Gli Proteins Research Articles

Mitochondrial Protection and Against Glutamate Neurotoxicity via Shh/Ptch1 Signaling Pathway to Ameliorate Cognitive Dysfunction by Kaixin San in Multi-Infarct Dementia Rats.

Multi-infarct dementia (MID), a prominent subtype of vascular dementia (VD), is responsible for at least 15 to 20 percent of dementia in the elderly. Mitochondrial dysfunctions and glutamate neurotoxicity due to chronic hypoperfusion and oxidative stress were regarded as the major risk factors in the pathogenesis. Kaixin San (KXS), a classic prescription of Beiji Qianjin Yaofang, was applied to treatment for “amnesia” and has been demonstrated to alleviate the cognitive deficit in a variety of dementias, including MID. However, little is known whether mitochondria and glutamate are associated with the protection of KXS in MID treatment. The aim of this study was to investigate the role of KXS in improving the cognitive function of MID rats through strengthening mitochondrial functions and antagonizing glutamate neurotoxicity via the Shh/Ptch1 signaling pathway. Our data showed that KXS significantly ameliorated memory impairment and hippocampal neuron damage in MID rats. Moreover, KXS improved hippocampal mitochondrial functions by reducing the degree of mitochondrial swelling, increasing the mitochondrial membrane potential (MMP), and elevating the energy charge (EC) and ATP content in MID rats. As expected, the concentration of glutamate and the expression of p-NMDAR1 were significantly reduced by KXS in the brain tissue of MID rats. Furthermore, our results showed that KXS noticeably activated the Shh/Ptch1 signaling pathway which was demonstrated by remarkable elevations of Ptch1, Smo, and Gli1 protein levels in the brain tissue of MID rats. Intriguingly, the inhibition of the Shh signaling pathway with cyclopamine significantly inhibited the protective effects of KXS on glutamate-induced neurotoxicity in PC12 cells. To sum up, these findings suggested that KXS protected MID rats from memory loss by rescuing mitochondrial functions as well as against glutamate neurotoxicity through activating Shh/Ptch1 signaling pathway.

Targeting Hedgehog Pathway and DNA Methyltransferases in Uterine Leiomyosarcoma Cells.

Uterine leiomyosarcoma (LMS) is an aggressive tumor that presents a poor prognosis, high rates of recurrence, and metastasis. Because of its rarity, there is no information available concerning LMS molecular mechanisms of origin and development. Here, we assessed the expression profile of Hedgehog (HH) signaling pathway markers and the effects of their pharmacological inhibition on uterine smooth muscle (UTSM), leiomyoma, and LMS cells. Additionally, we also evaluated the effects of DNMTs inhibition on LMS cell behavior. Cell proliferation, migration and apoptosis rates were evaluated by MTT, Scratch, and Annexin V assays, respectively. RNA expression and protein levels were assessed by qRT-PCR and Western blot. We found that SMO and GLIs (1, 2, and 3) expression was upregulated in LMS cells, with increased nuclear levels of GLI proteins. Treatment with LDE225 (SMOi) and Gant61 (GLIi) resulted in a significant reduction in Glis protein levels in LMS (p < 0.05). Additionally, the expression of DNMT (1, 3a, and 3b), as well as GLI1 nuclear expression, was significantly decreased after treatment with HH inhibitor in LMS cells. Our results showed that blocking of SMO, GLI, and DNMTs is able to inhibit LMS proliferation, migration, and invasion. Importantly, the combination of those treatments exhibited a potentiated effect on LMS malignant features due to HH pathway deactivation.

Xpo7 negatively regulates Hedgehog signaling by exporting Gli2 from the nucleus

Dynamic bidirectional transport between the nucleus and the cytoplasm is critical for the regulation of many transcription factors, whose levels inside the nucleus must be tightly controlled. Efficient shuttling across the nuclear membrane is especially crucial with regard to the Hedgehog (Hh) pathway, where the transcriptional signal depends on the fine balance between the amounts of Gli protein activator and repressor forms in the nucleus. The nuclear export machinery prevents the unchecked nuclear accumulation of Gli proteins, but the mechanistic insight into this process is limited. We show that the atypical exportin Xpo7 functions as a major nuclear export receptor that actively excludes Gli2 from the nucleus and controls the outcome of Hh signaling. We show that Xpo7 interacts with several domains of Gli2 and that this interaction is modulated by SuFu, a key negative regulator of Hh signaling. Our data pave the way for a more complete understanding of the nuclear shuttling of Gli proteins and the regulation of their transcriptional activity.

