PRC1 in mice and cell LF models was examined by qRT-PCR (quantitative real-time polymerase chain reaction), western blot and immunohistochemistry. MTT assay was used to detect cell viability, and western blot to determine the underlying mechanism. The effect of PRC1 on liver pathology was examined via measurement of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and hydroxyproline, as well as histopathological analysis.ResultsPRC1 was up-regulated in CCl4-induced mice LF model and activated HSC. Knockdown of PRC1 inhibited cell viability and promoted cell apoptosis of activated HSC. PRC1 expression was regulated by Wnt3a signaling, and PRC1 could regulate downstream β-catenin activation. Moreover, PRC1 could activate glioma-associated oncogene homolog 1 (GLI1)-dependent osteopontin expression to participate in LF. Adenovirus-mediated knockdown of PRC1 in liver attenuated LF and reduced collagen deposition.ConclusionsPRC1 aggravated LF through regulating Wnt/β-catenin mediated GLI1-dependent osteopontin expression, providing a new potential therapeutic target for LF treatment.

Lichen Secondary Metabolite Physciosporin Decreases the Stemness Potential of Colorectal Cancer Cells.

Secondary metabolites of lichens are promising bioresources for candidate anti-cancer drugs. Accordingly, several approaches have been proposed for screening these molecules for novel anti-cancer lead compounds. In this study, we found that a non-toxic concentration of physciosporin, a compound isolated from Pseudocyphellaria granulata, significantly decreased colony formation on soft agar and spheroid formation by CSC221 cancer stem-like cells. Physciosporin also decreased spheroid formation in other colorectal cancer cell lines, including DLD1, Caco2, and HT29. Aldehyde dehydrogenase-1 (ALDH1), the most important cancer stem marker, was sharply downregulated at both the protein and mRNA level following treatment with physciosporin. Physciosporin also decreased the transcriptional activity of the glioma-associated oncogene homolog zinc finger protein (Gli), as well as the Hes1 and CSL promoters, in reporter assays. Moreover, the drug significantly suppressed spheroid formation in CSC221 cells overexpressing Gli1/2 or ΔEN1 (an S2-cleaved but membrane-tethered form of human Notch1) but did not suppress spheroid formation in cells overexpressing both Gli1/2 and ∆EN1, suggesting that physciosporin suppresses colon cancer cell stemness through the Sonic hedgehog and Notch signaling pathways. Together, these results demonstrate for the first time that physciosporin is a potent inhibitor of colorectal cancer cell stemness.

STAT3-Induced Gli1 Promotes the Differentiation and Maturation of Myelofibrosis Fibrocytes

