Glioma-associated Oncogene Homolog Research Articles

Hh pathway expression in the blood, synovial cells and chondrocytes of different rheumatoid arthritis models

Purpose: To investigate the effect of the hedgehog (Hh) pathway inhibitor, cyclopamine, and activator purmorphamine on articular cartilage cell proliferation.
 Methods: Rats were subjected to AA and CIA models. Secondary paw swelling was measured at 12, 15, 18, 21, 24, 27, and 30 days. The rats were sacrificed on day 30. Tissues from the cartilage and knee joints were collected. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay while cell apoptosis was determined by annexin V-fluorescein isothiocyanate/propidium iodide assay. Protein expression levels of Shh, Ptch1 and Gli1 were determined by Western blotting.
 Results: Compared with the control group, arthritis index and secondary foot swelling of the adjuvant arthritis (AA) and collagen-induced arthritis (CIA) groups deteriorated significantly (p < 0.05). MTT data revealed that cyclopamine promoted articular cartilage cell proliferation of the AA and CIA groups. The cell proliferation rates of AA and CIA groups were significantly higher than that of control group (p < 0.05). Flow cytometry showed that the cell apoptosis rates of AA and CIA groups were significantly lower than that of control group (p < 0.05). Compared with control, cyclopamine decreased the protein expression levels of sonic Hh, patched homologue 1 and glioma-associated oncogene homologue, but the effect of purmorphamine was the reverse.
 Conclusion: Hh pathway inhibitor (cyclopamine) and activator (purmorphamine) affect the expression of Hh pathway. Disruption of the Hh pathway may be of potential therapeutic significance in protecting articular cartilage from rheumatoid arthritis.

Hedgehog signaling modulates cigarette-induced COPD development.

Hedgehog (Hh) signaling is involved in early embryogenesis and maintains quiescence in the adult lungs. The interruption of Hh signaling may lead to the development of chronic obstructive pulmonary disease (COPD). The current study aimed to assess whether the Hh pathway affects cigarette-induced emphysema and airway inflammation by regulating inflammatory cytokines. C57BL/6J mice were randomized into control, cigarette smoke (CS) or CS + cyclopamine (CSC) groups. Control mice were exposed to normal room air, CS mice were exposed to tobacco smoke and CSC mice were exposed to CS and received cyclopamine treatment. Histopathological examination of lung tissues was performed, and the expression of sonic hedgehog (HH), glioma-associated oncogene homolog 1 (Gli1), hedgehog-interacting protein (HIP) and several inflammatory mediators (intracellular adhesion molecule-1, IL-6, IL-8 and TNF-α) were compared using reverse transcription-quantitative PCR and western blotting. The emphysema of lung tissues by histopathological examination demonstrated partial amelioration in the CSC group compared with that in the CS group. Additionally, expression levels of SHH, Gli1 and inflammatory mediators were significantly higher in the CS group compared with the control group but were significantly decreased in the CSC group. The expression of HIP was decreased in the CS group, but significantly increased in the CSC group. Hh signaling may serve an important role in cigarette-induced emphysema and airway inflammation by regulating inflammatory cytokines in animal models. Therefore, diminishing the activation of the Hh signal may serve as a novel therapeutic strategy for patients suffering from smoking-related COPD.

Gene polymorphisms in odontogenic keratocysts and ameloblastomas: A systematic review.

The aim of this systematic review was to critically analyze available data on gene polymorphisms in odontogenic keratocysts (OKC) and ameloblastomas, including their possible relationship with clinical and histological features of these lesions. A comprehensive search of Web of Science Scopus, PubMed, Cochrane Central Register of Controlled Trials and EMBASE was conducted using relevant key terms and supplemented by a gray literature search. Quality assessment of included studies was performed using criteria from the Strengthening the Reporting of Genetic Association (STREGA) statement. Ten studies were included in the final review. Survivin -31G/C, interleukin IL-1α -889 C/T, p53 codon 72 G/C, tumor necrosis factor TNF-α (-308G>A) and its receptor TNF-R1 (36A>G), glioma-associated oncogene homolog 1 rs2228224 and matrix metalloproteinase 2 rs243865 gene polymorphisms were reported to be associated with OKC. For ameloblastomas, p53 codon 72 G/C, X-ray repair cross-complementing protein 1-codons 194 and 399 and matrix metalloproteinase 9 rs3918242 gene polymorphisms were identified as risk factors. It was not possible to establish a relationship between specific polymorphisms and clinical and histological features of investigated lesions. Several gene polymorphisms might be considered as a risk factor for the development of these lesions. Future studies should investigate whether these polymorphisms might be used to identify patients with increased risk of recurrence or aggressive disease.