Gli affects the stemness and prognosis of epithelial ovarian cancer via homeobox protein NANOG.

Gli proteins are key transcription factors of the Hedgehog (HH) signaling pathway, which is associated with tumorigenesis and drug resistance. However, the role of the HH signaling pathway in epithelial ovarian cancer (EOC) remains unclear. Studies have demonstrated that in some tumors, homeobox protein NANOG (NANOG), a known stem cell marker, is a downstream effector of Gli. However, limited research has been conducted on the association between Gli and NANOG in EOC, particularly regarding their roles in the tumor stemness, such as tumor development, drug resistance and patient prognosis. Thus, the aim of the present study was to explore the aforementioned issues. In this study, Gli1, Gli2 and NANOG expression in EOC tissues was assessed using immunohistochemistry. Gene expression was also assessed using western blotting and reverse transcription-quantitative PCR in SKOV3 cells treated with a Gli inhibitor and an HH agonist. Furthermore, cell proliferation, colony-forming ability and cisplatin sensitivity were assessed using Cell Counting Kit-8 and colony formation assays. The results showed that both Gli1 and NANOG were associated with cisplatin resistance and EOC disease stage, while the nuclear expression of Gli2 was significantly associated with cisplatin resistance. Together, the expression of Gli and NANOG predicted poor patient prognosis. Targeting Gli with GANT61 impeded tumor proliferation, reversed cisplatin resistance and colony formation, and reduced NANOG expression. To conclude, Gli and NANOG may be effective indicators of platinum resistance and prognosis in EOC. Targeting Gli may reduce the stemness of ovarian cancer cell, which may be achieved via indirect targeting of NANOG.

Qingjie Fuzheng Granules regulates cancer cell proliferation, apoptosis and tumor angiogenesis in colorectal cancer xenograft mice via Sonic Hedgehog pathway.

Sonic Hedgehog (SHh) signaling pathway plays a critical role in cell proliferation, apoptosis, and tumor angiogenesis in various types of malignancies including colorectal cancer (CRC). Qingjie Fuzheng Granules (QFG) is a traditional Chinese medicinal formula, which has been clinically used in various cancer treatments, including CRC. In this study, we explored the potential molecular mechanisms of QFG treatment effects on CRC via the SHh pathway. A CRC HCT-116 xenograft mouse model was utilized for all experiments. Mice were treated with intra-gastric administration of 1 g/kg of QFG or saline 6 days a week for 28 days (4 weeks). Body weight, length and shortest diameter of the tumor were measured every 3 days. At the end of the treatment, the tumor weight was measured. TUNEL staining assays were used to detect tumor apoptosis. Western blot and immunohistochemistry (IHC) assays were used to detect the expression of relative proteins. In our results, QFG inhibited the increase of tumor volume and weight, and exhibited no impact on mouse body weight. Furthermore, QFG significantly decreased the expression of SHh, Smo and Gli proteins, indicating the action of SHh signaling. Consequently, the expression of pro-proliferative survivin, Ki-67, Cyclin-D1 and CDK4 were decreased and expression of anti-proliferative p21 was increased. The pro-apoptotic Bax/Bcl-2 ratio, cle-caspase-3 and TUNEL-positive cell percentage in tumor tissues were increased. Meanwhile, the pro-angiogenic VEGF-A and VEGFR-2 expression was down-regulated. QFG inhibited CRC cell proliferation and promoted CRC cell apoptosis and tumor angiogenesis in vivo through the suppression of SHh pathway, suggesting that QFG could be a potential therapeutic drug for CRC.