Myelofibrosis (MF) is characterized by constitutive activation of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway and progressive bone marrow (BM) fibrosis. We previously found that the BM fibrosis in MF is induced by clonal monocyte-derived fibrocytes. However, whether or how the JAK-STAT pathway affects MF fibrocyte differentiation and maturation has not been elucidated. A recent study suggested that the transcription factor glioma-associated oncogene homolog 1 (Gli1) plays a significant role in the induction of BM fibrosis in MF (Schneider et al. Cell Stem Cell 2017). We have recently found that MF BM fibrocytes express high levels of Gli1 both in situ and in vitro (ASH 2018: Abstract 1793). Therefore, we sought to determine whether Gli1 affects the differentiation and maturation of MF fibrocytes and whether Gli1 expression is induced by the constitutively activated JAK-STAT pathway. To test our hypothesis, we first cultured normal donor and MF patients' BM CD14+ monocytes in fibrocyte differentiation-supporting medium and then transfected them using Gli1-siRNA. After 14 days we quantitated the number of mature fibrocytes using F-actin and DAPI staining. We found that the numbers of BM monocyte-derived fibrocytes in cultures containing Gli1-siRNA were significantly lower compared to mock-transfected cells. To study downstream effects of Gli1 silencing, we transfected mature fibrocytes with Gli1-siRNA and found that the expression of Gli1 was reduced by 137-fold (P<0.0001) and that Gli1 protein level was significantly reduced as assessed by western immunoblotting. Then, to determine whether Gli1 affects the differentiation and function of BM fibrocytes we assessed the levels of matrix metalloproteinases (MMPs) 2 and 9, known to activate TGF-β that induces collagen expression in fibrocytes. Using magnetic bead-based flow immunoassay we found a 5.2-fold increase in the plasma levels of MMP-2 of 59 treatment-naïve MF patients compared to healthy donors (P<0.0001). Therefore we wondered whether Gli1 silencing downregulates expression of both MMP-2 and MMP-9 in MF patients' fibrocytes. Then, using quantitative PCR we detected 2.5- and 2.7-fold lower expression of both MMP-2 and MMP-9 genes in MF fibrocytes transfected with Gli1-siRNA (P=0.0006 and P=0.0018, respectively). These findings were further corroborated on the protein level using western blot analysis. Remarkably, unlike Gli1-siRNA, the Gli1 antagonist GANT61 did not change the expression of either MMP-2 or MMP-9, suggesting that this inhibitor acts through a Gli1-independent mechanism. Because JAK-STAT pathway is constitutively activated in MF patients' fibrocytes, we sought to determine whether Gli1 expression is induced by STAT3. Using sequence analysis we identified 5 putative STAT3-binding sites in the Gli1 gene promoter region and designed probes that target these sites. Using chromatin immunoprecipitation (ChIP) assay we found that STAT3 protein co-precipitated all 5 STAT3-putative binding sites at high affinity. Then, by performing an electromobility shift assay (EMSA) of the fibrocyte nuclear extracts using DNA probes against Gli1 gene promoter STAT3-binding sites we detected STAT3-Gli1 DNA complexes, suggesting that STAT3 binds to the Gli1 gene promoter. We further confirmed these findings using anti-STAT3 antibodies which significantly attenuated the binding. Finally, we transfected MF fibrocytes with STAT3-siRNA and found that STAT3 siRNA significantly decreased Gli1 mRNA levels. In conclusion, we found that STAT3, phosphorylated by constitutively activated JAK2, induces the expression of Gli1 which in turn promotes the differentiation and maturation of MF fibrocytes. Downregulation of Gli1 levels halted fibrocyte development, suggesting that inhibition of Gli1 might alleviate BM fibrosis in patients with MF. Disclosures Verstovsek: Incyte: Research Funding; Roche: Research Funding; NS Pharma: Research Funding; Celgene: Consultancy, Research Funding; Gilead: Research Funding; Promedior: Research Funding; CTI BioPharma Corp: Research Funding; Genetech: Research Funding; Blueprint Medicines Corp: Research Funding; Novartis: Consultancy, Research Funding; Sierra Oncology: Research Funding; Pharma Essentia: Research Funding; Astrazeneca: Research Funding; Ital Pharma: Research Funding; Protaganist Therapeutics: Research Funding; Constellation: Consultancy; Pragmatist: Consultancy.

Testis-enriched circular RNA circ-Bbs9 plays an important role in Leydig cell proliferation by regulating a CyclinD2-dependent pathway.

Circular RNAs belong to a new category of non-coding RNAs, characterised by a circular structure, conservation, stability and high expression in eukaryotes. They often show tissue- or cell-specific expression. Here, we identified a testis-enriched circular RNA (circRNA), circular Bbs9 (circ-Bbs9) that is highly expressed in mouse testis. An RNase R treatment experiment confirmed that circ-Bbs9 is indeed a circRNA. In situ hybridisation experiments showed that circ-Bbs9 is expressed in Leydig cells along seminiferous tubules and in the cytoplasm of the TM3 Leydig cell line. Knocking down the circ-Bbs9 in TM3 cells by lentivirus vectors arrested cell proliferation, whereas overexpression of circ-Bbs9 induced cell proliferation significantly. Knocking down circ-Bbs9 inhibited the protein level of cyclin D2 (Ccnd2) and RNA immunoprecipitation results showed that circ-Bbs9 interacts with Ccnd2. Our results show that use of the Hedgehog pathway Smoothened Agonist (SAG) HCl and antagonists cyclopamine and gant6 affects the expression levels of Glioma-Associated Oncogene Homolog 1 (Gli1), Ccnd2 and other genes in this pathway. Our research reveals that a Leydig cell-specific circRNA, circ-Bbs9, plays a critical role in Leydig cell proliferation through regulating the levels of cell cycle-related Ccnd2. Thus, our results emphasise the important role of circRNA in the male reproductive system.