Effects of SET7 on angiotensin II-mediated proliferation and collagen synthesis of myocardial fibroblasts and its mechanisms.

Silence of SET domain containing lysine methyltransferase 7 (SET7) alleviates myocardial tissue injury caused by ischemia-reperfusion. But the effects of SET7 on angiotensin II (Ang II)-induced myocardial fibroblast proliferation and the collagen synthesis are not clear. The purpose of this study was to explore the effect of SET7 on the proliferation and collagen synthesis of myocardial fibroblasts and its mechanisms. Myocardial fibroblasts were isolated and identified by immunofluorescence. Myocardial fibroblasts were randomly divided into 4 groups: a control group (cells were normally cultured), an Ang II group (cells were treated with 100 nmol/L Ang II for 24 h), a siCtrl group (cells were transfected with siRNA control and were then treated with 100 nmol/L Ang II for 24 h), and a siSET7 group (cells were transfected with siRNA SET7 and were then treated with 100 nmol/L Ang II for 24 h). Cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) assay were used to evaluate cell proliferation. Real-time PCR was used to detect the mRNA levels of SET7, collagen I, collagen III, and α-smooth muscle actin (α-SMA). Western blotting was used to detect the protein expression of SET7, collagen I, collagen III, α-SMA, sonic hedgehog (Shh), ptched1 (Ptch1), and glioma-associated oncogene homolog 1 (Gli1). Fluorescence microscopy showed positive vimentin staining, and myocardial fibroblasts were in good condition. As compared to the control group, the mRNA and protein levels of SET7 in the Ang II group were significantly upregulated; cell proliferation rate and EdU fluorescence intensity in the Ang II group were significantly increased; the mRNA and protein levels of collagen I, collagen III, and α-SMA were significantly upregulated (all P<0.05). As compared to the siCtrl group, the mRNA and protein levels of SET7 in the siSET7 group were significantly downregulated; cell proliferation rate and EdU fluorescence intensity in the siSET7 group were significantly decreased; the mRNA and protein levels of collagen I, collagen III, and α-SMA in the siSET7 group were significantly downregulated (all P<0.05). Silence of SET7 gene inhibits Ang II-induced proliferation and collagen synthesis of myocardial fibroblasts. Shh signaling pathway may be involved in this process.

The value of the hedgehog signal in osteoblasts in fluoride-induced bone-tissue injury

ObjectiveThis study was designed to observe the expression of important hedgehog (Hh) signal factors in the bone tissue of rats with chronic fluorosis and cultured osteoblasts in order to investigate the role and significance of the Hh signal in fluoride-induced bone injury.MethodsHealthy Sprague-Dawley (SD) rats were randomly divided into four groups: the control group, the fluorosis group (F Group), the fluoride + blocker group (F + Cycl group: rats were treated with fluoride + cyclopamine), and the fluoride + blocker control group (F + DMSO group). After 6 months of intervention, the urinary fluoride content of rats in each group was detected. The primary osteoblasts of rats were selected for cell experiment, and the experiment was carried out after the cells were passaged from the second to the fourth generation.ResultsThe proliferation rate of primary rat osteoblasts presented time-affected and dose-affected relationships in a short time under treatment with a low dose of sodium fluoride (NaF), but the proliferation of osteoblasts was inhibited by long-term and high-dose NaF exposure. In the F group, the alkaline phosphatase (ALP) activity of osteoblasts increased gradually. The ALP activity was lower in the F + Cycl group than in the F group, and there was no significant difference between the F + DMSO group and F group. With the increase in fluoride exposure, the expression of Hh signal factors and osteogenic-related factor proteins increased gradually. The expressions of Indian hedgehog (Ihh), smoothened (Smo), Glioma-associated oncogene homolog (Gli) 2, and Runt-related transcription factor 2 (Runx2)in the F + Cycl group increased with the dose of fluoride but they were significantly inhibited compared with the F group. Compared with the control group, the content of urinary fluoride in the F group was significantly higher (P < 0.05), but there was no significant change in urinary fluoride content in the F + Cycl group and the F + DMSO group. Compared with the control group, the serum bone alkaline phosphatase (BALP) contents of rats in the other groups increased after 6 months’ intake of fluoride water (P < 0.05). After drug blocking, the serum BALP content in the F + Cycl group was lower than that in the F + DMSO group (P < 0.05). The BALP content in the F + DMSO group was similar to that in the F group: it did not decrease. The mRNA expressions of Ihh, Smo, Gli2, and Runx2 in bone tissue of the F group were significantly higher than those in the control group (P < 0.05). After cyclopamine blocking, the expressions decreased (P < 0.05), but the differences between the F + DMSO group and F group were not statistically significant.ConclusionHh signal plays an important role in fluoride-induced bone injury. The effective inhibition of cyclopamine is expected to be a new target for the treatment of skeletal damage caused by fluorosis.