A Novel Nonsense GLI3 Variant Is Associated With Polydactyly and Syndactyly in a Family by Blocking the Sonic Hedgehog Signaling Pathway.

Polydactyly and syndactyly are congenital limb malformations that may occur either as non-syndromic or syndromic forms. In the present study, massively parallel sequencing was performed on a proband in a four-generation family with polydactyly and syndactyly to identify disease-causing variant(s). A pathogenic variant c.739C > T (p.Gln247∗) in the glioma-associated oncogene family zinc finger 3 (GLI3) gene was identified and co-segregated with the affected members of the family. Firstly, we examined GLI3 mRNA and GLI3 protein levels in peripheral blood mononuclear cells (PBMCs) of patients carrying this variant. The results showed that the truncated GLI3 p.Gln247∗ (c.739C > T) protein was detectable in patients and the GLI3 transcript and protein levels were not significantly altered in the PBMCs of patients compared with healthy controls. Furthermore, functional analysis showed that the truncated GLI3 p.Gln247∗ (c.739C > T) protein variant could lead to cytoplasmic accumulation of mutant protein and loss of ability to bind to the Suppressor of Fused protein. Alterations in protein expression levels of core components of the Sonic hedgehog signaling pathway were also observed. Our study shows that this novel GLI3 variant contributes to the malformations in this family and provides evidence for the mechanism by which GLI3 c.739C > T (p.Gln247∗) was implicated in the pathogenesis of polydactyly and syndactyly.

Trial in Progress: Glad-AML - a Randomized, Phase 2 Trial of Glasdegib with Two Standard Decitabine Regimens for Older Patients with Newly-Diagnosed, Poor-Risk Acute Myeloid Leukemia

Trial in Progress: Glad-AML - a Randomized, Phase 2 Trial of Glasdegib with Two Standard Decitabine Regimens for Older Patients with Newly-Diagnosed, Poor-Risk Acute Myeloid Leukemia

MiR-214-3p-Sufu-GLI1 is a novel regulatory axis controlling inflammatory smooth muscle cell differentiation from stem cells and neointimal hyperplasia

BackgroundInflammatory smooth muscle cells (iSMCs) generated from adventitial stem/progenitor cells (AdSPCs) have been recognised as a new player in cardiovascular disease, and microRNA-214-3p (miR-214-3p) has been implicated in mature vascular SMC functions and neointimal hyperplasia. Here, we attempted to elucidate the functional involvements of miR-214-3p in iSMC differentiation from AdSPCs and unravel the therapeutic potential of miR-214-3p signalling in AdSPCs for injury-induced neointimal hyperplasia.MethodsThe role of miR-214-3p in iSMC differentiation from AdSPCs was evaluated by multiple biochemistry assays. The target of miR-214-3p was identified through binding site mutation and reporter activity analysis. A murine model of injury-induced arterial remodelling and stem cell transplantation was conducted to study the therapeutic potential of miR-214-3p. RT-qPCR analysis was performed to examine the gene expression in healthy and diseased human arteries.ResultsmiR-214-3p prevented iSMC differentiation/generation from AdSPCs by restoring sonic hedgehog-glioma-associated oncogene 1 (Shh-GLI1) signalling. Suppressor of fused (Sufu) was identified as a functional target of miR-214-3p during iSMC generation from AdSPCs. Mechanistic studies revealed that miR-214-3p over-expression or Sufu inhibition can promote nuclear accumulation of GLI1 protein in AdSPCs, and the consensus sequence (GACCACCCA) for GLI1 binding within smooth muscle alpha-actin (SMαA) and serum response factor (SRF) gene promoters is required for their respective regulation by miR-214-3p and Sufu. Additionally, Sufu upregulates multiple inflammatory gene expression (IFNγ, IL-6, MCP-1 and S100A4) in iSMCs. In vivo, transfection of miR-214-3p into the injured vessels resulted in the decreased expression level of Sufu, reduced iSMC generation and inhibited neointimal hyperplasia. Importantly, perivascular transplantation of AdSPCs increased neointimal hyperplasia, whereas transplantation of AdSPCs over-expressing miR-214-3p prevented this. Finally, decreased expression of miR-214-3p but increased expression of Sufu was observed in diseased human arteries.ConclusionsWe present a previously unexplored role for miR-214-3p in iSMC differentiation and neointima iSMC hyperplasia and provide new insights into the therapeutic effects of miR-214-3p in vascular disease.