Icariin-induced miR-875-5p attenuates epithelial-mesenchymal transition by targeting hedgehog signaling in liver fibrosis.

Hepatic fibrosis is the final endpoint for most chronic liver diseases and remains a significant public health problem worldwide. Icariin, a naturally occurring flavonol glucoside, has been reported to exhibit protective effects on liver injury and alleviate liver fibrosis. However, the underlying detail molecular mechanism is not fully revealed. Mouse primary hepatic stellate cells (HSCs) and carbon tetrachloride (CCL4 )-induced liver fibrosis model in mice were used as in vitro and in vivo models in this study. The expression levels of miR-875-5p were detected by quantitative reverse transcription-PCR. The validation of the direct target of miR-875-5p was through dual-luciferase reporter assay and western blotting assay. The cell proliferation and cell mobility were determined using MTT assay and Transwell migration assay, respectively. We found that icariin inhibited epithelial-mesenchymal transition and collagen protein section of HSCs. Icariin exerted hepatoprotective effects on mice model of CCL4 -induced liver fibrosis. Our further results revealed that miR-875-5p was downregulated in human cirrhosis tissues and activated murine HSCs. Icariin induced miR-875-5p upregulation and subsequently decreased glioma-associated oncogene homolog 1 (GLI1) expression through direct binding to the three prime untranslated region of GLI1 mRNA. Our study highlighted the potential therapeutic application of icariin for liver fibrosis management.

GLI1 is increased in ovarian endometriosis and regulates migration, invasion and proliferation of human endometrial stromal cells in endometriosis.

Endometriosis is a benign gynecological disorder which shares certain characteristics with malignant tumor like migration, invasion and proliferation. Glioma-associated oncogene homolog 1 (GLI1) has been implicated in some cancers including endometrial cancer, however, its role in endometriosis remains unknown. The aim of this study was to explore the expression pattern of GLI1 in endometriosis, and further investigate the effect of GLI1 regulation on human endometrial stromal cells. The expression of GLI1 in normal endometrium and ectopic tissues was analyzed by immunohistochemistry, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot. The Short hairpin RNA (ShRNA) intervention technique and GLI1 inhibitor GANT-61 were used to silence GLI1. The expression levels of GLI1, MMP2 and MMP9 was detected by qRT-PCR and western blot. The migration and invasion ability of human endometrial stromal cells was determined by wound healing assay and transwell migration/invasion assay. The viability and proliferation potentiality of cells was detected by MTT assays and colony formation assay, respectively. We found that the expression of GLI1 mRNA and protein were significantly higher in ectopic endometrium from patients with endometriosis. Our analyses also show that GLI1 downregulation attenuated cells migration, invasion and proliferation abilities. What's more, reduced expression of GLI1 inhibited the expression of matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9). Our findings suggest that high levels of GLI1 may contribute to the development of endometriosis by promoting cell migration, invasion and proliferation involving regulation of MMP2 and MMP9 expression. Therefore, inhibition of GLI1 might be a novel potential therapeutic approach to the treatment of endometriosis, which sheds new light on our understanding of the pathogenesis of endometriosis.