Targeted Dual Small Interfering Ribonucleic Acid Delivery via Non-Viral Polymeric Vectors for Pulmonary Fibrosis Therapy.

Inhibiting the myofibroblast differentiation of lung-resident mesenchymal stem cells (LR-MSCs) is a promising yet challenging approach for pulmonary fibrosis (PF) therapy. Here, micelles formed by a graft copolymer of multiple PEGs modified branched polyethylenimine are used for delivering runt-related transcription factor-1 (RUNX1) small interfering RNA (siRNA) (siRUNX1) to the lung, aiming to inhibit the myofibroblast differentiation of LR-MSCs. LR-MSC targeting is achieved by functionalizing the micelle surface with an anti-stem-cell antigen-1 antibody fragment (Fab'). Consequently, therapeutic benefits are obtained by successful suppression of myofibroblast differentiation of LR-MSCs in bleomycin-induced PF model mice treated with siRUNX1-loaded micelles. Furthermore, an excellent synergistic effect of PF therapy is achieved for this micelle system loaded siRUNX1 and glioma-associated oncogene homolog-1 (Gli1) small interfering RNA (siGli1), a traditional anti-PF siRNA of glioma-associated oncogene homolog-1. Hence, this work not only provides RUNX1 as a novel PF therapeutic target, but also as a promising dual siRNA-loaded nanocarrier system for the therapy of PF.

The vital role of Gli1+ mesenchymal stem cells in tissue development and homeostasis.

The hedgehog (Hh) signaling pathway plays an essential role in both tissue development and homeostasis. Glioma-associated oncogene homolog 1 (Gli1) is one of the vital transcriptional factors as well as the direct target gene in the Hh signaling pathway. The cells expressing theGli1 gene (Gli1+ cells) have been identified as mesenchymal stem cells (MSCs) that are responsible for various tissue developments, homeostasis, and injury repair. This review outlines some recent discoveries on the crucial roles of Gli1+ MSCs in the development and homeostasis of varieties of hard and soft tissues.

Glypican-1, -3, -5 (GPC1, GPC3, GPC5) and Hedgehog Pathway Expression in Oral Squamous Cell Carcinoma.

Proteoglycans are involved in tumor development and may regulate the Hedgehog (HH) pathway. This study aimed to investigate the gene and protein expression of glypican-1 (GPC1), -3 (GPC3), and -5 (GPC5) in oral squamous cell carcinoma (OSCC) and tumor-free lateral margins (TM) and their association with the HH pathway. Quantitative PCR was performed for GPC1, GPC3, GPC5, SHH, PTCH1, SMO, and GLI1 genes in samples of OSCC (n=31), TM (n=12), and non-neoplastic oral mucosa (NNM) of healthy patients (n=6), alongside an immunohistochemical evaluation of GPC1, GPC3, and GPC5 proteins and HH proteins SHH and glioma-associated oncogene homolog 1 (GLI1). Double staining for GPC3/SHH, GPC5/SHH, GPC3/tubulin [ac Lys40], GPC5/Tubulin [ac Lys40], and GPC5/GLI1 was also performed. Overexpression of GPC1 and GPC5 in tumor samples and underexpressed levels of GPC3 gene transcripts were observed when compared with TM (standard sample). HH pathway mRNA aberrant expression in OSCC samples and a negative correlation between GPC1 and GPC5 at transcription levels were detected. GPC1 staining was rare in OSCC, but positive cells were found in NNM and TM. Otherwise positive immunostaining for GPC3 and GPC5 was observed in OSCCs, but not in NNM and TM. Blood vessels adjacent to tumor islands were positive for GPC1 and GPC5. Co-localization of GPC3-positive and GPC5-positive cells with SHH and Tubulin [ac Lys40] proteins was noted, as well as of GPC5 and GLI1. The absence of the GPC1 protein in neoplastic cells, underexpression of the GPC3 gene, and co-localization of GPCs and HH proteins may indicate the maintenance of aberrant HH pathway activation in OSCC.