TMEM107 inhibits EMT and invasion of NSCLC through regulating the Hedgehog pathway.

BackgroundTransmembrane protein 107 (TMEM107) is a key regulator of the cilium composition and Hedgehog signaling. Lower TMEM107 gene copies are correlated with poor prognosis in non‐small cell lung carcinoma (NSCLC). However, TMEM107 protein expression, localization, and function in NSCLC remain unclear.MethodsWe first evaluated TMEM107 expression in 12 newly diagnosed cases of NSCLC and paired adjacent healthy tissues by western blotting. We then used an immunohistochemical method to detect TMEM107 expression in 106 paraffin‐embedded NSCLC and corresponding normal samples and analyzed its relationship with clinicopathological parameters. Moreover, we determined the impact of TMEM107 upregulation and downregulation on invasion, EMT and Hedgehog pathway in NSCLC cells.ResultsOur results showed that TMEM107 is localized in the cytoplasm and that its expression was lower in NSCLC. TMEM107 expression was positively correlated with cell differentiation and negatively correlated with lymph node metastasis. In A549 and HCC460 cells, downregulation of TMEM107 facilitated cell invasion and upregulated the expression of the Hedgehog pathway target protein Gli1, invasion‐associated proteins N‐cadherin, vimentin, MMP2, and MMP9, and epithelial‐mesenchymal transition (EMT), and inhibited the expression of E‐cadherin. Treatment with the Hedgehog pathway inhibitor GANT61 attenuated TMEM107‐knockdown–induced EMT and invasiveness.ConclusionsThese results indicate that TMEM107 inhibits EMT and invasion by negatively regulating Hedgehog signaling and that it is downregulated in NSCLC.Key points TMEM107 expression is lower in NSCLC tissues and correlates with poor prognosisTMEM107 inhibits invasion of NSCLC cellsTMEM107 inhibits EMT of NSCLC cellsDownregulation of TMEM107 activates the Hedgehog signaling pathwayDownregulation of TMEM107 promotes EMT and migration in NSCLC by activating the Hedgehog signaling pathway

Drosophila hedgehog can act as a morphogen in the absence of regulated Ci processing.

Extracellular Hedgehog (Hh) proteins induce transcriptional changes in target cells by inhibiting the proteolytic processing of full-length Drosophila Ci or mammalian Gli proteins to nuclear transcriptional repressors and by activating the full-length Ci or Gli proteins. We used Ci variants expressed at physiological levels to investigate the contributions of these mechanisms to dose-dependent Hh signaling in Drosophila wing imaginal discs. Ci variants that cannot be processed supported a normal pattern of graded target gene activation and the development of adults with normal wing morphology, when supplemented by constitutive Ci repressor, showing that Hh can signal normally in the absence of regulated processing. The processing-resistant Ci variants were also significantly activated in the absence of Hh by elimination of Cos2, likely acting through binding the CORD domain of Ci, or PKA, revealing separate inhibitory roles of these two components in addition to their well-established roles in promoting Ci processing.

Tumor-associated macrophages promote human hepatoma Huh-7 cell migration and invasion through the Gli2/IGF-II/ERK1/2 axis by secreting TGF-β1