Hypoxia induced Sonic Hedgehog signaling regulates cancer stemness, epithelial-to-mesenchymal transition and invasion in cholangiocarcinoma

Aberrant activation of Sonic Hedgehog (SHH) pathway has been implicated in a variety of cancers including cholangiocarcinoma (CC); however, the influencing factors are still unknown. Additionally, intratumoral hypoxia is known to contribute towards therapeutic resistance through modulatory effects on various pathways. In this study, we investigated the relationship between hypoxia and SHH pathway activation and the effect of this interplay on cancer stemness and epithelial-to- mesenchymal transition (EMT) during cholangiocarcinogenesis. Hypoxia promoted SHH pathway activation, evidenced by upregulated SHH and SMO levels, and enhanced glioma-associated oncogene homolog 1 (GLI1) nuclear translocation; whereas silencing of HIF-1α impaired SHH upregulation. Hypoxia also enhanced the expression of cancer stem cell (CSC) transcription factors (NANOG, Oct4, SOX2), CD133 and EMT markers (N-cadherin, Vimentin), thereby supporting invasion. Cyclopamine treatment suppressed hypoxia induced SHH pathway activation, consequently reducing invasiveness by downregulating the expression of CSC transcription factors, CD133 and EMT. Cyclopamine induced apoptosis in CC cells under hypoxia, suggesting that hypoxia induced activation of SHH pathway has modulatory effects on CC progression. Therefore, SHH signaling is proposed as a target for CC treatment, which is refractory to standard chemotherapy.

TGLI1 transcription factor mediates breast cancer brain metastasis via activating metastasis-initiating cancer stem cells and astrocytes in the tumor microenvironment.

Mechanisms for breast cancer metastasis remain unclear. Whether truncated glioma-associated oncogene homolog 1 (TGLI1), a transcription factor known to promote angiogenesis, migration and invasion, plays any role in metastasis of any tumor type has never been investigated. In this study, results of two mouse models of breast cancer metastasis showed that ectopic expression of TGLI1, but not GLI1, promoted preferential metastasis to the brain. Conversely, selective TGLI1 knockdown using antisense oligonucleotides led to decreased breast cancer brain metastasis (BCBM) in vivo. Immunohistochemical staining showed that TGLI1, but not GLI1, was increased in lymph node metastases compared to matched primary tumors, and that TGLI1 was expressed at higher levels in BCBM specimens compared to primary tumors. TGLI1 activation is associated with a shortened time to develop BCBM and enriched in HER2-enriched and triple-negative breast cancers. Radioresistant BCBM cell lines and specimens expressed higher levels of TGLI1, but not GLI1, than radiosensitive counterparts. Since cancer stem cells (CSCs) are radioresistant and metastasis-initiating cells, we examined TGLI1 for its involvement in breast CSCs and found TGLI1 to transcriptionally activate stemness genes CD44, Nanog, Sox2, and OCT4 leading to CSC renewal, and TGLI1 outcompetes with GLI1 for binding to target promoters. We next examined whether astrocyte-priming underlies TGLI1-mediated brain tropism and found that TGLI1-positive CSCs strongly activated and interacted with astrocytes in vitro and in vivo. These findings demonstrate, for the first time, that TGLI1 mediates breast cancer metastasis to the brain, in part, through promoting metastasis-initiating CSCs and activating astrocytes in BCBM microenvironment.

Concomitant targeting of Hedgehog signaling and MCL-1 synergistically induces cell death in Hedgehog-driven cancer cells.

In the present study, we show that concomitant inhibition of Hedgehog (HH) signaling by the glioma-associated oncogene homolog1 (GLI1)-targeting agent GANT61 and the antiapoptotic BCL-2 protein family member MCL-1 by A-1210477 synergistically induces cell death in HH-driven cancers, i.e. rhabdomyosarcoma (RMS) and medulloblastoma (MB) cells. Combined genetic and pharmacological inhibition emphasized that co-treatment of GANT61 and A-1210477 indeed relies on inhibition of GLI1 (by GANT61) and MCL-1 (by A-1210477). Mechanistic studies revealed that A-1210477 triggers the release of BIM from MCL-1 and its shuttling to BCL-xL and BCL-2. Indeed, BIM proved to be required for GANT61/A-1210477-induced cell death, as genetic silencing of BIM using siRNA significantly rescues cell death upon GANT61/A-1210477 co-treatment. Similarly, genetic silencing of NOXA results in a significant reduction of GANT61/A-1210477-mediated cell death. Also, overexpression of MCL-1 or BCL-2 significantly protects RMS cells from GANT61/A-1210477-triggered cell death. Addition of the pan-caspase inhibitor zVAD.fmk significantly decreases GANT61/A-1210477-stimulated cell demise, indicating apoptotic cell death. In conclusion, GANT61 and A-1210477 synergize to engage mitochondrial apoptosis. These findings provide the rationale for further evaluation of dual inhibition of HH signaling and MCL-1 in HH-driven cancers.