GLI2 but not GLI1/GLI3 plays a central role in the induction of malignant phenotype of gallbladder cancer.

We previously reported that Hedgehog (Hh) signal was enhanced in gallbladder cancer (GBC) and was involved in the induction of malignant phenotype of GBC. In recent years, therapeutics that target Hh signaling have focused on molecules downstream of smoothened (SMO). The three transcription factors in the Hh signal pathway, glioma-associated oncogene homolog 1 (GLI1), GLI2, and GLI3, function downstream of SMO, but their biological role in GBC remains unclear. In the present study, the biological significance of GLI1, GLI2, and GLI3 were analyzed with the aim of developing novel treatments for GBC. It was revealed that GLI2, but not GLI1 or GLI3, was involved in the cell cycle-mediated proliferative capacity in GBC and that GLI2, but not GLI1 or GLI3, was involved in the enhanced invasive capacity through epithelial-mesenchymal transition. Further analyses revealed that GLI2 may function in mediating gemcitabine sensitivity and that GLI2 was involved in the promotion of fibrosis in a mouse xenograft model. Immunohistochemical staining of 66 surgically resected GBC tissues revealed that GLI2-high expression patients had fewer numbers of CD3+ and CD8+ tumor-infiltrating lymphocytes (TILs) and increased programmed cell death ligand 1 (PD-L1) expression in cancer cells. These results suggest that GLI2, but not GLI1 or GLI3, is involved in proliferation, invasion, fibrosis, PD-L1 expression, and TILs in GBC and could be a novel therapeutic target. The results of this study provide a significant contribution to the development of a new treatment for refractory GBC, which has few therapeutic options.

Hedgehog Signaling: Implications in Cancers and Viral Infections.

The hedgehog (SHH) signaling pathway is primarily involved in embryonic gut development, smooth muscle differentiation, cell proliferation, adult tissue homeostasis, tissue repair following injury, and tissue polarity during the development of vertebrate and invertebrate organisms. GLIoma-associated oncogene homolog (GLI) family of zinc-finger transcription factors and smoothened (SMO) are the signal transducers of the SHH pathway. Both SHH ligand-dependent and independent mechanisms activate GLI proteins. Various transcriptional mechanisms, posttranslational modifications (phosphorylation, ubiquitination, proteolytic processing, SUMOylation, and acetylation), and nuclear-cytoplasmic shuttling control the activity of SHH signaling pathway proteins. The dysregulated SHH pathway is associated with bone and soft tissue sarcomas, GLIomas, medulloblastomas, leukemias, and tumors of breast, lung, skin, prostate, brain, gastric, and pancreas. While extensively studied in development and sarcomas, GLI family proteins play an essential role in many host-pathogen interactions, including bacterial and viral infections and their associated cancers. Viruses hijack host GLI family transcription factors and their downstream signaling cascades to enhance the viral gene transcription required for replication and pathogenesis. In this review, we discuss a distinct role(s) of GLI proteins in the process of tumorigenesis and host-pathogen interactions in the context of viral infection-associated malignancies and cancers due to other causes. Here, we emphasize the potential of the Hedgehog (HH) pathway targeting as a potential anti-cancer therapeutic approach, which in the future could also be tested in infection-associated fatalities.

GLI3Is Associated With Neuronal Differentiation in SHH-Activated and WNT-Activated Medulloblastoma.

Glioma-associated oncogene homolog 3 (GLI3), whose main function is to inhibit GLI1, has been associated with neuronal differentiation in medulloblastoma. However, it is not clear what molecular subtype(s) show increased GLI3 expression. GLI3 levels were assessed by immunohistochemistry in 2 independent cohorts, including a total of 88 cases, and found to be high in both WNT- and SHH-activated medulloblastoma. Analysis of bulk mRNA expression data and single cell RNA sequencing studies confirmed that GLI1 and GLI3 are highly expressed in SHH-activated medulloblastoma, whereas GLI3 but not GLI1 is highly expressed in WNT-activated medulloblastoma. Immunohistochemical analysis has shown that GLI3 is expressed inside the neuronal differentiated nodules of SHH-activated medulloblastoma, whereas GLI1/2 are expressed in desmoplastic areas. In contrast, GLI3 is diffusely expressed in WNT-activated medulloblastoma, whereas GLI1 is suppressed. Our data suggest that GLI3 may be a master regulator of neuronal differentiation and morphology in these subgroups.