ABSTRACT Aim In this study, we explored the ability of TAMs to affect the malignant phenotype of human hepatoma Huh-7 cells through the Gli2/IGF-II/ERK1/2 pathway. Methods The TAMs were characterized by flow cytometry and ELISA assays. Huh-7 cells were treated with conditioned medium of TAMs (TAMs-CM), and the proliferation, migration and invasion abilities were measured by CCK-8, Transwell and scratch assays. The levels of TGF-β1, Gli2, IGF-II and related proteins in the ERK1/2 pathway and the epithelial-mesenchymal transition (EMT) process were examined by RT-qPCR and western blot. Huh-7 cells were injected subcutaneously into nude mice with TAMs to explore the role of TAMs in tumor growth. Results The expression levels of TGF-β1, Gli2 and IGF-II and the cell proliferation, migration and invasion abilities were elevated in Huh-7 cells treated with TAMs-CM. TGF-β1 was upregulated in the conditioned medium and was found to be involved in the promotion of migration, invasion and the EMT of Huh-7 cells. The activation of TGF-β1 signaling increased the expression of Gli2. Knockdown of Gli2 decreased the expression of IGF-II and also reversed the promotional effect of the conditioned medium on migration, invasion and the EMT of Huh-7 cells. TGF-β1/Gli2/IGF-II signaling was shown to promote the malignant phenotype of Huh-7 cells by activating the ERK1/2 signaling pathway. Further, TGF-β1 knockdown attenuated the influence of TAMs on tumor growth in mouse model. Conclusion The TGF-β1 secreted by TAMs promotes the migration, invasion and EMT of human hepatoma Huh-7 cells through the Gli2/IGF-II/ERK1/2 pathway.

Overexpressed Gliotactin activates BMP signaling through interfering with the Tkv-Dad association.

Epithelial junctions ensure cell-cell adhesion and establish permeability barriers between cells. At the corners of epithelia, the tricellular junction (TCJ) is formed by three adjacent epithelial cells and generates a functional barrier. In Drosophila, a key TCJ protein is Gliotactin (Gli) where loss of Gli disrupts barrier formation and function. Conversely, overexpressed Gli spreads away from the TCJ and triggers apoptosis, delamination, and cell migration. Thus, Gli protein levels are tightly regulated and by two mechanisms, at the protein levels by tyrosine phosphorylation and endocytosis and at the mRNA level through microRNA-184. Regulation of Gli mRNA is mediated through a Gli-BMP-miR184 feedback loop. Excessive Gli triggers BMP signaling pathway through the activation of Tkv type-I BMP receptor and Mad. Elevated level of pMad induces micrRNA-184 expression which in turn targets the Gli 3'UTR and mRNA degradation. Gli activation of Tkv is not through its ligand Dpp but rather through the inhibition of Dad, an inhibitory-Smad. Here, we show that ectopic expression of Gli interferes with Tkv-Dad association by sequestering Dad away from Tkv. The reduced inhibitory effect of Dad on Tkv results in the increased Tkv-pMad signaling activity, and this effect is continuous through larval and pupal wing formation.

Novel GLI3 pathogenic variants in complex pre- and postaxial polysyndactyly and Greig cephalopolysyndactyly syndrome.

Polydactyly is a limb malformation and can occur as nonsyndromic polydactyly, syndromic polydactyly, or along with other limb defects. A few genes have been identified that cause various forms of syndromic and nonsyndromic polydactyly, of which GLI3 has been extensively explored. In the present study, GLI3 gene was screened by direct resequencing in 15 polydactyly cases with or without other anomalies. GLI3 screening revealed two novel pathogenic variants, NM_000168.6:c.3414delC [p.(H1138Qfs*68)] and NM_000168.6:c.1862C>T [p.(P621L)], found in two unrelated cases of familial complex pre- and postaxial polysyndactyly and sporadic Greig cephalopolysyndactyly syndrome (GCPS), respectively. The first pathogenic GLI3 variant, NM_000168.6:c.3414delC, causes premature protein truncation at the C-terminal domain of GLI3. Alternatively, the second pathogenic variant, NM_000168.6:c.1862C>T, lies in the DNA binding domain of GLI3 protein and may affect its hydrophobic interaction with DNA. Both pathogenic GLI3 variants had reduced transcriptional activity in HEK293 cells that likely had led to haploinsufficiency and, consequently, the clinical phenotypes. Overall, the present study reports a novel familial case of complex pre- and postaxial polysyndactyly and the underlying novel pathogenic GLI3 variant expanding the clinical criteria for GLI3 mutational spectrum to complex pre- and postaxial polysyndactyly. Furthermore, this study also reports a novel GLI3 pathogenic variant linked to GCPS, highlighting the known genotype-phenotype correlation.