BSCI-02. tGLI1 IS A NOVEL, ACTIONABLE TARGET FOR THE TREATMENT OF BREAST CANCER BRAIN METASTASES

Despite improvements in early detection and intervention, breast cancer remains the second leading cause of cancer-related death in women and the second most common cancer to metastasize to the brain. Current standard of care options for breast cancer brain metastases (BCBM) include stereotactic radiosurgery, whole-brain radiotherapy, and surgical resection. Local and distant recurrences are common leading to significant morbidity; effective FDA-approved drugs for these patients remain a significant unmet need. Our laboratory discovered an alternative splice variant of glioma-associated oncogene homolog 1 (GLI1), termed truncated GLI1 (tGLI1) that is a tumor-specific gain-of-function transcription factor preferentially expressed in most BCBM samples and recurrent gliomas. Recent results established that tGLI1 promotes breast cancer stem cells (BrCSCs) and is associated with preferential metastasis to the brain and radioresistance, justifying tGLI1 as an ideal therapeutic target for BCBM patients. To identify tGLI1-targeting agents, we screened 1,520 compounds across three commercial drug libraries and found ketoconazole, an FDA-approved azole antifungal and component of previously studied anti-neoplastic regimens, selectively killed tGLI1-expressing breast cancer cells with heightened efficacy against the CSC subpopulation in vitro. tGLI1 knockdown abolished the ability of ketoconazole to target BrCSCs, indicating that ketoconazole effect is dependent on tGLI1. Intracardiac mouse studies showed ketoconazole selectively inhibited circulating tGLI1-positive breast cancer cells from developing into brain metastases and suppressed the progression of existing brain metastases. Mass spectrometry demonstrated ketoconazole effectively penetrated the blood-brain barrier (BBB) and blood-tumor barrier (BTB). Mechanistic studies suggest that ketoconazole-dependent cell kill is, in part, mediated through disruption of the tGLI1-STAT3 interaction. Collectively, our preclinical results demonstrate that ketoconazole is an effective inhibitor of BrCSCs and brain metastasis of tGLI1-positive breast cancer. Based on these promising preclinical data, we opened a window-of-opportunity study in patients with BCBM and recurrent gliomas to determine if ketoconazole treatment alters tGLI1 signaling in humans (NCT03796273).

BSCI-13. TUMOR-SPECIFIC tGLI1 TRANSCRIPTION FACTOR MEDIATES BREAST CANCER BRAIN METASTASIS VIA ACTIVATING METASTASIS-INITIATING CANCER STEM CELLS AND ASTROCYTES IN THE TUMOR MICROENVIRONMENT