Jervine inhibits non-small cell lung cancer (NSCLC) progression by suppressing Hedgehog and AKT signaling via triggering autophagy-regulated apoptosis

Non-small cell lung cancer (NSCLC) has been identified as a leading cause of tumor-associated death around the world. Presently, it is necessary to find effective and safe therapy for its treatment in clinic. Jervine (Jer), a sterodial alkaloid from rhizomes of Veratrum album, exhibits anti-inflammatory and anti-cancer effects. However, its effects on lung cancer progression are still unknown. In this study, we explored if Jer showed any influences on NSCLC development, as well as the underlying molecular mechanisms. The results showed that Jer time- and dose-dependently reduced the proliferation of NSCLC cells, along with inhibited colony formation capacity. Apoptosis was highly induced by Jer in NSCLC cells through promoting the expression of cleaved Caspase-3. Furthermore, Jer treatment led to autophagy in cancer cells, as evidenced by the fluorescence microscopy results and increases of LC3II. Autophagy inhibitor bafilomycinA1 (BafA1) abrogated the inhibitory effects of Jer on cell proliferation and apoptosis induction, showing that Jer triggered autophagy-mediated apoptosis in NSCLC cells. Additionally, AKT and mammalian target of Rapamycin (mTOR) signaling pathway was highly repressed in cancer cells. Importantly, promoting AKT activation greatly rescued the cell survival, while attenuated autophagy and apoptosis in Jer-incubated NSCLC cells, revealing that Jer-modulated autophagic cell death was through the blockage of AKT signaling. Hedgehog signaling pathway was then found to be suppressed by Jer, as proved by the decreased expression of Sonic Hedgehog (Shh), Hedgehog receptor protein patched homolog 1 (PTCH1), smoothened (SMO) and glioma-associated oncogene homolog 1 (Gli1) in NSCLC cells. Of note, enhancing Shh signaling dramatically diminished the stimulative effects of Jer on autophagy-mediated apoptosis in vitro, demonstrating the importance of Hedgehog signaling in Jer-regulated cell death. Moreover, Jer treatment effectively reduced tumor growth in A549-bearing mice with few toxicity. Together, Jer may be a promising and effective therapeutic strategy for NSCLC treatment.

Hedgehog Signaling and Truncated GLI1 in Cancer.

The hedgehog (HH) signaling pathway regulates normal cell growth and differentiation. As a consequence of improper control, aberrant HH signaling results in tumorigenesis and supports aggressive phenotypes of human cancers, such as neoplastic transformation, tumor progression, metastasis, and drug resistance. Canonical activation of HH signaling occurs through binding of HH ligands to the transmembrane receptor Patched 1 (PTCH1), which derepresses the transmembrane G protein-coupled receptor Smoothened (SMO). Consequently, the glioma-associated oncogene homolog 1 (GLI1) zinc-finger transcription factors, the terminal effectors of the HH pathway, are released from suppressor of fused (SUFU)-mediated cytoplasmic sequestration, permitting nuclear translocation and activation of target genes. Aberrant activation of this pathway has been implicated in several cancer types, including medulloblastoma, rhabdomyosarcoma, basal cell carcinoma, glioblastoma, and cancers of lung, colon, stomach, pancreas, ovarian, and breast. Therefore, several components of the HH pathway are under investigation for targeted cancer therapy, particularly GLI1 and SMO. GLI1 transcripts are reported to undergo alternative splicing to produce truncated variants: loss-of-function GLI1ΔN and gain-of-function truncated GLI1 (tGLI1). This review covers the biochemical steps necessary for propagation of the HH activating signal and the involvement of aberrant HH signaling in human cancers, with a highlight on the tumor-specific gain-of-function tGLI1 isoform.

Expression of Glioma-associated oncogene homolog 1 as biomarker with sonidegib in advanced basal cell carcinoma.