Acute Alcohol Intoxication Impairs Sonic Hedgehog-Gli1 Signaling and Activation of Primitive Hematopoietic Precursor Cells in the Early Stage of Host Response to Bacteremia.

Activation of hematopoietic stem cells [HSCs, lineage(lin)- stem cell growth factor receptor (c-kit)+ stem cell antigen-1(Sca-1)+ , or LKS cells in mice] is critical for initiating the granulopoietic response. This study determined the effect of alcohol exposure on sonic hedgehog (SHH) signaling in the regulation of HSC activation during bacteremia. Acute alcohol intoxication was induced in mice by intraperitoneal (i.p.) injection of 20% alcohol (5g alcohol/kg body weight). Control mice received i.p. saline. Thirty minutes later, mice were intravenously (i.v.) injected with Escherichia coli (E.coli, 1 to 5×107 CFUs/mouse) or saline. SHH expression by lineage-negative bone marrow cells (BMCs) was significantly increased 24hours after E.coli infection. Extracellular signal-regulated kinase 1/2 (ERK1/2)-specificity protein 1 (Sp1) signaling promotes SHH expression. ERK1/2 was markedly activated in BMCs 8hours following E.coli infection. Alcohol suppressed both the activation of ERK1/2 and up-regulation of SHH expression following E.coli infection. E.coli infection up-regulated GLI family zinc finger 1 (Gli1) gene expression by BMCs and increased Gli1 protein content in LKS cells. The extent of Gli1 expression was correlated with the activity of proliferation in LKS cells. Alcohol inhibited up-regulation of Gli1 expression and activation of LKS cells in response to E.coli infection. Alcohol also interrupted the granulopoietic response to bacteremia. These data show that alcohol disrupts SHH-Gli1 signaling and HSC activation in the early stage of the granulopoietic response, which may serve as an important mechanism underlying the impairment of immune defense against bacterial infection in host excessively consuming alcohol.

GLI1/GLI2 functional interplay is required to control Hedgehog/GLI targets gene expression.

The Hedgehog-regulated transcription factors GLI1 and GLI2 play overlapping roles in development and disease; however, the mechanisms underlying their interplay remain elusive. We report for the first time that GLI1 and GLI2 physically and functionally interact in cancer cells. GLI1 and GLI2 were shown to co-immunoprecipitate in PANC1 pancreatic cancer cells and RMS13 rhabdomyosarcoma cells. Mapping analysis demonstrated that the zinc finger domains of both proteins are required for their heteromerization. RNAi knockdown of either GLI1 or GLI2 inhibited expression of many well-characterized GLI target genes (BCL2, MYCN, PTCH2, IL7 and CCND1) in PANC1 cells, whereas PTCH1 expression was only inhibited by GLI1 depletion. qPCR screening of a large set of putative canonical and non-canonical Hedgehog/GLI targets identified further genes (e.g. E2F1, BMP1, CDK2) strongly down-regulated by GLI1 and/or GLI2 depletion in PANC1 cells, and demonstrated that ANO1, AQP1 and SOCS1 are up-regulated by knockdown of either GLI1 or GLI2. Chromatin immunoprecipitation showed that GLI1 and GLI2 occupied the same regions at the BCL2, MYCN and CCND1 promoters. Furthermore, depletion of GLI1 inhibited GLI2 occupancy at these promoters, suggesting that GLI1/GLI2 interaction is required for the recruitment of GLI2 to these sites. Together, these findings indicate that GLI1 and GLI2 co-ordinately regulate the transcription of some genes, and provide mechanistic insight into the roles of GLI proteins in carcinogenesis.

GANT61 and Lithium Chloride Inhibit the Growth of Head and Neck Cancer Cell Lines Through the Regulation of GLI3 Processing by GSK3β.