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. Mechanisms for BCBM remain unclear, which contributes to ineffective treatments and dismal prognosis. Truncated glioma-associated oncogene homolog 1 (tGLI1) belongs to the GLI1 family of zinc-finger transcription factors and functions as a tumor-specific gain-of-function mediator of tumor invasion and angiogenesis. Whether tGLI1 plays any role in metastasis of any tumor type remains unknown. Using an experimental metastasis mouse model, via intracardiac implantation, we showed that ectopic expression of tGLI1, but not GLI1, promoted preferential metastasis to brain. Conversely, selective tGLI1 knockdown using tGLI1-specific antisense oligonucleotides led to decreased brain metastasis of intracardially inoculated breast cancer cells. Furthermore, intracranial implantation mouse study revealed tGLI1 enhanced intracranial colonization and growth of breast cancer cells. Immunohistochemical staining of patient samples showed that tGLI1, but not GLI1, was increased in lymph node metastases compared to matched primary tumors, and that tGLI1 was expressed at higher levels in BCBM specimens compared to primary tumors. Whether tGLI1 plays any role in radioresistance is unknown; we found radioresistant BCBM cell lines and patient specimens expressed higher levels of tGLI1 than radiosensitive counterparts, and that tGLI1 promotes radioresistance. Since cancer stem cells (CSCs) are highly metastatic and radioresistant, we examined whether tGLI1 promotes BCBM and radioresistance through activating CSCs. Results showed that tGLI1 transcriptionally activates stemness genes CD44, Nanog, Sox2, and OCT4, leading to stem cell activation. Furthermore, we observed that tGLI1-positive CSCs strongly activated and interacted with astrocytes, the most abundant brain tumor microenvironmental cells known to promote tumor growth, in vitro and in vivo. Collectively, our findings establish a novel role of that tGLI1 plays in promoting breast cancer preferential metastasis to brain, radioresistance, and astrocytes in the metastatic niche.

Abstract 3689: Tumor-specific gain-of-function tGLI1 transcription factor is a novel mediator of breast cancer stem cells and a novel transcriptional activator of cancer stemness genes

Abstract Breast cancer is the second leading cause of cancer-related mortality in women; metastasis to distant organs results in 90% of deaths for these patients. Cancer stem cells (CSCs) are considered the drivers of metastasis. Despite our current knowledge of breast CSCs, there still remains a significant challenge in managing patients with the metastatic breast cancer, underscoring the need for identifying novel regulators of breast CSCs. The hedgehog pathway is an important mediator of stem cells; however, the effect of truncated glioma-associated oncogene homolog 1 (tGLI1), a nuclear effector of the hedgehog pathway and a gain-of-function GLI1 transcription factor, on breast CSCs has never been investigated. Herein, we investigated whether tGLI1 is implicated in breast CSCs by evaluating tGLI1 expression levels in cells grown as monolayer versus mammospheres, as a representation of the stem cell population, and found tGLI1 to be induced in mammosphere culture. Overexpression of tGLI1 promoted mammosphere-forming ability of breast cancer cells, as well as, increased the breast CSC population defined by CD44high/CD24low expression. Further, tGLI1 overexpression transformed normal mammary epithelial cells resulting in increased mammosphere formation and enhanced anchorage-independent growth of immortalized human mammary epithelial HMLE cells. Functional and biochemical assays further showed that tGLI1 promotes breast CSC self-renewal by transcriptional activation of stemness genes including a novel tGLI1 target gene, OCT4, a recently reported tGLI1 target gene (CD44), and known GLI1 target genes (Nanog and SOX2). Bioinformatic analysis of breast cancer patient datasets revealed that activated tGLI1 is associated with shortened time to develop metastasis to the lung, bone, and brain. Furthermore, tGLI1 activation is enriched in HER2-enriched and triple-negative breast cancers, the subtypes with the highest propensity to metastasize, compared to luminal subtypes. Gene Set Enrichment Analysis showed that high tGLI1 activation is enriched in breast cancer with high gene signatures of breast CSCs, radioresistance, and metastasis. We further validated these results by immunohistochemical staining of paired primary breast tumors with lymph node metastases and found that that expression of tGLI1, but not GLI1, was increased in lymph node metastases and that tGLI1 was expressed at higher levels (84-91%) of lymph node-positive metastatic HER2-enriched and triple-negative breast tumors. Lastly, tGLI1 knockdown resulted in decreased mammosphere formation of breast cancer cells and decreased expression of stemness genes, OCT4, CD44, and Nanog. Taken together, these findings establish a novel role that tGLI1 plays in mediating breast CSCs and implicate tGLI1 in facilitating breast cancer metastasis. Citation Format: Sherona R. Sirkisoon, Richard L. Carpenter, Tadas Rimkus, Daniel Doheny, Dongqin Zhu, Noah R. Aguayo, Marlyn Anguelov, Austin Arrigo, Fei Xing, Michael Chan, Jimmy Ruiz, Linda J. Metheny-Barlow, Roy Strowd, Jiayuh Lin, Boris C. Pasche, Waldemar Debinski, Kounosuke Watabe, Hui-Wen Lo. Tumor-specific gain-of-function tGLI1 transcription factor is a novel mediator of breast cancer stem cells and a novel transcriptional activator of cancer stemness genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3689.