The pivotal BOLT (Basal cell carcinoma Outcomes with LDE225 [sonidegib] Treatment) study established the durable efficacy and manageable toxicity of sonidegib 200 mg once daily (QD) through 42 months in patients with advanced basal cell carcinoma (BCC). This secondary analysis used expression of Glioma-associated oncogene homolog 1 (GLI1) as a biomarker to assess the extent of Hedgehog pathway inhibition by sonidegib in patients with locally advanced BCC (laBCC) and metastatic BCC (mBCC). The study enrolled 230 patients, 79 and 151 receiving sonidegib 200 and 800 mg QD, respectively. At week 17, GLI1 expression was reduced from baseline by a median percentage (95% confidence interval) of 88.7% (54.6%–93.0%) and 97.0% (77.5%–98.9%) for aggressive laBCC, 97.5% (80.3%–98.8%) and 95.0% (80.7%–97.5%) for nonaggressive laBCC, and 99.1% (96.4%–99.6%) and 99.3% (95.9%–99.9%) for mBCC in the 200 and 800 mg groups, respectively. Substantial repression of GLI1 was observed in patient subgroups stratified by age, sex, BCC cytological subtype, Eastern Cooperative Oncology Group performance status, lesion site, baseline number of BCCs, and prior radiotherapy. Results support further studies on the inhibition of Hedgehog pathway genes by sonidegib in patients with laBCC and mBCC.

Silencing glioma-associated oncogene homolog 1 suppresses the migration and invasion of hepatocellular carcinoma in vitro.

Hepatocellular carcinoma (HCC) is the fourth most common cause of cancer-associated death worldwide. Glioma-associated oncogene homolog 1 (Gli1) is a key component and functions as a reliable marker of Hedgehog signaling pathway activation. Previous studies have demonstrated that Gli1 serves important roles in the progression of various types of cancer, including HCC. However, its effect on HCC invasion and metastasis and the underlying mechanism remain to be elucidated. Small interference RNA was employed to silence the Gli1 gene in liver cancer cells. Reverse transcription-quantitative PCR and western blot analysis were performed to evaluate the mRNA and protein expression of Gli1, respectively. A series of assays, including Cell Counting Kit-8, adhesion, wound healing and Matrigel invasion were performed to investigate cell viability, adhesive, migratory and invasive capabilities of liver cancer cells, respectively. In addition, immunofluorescence staining was performed to determine the cellular localization of focal adhesion kinase (FAK), phosphorylated (p-)FAK and p-AKT. The mRNA and protein expression of Gli1 in liver cancer cells (HepG2 and SK-Hep1) were markedly decreased in a dose-dependent manner following Gli1-knockdown. Gli1 silencing significantly inhibited the adhesion, migration and invasion of SK-Hep1 cells. Additionally, knockdown of Gli1 markedly suppressed the expression of metalloproteinase (MMP)-2 and MMP-9. Furthermore, downregulation of Gli1 blocked the FAK/AKT signaling pathway. Gli1 serves significant roles in the migration and invasion of HCC cells through activation of the FAK/AKT signaling pathway and subsequent upregulation of MMP-2 and MMP-9 expression. Thus, Gli1 may be a potential protein target for the regulation of HCC migration and invasion.

Structural insight into the recognition between Sufu and fused in the Hedgehog signal transduction pathway

Hedgehog signaling plays a crucial role in embryogenesis and adult tissue homeostasis, and mutations of its key components such as Suppressor of fused (Sufu) are closely associated with human diseases. The Ser/Thr kinase Fused (Fu) promotes Hedgehog signaling by phosphorylating the Cubitus interruptus (Ci)/Glioma-associated oncogene homologue (Gli) family of transcription factors. Sufu associates with both Fu and Ci/Gli, but the recognition mechanism between Sufu and Fu remains obscure. Here, our structure of the N-terminal domain (NTD) of Drosophila Sufu (dSufu) in complex with the Sufu-binding site (SBS) of Fu reveals that both main-chain β sheet formation and side-chain hydrophobic interactions contribute to the recognition between Sufu and Fu, and point mutations of highly conserved interface residues eliminated their association. Structural comparison suggests that Fu and Ci/Gli bind on opposite sides of dSufu-NTD, allowing the formation of a Fu-dSufu-Ci ternary complex which facilitates the phosphorylation of Ci/Gli by Fu. Hence, our results provide insights into the Sufu-Fu recognition mechanism.

A Novel Therapeutic siRNA Nanoparticle Designed for Dual-Targeting CD44 and Gli1 of Gastric Cancer Stem Cells.