Several signaling pathways are aberrantly activated in head and neck squamous cell carcinoma (HNSCC), including the Hedgehog-Gli (HH-GLI), WNT, EGFR, and NOTCH pathways. The HH-GLI pathway has mostly been investigated in the context of canonical signal transduction and the inhibition of the membrane components of the pathway. In this work we investigated the role of downstream inhibitors GANT61 and lithium chloride (LiCl) on cell viability, wound closure, and colony forming ability of HNSCC cell lines. Five HNSCC cell lines were treated with HH-GLI pathway inhibitors affecting different levels of signal transduction. GANT61 and LiCl reduce the proliferation and colony formation capabilities of HNSCC cell lines, and LiCl has an additional effect on wound closure. The major effector of the HH-GLI signaling pathway in HNSCC is the GLI3 protein, which is expressed in its full-length form and is functionally regulated by GSK3β. LiCl treatment increases the inhibitory Ser9 phosphorylation of the GSK3β protein, leading to increased processing of GLI3 from full-length to repressor form, thus inhibiting HH-GLI pathway activity. Therefore, downstream inhibition of HH-GLI signaling may be a promising therapeutic strategy for HNSCC.

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.

Fine-Tuning of GLI Activity through Arginine Methylation: Its Mechanisms and Function

The glioma-associated oncogene (GLI) family consists of GLI1, GLI2, and GLI3 in mammals. This family has important roles in development and homeostasis. To achieve these roles, the GLI family has widespread outputs. GLI activity is therefore strictly regulated at multiple levels, including via post-translational modifications for context-dependent GLI target gene expression. The protein arginine methyl transferase (PRMT) family is also associated with embryogenesis, homeostasis, and cancer mainly via epigenetic modifications. In the PRMT family, PRMT1, PRMT5, and PRMT7 reportedly regulate GLI1 and GLI2 activity. PRMT1 methylates GLI1 to upregulate its activity and target gene expression. Cytoplasmic PRMT5 methylates GLI1 and promotes GLI1 protein stabilization. Conversely, nucleic PRMT5 interacts with MENIN to suppress growth arrest-specific protein 1 expression, which assists Hedgehog ligand binding to Patched, indirectly resulting in downregulated GLI1 activity. PRMT7-mediated GLI2 methylation upregulates its activity through the dissociation of GLI2 and Suppressor of Fused. Together, PRMT1, PRMT5, and PRMT7 regulate GLI activity at multiple revels. Furthermore, the GLI and PRMT families have strong links with various cancers through cancer stem cell maintenance. Therefore, PRMT-mediated regulation of GLI activity would have important roles in cancer stem cell maintenance.

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

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

GLI1 activation is a key mechanism of erlotinib resistance in human non-small cell lung cancer.

Lung cancer is the leading cause of cancer-associated death worldwide. In recent years, the advancement of epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) targeted therapies has provided clinical benefits for lung cancer patients with EGFR mutations. The response to EGFR-TKI varies in patients with lung cancer, and resistance typically develops during the course of the treatment. Therefore, understanding biomarkers which can predict resistance to EGFR-TKI is important. Overexpression of GLI causes activation of the Hedgehog (Hh) signaling pathway and plays a critical role in oncogenesis in numerous types of cancer. In the present study, the role of GLI1 in erlotinib resistance was investigated. GLI1 mRNA and protein expression levels were determined using reverse transcription-quantitative PCR and immunohistochemistry (IHC) in lung cancer cell lines and tumor specimens, respectively. GLI1 mRNA expression levels were found to be positively correlated with the IC50 of erlotinib in 15 non-small cell lung cancer (NSCLC) cell lines. The downregulation of GLI1 using siRNA sensitized lung cancer cells to the erlotinib treatment, whereas the overexpression of GLI1 increased the survival of lung cancer cells in the presence of erlotinib, indicating that Hh/GLI activation may play a critical role in the development of TKI resistance in lung cancer. Combined treatment with erlotinib and a GLI1 inhibitor reduced the cell viability synergistically. A retrospective study of patients with NSCLC treated with erlotinib revealed that those with a high IHC score for GLI1 protein expression had a poorer prognosis. These results indicated that GLI1 is a key regulator for TKI sensitivity, and patients with lung cancer may benefit from the combined treatment of TKI and GLI1 inhibitor.