Targeting the RAS axis alleviates silicotic fibrosis and Ang II-induced myofibroblast differentiation via inhibition of the hedgehog signaling pathway

The hedgehog (HH) signaling pathway plays an important role in lung development, but its significance in silicosis is unclear. We showed that in human coal pneumoconiosis autopsy specimens, Sonic Hedgehog (SHH) and the Glioma-associated oncogene homolog transcription factors family (GLI) 1 proteins were up-regulated, whereas Patch-1 (PTC) was down-regulated. The protein levels of SHH, smoothened (SMO), GLI1, GLI2, α-smooth muscle actin (α-SMA) and collagen type Ⅰ (Col Ⅰ) were also elevated gradually in the bronchoalveolar lavage fluid (BALF) of different stages of coal pneumoconiosis patients, dynamic silica-inhalation rat lung tissue and MRC-5 cells induced by Ang II at different time points, whereas the PTC and GLI3 levels were diminished gradually. Ac-SDKP, an active peptide of renin-angiotensin system (RAS), is an anti-fibrotic tetrapeptide. Targeting RAS axis also has anti-silicotic fibrosis effects. However, their roles on the HH pathway are still unknown. Here, we reported that Ac-SDKP + Captopril, Ac-SDKP, Captopril, or Ang (1–7) could alleviate silicotic fibrosis and collagen deposition, as well as improve the lung functions of silicotic rat. These treatments decreased the expression of SHH, SMO, GLI1, GLI2, α-SMA, and Col Ⅰ and increased the expression of PTC and GLI3 on both the silicotic rat lung tissue and MRC-5 cells induced by Ang II. We also reported that Ang II may promote myofibroblast differentiation via the GLI1 transcription factor and independently of the SMO receptor.

RETRACTED: Silencing of the MEKK2/MEKK3 Pathway Protects against Spinal Cord Injury via the Hedgehog Pathway and the JNK Pathway

Spinal cord injury (SCI) is a devastating medical condition, often accompanied by motor and sensory dysfunction. The Hedgehog (Hh) pathway has a protective role in pathological injury after SCI. However, the specific mechanism remains unclear. The present study aimed to confirm the effects of the mitogen-activated protein kinase kinase-2 (MEKK2)/MEKK3/JNK/Hh pathway on SCI. SCI rat models were established and then inoculated with plasmids overexpressing MEKK2/MEKK3 or with small interfering RNA (siRNA) against MEKK2/MEKK3. The expression of MEKK2 and -3 was detected in dorsal root ganglia (DRG) cells. The motor function of hindlimbs, the expression of the c-Jun N-terminal kinase (JNK)- and Hh-pathway-related genes, and the level of neurofilament-200 (NF-200) and glial fibrillary acidic protein (GFAP) were measured. MEKK2 and -3 were expressed at a high level in DRG cells. The silencing of MEKK2/MEKK3 in rats caused an increase in the expression of glioma-associated oncogene homolog-1 (Gli-1), Nestin, smoothened (Smo), and Sonic Hedgehog (Shh). The Basso, Beattie, and Bresnahan (BBB) rating and the level of NF-200 protein also increased. However, the expression of monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1β (MIP-1β), MIP-3α, p-JNK/JNK, and p-c-Jun/c-Jun and the level of GFAP were reduced. Downregulation of MEKK2/MEKK3 ameliorated the symptoms of SCI by promoting neural progenitor cell differentiation via activating the Hh pathway and disrupting the JNK pathway. The findings in this study reveal a potential biomarker for SCI treatment.