PurposeGastric cancer stem cells (CSCs) are important for the initiation, growth, recurrence, and metastasis of gastric cancer, due to their chemo-resistance and indefinite proliferation. Herein, to eliminate gastric CSCs, we developed novel CSC-targeting glioma-associated oncogene homolog 1 (Gli1) small interfering RNA (siRNA) nanoparticles that are specifically guided by a di-stearoyl-phosphatidyl-ethanolamine- hyaluronic-acid (DSPE-HA) single-point conjugate, as an intrinsic ligand of the CD44 receptor. We refer to these as targeting Gli1 siRNA nanoparticles.MethodsWe used the reductive amination reaction method for attaching amine groups of DSPE to aldehydic group of hyaluronic acid (HA) at the reducing end, to synthesize the DSPE-HA single-point conjugate. Next, targeting Gli1 siRNA nanoparticles were prepared using the layer-by-layer assembly method. We characterized the stem cellular features of targeting Gli1 siRNA nanoparticles, including their targeting efficiency, self-renewal capacity, the migration and invasion capacity of gastric CSCs, and the penetration ability of 3D tumor spheroids. Next, we evaluated the therapeutic efficacy of the targeting Gli1 siRNA nanoparticles by using in vivo relapsed tumor models of gastric CSCs.ResultsCompared with the multipoint conjugates, DSPE-HA single-point conjugates on the surface of nanoparticles showed significantly higher binding affinities with CD44. The targeting Gli1 siRNA nanoparticles significantly decreased Gli1 protein expression, inhibited CSC tumor spheroid and colony formation, and suppressed cell migration and invasion. Furthermore, in vivo imaging demonstrated that targeting Gli1 siRNA nanoparticles accumulated in tumor tissues, showing significant antitumor recurrence efficacy in vivo.ConclusionIn summary, our targeting Gli1 siRNA nanoparticles significantly inhibited CSC malignancy features by specifically blocking Hedgehog (Hh) signaling both in vitro and in vivo, suggesting that this novel siRNA delivery system that specifically eliminates gastric CSCs provides a promising targeted therapeutic strategy for gastric cancer treatment.

Selective Targeting of the Hedgehog Signaling Pathway by PBM Nanoparticles in Docetaxel-Resistant Prostate Cancer.

An abnormality in hedgehog (Hh) signaling has been implicated in the progression of prostate cancer (PCa) to a more aggressive and therapy-resistant disease. Our assessments of human PCa tissues have shown an overexpression of the Hh pathway molecules, glioma-associated oncogene homolog 1 (GLI-1), and sonic hedgehog (SHH). The effect of the natural compound thymoquinone (TQ) in controlling the expression of Hh signaling molecules in PCa was investigated in this study. We generated planetary ball-milled nanoparticles (PBM-NPs) made with a natural polysaccharide, containing TQ, and coated with an RNA aptamer, A10, which binds to prostate-specific membrane antigen (PSMA). We prepared docetaxel-resistant C4-2B-R and LNCaP-R cells with a high expression of Hh, showing the integration of drug resistance and Hh signaling. Compared to free TQ, A10-TQ-PBM-NPs were more effective in controlling the Hh pathway. Our findings reveal an effective treatment strategy to inhibit the Hh signaling pathway, thereby suppressing PCa progression.

Abstract 5025: Antifungal ketoconazole inhibits tumor-specific transcription factor tGLI1 leading to suppression of breast cancer stem cells and brain metastasis

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. 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 stem cells, however, FDA-approved therapies targeting this pathway have demonstrated limited efficacy in breast cancer clinical trials. Despite our current knowledge of breast cancer stem cells (BCSC), there remains a significant challenge in managing patients with metastatic breast cancer, underscoring the need to identify novel, actionable targets. 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 and terminal effector of the hedgehog pathway that is preferentially expressed in most BCBM samples and recurrent gliomas. Recent results established that tGLI1 promotes BCSC 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 (KCZ), an FDA-approved imidazole antifungal and component of previously studied anti-neoplastic regimens, selectively killed tGLI1-expressing breast cancer cells with increased efficacy against BCSC in vitro. tGLI1 knockdown abolished the ability of KCZ to target BCSC, indicating that KCZ-mediated suppression of BCSC is dependent on tGLI1. Intracardiac mouse studies showed KCZ selectively inhibited circulating tGLI1-positive breast cancer cells from developing into brain metastases and suppressed the progression of existing brain metastases. Mass spectrometry demonstrated KCZ effectively penetrated the blood-brain barrier (BBB) and blood-tumor barrier (BTB). Mechanistic studies suggest that KCZ-dependent cell kill is, in part, mediated through disruption of the functional tGLI1-STAT3 interaction. Furthermore, 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 BBB penetrance. Collectively, our preclinical results demonstrate that KCZ is an effective inhibitor of BCSC 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 KCZ treatment alters tGLI1 signaling in humans (NCT03796273). Citation Format: Daniel L. Doheny, Sherona R. Sirkisoon, Tadas Rimkus, Dongqin Zhu, Noah R. Aguayo, Alexandria Harrison, Marlyn Anguelov, Sara Manore, Fei Xing, Linda J. Metheny-Barlow, Kounosuke Watabe, Alexandra Thomas, Adrianna Henson Masters, Roy Strowd, Hui-Wen Lo. Antifungal ketoconazole inhibits tumor-specific transcription factor tGLI1 leading to suppression of breast cancer stem cells and brain metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5025.

Abstract 425: Novel transgenic mouse model of tGLI1 for in vivo studies of breast cancer and glioblastoma

Abstract Truncated glioma-associated oncogene homolog 1 (tGLI1) was first identified in 2009 as a gain-of-function isoform of the GLI1 transcription factor. tGLI1 isoform lacks exon 3 and part of exon 4, but retains all functional GLI1 domains to promote expression of GLI1 target genes. Since its discovery, we have shown that tGLI1 expression is unique to tumor tissue of breast and brain cancers. In glioblastoma (GBM), we show that tGLI1 is more potent in driving an aggressive phenotype compared to GLI1. We observed increased growth rate and vascularity in tGLI1-positive GBM xenografts along with increased tubule formation by surrounding vascular endothelial cells. We also found that tGLI1 expression in GBM promotes expression of heparanase, VEGF-A, VEGF-C, and TEM7, all of which can mediate GBM angiogenesis and vascularity. Most recently, we show that tGLI1 is enriched in mesenchymal GBM and that tGLI1 drives formation of glioma stem cells. Similarly, we have shown that tGLI1 drives breast cancer brain metastasis (BCBM) and supports radioresistance of breast cancer stem cells (CSCs). We found that tGLI1-expressing breast cancer cells secrete more VEGF-A and exhibit increased cell motility, invasion, and anchorage-independent growth. Additionally we observed that tGLI1 expression promotes breast CSCs through upregulation of stemness genes such as Nanog, Sox2, CD44, and Oct4, ultimately activating astrocytes. tGLI1-mediated astrocyte activation renders the brain more susceptible to and supportive of breast cancer cell invasion. Indeed, we show that tGLI1 loss suppresses breast CSCs and reduces BCBM in vitro, providing a mechanism for tGLI1-dependent breast cancer brain metastasis. While we have shown the oncogenic role of tGLI1 in vitro, no studies show whether tGLI1 is adequate for cell transformation or tumor initiation and progression in vivo. Thus, we generated the first conditional tGLI1 knock-in (KI) mouse model to study the role of tGLI1 in tumor initiation, progression, and metastasis. Our tGLI1-KI model was generated such that the human tGLI1 gene was inserted in an inverted orientation and flanked with loxP sites, yielding no tGLI1 product. Cre-mediated recombination reorients tGLI1 gene, allowing for its transcription. Cre-mediated knock-in of tGLI1 also induces concomitant luciferase gene transcription to allow for imaging of animals. To model GBM tumorigenesis, we crossed tGLI1-KI mice to the GFAP-cre model, inducing tGLI1 expression in astrocytes. We will also cross tGLI1-KI mice with MMTV-cre, inducing tGLI1-KI in mammary epithelial cells to model breast tumorigenesis. Our transgenic tGLI1-KI mouse is the first to model the oncogenic impact of tGLI1 in vivo. We anticipate our tGLI1-KI mouse model to expand our understanding of tGLI1-driven tumorigenesis and support therapeutic development in GBM and breast cancer. Citation Format: Angelina T. Regua, Dongqin Zhu, Austin Arrigo, Daniel Doheny, Hui-Wen Lo. Novel transgenic mouse model of tGLI1 for in vivo studies of breast cancer and glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 425.